Antimicrobial peptides with therapeutic potential from skin secretions of polyploid frogs of the Pipidae family

Milena Mechkarska Thesis committee

Promotor

Prof. dr. Jerry M. Wells Professor of Host Microbe Interactomics, Wageningen University

Co-promotor

Prof. J. Michael Conlon Professor of Biochemistry College of Medicine and Health Sciences (CMHS) United Arab Emirates University (UAEU), Al Ain, UAE

Other members Prof. Sylvie Dufour, Museum National d’Histoire Naturelle, Paris, France Prof. Eric W. Roubos, Radboud University, Nijmegen, The Netherlands Prof. dr. ir. Huub Savelkoul, Wageningen University Prof. Liliane Schoofs, KU Leuven, Belgium

This research was conducted under the auspices of the Graduate School of Wageningen Institute of Animal Sciences (WIAS) Antimicrobial peptides with therapeutic potential from skin secretions of polyploid frogs of the Pipidae family

Milena Mechkarska

Thesis submitted in fulfillment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. dr. M.J. Kropff, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Tuesday 7 May 2013 at 4 p.m. in the Aula. Milena Mechkarska Antimicrobial peptides with therapeutic potential from skin secretions of polyploid frogs of the Pipidae family, 226 pages.

Thesis, Wageningen University, Wageningen, NL (2013) With references, with summaries in English and Dutch

ISBN: 978-94-6173-550-8 To my family:

The ones who love, support, care and forgive

Table of contents

Chapter 1 General introduction 9

Chapter 2 Antimicrobial peptides with therapeutic potential from frog skin 47 secretions of the Marsabit clawed frog Xenopus borealis (Pipidae)

Chapter 3 Purification and properties of antimicrobial peptides from skin 65 secretions of the Eritrea clawed frog Xenopus clivii (Pipidae)

Chapter 4 Genome duplications within the Xenopodinae do not increase the 83 multiplicity of antimicrobial peptides in Silurana paratropicalis and Xenopus andrei skin secretions

Chapter 5 Peptidomic analysis of skin secretions demonstrates that the 107 allopatric populations of Xenopus muelleri (Pipidae) are not conspecific

Chapter 6 The hymenochirins: a family of antimicrobial peptides from the 127 Congo dwarf clawed frog Hymenochirus boettgeri (Pipidae)

Chapter 7 Hybridization between the tetraploid African clawed frogs 147 Xenopus laevis and Xenopus muelleri (Pipidae) increases the multiplicity of antimicrobial peptides in the skin secretions of female offspring

Chapter 8 General discussion 169

Summary 203

Samenvatting 209

Acknowledgements 215

Personalia 219 Curriculum Vitae List of publications

Chapter 1

General introduction

Milena Mechkarska

“I don’t see no p’ints about that frog that’s any better’n any other frog.” Mark Twain, 1867, The Celebrated Jumping Frog of Calaveras County Chapter 1 different mechanisms (Fig. 1). Bacteria areeither non-responsive or resistan alteration of target .Kindl target. Key: enzyme (proteases) ;antibiotic (5) Protectionof intracellular ta threat to public health.This antibiotic resistance had alreadybegun toemerge significant discoverymedicine. in However, ev most single to be the considered been have Antibiotics antibiotic. an to exposure survive modification of(4)Over-exp membrane fluidity; intracellular molecules; (3)Reduction ofpermeability by changing trans-membrane potential or production of proteases; (2) Target alteration suchascomposition ofthemembrane, LPS or Fig. 1. heavily on healthcare costs commonly usedantibiotics, which causeincr A global problem is rapidly evolving and expanding to the point of posing a serious Worldwide spread of infections caused Antibiotic resistance is atype of drug resi Bacterial resistance mechanisms toantibiotics. (1) Enzymatic degradation as aresult of y y provided by Prof. T. Pal(2012).

(Livermore, 2009). 2009). (Livermore, is theemergence of pathogenic rget; (6)Overproduction of target; and (7)Bypassing theaction on E 10 ression ofefflux pumps by multidrug resistant bymultidrug bacteria t to the action of antibiotics by arange of eased morbidity andmortality, andimpact stance in which amicroorganism isableto ; intracellular ; protection target ; en at the early stageaftertheirdiscovery, en atthe ( Rammelcamp and Maxon, Maxon, 1942). and Rammelcamp bacteria andfung causing immediate export; i resistant to

Chapter 1 sistant bacteria are are sistant bacteria (MRSA) quickly quickly (MRSA) new antibiotics despite the the despite antibiotics new is summarized in Fig. 2. S. aureus levels of pre-existing antibiotic- . This microorganism is carried by microorganism . This S. aureus acterial genes, or even more frequently by by frequently more or even genes, acterial in sharing of resistance between bacterial between of resistance in sharing the emergence of antibiotic resistance “the “the resistance antibiotic of emergence the iers of the host defence system and it can can and it system defence host the of iers urlenda and Grinholc, 2012; and references tible to only oneor two categories) and PDR the population(s).the The re ., 2012). MDR is defined as acquired non- as acquired defined is MDR ., 2012). 11 posons or acquired by natural competence for competence by natural acquired or posons extensively drug-resistant (XDR) and pandrug- and (XDR) drug-resistant extensively documented. The problem is made worse by the the by worse is made problem The documented. industry involving release involving industry of Staphylococcus aureus ., 2010).., Although there were low possesses a wide spectrum of virulence factors (Feng et al., 2008; Plata Plata 2008; al., et (Feng factors of virulence spectrum a wide possesses was was one of the first bacteria in which penicillin wasresistance found S. aureus The development of antibiotic resistance by resistance of antibiotic development The One of the major drug-resistant pathogens human of theOne major is a strainGram-positive of the Resistance can arise either by mutations of b of mutations can arise either by Resistance (Rammelcamp and1942) Maxon, andspecific the role of penicillinase was subsequently use of clinical the necessitated This 1945). Dietz, and Bondi 1944; (Kirby, identified penicillinase-resistant derivatives such as methicillin as the antibiotics of choice.However, reports the describing emergence of methicillin-resistant et al., 2009) that enable it to bypass the barr bypass the it to enable et al., 2009) that survive hostile environmental conditions due its extraordinary to versatility and adaptability to antibiotic pressure1998; (Lowy, Lowy, 2003). S. aureus fact that resistant strains have already been already been have fact that resistant strains (Cohen, populations human among travel and international regional for trend increasing 1992; Tomasz, 1994; Swartz,1997). This makes colonizing by infections local causes It population. human of the third one approximately mucous which and membranes skin can, in later stages, develop infections, into systemic is infections of group second The children. and adults both in and sepsis bacteremia as such (K diseases superantigen-mediated and toxin- therein). species is the widespread use of antibiotics (Hawkey and Jones, 2009). Examples of the the of Examples 2009). Jones, and (Hawkey of antibiotics use widespread the is species misuse of antibiotics includesales over their the counter without a prescription; in practices unsound and feed; livestock to addition doctors; by prescription inappropriate manufacturing the pharmaceutical 2009). (Livermore, evolution” of principle Darwin’s of example eloquent most coccus: aerobic, non-motile resistant bacteria (Nelson, 2009; Caldwell and Lindberg, 2011), the majorfactor in and strains MDR of selection and development classified into multidrug-resistant (MDR), (MDR), multidrug-resistant classified into et al (Magiorakos resistant (PDR) categories susceptibility to at least one is agentXDR categories, in or antimicrobial three more definednon-susceptibility as to at least oneagent in all butor twofewer antimicrobial suscep bacterial isolates remain categories (i.e. is defined as non-susceptibility to all agents in all antimicrobial categories (Magiorakoset al., 2012).resistance The genesfor to antibiotics, the like antibioticsare themselves, et al (Donadio ancient genes may be mobilized between the same or or same the between be mobilized horizontalAntibiotic may gene transfer. resistance genes species by plasmids or even trans different via evolves that resistance antibiotic for gene a Thus, DNA. exogenous by transformation natural be selectionor across shared in may Chapter 1 gentamicin (Feng etal., 2008). (Feng gentamicin may not beresponsive to macrolides, clindamycin, fluoroquinolones, tetracycline and (Bozdogan et al., 2003). Thus, apart from being resistant to all provided by Dr. A. Sonnevend (2012). infections ispresentedinparallel. CA–co of methicillin-resistant Fig. 2. Timeline for theintroduction ofantibiotics intomedicalpractice. quic also antibiotics classes newer of poses asignificant problemworldwide (Kreiswirt ha MRSA Since then, 1961). (Jevons, followed bacteria such as MRSA haveintroducedsuch as been bacteria Livermore, 2009). and Woodford and Daum, 2007; fasciitis(Boyle-Vavra necrotizing for rapidly progressive, fatal diseases, including necrotizing pneumonia, severe sepsis and lesions. and pus-filled other boils, abscesses, procedure (e.g. dialysis, surgery, catheter) and usually involve skin infections, such as occur in otherwise healthy people who have not been recently hospitalized or had amedical community-acquired MRSA (CA-MRSA) hasbecome prevalent. CA-MRSA infections (Chambers and Deleo, 2009; Rosenthal et al., 2010); however, in the past 10 years, MRSA was associated predom Although effective new types of antibiotics against multidrug-resistant Gram-positive Gram-positive multidrug-resistant against newtypesofantibiotics effective Although The epi Penicillin 1940’s penicillinase 1940 -1960 Spread of plasmid- demiology of infections caused by MRSA is rapidly changing. Originally, borne Methicillin S. aureus 1960 reported MRSA 1961 First (MRSA) with thesubsequentch (MRSA)with inantly with with hospital-acquir inantly new classes classes new kly caused appearance ofresistant antibiotics Several Several 1980 Epidemic MRSA mmunity-acquired; HA – hospital-acquired. HA– Kindly mmunity-acquired; strains reported reported strains from 12 in the UK Frequently though, CA-MRSA is responsible CA-MRSA isresponsible though, Frequently 1980’s

hospitals s become endemicmanyin countries and or are in clinical trials (Kurlenda and trials(Kurlenda or are clinical in h etal.,1993).The subsequent usageof First CA-MRSA

reported from Australia and infections 1990’s USA ange inepidemiology ofMRSA ed infections (HA-MRSA) infections ed -lactam antibiotics, MRSA β-lactam antibiotics, soitdinfections associated The history ofthe emergence many countries many CA-MRSA causes widespread in 90-2000 1990 - HA-MRSA HA-MRSA infections healthcare ahcontinent each reported from CA-MRSA infections S. aureus 2012 strains Chapter 1 , , and other other , and Pseudomonas A. baumannii an increasingly an increasingly

), -lactamase (VIM type (VIM type β-lactamase β-lactamantibiotics are K. pneumoniae , survivefor a long on time acteria havea greatacteria ability for -lactamase (ESBL)-producing (ESBL)-producing β-lactamase coli

E. wounds, catheters and ventilators. and ventilators. wounds, catheters inoglycosides, cephalosporins, and and cephalosporins, inoglycosides, -lactam antibioticswithβ-lactam the exception -lactamases which, depending on the the on depending β-lactamases which, emases] (Bradford et al., 2004) and thusemases] and Pachón, 2008). During the last decade, the last decade, 2008). During and Pachón, ). The prevalence of ESBL-producing strains strains of ESBL-producing prevalence ). The ative bacteria threatenbacteria ative hospitalized mainly timicrobials via a wide range of mechanisms, of mechanisms, range via a wide timicrobials 13 -lactamase enzymes that are mostly encoded by encoded that are mostly enzymes -lactamase β -lactamase inhibitors, and monobactams (Jacoby (Jacoby monobactams and inhibitors, β-lactamase sistant HA- and CA-pathogens is is CA-pathogens and HA- sistant -lactam antibioticsβ-lactam (Jacobyand Munoz-Price, 2005). that are resistant to all that are resistant carbapenemase (KPC), metallo- (KPC), carbapenemase such as extended-spectrum as extended-spectrum such Escherichia coli and Klebsiella pneumoniae Klebsiella and coli Escherichia enter the body through spp. have emerged as an important pathogen in the hospital hospital the in pathogen as an important emerged have spp. ough the production of -lactam antibiotics.-lactam (MDRAB) have emerged to become a major phenotype in hospitals hospitals in phenotype a major become to emerged have (MDRAB) K. pneumoniae K. spp. are resistant that to all am Acinetobacter long thoughtbe relatively to avirulent (Talbotet al., 2006).It is A. baumanii The most common mechanism of resistance among among of resistance mechanism common The most One of the emerging multidrug re multidrug of the emerging One Enterobacteriaceae (primarily (primarily Enterobacteriaceae Pulmonary infections, particularly hospital-acquired pneumonia, caused by these these by caused pneumonia, hospital-acquired particularly infections, Pulmonary bacteria carbapenemase-producing particularly difficult(Kollef, b toGram-negative 2010). treat exchanging genetic material among strains of the same species and even among different different among even and species same of the strains among material genetic exchanging species. Grinholc, 2012), the situation regarding options treatment new infections for produced by pathogens MDR Gram-negative is less encouraging (Giamarellou and Poulakou, Poulakou, and (Giamarellou and Acinetobacter baumannii encouraging less aeruginosa is 2009).drug-resistant In general, Gram-neg are can weak. These bacteria systems immune whose patients and surfaces in the hospital was which Enterobacteriaceae is thr of group a heterogeneous are The ESBLs of majority the hydrolyse and genes plasmid-bourne of degrees varying and convey enzymes distinct 532 number now ESBLs of carbapenems. resistance to cephalosporins, penicillins, 2006 et al., Bhavnani 2005; and Munoz-Price, age. patient and hospital, of type areas), urban in (particularly geography by varies Importantly, there havebeenreports of ESBL-producingstrains that also produce [ carbapenemases and NDM1 type) and oxacillinase(OXA) carbapen resistant to all β became worldwidehigh rate (Dijkshoorn with a mortality 2008). et al., 2007; Peleg et al., enzyme, inactivate a wide range of range wide a inactivate enzyme, fluoroquinolones (Landman et al., 2002; Vila fluoroquinolones (Landman MDR settings and even in war zones. In many regions it is common to find strains of strains find to common is it regions many In zones. war even in and settings Acinetobacter common cause ofventilator-associated pneumonia. This hasmicroorganism rapidly available an to currently resistance developed and carbapenemases, ESBLs, enzymes, aminoglycoside-modifying of production including through changesouter in proteins, membrane penicillin-bindingproteins, and topoisomerases (Gales 2001; et al., and2006). Szabo, Bonomo is not It surprising, therefore, that Chapter 1 infection. In a healthy host, host, a In healthy infection. muchcells are more virulent than White 1988). (Soll, niches This switching of phenotypes contributes to abetter adaptation of phenotype factors of virulence major The 2007). Bachewich, and (Whiteway hyphae totrue yeast, pseudohyphae (Janeway and Medzhitov, 2002; Beutler, 2004). The key features of an innate immune inorder required urgently antibiotic combined withPDR their pathogens In view ofthe successful spread and the not albicans most tractsof the genitourinary and gastrointestinal the 2011). McCollister, of response biofilm formation or the emergence of small-colony-variants may beimportant in the (Lambert, 2002).Recentstudieshave shownth channels porin in areduction to leading mutations efflux andcellwall pumps, multidrug of production including mechanisms, (McCallum,2010). However, it resistance, itsintrinsic to addition treat andmay belife-threatening, difficultto areoften infections itscellwall(Lambert, Consequently, 2002). permeability of intrinsically susceptible to onlyalimitednumber ofantibacterial agen tract infections, and bacteremia (Talbot et al., 2006). Importantly, this pathogen is that is responsible for awide range of severe HA-infections, including pneumonia, urinary increasing population at risk of inva at of risk population that thereisaneverincreasing infection or receiving immunosuppressive therapy for cancers and organ transplants, mean significantly. Increasing numbers of patients with immune deficiencies, such as HIV of resistant strains are emerging (S towards antifungal agents is less frequent than in many other species, an increasing number immunocompromised patients (Eggimanal., et 2003) Innate immunity is the initial response of a host in defending against pathogen invasion invasion pathogen against defending in ofahost response initial the immunity is Innate immunityInnate and antimicrobial peptides(AMPs) Another important opportunistic human pathogen, an inhabitant ofthe oral cavity and P. aeruginosa In thepast decade, the situation regard . Thismicroorganism

switching between two distinct types of cells, white and opaque (Yang, 2003). C. albicans P. aeruginosa treatments (Cornelis, 2008; antibiotic to populations , invasive Gram-negativeopportunistic prevalent, pathogenis ahighly are proteinase secretion, hyphal formation, andadhesion and to control the infections. the infections. to control C. albicans

is a can causeserious disseminated infections particularly if the causative strain is drug resistant. In resistant. isdrug strain causative ifthe particularly lutskyet1985; al., Soll, 2002). P. aeruginosa polymorphic fungus, which transitions from budding β persists in equilibrium with the host’s microflora host’s equilibrium the persists in with -lactamases and carbapenemases, upregulation of able virulence capacity ofthe MDR, XDR and 14 ing antimicrobial resistance hasworsened resistance, novel treatment approaches are at phenotypic resistance associated with has also acquired resistance viamultiple . Although resistance of sive bacterialand

opaque cells after intravenous cells afterintravenous opaque healthy people, is C. albicans ts becauseofthelow

in critically ill or criticallyill in fungal infection. to new host new host to C. albicans Candida Candida

Chapter 1 proline, particularly acids mescale, mescale, rather than over a -sheet conformation; β-sheet amino nature nature and the most intensively studied composed of 8 - 50 amino acid residues, of 8 composed - 50 acid amino residues, divided divided into three major classes based on t-defense t-defense effector molecules present in entalization of cells and homeostasis. The certain rum), rum), rapid (often within minutes to hours) Yeaman and Yount, 2003). Membranes are the are Membranes Yount, 2003). and Yeaman of 15

adapt on an evolutionary ti percentage high a tryptophane. -helical AMPs with biological membranes membranes biological with AMPs -helical α with and -helical peptides are the most common in α peptideswith disulfidethree bonds adopting flat a α–helical conformation; cationic peptides with and without cysteine amino acids that adopt an amphipathic peptides with and without cysteine amino acids cationic arginine peptides The General of characteristics AMPs AMPs are typically small molecules generally Structures of AMPs Although exceptions exist, the AMPs can be One One of the central components of the innate immune response is the production of Interaction of AMPs target the cell membranes and their action ultimately leads to bacterial death i) iii) ii) response are that it is non-specific (broad-spect and conserved in its pattern of recognition, meaning that this system responds to pathogens in a generic way. Innate immunity can be constitutively expressed or be characterized inducible by and mechanisms is that lifespan. single antimicrobial peptides (AMPs). AMPs are virtually hos all life forms, including vertebrates, defense. host in importance invertebrates, evolutionary their emphasizing and plants (Zasloff, 2002), mostly in the usual L configuration, with a net positive charge Pukala of et +2 al., to 2006; Hancock +6 and (reviewed Sahl, in 2006; Conlon, 2011). They are high cationic percentage due to of a the basic amino acids, arginine, Diamond, 2000; lysine, Boman, and 1998; histidine Boman, (Hancock 2003). and Typically, more 2000). Hancock, and than (Scott hydrophobic are acids amino 50% of an AMP’s (Matsuzaki et al., 1989; Williams et al., 1990; Matsuzaki et al., 1991; Grantet Vaz al., Gomez 1993; Blazyk et al., 2001; et al., 1992; of the three classes. of the three fundaments maintaining life through compartm their three dimensional structure (Boman, 2003): 2003): (Boman, structure dimensional three their Chapter 1 open, glycerophosphocholine (SM)andst (PC),sphingomyelin neutral (right) are cardiolipin (CL), glycerophosphogl with that ofhuman erythrocytes. Me Fig. 3. 3). (Fig. 2005) mostimportant difference between eukaryotic and prokaryoticmembranes (Fahy et al., the constitutes composition their and andpolarity size,charge, varyin headgroups lipid glycerophosphocholines, glycerophosphoethanolamines, like lipids ofzwitterionic proportion high mammalian cytoplasmicmembranes. On the other hand, eukaryotic membranes possess a and as glycerophosphoglycerols the intrinsic characteristics of properties, mainly their cationic and amphipathic nature (Fig. 4) (Chapter 2004). Kalinowski, and Koronkiewicz 2003; al., layers Almeida et lipid (de ofthe integrity the and dynamics for areimportant which lipids erythrocyte (fromerythrocyte Yeamanand Yount, 2003). Pr The mode of action of AMPs on membranes is dictated by their physicochemical physicochemical their by isdictated membranes of on AMPs mode of action The E. coli E. Asymmetric lipid composition of cytoplasmic membranes from several bacteria compared compared bacteria several from membranes ofcytoplasmic composition lipid Asymmetric okaryotic cytoplasmic membranes contain a high proportion of anionic lipids, such such lipids, anionic of a proportion high contain cytoplasmicmembranes okaryotic ; horizontal hatching,

sphingomyelin (or ceramide phosphocholines) and the the sterol and phosphocholines) ceramide (or sphingomyelin the pathogens (Fig. 5). (Fig. pathogens the S. aureus mbrane constituentsranging from an cardiolipins, whereas these ; shaded, 16 B. subtilis ycerol (PG),glycerophos ycerol erols (cholesterolorergesterol) (ST). Key: ; checkered, lipids are rarely found in arerarelyfound lipids ionic (left) tozwitterionic or C. albicans phoethanolamine (PE), 8 ), aswellby ; solid,human Chapter 1 ity and hydrophobicity and magnesium ions and magnesium conditions, the AMPs eria (Fig. 5, step 1). In Gram-negative 1). In Gram-negative 5, step eria (Fig. -helicity), amphipathic aqueous environment but adopt a stable but adopt a stable environment aqueous α vant target of AMPs is the cytoplasmic AMPs vant target of from several interdependent physicochemical physicochemical interdependent several from respiring prokaryotic cells but not in not but cells prokaryotic respiring selective electrostatic interactions between selective electrostatic interactions between ort processes. Thus, the presence of large of large presence the Thus, ort processes. re binding to the membrane (Urrutia et al., al., et (Urrutia membrane the to binding re rted that under certain 17 lipopolysaccharide (LPS) al determinants in AMPs. The activity and selectivity of the the selectivity of and al determinants in AMPs. activity The embrane architectures prevail in bact prevail architectures embrane Interrelationship among structur Interrelationship ) is followed by a 2 nm thick peptidoglycan wall that protects the cytoplasmic cytoplasmic the protects that wall peptidoglycan thick nm 2 a by followed ) is . Two m Bacterial are characterized bymembranes a highly asymmetric lipid charge in an are usually unstructured The AMPs 2+ -helical conformation upon contact with the lipid bilayer. At low concentrations the AMPs AMPs the concentrations low At bilayer. lipid the with contact upon conformation -helical (modified from Yeaman and Yount, 2003). Yount, 2003). Yeaman and (modified from peptides on phospholipid membranes originates membranes originates on phospholipid peptides ( conformation charge (cationicity), as such parameters Fig. 4 (Mg bacteria, an outer membrane, rich in PG, rich in outer membrane, bacteria, an can aggregate or assemble together even befo even together or assemble can aggregate distribution generating a transmembrane potential. potential This electrical is necessary for and transp catalytic energy-dependent several membrane (Fig. 5, step 3). 3). 5, step (Fig. membrane inside-negative transmembrane potentials in 1995a) leads to erythrocytes (Matsuzaki et al., AMPs. and bacterial membranes α repo has been It surface. on the membrane lie membrane. In Gram-positive bacteria, the cytoplasmic membrane is directly surrounded by by surrounded directly is membrane cytoplasmic the bacteria, Gram-positive In membrane. a 2040 to nm thick peptidoglycanwall featuring anionic lipoteichoic acid groups that The rele surface. charged provide a negatively Chapter 1 Fig. 5. according to threemodels: membrane structure appear and leakage occurs through themembrane in differentmanners 1989). After reaching a“threshold” concentration size increases. Progressively, new AMPs are recruited and, through aprocess ofself-aggregation, the pore the hydrophilic and the bilayer of region core lipid the with align peptides inserted ofthe regions hydrophobic the which in membrane, assembly peptide packed tightly The pore. itsorientation assembly changes peptide the accumulate asmonomers alongside the bacterial AMPs the Initially, 1977). Lehar, and AMPs(Ehrenstein by bacteria of killing explain to (G+) bacteria:peptide insertion and membrane pe Tossi, University of Trieste, Italy. Italy. of Trieste, University Tossi, The barrel-staveporemodel Proposed mechanisms of action for mechanismsof (Fig. 5 α -helical AMPsGram-negative on (G-)andGram-positive , step 5a): This was the first mechanism proposed mechanism proposed wasthefirst 5a):This step , 18 rmeability models. Kindly forms structure abarrel-like spanning the to transmembrane thusforming aclassical peptide regions constitute the pore lining. lining. the pore constitute regions peptide surface. When concentration is increased, of peptide (Fig. 5, step 4), defects in the provided by Prof. A. Chapter 1 rron2006). et al., c lipid headgroups to -helical frog peptide from from peptide frog -helical to translocateboth acrossthe α , AMPs accumulate on the lipid lipid on the accumulate , AMPs esterhoff et al., 1989; Wieprecht et al., esterhoff et al., 1989; tics (Ge et al., 1999; Pe tics (Ge et intercalated anioni rane without causingdamage to the lipid ch in arginine, such as buforin II isolated II isolated buforin as such arginine, ch in et al., 1996). This can dissipate the electric the electric dissipate et al., 1996). This can uence of this pore formation, the peptides peptides the formation, pore of this uence the membrane is weakened by unfavorable unfavorable by weakened is membrane the 19 llel to the membraneduringprocess. When the mbranes occurs. The AMPs then re-orient and and re-orient then AMPs The occurs. mbranes ly different and highly destructive mode of action, are are of action, mode destructive highly and different ly (Fig. 5, step 5b): This mechanism combines components components combines mechanism This 5b): 5, step (Fig. r a resistance phenomenon to phenomenon unlikely resistance very Therefore, it is for a

(Park et al., 1996), are reported 1996), are (Park et al., ) (Pouny et al., 1992). Locally ) (Pouny (Fig. 5, steps 5c and 5d): This model was proposed on the basis of of basis the on proposed was This model 5d): 5c and 5, steps (Fig. the development of resistance against AMPs has been shown to be a much be a much to shown has been AMPs against of resistance the development

Bufo bufo gargarizans The AMPs, due to their marked AMPs, due to The The carpet model The carpet The toroidal wormhole model Althoughdisruption integrity of membrane is the principal mechanism of action of cellular and nuclear membranes (Futaki al., 2001).cellular membranes Oncenuclear and et inside the can cell, the AMPs and enzymatic synthesis cell-wall of inhibition in resulting targets RNA and DNA to bind activity (Fig.(Brogden, 7)5, step 2005). Phyllomedusa sauvagei AMPs ri example, bilayer (Fig. 5, step 6). For from usually active that against microorganisms are resistant currently to licensed antibiotics. organisms host between a co-evolution of consequence a is AMPs cationic of plethora The 2006). Sahl, and (Peschel AMPs of the diversity the shaped has which and microbes Despitefact that the bacteria have had a huge time-spanadapt to to their killing mechanisms, studies with dermaseptin S (a cationic amphipathic amphipathic S (a cationic dermaseptin with studies slower process than against conventional antibio bilayer in surface, orientation an is para that is attained, concentration peptide a high energeticinfluences and thinningthe of me peptideform aggregates that intercalate into the and membrane disrupt it by producing micelles in a detergent like manner. (Hancock 5a) step 5, (Fig. models carpet and 5d) 5c and steps 5, (Fig. barrel-stave of the with 1999). The AMPs assemble and Chapple, (Duclohier, pores form and 2000) al., et (Wieprecht repulsion charge the for compensate the in of lipids involvement direct The 1996). al., et Ludtke 1995b; al., et Matsuzaki 1994; the than radius have a larger pores The hole. a toroidal-shaped to leads structure pore conseq 5a). As a 5, step (Fig. classical pore rate (Matsuzaki translocation the lipid enhance (W of cells respiration the uncouple and potential 2005). al., et 2000; Glaser the cell memb can cross members AMPs, some Sincepathogen thewould primary target to cell have the of AMPs is the membrane, a develop to order in – process lengthy a very – membrane cell whole its redesign action. their resistance against arise rapidly. Chapter 1 Fig. 6.Co ms etal.,2007).Th Woodha etal.,1989; Pasquier (Du immunity innate and adaptive both including vertebrates higher in systems immune found the organism from invasion by pathogenic microorganisms. Frogs have the full complement of fungi. Therefore, these animals haveevolved and harmful bacteria of growth potentially to the conductive moist is environment that and epithelial cells (Hancock and Diamond, 2000) (Fig. 6). and mast basophils eosinophils, cells, neutrophils, al., 1996), et cells (NKcells)(Horton killer natural 1982), Horton, and (Manning granulocytes and cells dendritic macrophages, (Green and Cohen, 1977), cytokines and various types of cells, such as phagocytic Rollins-Smith AMPs etal.,1998; (Simmaco and Hancock, 2000). infection (Scott an from to athreat pathogen ofapotential the progression avoid to ability the host’s in role key a system plays immune theinnate respond, to first it isthe Because pathogens. various against frogs defenses of skin (beingrelativelytemperature- and Cohen, 1977; Simmaco et al., 1998). Th immunity, including complementmediated effect temperature-dependent andsuppressed if temperature decreases. Similarly, humoral innate etal., (Simmaco 19 skin inthe infections environment. Inaddition to being activated too slowly to provide sufficient protection for organisms whose internal temperature relie Anurans (frogs and toads) are AMPs in Innate immune defenses in amphibians include: pattern-recognition receptors (PRRs), recognition receptors receptors Pattern- mponents ofth frogs Dendritic cells Dendritic cells Macrophages Granulocytes e innateimmunity inam Leukocytes confined, for at leastpartoftheirlifecycle,toa warm Mast cells Mast cells Tissue IMMUNITY IMMUNITY INNATE INNATE 98), the adaptive immune system of frogsis system of immune adaptive 98), the phibians. 20 s heavily upon the te the upon s heavily independent) are essential in protecting the the areessential in protecting independent) innate defenses to protect the skin of the et al., 2005), a complement lytic system acomplement lyticsystem et al.,2005), Complement erefore, the specializedinnateimmune pathway s, isalso temperature-dependent (Green Antimicrobial peptides ey are poikilothermic mperature oftheir Cytokines Chapter 1 The scale bar is equivalent to bar is equivalent to scale The -adrenergic nerve terminals, causing causing terminals, nerve -adrenergic 24h after injection with amphibian α . Dermal granular glands are surrounded X. laevis granular gland (G) and two mucus glands (M) two granular gland (G) and illed in with AMP granules (depicted in pink); pink); in AMP granules (depicted in with illed 21 glands secrete AMPs lid and open arrows, respectively). lid and open through a gland duct onto the epidermal surface. (A) Schematic the duct onto through a gland ontract upon stimulation of local ontract ult frogs have two types of skin glands that are located in the dermis: granular and and granular dermis: the in are located that glands skin of types two have frogs ult Stimulated amphibian granular In general, AMPs are encoded by single genes or homologous gene families. These families. gene homologous or genes single by encoded are AMPs In general, Ad gene families geneare locatedclustered families in arrangements in themapped genome and syntenic to different of co-evolution suggests which regions, chromosomal loci) genetic (co-localized subclasses of AMPs (Scott andHancock, 2000; Bals, 2000). The expressionof AMPs the by induced and is changes metamorphic with correlates development amphibian during (Reilly et al., 1994;thyroid hormone treatment generally Ohnuma et al., 2006).AMPs are foundof ingranular cells the glands in adult frogskin7) (Fig. (Hoffmann 1983a; et al., et al., 1983b),Hoffmann in and sometimes the ofthe mucosa gastrointestinal tract (Moore synthesize to able glands granular of devoid is skin Larval 1998). al., Wang et 1991; al., et pro- pre and during of AMPs expression the for no evidence is there and AMPs and secrete become and rapidly develop glands these Afterwards, 2010). (Pollet, stages metamorphic functional of at the climax metamorphosis. mucus glands (Duellmanmucus glands and 1986; Trueb, Stebbins and Cohen,1995; Shepherdal., et Fig. 7. by cells that c myoepithelial al., 2012). 500 µm (Gammillet secretion of protective AMPs secretion of protective f gland granular amphibian dermal an depiction of (B) A hematoxylin and eosin stained skin section of one of active secretion saline with phosphate buffer so (indicated by through gland ducts Chapter 1 the peptidescleavedand are polyadenylatedmRNAs encoding the peptides (Chen et al., 2003). Within 15-30 minutes, et al., 1992; Flucher et al., 1986). conserved signal peptide followed (prepropeptides). All havea common “signal-spacer-bioactive peptide” organization with a etal., 1998). Shepherd Erspamer, 1994; 1986; structures in which a variety of biologically- and innervated by sympathetic fibers (Simmaco myocytes by cells,surrounded granule-filled multinucleated arelarge glands granular The Stebbins and Cohen, 1995), which is vital forefficient respiration and thermoregulation. 1986; Trueb, and (Duellman moist continuously the skin to keep components secreting Trueb, 1986; Steinborner et al., 1997). The mucus glands aremainly responsible for and (Duellman integument the throughout evenly are distributed species glands these in and these aggregations, lack species frog Many 1987). Tyler, 1986; Trueb, and (Duellman areas ofskin, described variously asparotid glands or dorsolateral folds in some species 1998). Both types of glands may occur in aggregations, or asmacroglands visible as raised AMPs have been isolated from skin secretions and/or skin extracts from frog species etal.,2012). (Novkovic function biological by peptides of the classification on based etal.,2005). Rollins-Smith deposits of skin peptides may berestored within 6 days to 2 weeks (Erspamer, 1994; (Schadich, 2009; SchadichCole, and 2009).After depletionthegranular of glands,the peptides skin the degrading by activity antimicrobial the inhibit that enzymes proteolytic microbiota of healthy frogssuchas which microorganisms, Some concentrations. self-injur orderto prevent in detoxification Steinborner et al.,1997;Bowieet 1999 storage vesicles compression of the peptide-containing serous ce stressors Rollins-Smith (Tyler etal., 1992; electrical excitation, administration of norepinephrine, injury, predation, handling or other precursor peptides by an endoprotease. Adrenergic stimulation of myocytes, such as by synthesis,the signalsequenceisremoved After etal.,2003). from the(Vanhoye sequences As aresult, secretions contain not only AMPs but also cytosolic components and intact AMPs from thedifferent frog families are In the most In the class common Anuran Database, theAMPsconstitute Defense Peptide in differentfrogfamilies AMPs Distribution of

by aholocrine-like mechanism involving the rupture of the glands (Moore deactivated onthe skin surf Proteus mirabilis Proteus by acidicspacer sequences flanking 22 active AMPs are stored(Duellman andTrueb, y, as AMPs arealsocytotoxic athigher et al., 2002; Woodhams, 2003), causes Woodhams, 2003), etal.,2002; e the mainet al.,1998).Theyarethe anatomical lls. The cells discharge the contents of the of contents the cellsdischarge lls. The are an abundant component of the skin ). Thisis assumed tobeameans of synthesized as large precursorpeptidessynthesized as and Aeromonas hydrophilia, Aeromonas ace (Resnick etal.,1991;

the AMPs AMPs the secrete

Chapter 1 Neobatrachia Archeobatrachia Archeobatrachia nidae idae y odidae y p hr hr tidae p elmatidae p y hio p o p tidae idae g -helical peptides-helical with antimicrobial y p Sca Rhino Pi Leio Al Bombinatoridae Pelod Pelobatidae Me ease cytotoxic peptides into their skin into their skin peptides ease cytotoxic 23 odified from Konig et al., 2012). al., 2012). odified from Konig et ylogeny of amphibians anuran (m ong the more ancient Archeobatrachia, α Archeobatrachia, ancient more ong the Ph Am However, not all frogs synthesize and rel and synthesize frogs all not However, activity have been identifiedbelonging species in the to families Leiopelmatidae, Alytidae, to examined species those in present not are but 1), (Table Pipidae and Bombinatoridae, datebelong that families Pelobatidae to the Scaphiopodidae and 2009; (Conlon et al., 2011). Conlon, Fig. 8. belonging to both the primitive Archeobatrachia and the more highly-derived Neobatrachia Neobatrachia highly-derived more and the Archeobatrachia primitive the both to belonging (Pukala2006; at el., Conlon,2011) (Fig.8). secretions (Conlon, 2010) making the distribution of dermal AMPs among anuran families families anuran among AMPs dermal of distribution the making 2010) (Conlon, secretions sporadic.In some species the production of AMPs is seasonal and influenced by thyroid bacteria to exposure by stimulated be either may and 2006), al., et (Ohnuma status hormone pesticides with contact as such factors environmental by or inhibited 2000) al., et (Matutte 2007). et al., (Davidson Chapter 1 for swimmingandlungingafter fo because theyare the onlyamphibians to have surface to breathe air because theyhave lungs frogs inthefamily Pipidae are exclusively a in and motion water (Cannatella Trueb, 1988). The wave to detect body, the on as stitches system, organ visible alateralline and feet,flattened body, –webbed their habitat befitting multipleadaptations show but (Fig. 8) Sa,1993) de and (Cannatella Archaeobatrachia 2011). etal., Conlon, 2009; (Conlon the in Neobatrachia Rhacophoridae and families Bufonidae,Ceratophyridae, Eleuth *- described in section 6 Alytidae Leiopelmatidae Family 1. Table However, AMPs have not been detected in those Myobatrachidae, Ranidae, and Leptodactylidae the six to Hylidae, families Hyperoliidae, Species, Discoglossidae, Neobatrachia. belonging Pipidae Bombinatoridae Bombinatoridae Pipid frogs are indisputably basal, belonging to the phylogenetically more ancient ancient more to the phylogenetically basal, belonging areindisputably frogs Pipid Frogs of the Pipidae family A sim Examples ofAMPsfrom skin secretions of frogs the of Archaeobatrachia ilar sporadic distribution of moreAMPs derived the is distribution in highly found ilar sporadic Alytes Ascaphus Genus Xenopus Xenopus Bombin a od and alsousetheclawsontheirfeet totearpiecesof 24 erodactylidae, Microhylidae, Pyxicephalidae actual claws. Pipid frog and not gills. Pipids are called clawed frogs, quatic. However, they need to rise to the the riseto to need However, they quatic.

Bom Species Xenopus laevis Xenopus Alytes Ascaphus release AMPs into th release AMPsinto species analysed to date belongingtothe bina bina obstetricans bombina bombina truei s havepowerful legs eir skinsecretions. ascaphins Peptides XPF* CPF* PGLa* magainins* bombinins alyteserins .

Chapter 1 , . ; Pipinae Silurana in genus in

is found in in found is P. carvalhoi Pipa g speciation such as as g speciation such that is monophyletic monophyletic is that

and H. feae allopolyploidization descendant descendant speciation by 1 2 Pseudhymenochirus, , H. curtipes varies (2n = 20 for for 20 = (2n varies , Speciation Allopolyploid Bifurcating Descendant Descendant are unitedin the sub-family ) (Canatella andTrueb, 1988; Cannatella 9). One is bifurcatin Pipinae er, 1991; Kobel et al., 1996; Evans, 2008). an ancestral species become geographically geographically become species an ancestral 25 Hymenochirus H. boulengeri , ily Xenopodinae (Conlon et al., 2012). et Xenopodinae (Conlon ily Pseudhymenochirus – – genus Pseudhymenochirus the species in a single Hymenochirus and Pipa, H. boettgeri are united in the subfamily Xenopodinae and 2n = 24 for H. boettgeri for = 24 2n and Xenopus 2 1 r r r . The number of chromosomes in in chromosomes of number The . in sub-Saharan Africa. They are proposed to have diverged approximately approximately diverged have to are proposed They Africa. sub-Saharan in and Pseudhymenochirus

Ancesto Ancesto Ancesto ilurana . Modes of speciation acting on subfam acting speciation . Modes of Xenopus ly Pipidae comprise five genera. genera. five comprise Pipidae ly fami of the frogs clawed The extant Pipa, S Fig. 9 tongue and vocal cords, instead having bony rods in the the in rods bony having instead cords, vocal and lack a tongue These frogs food. large frogs other the from way different a radically in sound clicking produce help that larynx (Rabb, 1960; Yager, 1996). South America with the other four genera genera four other the with America South and Hymenochirus P. parva 2n = 30 for 100 million years ago (MYA) (Cannatella and de Sa, 1993; Roelants et al., 2007). 2007). al., et Roelants 1993; de Sa, and (Cannatella (MYA) ago years million 100 There are 7 species in genus genus in 7 species are There – 4 species P. merlini, and based uponnucleotide sequences ofmitochondrial the genes. However,use the of two clades” two the between differences historical and biological trenchant “underscores genera are complex taxon of this history evolutionary and The cytogenetics 2004). al., et (Evans (de Sa andHillis, 1990; Kobel and Du Pasqui of modes different two that assumed is it if be explained best can distribution The species (Fig. Xenopodinae on the have acted speciation and de Sa, 1993). allopatric speciation whenpopulations two of separated (de Queiroz, 1998). The otherone is reticulate sympatric speciation involving two between combination genetic which in allopolyploidization, by duplication genome ancestral results species in a descendantwith the full complement of chromosomes from 1996). (Kobel, ancestors both Chapter 1 X. largeni 2008; Evans et al., 2004; Tobias et al., 2011): the basis of their morphology, advertisement calls, and proposed evolutionary history (Evans, chromosomes(Fig. 10). These species have been divided further into three groups on the tetraploid the ancestor of extant all the produced 1982). Between 21 and 41 MYA, agenome duplication event in the = with 2n species tetraploid (Fig. 11). (2n = 72)and the tetraploid species produc comprising 7 species and in a similar fashion, genetic combination between the octoploid 10). (Fig. discovered been yet not has would appear that the original X. borealis events haveproduced diploid statuswithchromoso Silurana sequences of mitochondrialgenes discovered (indicatedby level (2n).The diploid Fig. 10.Anevolutionary scenarioXenopodi forsubfamily At least two further allopolyploidization events produced two groups octoploid frogs Silurana The genusSilurana An evolutionary scenario is proposed in which adiploid ancestor diverged into and Xenopus and , and X.clivii and X. petersii betweenandMYA 53 64 (Fig. 10) Xenopus X S. epitropicalis , and ). The figure isbased uponthe resultsofcomparisons ofthenucleotide ; and the fraseri the ; and comprise atleastthree extant species – 40 chromosomes (Tymowska, 1991; Tymowska and Fischberg, Fischberg, and Tymowska 1991; (Tymowska, chromosomes 40 me number 2n=20,whereasmore recent polyploidization species (2n = 18)has either disappeared or has not yet been provided by Evans etal.(2004). X. victorianus Xenopus andatleasttwo other incompletely characterised diploid species (2n =18) has either disappeared or group includes 26 ; the ; the ed the 2 dodecaploid (2n = 108) species = (2n 108) ed the 2 dodecaploid laevis group includes muelleri nae, including specieswithdifferent ploidy Xenopus Xenopus .

X. fraseri X.fraseri group includes S. tropicalis species with 2n =36 2n with species and X. laevis, X. pygmaeus Xenopus has has retained X. muelleri lineage lineage X. gilli . It , , Chapter 1 2n = 72 2n = 72 2n = 72 2n = 72

a

wittei vestitus itombwensis amieti lenduensis andrei boumbaensis X. X. X.

X. X. X. X.

ruwenzoriensis longipes e a basis for comparison of the amino acid of the amino basis for comparison e a X. 2n = 108 X. 2n = 108 27

Peptide sequence Peptide sequence GLASFLGKALKAGLKIGAHLLGGAPQQ GLASFLGKALKAGLKIGAHLLGGAPQQ GWASKIGQTLGKIAKVGLQGLMQPK GIGKFLHSAGKFGKAFVGEIMKS GIGKFLHSAGKFGKAFVGEIMKS GIGKFLHSAKKFGKAFVGEIMNS GMASKAGAIAGKIAKVALKAL s et al. (2004) (Conlon et al., 2012). et al., 2012). (Conlon (2004) et al. s a Octoploid II ancestor Octoploid I ancestor X. laevis. Allopolyploidization Allopolyploidization . The figure is based upon the results of comparisons of the nucleotide sequences sequences nucleotide of the results of comparisons the is based upon . The figure

was the first species in which amphibian AMPs were unambiguously unambiguously were AMPs amphibian which in species first the was X. laevis X. 4 3 1 2 -amidation is denoted by Xenopus α Ps in Peptides from Peptides isolated A simplified schematic representation of the origin of the octoploid and dodecaploid species species and dodecaploid octoploid of the the origin of representation schematic A simplified . Tetraploid Tetraploid X. laevis AM Tetraploid Tetraploid Magainin 1 Magainin 2 PGLa CPF-1 XPF-1 Peptide name Peptide name C-terminal C-terminal Fig. 11 in the genus of mitochondrial genes provided by Evan by of mitochondrial genes provided Table 2. identified(Zasloff,provid 1987). These AMPs family Pipidae to belonging species other from isolated AMPs novel of sequences (Table 2). Chapter 1 in the three signal peptides. acid deletions in the N-terminal region (residues 1-14) result in the complete loss of of complete loss the in result (residues 1-14) region N-terminal the in deletions acid species ofbacteriaand fungi induceand osmotic lysis ofprotozoa (Zasloff, 1987). Amino step 5b) Chapter in (described the disrupt membrane, and cell bacterial the unstructured in solution, form anantiparallel dimer ofamphipathic helices upon binding to Giovannini et al, 1987). Biophysical studies have shown that these peptides are pH(Zasloff, atneutral 1987; +4 of charge anet exhibit 2)and (Table 22 and 10 positions (Fig. 13). The two closely related peptides – magainin 1 and 2, differ by two residues at sequence and five magainin 2 sequences flan magainin prepropeptideis predicted to be303 residueslong. It contains magaininone 1 Fig. 12. Barker, 1988; Kuchler et al., 1989). al., 1989). et Kuchler 1988; Barker, common ancestral gene by a series of duplication events and exon shuffling (Hunt and AMPsin the that segments,suggests spacer-peptide of N-terminal pro-regions the in similarity (Zasloff, 1987; Hunt and Barker, 1988; Gibson et al., 1991). The significant structural of the spacer-peptide subunits, anotable feature for many archaeobatrachian AMP genes contrast, thegenes for magainin- andcaerulin-precursor in Konig and Bininda-Emonds, 2011) (Fig. 12). peptide” with a partially conserved 20 amino acid signal peptide (Hunt andBarker, 1988; Both magainin 1and 2 have low haemolytic activity, inhibit growth of numerous The name Magainins The The AMP genesin Alignment ofthe AMPsignalpeptides in

(Matsuzaki, 1999) prepropeptide for PGLa is one of the simplest comprising only 64 amino acids. In magainin comes from Hebrew word PGLa XPF-1 Magainin 1 , X. laevis display thestereotypical stru Interaction of of Interaction . MYKQIFLCLIIAALCATIMA MYKGIFLCVLLAVICANSLA MFKGLFICSLIAVICANALP α -helical AMPswith biological membranes 28 X. laevis the precursor, aswelltherepeats of the membrane via toroidal-type pore formation ked by almost identicalacidicspacers . The shaded residues magain X. laevismay haveevolved from a X. laevisshow tandem repeats which means “a shield”. The cture “signal-spacer-bioactive

have beenconserved ; Fig. 5, Chapter 1

72 72 26 26 174 S. aureus skin cDNA 303 128 210 256 m of action. Thus, Thus, of action. m X. laevis KRDAEAVGPEAFA KRDAEAVGPEAFA KRDAEAVGPEAFA KRDAEAVGPEAFA KRDAEAVDDRRWVE KRDAEAVGPEAFA KRDAEAVGPEAFA (Zasloff et al., 1988) and (Zasloff et al., 1988) and bian AMPs to be identified, there has there to be identified, AMPs bian but at higher concentrations, against against but at higher concentrations, 2 (five copies) depicted in bold (Terry et al., in bold (Terry copies) depicted 2 (five structure and mechanis E. coli with almost identical sequences are flanking the with almost identical sequences alian cells (Ge et al., 1999). The bactericidal alian cells (Ge bactericidal et al., 1999). The positive and anaerobic Gram-negative, and 29 . Pexiganan showed distinct promise as a topical as a topical promise distinct showed . Pexiganan 1 and 2 peptides deduced from MFKGLFICSLIAVICANALPQPEASA MFKGLFICSLIAVICANALPQPEASA of infected foot ulcers in diabetic patients and as a a and as patients diabetic in ulcers foot of infected activity against al., 1988). In contrast, analogues with deletions in the C- the in deletions with analogues In contrast, 1988). al., sociated with their C. albicans C. vitro

in GIGKFLHSAKKFGKAFVGEIMNS GIGKFLHSAKKFGKAFVGEIMNS GIGKFLHSAKKFGKAFVGEIMNS GIGKFLHSAKKFGKAFVGEIMNS GIGKFLHSAKKFGKAFVGEIMNS GIGKFLHSAGKFGKAFVGEIMKS (Zasloff et al., 1988; et al., 2000).(Zasloff et al., analogueshave Giacometti Synthetic been DEDLDEREVR Prepropetide sequence for magainin Prepropetide DEDLDEREVR DEDLDEREVR DEDFDEREVR DEDLDEREVR Because magainins were among the first amphi the were among Because magainins DEDMDEREVR library. Magainin 1 peptide is underlined. Spacers 1 peptide is underlined. Magainin library. magainin and copy) magainin 1 (one sequences of the 1988). been considerable research as magainin 2 shows potent potent 2 shows magainin Fig. 13. terminal region (Ala-15, Cly-18 or Clu-19) have equal or even increased antimicrobial antimicrobial increased even or equal have or Clu-19) Cly-18 (Ala-15, region terminal activity. antimicrobial activity (Cuervo et activity antimicrobial (Giacometti etand al., 2000), is also active, P. aeruginosa An cytotoxicity. and minimize effects antimicrobial the maximize to order in developed extensive structure-activity investigation on magainin 2 resulted in the development of the broad- with acetate pexiganan or MSI-78 analogue linear lysine-containing, 22 amino-acid, spectrum antibacterial activity against Gram- to mamm but low cytotoxicity aerobic bacteria ofloxacin of that to equivalent is anaerobes and aerobes against of pexiganan activity (FuchsIn et al., 1998). addition, it is active clinical against some isolates of the pathogen yeast opportunistic anti-infective agent for treatment receive not did but 2008) al., et Lipsky 1999; al., et (Ge impetigo for treatment possible FDA approval. Chapter 1 E. coli, of natural counterpart of PYLa named PGLa was is cyto with bacteriostatic, similar peptides to al., 1983b). It was concluded that PYLa could form amembrane-active amphipathic helix X. laevis, the PYL precursor mRNA is one of the most abundant components (Hoffman et from mRNAisolated polyadenylated total in that shown Itwasfurthermore al., 1983a). monomeric state, via tilted dimers to anupright inserted state (described in Chapter PGLa adopts different orientations in the Interestingly, etal.,1998). Matsuzaki etal.,1995; (Westerhoff activities antimicrobial resulti 2 magainin forms with heterodimers P. aeruginosa, Interaction of club-shaped cells of the large intestine (Sad largeintestine the cellsof club-shaped immunoreactivity, butnotXPF, wasalso detected from specificgranular cells inthe lowe components can beisolated from extracts of protein contains xenopsin at the COOH terminus (Sures and Crippa, 1984). Both processing of a single precursor molecule (Gib al., 2006b). withmagainin interactions and concentration cells (Bevins and Zasloff, 1990). In particular, PGLa shows potent eukaryotic against minimal toxicity has but protozoa and fungi bacteria, Gram-negative a or micelles (Jackson etal., 1992; Latal et ispredominantly but solution inaqueous is that PGLa unstructured indicate Biophysical studies 2009). Prossnigg, and (Lohner carboxypeptidases actionof the to resistance provides that C-terminus amidated lysine residues andtheamino groupatthe N- a ch PYLa, with of net N-terminal tripeptide the X. laevisskin cDNAlibrary forclones encoding X. laevis The octapeptide xenopsin and the related XPF are bioactive peptides derived from a Xenopsin precursor fragment (XPF) PGLa exhibits broad spectrum of antimicrobial activity against Gram-positive and Gram-positive against activity antimicrobial spectrum of broad PGLa exhibits Initially, peptidyl-tyrosine-leucine-amide (P peptidyl-tyrosine-leucine-amide Initially, Peptidyl-glycine-leucine-amide (PGLa) S. aureus by the same group (Andreu et al., 1985). PGLa is a21-residue peptide, lacking

Saccharomyces cerevisiae α , and -helical AMPs with biological membranes S. pyogenes α gatively-charged lipid bilayers bilayers lipid -helical inthepresence ofnegatively-charged ,

and is also active, but at higher concentrations, against al., 1997; Bechinger et al., 1998). etal., 1998). Bechinger al.,1997; , and bacterialmembrane, cell froma surface aligned 30 ler et al., 1992). XPF exhibits antimicrobial antimicrobial ler etal.,1992). XPF exhibits X. laevisskin (Araki et al., 1973), as well as ng in synergisticmembrane disruption and toxic and/or lytic properties. However, the and/or toxic lytic properties. 2 (Tremouilhac etal., 2006a; Tremouilhac et r esophagus and stomach. Xenopsin-like terminal glycine (Table 2). It also 2).Italso has an (Table terminal glycine son etal.,1986)(Tab C. albicans YLa) was predicted from the screening screening of from the YLa) waspredicted olated twoyearslaterfromskinsecretions arge of +5 at neutral pH because of four in tall,thincellsof the precursor ofcaerulein (Hoffmann et (Soravia et al., 1988). PGLa ), depending on peptide in the duodenum andin

vitro vitro leThe precursor 2). activity against against activity 1 , Chapter 1 , and the peptides Xenopus

Litoria caerulea Litoria ns the additional bioactive bioactive ns the additional (Richter et al., 1986; Gibson et al., 1986; 1986; al., et Gibson 1986; al., et (Richter d sequence shows a close resemblance to resemblance a close shows d sequence

31

le caerulein/CPF genes, genes, le caerulein/CPF multip contains genome X. laevis ) and so exert similar biological effects (Erspamer and and (Erspamer effects biological similar exert and so ) 2 (Anastasi et al., 1967). Caerulein-related peptides have been been have peptides Caerulein-related 1967). al., et (Anastasi X. laevis as well as in multiple species in Hylidae, the families skin (Wakabayashi et al., 1985) (Table 2). Subsequent cDNA cloning cloning cDNA 2). Subsequent (Table 1985) al., et (Wakabayashi X. laevis skin Hyla caerulea Hyla caerulea arose independently with the former arising from a duplication of the CCK gene and and gene CCK the of a duplication from arising former the with independently arose Caerulein-precursor fragment (CPF) fragment Caerulein-precursor aci amino whose decapeptide Caerulein is a containing one, three, or four copies of caerulein copies four or three, one, containing studies demonstrated that the that the demonstrated studies Giovannini et al., 1987). The antimicrobial activity of CPF has not been studied in detail. detail. in studied been not has of CPF activity antimicrobial The 1987). al., et Giovannini Melchiorri, 1983). It has been proposed that the genes encoding caeruleins in in caeruleins encoding genes the that proposed has been It 1983). Melchiorri, Litoria with As 2010). al., et (Roelants gene gastrin of the a duplication from latter the of caerulein contai precursor biosynthetic proxenopsin, the cDNA a using experiments cloning in predicted was a CPF of The structure CPF. peptide from library formerly formerly gastrin and CCK peptides mammalian the to common is that sequence a C-terminal contain (Gly-Trp-Met-Asp-Phe.NH identified in the skins of Leptodactylidae, Hyperoliidae, and Ranidae and (Bowie Tyler, 2006). Although the amino of variable, most caeruleins is the region of at N-terminal acid sequence activitiesagainst and Gram-positive Gram-negative to comparable those bacteria of the magainins(Soravia et al., 1988). the bioactive C-terminal sites of themammalian gastrointestinal cholecystokinin hormone was originally(CCK). It the isolated skin from Australian of thefrog Chapter 1 African clawed frogs with different level of polyploidy belonging to the Pipidae family. purify andcharacterize AMPswiththerapeutic had been isolated andcharacterized. The aimof et al.,2010)weretheonlyrepresentatives ofthe Pipidae family from which dermal AMPs X. laevis(Zasloff, 1987; Giovannini et al., 1987), and the octoploid frog       The specific objectives of the studywere: Until recently,thediploid frog Researchobjectiv aim, th affectsthe to interspecies number of extent hybridization To which determine the sk th the AMPs in multiplicityof affectthe whether genomeduplications analyse To t To clinicalisolates and strains To c analysis. degradation combined withmass spectrometry andamino acid compositi To from thePipidaefamily. To isolate and purify AMPs from skin secretions of various polyploid frog speci he taxonomiche relation of different frog species and genera. e AMPs in skin secretions fromfrog secretions e AMPsinskin hybrid in secretions investigate the possible application of of application possible the investigate determine thestructures of the newl haracterize the . es and thesisoutline in vitro in activity of the purified AMPs against various referen various AMPs against purified the of activity of a range of microorganisms. S. tropicalis 32 potential from skin secretions of species of species of secretions from skin potential y discovered AMPs by automated Edman this thesis was to isolate systematically, systematically, isolate thesis wasto this the AMPsasclassification tools (Ali et al., 2001), the tetraploid frog frog tetraploid the 2001), (Alietal., s. (Conlon X. amieti(Conlon tostudy on ce es e Chapter 1 2n = 20 20 = 2n Pipinae Pipinae H. boettgeri Hymenochirus Hymenochirus

2n = 72 72 = 2n X. andrei X. andrei Anura Anura Pipidae Pipidae Xenopus

x African clawed frogs frogs African clawed West muelleri 2n = 36 36 = 2n X. clivii 33 X. laevis X. laevis X. borealis X. borealis X. muelleri X. X. muelleri Xenopodinae

new new “S. Silurana 2n = 40 40 = 2n tetraploid 1” family family sub-family sub-family order order genus genus ploidy ploidy species species interspecies interspecies hybrid The following species have been studied: been species have The following studied: Chapter 1 AMPs genes is described. the of ofinheritance mode the asastudy to model hybrids the interspecies of suitability the the fateof addition, In outlined. elucidate the evolutionary relations among frogs fromdifferent genera and species is markers asreliable as towell astheir agents, application therapeutic into development skin secretions of Chapters Chapters reference strains of clinically relevant microorganisms wasinvestigated. effect the of puri Additionally, respectively. X. muelleri X. laevis AMPs ineach specieswas compared tothose described inChapters of multiplicity The microorganisms. a range of aretested against activities antimicrobial primary structures and physicochemical properties of the AMPs are determined and the “ characterized incompletely order to determine whether hybridization affects AMP multiplicity. affects AMP multiplicity. whether hybridization to determine order was studied inChapter between the two frog populations. primary sequences of the AMPs sets was perf reference using bacteria wasevaluated peptides corresponding profiles from both parent species from fema secretions skin in AMP profile rangereference of strains of microorganisms. wereagainst a activities determined their antimicrobial isdescribed and hymenochirins, The Chapter Chapter A comparative study of the AMPs in skin secretions from two discrete populations of populations discrete fromtwo secretions skin the AMPs in of study A comparative This thesis is completed by a general discussion general a by iscompleted thesis This The AMP profile from AMPThe profile The study in Chapter Chapter in study The

. isolation andstructural characterization of AMPs in norepinephrine-stimulated 2 is presented in Chapter inChapter is presented - 7 4 .

alysis of skin secretions from the presents secretions results from skin analysisof acomparative The potential of the newly charact newly the of potential The X. borealis and X. borealis 6 . A novel family of five structurally-related peptides, termed the termed the peptides, structurally-related five . familyA novel of 7 was focused on F1 hybrid hybrid F1 on focused was Silurana Silurana Hymenochirus boettgeri, X.clivii is described in 5 . The antimicrobial potential of the newly identified thenewlyidentified of . potential The antimicrobial duplicated AMPs isdiscussed. genes duplicated new tetraploid 1” new tetraploid 34 ormed to clarify the taxonomic relationship taxonomicrelationship clarifythe ormed to fied peptides on the growth and viability of of viability the growth and on fied peptides le hybrid frogs is compared with the withthe iscompared frogs le hybrid l strains. In addition, a comparison of the of acomparison addition, In l strains. X. laevisand

(Chapter subfamily Pipinae, Pipinae, aspecies inthesubfamily erised AMPs as candidates for

X. laevis and the octoploid

8 Chapter Chapter X. muelleri ) of theresults reported in xX.muelleri 2 2 (Chapter

and X. andrei and Moreover, the

frogs. The Chapter Chapter 3 , and in , andin . The 5 ) in ) in 3 Chapter 1

. species and species caerulein and and caerulein Xenopus laevis Acinetobacter lly Active Peptides, Elsevier lly Elsevier Active Peptides, Diagn Microbiol Infect Dis Diagn Microbiol

-phase synthesis of PYLa and of PYLa and -phase synthesis lian amphibians: , 1973. Isolation and structure of structure , 1973. Isolation and Proc Royal Soc Exper Biol Med Proc Royal Soc Exper Biol , especially on a strip of, especially on a strip fundus a rat from secretions of Australian amphibians. The amphibians. The of Australian secretions species as defined by CLSI reference methods: reference by CLSI species as defined evaluation of patients with ESBL- and non- with ESBL- of patients evaluation (Pipidae). Biochim Biophys Acta 1550:81-89. 1550:81-89. Biophys Acta Biochim (Pipidae). tics and concept of innate immunity: An update concept of innate immunity: An update tics and host defense against infection. Respir Res 1:141- infection. against host defense cture and dynamics of the antibiotic peptide PGLa of the antibiotic peptide cture and dynamics ., Dudley, M.N., Jones, R.N., 2006. SENTRY ., Dudley, M.N., 35 . Experientia 23:699-700. 23:699-700. . Experientia c facts and emerging concepts. J Intern Med 254:197- concepts. J c facts and emerging 43:S49-S56. 43:S49-S56. [PYLa-(4-24)] from [PYLa-(4-24)] skin secretion of

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41 e. Medicina Konyvki Acinetobacter baumannii its pathogenicity. Pol 56:597-612. Acta Biochim Xenopus laevis Xenopus Staphylococcus aureus Staphylococcus the skin of the hyperoliid frog, the skin of the hyperoliid e, magainin 2. Bi e, magainin antimicrobial peptides magain peptides antimicrobial V., Tossi, A., Juretic, D., 2012. A., Juretic, D., Tossi, V., ki, H., Iwamuro, S., 2006. Deve ki, H., Iwamuro, S., 2006. 1996. A novel antimicrobial peptide from Bufo bufo gargarizans peptide A novel antimicrobial 1996. N., Miyajima, K., 1996 D.L., 2008. G., 2006. Experimental evolution of resistance to an antimicrobial antimicrobial an of resistance to evolution Experimental G., 2006. infections and modelling disease. In: Ashbee, H.R., Bignell, E. E. Bignell, H.R., In: Ashbee, disease. modelling and infections Candida ., Akada, K.Y., Murase, O., Yoneyama, O., Yoneyama, ., Akada, K.Y., Murase, Microbiol 4:529-536. 4:529-536. Microbiol H., Paterson, E., Wegrzyn, G., 2009. . Gen Comp Endocrinol 146:242-250. 146:242-250. Endocrinol . Gen Comp resistance. Nat Rev resistance. Nat Rev Sci 273:251-256. Proc Biol peptide. induced expression of genes encoding preprotemporins in the skin of Tago's brown frog brown the skin of Tago's in preprotemporins encoding of genes expression induced tagoi of biochemistry and molecular genetics of of genetics and molecular of biochemistry selectivity of an antimicrobial peptid antimicrobial an selectivity of bilayers. magainin 2, in phospholipid peptide, Biochemistry 35:11361-11368. 35:11361-11368. Biochemistry induced rapid flip-flop of phospholipids coupled w induced rapid flip-flop Mechanism of synergism between of synergism Mechanism from isolated activity antimicrobial Chemother 55:2189-2196. Agents Antimicrob of drug uptake. phases dependent of stomach in the peptides Antimicrobial (Eds.), Pathogenic Yeasts, The Yeast Handbook Yeasts, (Eds.), Pathogenic cell in Zasloff, M., 1992. A novel peptide-producing 37:15144-15153. 268:433-436. Res Commun Biochem Biophys skin. of the gland granular the to similar strikingly cell pathogen. Clin Microbiol Rev 21:538-582. Microbiol Rev 21:538-582. Clin pathogen. Biochem Biophys Res Commun 218:408-413. Biochem Biophys defense peptides. Bioinformatics 28:1406-1407. Bioinformatics defense peptides. iUniverse (Self Published), p. 294. 294. Published), p. iUniverse (Self Perron, G.G., Zasloff, M., Bell, Bell, M., Zasloff, Perron, G.G., Peschel, A., Sahl, H.G., 2006. The co-evolution of The co-evolution H.G., 2006. Peschel, A., Sahl, Pal, T., 2012. Az orvosi microbiologia tankonyv Az orvosi microbiologia T., 2012. Pal, Ohnuma, A., Conlon, J.M., Kawasa Plata, K., Rosato, A. Matsuzaki, K., Sugishita, K., Fujii, N., Miyajim N., Fujii, K., Sugishita, K., Matsuzaki, Ki 1995b. K., Miyajima, O., Murase, K., Matsuzaki, Park, C.B., Kim, M.S., Kim, S.C., S.C., Kim, M.S., Kim, Park, C.B., Matsuzaki, K., Mitani, Y Matsuzaki, K., Moore, K.S., Bevins, C.L., Brasseur, M.M., Tomassini, N., Turner, K., Eck, H., Zasloff, M., 1991. 1991. M., Zasloff, H., Eck, K., Turner, N., Tomassini, M.M., Brasseur, C.L., Bevins, K.S., Moore, Matsuzaki, K., Murase, O., Fujii, Matsuzaki, K., aminoglyc from bacteria protects oxide 2011. Nitric B.D., McCollister, J., Reynolds, J.E., Moreira, K., Sadler, K.M., Huttner, N., Tomassini, C.L., Bevins, K.S., Moore, Mattute, B., Knoop, F.C., Conlon, J.M., 2000. Ka J.M., 2000. F.C., Conlon, Knoop, Mattute, B., MacCallum, D.M., 2010. Peleg, A.Y., Seifert, Nelson, R.W., 2009. Darwin, then and now: the most 2009. Darwin, Nelson, R.W., Novkovic, M., Simunic, J., Bojovic, Novkovic, M., Simunic,

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42

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44

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Chapter 2

Antimicrobial peptides with therapeutic potential from frog skin secretions of the Marsabit clawed frog Xenopus borealis (Pipidae)

M. Mechkarska, E. Ahmed, L. Coquet, J. Leprince, T. Jouenne, H. Vaudry, J.D. King, J.M. Conlon

Comparative Biochemistry and Physiology (2010) 152:467-472. Chapter 2 netv aet a CFB (LSLKFILT GMGARQ with GKMMGGAPREQ) (GLGSLLGKAFKIGLKTV CPF-B1 = MIC was agent infective anti- valuabletherapeutically a into development for potential greatest the with peptide baumannii (MDRAB)withMICvaluesintherange4-8 µM. methicillin-resistant pathogens, CPF-B1 secretions. skin (LC Candida borealis frogXenopus tetraploid Staphylococcus aureus Nine peptides ABSTRACT had only weak antimicrobial activity. activity. The weakantimicrobial that had only compared wasidentified with magainin-2 (G**KFLHSAGKFGKAFLGEVMIG) containing atwo amino acid residue deletion peptide a secondmagainin-related . Inaddition X. amieti and xenopsin-precursor fragments, XPF(2 peptides), previously isolated from X.laevisand glycine-leucine-amide, PGLa (2 peptides), c peptide (1 peptide), magainin-2 to were orthologous they that demonstrated peptides 50 >200 µM). This µM). >200 5 µM against µM 5 is hmn erythrocytes human against activity hemolytic low and albicans, with differential growth inhibitory activity against activity against inhibitory growth with differential , MIC = 5 µM against µM 5 = MIC E.coli, were isolated fromnorepinephrine-stimulated skin secretions of the peptide was also the most abundant antimicrobial peptide in the in peptide antimicrobial abundant most the also was peptide a atv aant lncl slts f h nosocomial the of isolates clinical against active was Parker, 1936 (Pipidae). Struct (MRSA) and multidrug-resistant and (MRSA) aureus S. aerulein-precursor fragme 48 and MIC = 25 µM against µM 25 = MIC and aureus, S. ural characterization of the Escherichia coli nts, CPF (4 peptides) Acinetobacter and Chapter 2

, only Pipa -helical -helical Silurana Xenopus  + Silurana agents (Norrby et et (Norrby agents Xenopus 1986; Soravia et al., al., 1986; Soravia et , Hymenochirus means all, species of all, species means compounds. Analysis Analysis compounds. awed frogs is complex awed dae (Frost, 2010). The e fact that the African clawed clawed e fact that the African deduced from the nucleotide from deduced ., 1986; Richter et al., ., 1986; Richter w types of antimicrobial of w types on and characterization of magainin-1 and - and magainin-1 of characterization on and S. epitropicalis (Tymowska and Fischberg, e amino acid sequences of the biosynthetic acid sequences of the biosynthetic e amino netics of the African cl netics of the ures in solvent a membrane-mimetic such as ith broad spectrum antimicrobial activityspectrum ith broad several, although by no lly useful antimicrobial lineage hasproduced tetraploid species that g potenciesg and specificitiesderived that are with 2nchromosomes = 20 is only the diploid 49 ) within familythe Pipi with 2n = 36 chromosomes (Evans et al., 2004). At At 2004). al., et (Evans chromosomes 36 2n = with have the propensity to form an amphipathic an amphipathic form to propensity the have Xenopus Xenopus ificant structural similarity inificant similarity the regions structural N-terminal of onlon et al., 2007). Despite th Despite 2007). onlon et al., . S. tropicalis c phospholipid vesicle (Powers of an anioni in the presence although several additional, as yet unnamed, species have have species unnamed, as yet additional, several although , and is controversial as cladistic analyses based upon nucleotide upon as cladistic analyses based is controversial X. laevis Xenopus borealis Silurana Silurana , and X. laevis and a duplication within the , Xenopuswas the first amphibian laevis species in which such peptides were identified Introduction comm to resistance with and fungi bacteria of strains of pathogenic The emergence The clawed frogs comprise 32 species distributed in five genera ( genera five in distributed species 32 comprise frogs The clawed X. laevis skinhas led secretions to isolati the sed antibioticsnecessitated has a search for ne (Kobel and Du Pasquier, 1991) Pasquier, Du and (Kobel andspecies Hillis,Sa (de 1990). A putativewholegenome duplication within the lineagehasgiven rise to tetraploid the species 1. u (Zasloff, 1987), relatively little work has been done to exploit other species belongingthe to of pharmaceutica as a source Pipidae family al., 2005;2009). Livermore, Peptidesw of skin the in glands granular in synthesized potential source of a therapeutically represent and toads) Anura (frogs valuable anti- infective drugs (Rinaldi, 2002; C frog 2, peptides that are encoded by the same gene (Giovannini et al., 1987; Zasloff, 1987) and and 1987) Zasloff, 1987; al., et gene (Giovannini same the by are encoded that 2, peptides multipleantimicrobial peptides with varyin xenopsin, caerulein, of, precursors biosynthetic the of processing post-translational the from and peptideglycine-leucine-amide al (Gibson et sign reveals of cDNAs, sequences ancestral a common from have evolved may peptides the that suggesting precursors the In 1989). al., et Kuchler 1988; and Barker, (Hunt events of duplication a series by gene common with the vast majority of frog skin antimicrobial peptides, all the active from isolated components of et1988; al., 1994) James . A comparisonof th and proxenopsin, promagainin, of procaerulein, precursors struct of their part least at over conformation 50% trifluoroethanol-water or and Hancock,2003). and biological trenchant genera "underscores two the use of that out was pointed it but genus The 2004). al., et (Evans clades" two the between differences historical 18 species currently contains et al., 2004). The cytoge (Evans reported been 1982) include Pseudhymenochirus of status taxonomic sequencesofmitochondrial genes strongly support the monophyly of least onegenome duplication further event within the tetraploid lineage has given rise to Chapter 2 concentration 1% v/v) and stored at -20 solution containing the secretions wasacidif norepinephrine bitartrate (40 nmol/g body mass) and immersed in water for 15 min. The the IUCNRed List ofThreatened streams in grasslands and pasturelands. It is cu (above altitude are high habitats whose natural Kenya and northern Tanzania andmay also befound in Uganda. It is anaquatic species 2 mL/min through 8 Sep-Pak C-18 dodecaploid species oftetraploid combination the involving octoploid species that include norepinephrine-stimulated skin secretions of the Marsabit clawed frog antimicrobial peptides by isolating and characterizing the components that arepresent in array Xenopus diverse of ofthe molecular evolution the into insight to and gain agents peptides novel study wasto identify present the X.amieti(Conlon and etal.,2001) (Ali al., tropicalis 2010). Consequently,S. the aim of (2 FL, (Brooksville, Inc. Express Xenopus by investigators. Adult specimens of No. (Protocol U.A.E.University of committee 2.1. 2. CPF-B2. CPF-B1and e.g. recognized. The speciesorigin isdenoted by precursor fragment (CPF), and xenopsin-precursor fragment (XPF) peptide families are etal.,2010). (Conlon study is that used previously for peptides from known asthe Kenyaclawed frog). v/v/v; 10 mL)freeze-dried.v/v/v; 10 and connected in series. Boundmaterialwaselut  20

All procedures with live animals were animals live ap with All procedures secretions skin of Collection Experimental inthis identified peptides theantimicrobial for designating used The nomenclature In addition to In addition N, 37 N,  59 E). Each animal was injected at two sites in the dorsal lymph sac lymph with dorsal the sitesin attwo wasinjected animal E). Each X. laevis X. ruwenzoriensis and The magainin, peptide glycine-leucine amide (PGLa),caerulein- glycine-leucine peptide The magainin, , antimicrobial peptides havebeenisolated only from Species (Tinsley etal., 2004). X. amieti, X. borealis X.

cartridges (Waters Associates, Milford, MA, USA) borealis borealis o C. Thepooled solutionflowa waspassedrateofat X. longipes 50 ed withacetonitrile/water/TFA(70.0:29.9:0.1, and octaploid genomeshave producedthe (n= 2; male23 g, female 33g) were supplied B andparalogs are differentiated by numerals 1500 m) freshwater ponds and slow-flowing m)and ponds freshwater 1500 and further independent polyploidizations USA). The frogs were collected in Kenya Kenya in frogs were collected USA). The rrently listedasaspecies of leastconcern in is widely distributed in central and northern A21-09) and were carried out by authorized ied with trifluoroacetic acid (TFA) acid (final trifluoroacetic with ied X. laevis(James 1994)and etal., with therapeutic potential as anti-infective asanti-infective potential therapeutic with proved by theAnimal ResearchEthics (2n = 108) (Evans et al., 2004). 2004). al., et (Evans 108) = (2n X. borealis X. amieti (also

Chapter 2

and L of  (ATCC S. aureus S. aureus (MDRAB) strains strains (MDRAB) ted with 0.1% (v/v) ted value was taken as the the as taken was value Escherichia coli Escherichia 50 C in a humidified atmosphere atmosphere humidified a C in o

L) with an inoculum (50 an inoculum L) with  L) of fractions the inhibit to the growth  (ATCC 25923), d, IL, USA) equilibra d, IL, titer plate reader. Minimum inhibitory titer plate reader. Minimum e clonal lineage of the strains have been of the strains have lineage e clonal Acinetobacter baumannii oncentrations of ampicillin and incubations incubations and ampicillin of oncentrations e concentration of acetonitrile in the eluting of acetonitrile in the eluting e concentration 51 t hospitalst in Abu Dhabi Emirate. The origin, were carried out in RPMI 1640 medium for 48 h at h at 48 for medium 1640 RPMI in out carried were CFU/mL. After incubation, the absorbance at630 nm of 4 (ATCC 90028) were purchased from the American Type Type American the from purchased were 90028) (ATCC Staphylococcus aureus Staphylococcus C. albicans with amphotericin as previously described (Conlon et al., 2007, 2010). 2010). 2007, et al., (Conlon described as previously amphotericin with Candida albicans Candida were determined as described in the previous section. Fractions Fractions section. previous the in as described determined and were E. coli M by standard double dilution methods (Clinical and Laboratory Standards Standards and Laboratory (Clinical methods dilution double standard by M albicans

in 96-wellin cell-culture microtiter for plates h at 37 18 C. Hemolytic activity against human erythrocytes taken from a healthy donor was was donor a healthy from taken erythrocytes human against activity Hemolytic Purification of the peptides was monitored by incubating lyophilized aliquots of Antimicrobial assays assays Antimicrobial of strains Reference Peptide purification in redissolved were cartridges, Sep-Pak on purification partial after secretions, The skin C with an inoculum of 5 x 10 x 5 of inoculum an with C

colony forming units/mL) from a log-phase culture of reference strains of strains reference of culture a log-phase from units/mL) forming colony o S. aureus S. 6 measured as previously described (Conlon et al., 2010). The LC 2010). al., et (Conlon described previously as measured 35 35 E. coli each well was determined using a micro a using each well was determined of range concentration the in measured was peptides the of (MIC) concentration μ 200 3 - with Institute,2008a,b) and taken was lowest the as concentration of peptide wherevisible no nm. 630 at of absorbance measurement by confirmed was value This observed. was growth In order to monitor the validity and reproducibility of the assays, incubationsbacteria with outwere carriedparallel with in increasing c of air. Incubations with with Incubations of air. 10 chromatographic effluent in Mueller-Hinton broth (50 (50 broth Mueller-Hinton in effluent chromatographic 25922), and 2.2. 0.1% (v/v) TFA/water (4 mL) and injected onto a (2.2 x 25-cm) Vydac 218TP1022 (C-18) (C-18) 218TP1022 Vydac x 25-cm) a (2.2 onto and (4 injected mL) TFA/water (v/v) 0.1% Deerfiel (Grace, column HPLC reversed-phase 2.3. mean concentration of peptide producing 50% hemolysis in two independent experiments. experiments. independent two in hemolysis 50% producing peptide of concentration mean Th 6.0 mL/min. rate of a flow TFA/water at solvent raised was 21% to (v/v) overmin 10 and 63% (v/v) to 60 using over min linear were min) (1 and fractions nm and 280 nm 214 at monitored was Absorbance gradients. collected.abilities The of freeze-dried aliquots (50 of (MRSA) strains andfiveresistant multi-drug differen four at patients from were isolated spectrumof resistance to antibiotics, and th described previously2006; (Pál Conlon et al., et al., 2009). Culture Collection (Rockville, MD, USA). Sixof methicillin-resistant strains Chapter 2 ability to inhibit the growth of growth the inhibit to ability and 8 with of were associated the 1-4 assay, peaks conditions Underfurther the purification. column (Fig. 1). The prominent peaks designated 1-8 were collected and subjected to cartridges, were chromatographed on aVyd CPF-B4. peak 6: amixture of CPF-B1, CPF-B2, and PGLa-B2, peak 7: CPF-B3, and peak 8 magainin-B1,magainin-B2, peak2: peak3:XPF-B1, XPF-B2, peak4: PGLa-B1, peak5: E. coliand extraction andtheaccelerating vo wasoperated inreflectormode that Biosystems) with (Applied instrument delayed USA). MALDI-TOF mass spectrometry wascarried outusing a Voyager DE-PRO degradation using an Applied Biosystemsmodel 494 Procise sequenator (Foster City, CA, 3.1. 3. USA). CoreFacility NE, (Omaha, Structure Protein Center Medical Nebraska of University the mass determinations was calibrated withpeptides of known molecular 2.4. 2.0mL/min. was rate the minflow and 50 over to 56% from was 21% raised solvent eluting columnEverest(1 x25-cm)(C-18) 238EV510 Vydac 214TP510 (C-4) column, a(1x25-cm) Vydac 208TP510 (C-8) column, anda associated with antimicrobial activitywere su

Purification of the peptides the of Purification The primarystructures of Structural characterization Results The pooled skin secretions from secretions skin pooled The S. aureus . Subsequent structural analysis de  0.02%. Amino acid composition analyses were performed by by wereperformed analyses composition Amino acid 0.02%. E. colionly whereas peaks 5-7 inhibited the growth of both

the peptides were determined by automated Edman Edman automated by weredetermined peptides the ltage inthe ionsource was20kV.The instrumentwas X. borealis 52 ccessively chromatographed on a (1 x 25-cm) massin the2- 4kDarange. Theaccuracy of ac C-18preparativereversed-phase HPLC , after partial purification on C-18 on Sep-Pak , afterpartial purification . Theconcentration of acetonitrileinthe monstrated that peak 1 contained Chapter 2 her. The dashed line shows the line her. The dashed The peaks designated 1-8 displayed 1-8 displayed designated peaks The . neity, as assessed by a symmetrical peak 53 activity and were purified furt after partial purification on Sep-Pak cartridges after partial purification e peptides were purified toe peptideswere purified near homoge Reversed-phase HPLC on a preparative Vydac C-18 column of skin Vydac HPLC on a preparative C-18 secretions from Reversed-phase Th shape and mass spectrometry, by further chromatography on semipreparative Vydac C-4, C-4, Vydac on semipreparative chromatography further by spectrometry, and mass shape Vydac C-8, andEverest C-18 The columns. methodology is illustrated bypartial the separationCPF-B1, of CPF-B2, and PGLa-B2Vydacon a C-42A), column (Fig. followed on a chromatographies successive by of CPF-B1 homogeneity near to purification by C-18and an Everest column 2B) 2C). (Fig. (Fig. column Vydac C-8 varying degrees of antimicrobial of antimicrobial degrees varying concentration of acetonitrile in the eluting solvent. solvent. eluting the in acetonitrile of concentration Fig. 1. X. borealis, Chapter 2 Fig. 2. B andC, the peakcontaining CPF-B1isdenoted by thesymbol panel A,peak1containsCPF-B1, C-8, and(c) Everest C-18 columns.Thearrowheads Purificationtonearhomogeneity peak 2containsPGLa-B2,and peak ofCPF-B1 54 show wherepeakcollection began and ended. In on semipreparative (a) Vydacon semipreparative C-4,(b)Vydac + . 3contains CPF-B3.Inpanels Chapter 2 skin calc 2194.2 2356.4 2408.5 2666.4 1955.2 2409.3 M+H X. borealis . obs X. borealis 2194.0 2356.2 M+H -amidated. -amidated. α obs), and calculated molecular masses masses molecular and calculated obs), r 2113.1 2113.1 2113.3 2 1955.2 2 secretions of secretions onfirm the proposed structures and to to and structures proposed the onfirm 55

VGKMMGGAPREQ 2844.5 2844.6 microbial peptides isolated from from peptides isolated microbial AKVGLKEFVQS 2563.6 2563.4 KTVGKMMGGAPR 2587.6 2587.5 KAFVGEIINPK 2666.7 KAFLGEVMKS 2409.5 isolated from skin from isolated GKAFLGEVMIG erved molecular masses (M Amino acid sequences, obs The approximate final yields ofpeptides purified magainin-B1 were (nmol) 120, Structural characterization characterization Structural structures of the anti The primary

calc) of the antimicrobial peptides peptides antimicrobial the of calc) r 2. CPF-B4Fig. 3. GLLTNVLGFLKKAGKGVLSGLLPL 2408.2 magainin-B2 590, XPF-B1 25, XPF-B2 80, PGLa-B1 35, PGLa-B2 510, CPF-B1 720, CPF-B4 35. 55, and CPF-B3 CPF-B2 20, 3. Magainin-B1 GKFLHSAGKF Magainin-B1 Magainin-B2 GIGKFLHSAGKFG Magainin-B2 XPF-B1 XPF-B2 GFKQFVHSMGKFG PGLa-B1 GWASKIGTQLGKM GMASKAGTIAGKIAKTAIKLAL.NH CPF-B1 CPF-B2 GLGSLLGKAFKIGLKT CPF-B3 GLGSLLGKAFKIGL GLGSLLGSLFKFIPKLLPSIQQ PGLa-B2 GMASKAGSIVGKIAKIALGAL.NH (M secretions were established by automated Edman degradation. As the C-terminal regions of of regions C-terminal As the degradation. Edman automated by established were secretions not was it hydrophobic, strongly are CPF-B4 and PGLa-B2, PGLa-B1, magainin-B1, the the from wash-out of because residue acid amino C-terminal their determine to possible acid amino the determining by was resolved ambiguity this However, disk. sequencer 3. Fig. in shown are structures primary complete and their peptides of the compositions to c was used spectrometry mass MALDI-TOF C-terminally are and PGLa-B2 that PGLa-B1 demonstrate Chapter 2 cleavage atArg pro processing post-translational alternative to isidentical CPF-B2, which whether unclear XPF displayed very low hemolytic activity (LC activity very low hemolytic displayed were within the range 4-8 µM for allstrain in Table 1.The MICvalues for CPF-B1agai negative bacterium 1986; James et al., 1994), and the octoploid frog frog the octoploid and James al.,1994), et 1986; the diploid frog S.tropicalis orthologs of previously described peptides (magainin, PGLa, CPF, and XPF) isolated from shown in Fig. 4,acomparison of their prim CPF secretions of activity in norepinephrine-stimulated sk norepinephrine-stimulated in activity 4. Data are expressed asµM.The values in pa PGLa Magainin Magainin ofreferencest growth the inhibit 3.3. familybasis ontheofstructural similarity contains but atwoamino acid residue (Ile (magainin-B1) was isolatedin relatively lowyiel activity against human erythrocytes(HC 1. Table CPF-B2, CPF-B3, CPF-B4, and XPF-B1. material pure determin to was insufficient ‐

‐ The abilities of the most abundant of the peptides in the in the the peptides mostof abundant the of abilities The activities Antimicrobial The Discussion B2 B1 ‐ B2

present study has identified ten peptides with varying degrees of antimicrobial ‐ ‐ Minimum inhibitory concentrations against microorganisms and hemolytic B2 B1

X. borealis X. borealis 26 or, less probably, is the product of a separate gene. In addition, a peptide a peptide In addition, ofaseparategene. isthe product lessprobably, or, E. coli,andthe opportunistic yeast pathogen 100 5 E. 25 50 >100

coli

(Ali et al., 2001), the tetraploid frog frog tetraploid the etal.,2001), (Ali

rains oftheGram-positive bacteriumrains S.aureus rentheses represent the %hemolysis at 200 µM. in secretions of the tetraploid frog frog thetetraploid of secretions in 25 5 S.aureus 50 >100 >100 50

50 ) of the endogenous peptides isolated from skin

s tested. Magainin-B2, PGLa-B2, andCPF-B1 PGLa-B2, s tested.Magainin-B2, >200 µM) against human erythrocytes. There 56 duct of the CPB-B1 precursor arising from e the antimicrobial activities of PGLa-B1, PGLa-B1, of activities antimicrobial e the

nst clinical isolates ofMRSA isolates MDRAB clinical and nst ary structuresreveals thatthey represent residues (1 -26) of CPB-B1, represents an an represents ofCPB-B1, -26) (1 residues d that is clearly amember ofthe magainin

X. amieti ND 25 C. 25 100 ND X. borealis (Conlon et al., 2010). It is

albicans

C.

X. laevis albicans

skin secretions to secretions skin X. borealis (Gibson etal., ND >200 HC >200 >200 ND arecompared , 50

the Gram- the

(18%) (27%) (0%) 2 -Gly . As . As

3 ) Chapter 2 -helix so that α ). A Schiffer- S. aureus S. residue in magainin-B2 by by magainin-B2 in residue 22 and E. coli a consequence of thepositive decreased 57 al., 1992; Dathe et al., 2001). Dathe al., 1992; GL*GSVLGKILKMGANLLGGAPKGA G**KFLHSAGKFGKAFLGEVMIG GIGKFLHSAGKFGKAFLGEVMKS GIGKFLHSAKKFGKAFVGEIMNS IC > 100 µM against µM IC > 100 GMATKAGTALGKVAKAVIGAALa GMATKAGTALGKVAKAVIGAALa GLLGPLLKIAAKVGSNLLa GFLGSLLKTGLKVGSNLLa GLLTNVLGFLKKAGKGVLSGLLPL GMASKAGTIAGKIAKTAIKLALa GMASKAGTIAGKIAKTAIKLALa GMASKAGSIVGKIAKIAL*GALa GL*GSVLGKALKIGANLLa GL*GSLVGNALRIGAKLLa GMASKAGSVLGKVAKVALKAALa GMASTAGSVLGKLAKAVAIGALa GMASKAGAIAGKIAKVALK*ALa GVSKILHSAGKFGKAFLGEIMKS GIGKFLHSAGKFGKAFVGEIMKS GIKEFAHSLGKFGKAFVGGILNQ Evolutionary pressure to conserve the primary structures of the antimicrobial peptides peptides antimicrobial the of structures primary the conserve to pressure Evolutionary Edmundson wheel (1967) representation of the magainin-B1 structure (not shown) shown) (not structure magainin-B1 of the representation (1967) wheel Edmundson demonstrates that the peptide retains the propensity toform an amphipathic the reduced antibacterial activity is probably chargeonpeptide the produced bysubstitution the Lys of the from the clawed frogs has not been particularly strong (Fig. 4). 4). (Fig. strong particularly been not has frogs clawed the from X. amieti S. tropicalis S. tropicalis Procaerulein-derived peptides X. borealis X. amieti X. amieti X. amieti X. laevis S. tropicalis X. borealis X. borealis X. amieti X. amieti X. laevis X. laevis X. borealis X. borealis X. amieti PGLa-related peptides Magainin-related peptides deletion compared with magainin-B2 (Fig. 3). This peptide had only very weak weak very only had peptide This 3). (Fig. magainin-B2 with compared deletion activity (M antimicrobial Ile (Fig. 3). Studies with analogs of magainin-2 have demonstrated that increasing the the increasing that demonstrated have magainin-2 of analogs with 3). Studies (Fig. Ile positive charge onpeptide the resulted in an increase in antimicrobial activity until a limit further any in result not did cationicity in increases further whereupon was reached, et (Bessalle activity increase in Chapter 2 reference strains of Gram-negative ( against spectrumactivity CPF-B1 showed broad potent, agent. growth-inhibitory infective component with the greatest potential for development into atherapeutically valuable anti- and ( frog orthologous peptides fromtheoctoploid frog Fig. 4. S. tropicalis X. laevis X. laevis X. amieti X. borealis X. borealis Proxenopsin-derived peptides S. tropicalis X. laevis X. laevis X. laevis X. laevis X. borealis X. borealis which antibiotic resistance has become a serious problem, notably methicillin-resistant methicillin-resistant problem, notably serious becomea has resistance antibiotic which (MIC =4-8 µM) against isolates oftwo groups ofclinicallyimportant pathogens for potency high displayed the peptide addition, In 1). (Table erythrocytes human against pathogen yeast opportunistic the against some into sequences in order tosimilarity.introduced maximize structural group and 5 amino acids in the second group are invariant, even after deletions have been suggests that they divide naturally into two sub-groups but only 3 amino acids in the first the XPFfamily. Acomparison of the primary proxenopsin-related peptides are highly variable. Only 5residues out of 24 areconserved in 11 out of22residues are invariant. Incont 10 out of 23 amino acids are invariant and in the case of peptides fromthe PGLa family, S. tropicalis The most abundant antimicrobial peptide in the to-date, familystudied species magainin fromfour the the to belonging In peptides a ) indicates that the peptide is C-terminally Acomparison oftheprimary structures oftheantimicrobial peptides from GWASKIGQTLGKIAKVGLKELIQPK GWASKIGQTLGKIAKVGLQGLMQPK GWASKIAQTLGKMAKVGLQELIQPK . Structural similarity is emphasized by GWASKIGTQLGKMAKVGLKEFVQS GFKQFVH*SMGKFGKAFVGEIINPK GLGSLLGSLF*KFIPK******LLPSIQQ GL*GSLLGKAFKIGLKTVGKMMGGAPREQ GLASTLGSFLGKFAKGGAQAFLQPK GFLGPLLKLAAKGVAK**VIPHLIPSRQQ GLASLLGKAL*KAGLK*IGTHFLGGAPQQ GFGSFLGKAL*KAALK*IGANALGGSPQQ GFASFLGKAL*KAALK*IGANMLGGTPQQ GLASFLGKAL*KAGLK*IGAHLLGGAPQQ E. coli E. C. albicans X. amieti  ) and Gram-positive ( Gram-positive ) and 58 -amidated. rast, sequences ofth , the tetraploid frog structures ofthe CPF family peptides the shading. (*) denotes aresidue deletion but only very low hemolytic activity activity lowhemolytic very only but X. borealis skin secretions was the the was secretions skin S. aureus X. laevis e procaerulein-and , andthe diploid ) bacteria,and X. borealis with

Staphylococcus aureus (MRSA) and multidrug-resistant Acinetobacter baumannii (MDRAB). During the last decade, MRSA (Chambers and Deleo, 2009) and MDRAB (Dijkshoorn et al., 2007; Peleg et al., 2008) have emerged to become a major phenotype in hospitals worldwide with a high rate of mortality. More recently, new strains of the pathogens have emerged in the community, causing infections in young, otherwise healthy, people (Gootz and Marra, 2008; Cooke and Brown, 2010; Deleo et al., 2010). In addition to Cha -lactam resistance, MRSA strains may exhibit multi-drug resistance including non- p susceptibility to quinolones, macrolides and sulfonamides (Garau et al., 2009). Similarly, ter 2 among strains causing nosocomial outbreaks, resistance of MDRAB to fluoroquinolones, aminoglycosides, sulphonamides, third-generation cephalosporins and even carbapenems is common (Vila and Pachón, 2008). Treatments of MDRAB with alternative drugs such as polymyxins, particularly colistin (polymyxin E), and the glycylcycline, tigecycline are far from optimal due to concerns with nephrotoxicity regarding colistin and the bacteriostatic nature of tigecycline (Karageorgopoulos and Falagas, 2008). There is clearly a need for new anti-infective agents to which these pathogens have not been exposed and CPF-B1 represents a promising candidate. A major obstacle to the development of peptide-based anti-infective drugs, particularly if they are to be administered systemically, is their short half-lives in the circulation (Zhang and Falla, 2010). However, peptides applied to infected skin or skin lesions in the form of sprays or ointments can penetrate into the stratum corneum to kill microorganisms. MRSA is a serious problem in infections of the superficial epidermis such as impetigo in infants and children (Geria and Schwartz, 2010) and in colonization of surface lesions such as the foot ulcers of diabetic patients (Bowling et al., 2009). Similarly, MDRAB infection of wounds, particularly associated with natural disasters and conflicts in war zones, is becoming increasingly common and can lead to necrotizing lesions that do not heal (Sebeny et al., 2008). The high potency of CPF-B1 against MRSA and MDRAB together with its low toxicity suggests that topical applications of the peptide may have a role to play as a treatment option in combating skin infections caused by these pathogens and in therapeutic regimes to promote wound healing.

Acknowledgments This work was supported by a Faculty Support Grant (NP/10/05) from United Arab Emirates University. The authors thank Laurey Steinke and Michele Fontaine (University of Nebraska Medical Center, Omaha, NE, USA) for amino acid composition analysis.

59

Chapter 2 de Sa,R.O., Hillis,D.M.,1990. Phylogene Dathe, M.,Nikolenko, J.,Beyerma H.,Meyer, Cooke, F.J., Brown, N.M., 2010. Community-associated meth Cooke, F.J.,Brown, N.M.,2010.Community-associated Conlon, J.M.,Al-Ghaferi,N.,Ahme Conlon, J.M., Ahmed, E. Conlon, J.M., Al-Ghaferi, N.,Abraham,B., Leprince, J.,2007. Strategiesfor transformation of Clinical Laboratory andStandards Institute,2008b. Clinical Laboratory andStandards Institute,2008a.Methods for Dilution Antimicrobial Susceptibility Ali, M.F.,Soto,A.,Knoop,F.C REFERENCES Chambers, H.F.,Deleo,F.R. Bowling, F.L.,Jude, E.B.,Boult Bessalle, R., Haas, H., Goria, A., Shalit, I., Fridkin, M., 1992. Augmentation oftheantibacterial Deleo, F.R., Otto, M.,Kreiswirth,B.N.,Chambe Evans, B.J., Kelley, D.B., R.C.,Evans, B.J.,Tinsley, Melnic Kelley, Dijkshoorn, L., Nemec, A.,Seifert, H.,2007.An antimicrobial activity ofantimicrobial activity magain infections. Br. Med. Bull.94,215-227. of theoctoploid frog of magainin, PGLa, procaerulein-derived, and pr Biol. DrugDes. 74, 488-493. peptide andits analogs: Efficacy against multidrug-resistant agents. Methods 42, 349-357. am naturally-occurring Susceptibility Testing ofYeast. Appr Tests forBacteria that GrowAerobically. Nat. Rev. Microbiol. 7,629-641. of thediploidfrog, secretions infecting organisms? Curr.Diab. Rep. 9, 440-444. 313-317. activity ofmagainin by positive-charge chain extension. Antimicrob. Agents Chemother. 36, resistant resistant an integration of ribosomalDNAandmorphology. Mol.Biol. Evol. 7, 365-376. Mol. Phylogenet. Evol. 33, 197-213. phylogeny ofAfricanclawedfrogs: phylogeograp Acinetobacter baumannii Staphylococcus aureus Xenopus amieti , Condamine, E., 2009. Antimicrobial phibian antimicrobial peptides into therapeutically valuable anti-infective , 2009.Wavesofresistance: . Nat. Rev. Microbiol. 5, 939-951. on, A.J.,2009. MRSA anddiabetic ., Conlon, J.M., 2001. Antimicrobialpeptides isolatedfromskin Xenopus tropicalis d, E.,Meetani, M.A., Leprince, . Lancet 375, 1557-1568. in peptidesby modification of (Pipidae). Peptides 31, 989-994. oved Standard M27-A3.CLS1, PA. Wayne, tic relationshipsofthe pipidfrogs Approved Standard PA. M07-A8. CLSI,Wayne, k, D.J.,Cannatella, D.C., 2004. A mitochondrial DNA 60 nn, M.,Bienert, M.,2001. Optimization ofthe rs, H.F., 2010. Community-associated meticillin- rs, H.F.,2010. Community-associated Reference MethodforBroth Dilution Antifungal increasing threat in hospitals: multidrug-resistant oxenopsin-derived peptides from skinsecretions (Pipidae). Biochim. Biophys. Acta1550,81-89. hy hy and implications fo Staphylococcus aureusintheantibiotic era. icillin-resistant charge. FEBSLett.501, 146-150. Acinetobacter baumannii J., Nielsen,P.F propertiesbrevinin-2-related of footwounds:contaminating or Staphylococcus Xenopus r polyploid evolution. ., 2010.Orthologs and Silurana: . Chem.

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Frost, D.R., 2010. Amphibian species of the world: an online reference. Version 5.4. American Museum of Natural History, New York, USA. Electronic database accessible at http://research.amnh.org/ herpetology/ amphibia /index.php. Garau, J., Bouza, E., Chastre, J., Gudiol, F., Harbarth, S., 2009. Management of methicillin-resistant Staphylococcus aureus infections. Clin. Microbiol. Infect. 15, 125-136. Cha Geria, A.N., Schwartz, R.A., 2010. Impetigo update: new challenges in the era of methicillin

resistance. Cutis 85, 65-70. p ter 2 Gibson, B.W., Poulter, L., Williams, D.H., Maggio, J.E., 1986. Novel peptide fragments originating from PGLa and the caerulein and xenopsin precursors from Xenopus laevis. J. Biol. Chem. 261, 5341-5349. Giovannini, M.G., Poulter, L., Gibson, B.W., Williams, D.H., 1987. Biosynthesis and degradation of peptides derived from Xenopus laevis prohormones. Biochem. J. 243, 113-120. Gootz, T.D., Marra, A., 2008. Acinetobacter baumannii: an emerging multidrug-resistant threat. Expert Rev. Anti Infect. Ther. 6, 309-325. Hunt, L.T., Barker, W.C., 1988. Relationship of promagainin to three other prohormones from the skin of Xenopus laevis: a different perspective. FEBS Lett. 233, 282-288. James, S., Gibbs, B.F., Toney, K., Bennett, H.P., 1994. Purification of antimicrobial peptides from an extract of the skin of Xenopus laevis using heparin-affinity HPLC: characterization by ion-spray mass spectrometry. Anal. Biochem. 217, 84-90. Karageorgopoulos, D.E., Falagas, M.E., 2008. Current control and treatment of multidrug-resistant Acinetobacter baumannii infections. Lancet Infect. Dis. 8, 751-762. Kobel, H.R., Du Pasquier, L., 1991. Genetics of Xenopus laevis. Methods Cell Biol. 36, 9-34. Kuchler, K., Kreil, G., Sures, I., 1989. The genes for the frog skin peptides GLa, xenopsin, levitide and caerulein contain a homologous export exon encoding a signal sequence and part of an amphiphilic peptide. Eur. J. Biochem. 179, 281-285. Livermore, D.M., 2009. Has the era of untreatable infections arrived? J. Antimicrob. Chemother. 64 Suppl. 1, i29-36. Norrby, S.R., Nord, C.E., Finch, R., 2005. Lack of development of new antimicrobial drugs: a potential serious threat to public health. Lancet Infect. Dis. 5, 115-119. Pál, T., Abraham, B., Sonnevend, Á., Jumaa, P., Conlon, J.M., 2006. Brevinin-1BYa: a naturally occurring peptide from frog skin with broad spectrum anti-bacterial and anti-fungal properties. Int. J. Antimicr. Agents 27, 525-529. Peleg, A.Y., Seifert, H., Paterson, D.L., 2008. Acinetobacter baumannii: emergence of a successful pathogen. Clin. Microbiol. Rev. 21, 538-582. Powers, J.P., Hancock, R.E., 2003. The relationship between peptide structure and antibacterial activity. Peptides 24, 1681-1691.

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Chapter 2 Zhang, M.,Falla, T.J., 2010. Potential therapeutic Zasloff, M.,1987.Magainins,acla Vila, J.,Pachón, J.,2008. Tymowska, J., Fischberg, M., Tymowska, 1982. Acomparisonof Tinsley, R., Measey, J., R.,Measey, Tinsley, Ho Soravia, E., Martini,G.,Zasloff, M.,1988. Sebeny, P.J.,Riddle,M.S.,Petersen, K.,2008. Schiffer, M.,Edmundson, A.B.,1967.Useofhelicalwh Rinaldi, A.C. 2002. Antimicrobialpeptides from Richter, K.,Egger, R., Kreil, G., 1986.Sequenceofpreprocaerulein cDNAsclonedfromskinof Mol. Biol. 618, 303-327. Sci. USA84,5449-5453. characterization of twoactiveformsa Pharmacother. 9, 587-599. 49-157. and Picardwith those of and nucleolarorganizer regions Version201 Threatened Species. granular glandsecretions associated with war trauma. to identify segments with helica Chem. Biol.6, 799-804. product. J.Biol. Xenopus laevis . A smallfamily ofprecursorscontaining Chem. 261,3676-3680. Therapeutic optionsfor . FEBS Lett. 228, 337-340. well, K., Lött Xenopus tropicalis Clin. Infect. Dis.47:444-449. l potential. Biophys. J.7, 121-135. 0.1. Electronicdatabase 0.1. acce of thenew tetraploid species ss ofantimicrobialpeptidesfrom nd partial cDNA sequence nd partialcDNAofaprecursor. ers, S.,2004. Acinetobacter baumannii Antimicrobial properties ofpeptides from Gray (Anura,Pipidae).Cytogenet. Gray Cell. Genet.34, 62 amphibian skin: anexpanding scenario. Curr. Opin. Acinetobacter baumannii applications ofhostdefense peptides. Methods the karyotype, constitutiveheterochromatin, thekaryotype, eels to represent the structures of proteins and eels torepresentthestructures ofproteinsand Xenopus borealis one, three, orfour copies ofthefinal ssible at www.iucnredlist.org. ssible atwww.iucnredlist.org. Xenopus epitropicalis skin and soft-tissue infection infection soft-tissue skinand Xenopus infections. Expert Opin. . IUCN RedListof Proc. Natl. Acad. skin:isolation, Fischberg Xenopus

Chapter 3

Purification and properties of antimicrobial peptides from skin secretions of the Eritrea clawed frog Xenopus clivii (Pipidae)

J.M. Conlon, M. Mechkarska, E. Ahmed, J. Leprince, H. Vaudry, J.D. King, K. Takada

Comparative Biochemistry and Physiology (2011) 153:350–354.

ABSTRACT

Five peptides with antimicrobial activity were isolated from norepinephrine-stimulated skin secretions of the tetraploid frog Xenopus clivii Peracca, 1898 (Pipidae). Characterization of the peptides demonstrated that they are structurally similar to magainins (2 peptides), caerulein-precursor fragments, CPF (2 peptides), and xenopsin-precursor fragments, XPF (1 peptide) that have been previously isolated from other species of the genus Xenopus. The magainins and the XPF peptide were active only against the Gram-negative microorganism Escherichia coli whereas the CPF peptides were also active against the Gram-positive Staphylococcus aureus. The most abundant antimicrobial peptide in

the secretions, CPF-C1 (GFGSLLGKALRLG ANVL.NH2) inhibited the growth of the 3 r

te Gram-negative bacteria Acinetobacter baumannii, Klebsiella pneumoniae, and p Pseudomonas aeruginosa (MIC ≤ 25 µM) suggesting potential for development into an

Cha anti-infective agent for use against these emerging antibiotic-resistant pathogens.

66 1. Introduction The skin secretions of many species of frogs contain biologically active peptides with the potential for development into pharmaceutically useful compounds (Clarke, 1997). The family Pipidae is composed of 32 species in 5 genera (Hymenochirus, Pipa, Pseudhymenochirus, Silurana, and Xenopus) (Frost, 2010). All are found in Africa except for members of the genus Pipa which are found in South America. The genus Xenopus (commonly known as African clawed frogs) currently contains 18 species although several additional, as yet unnamed, species have been reported (Evans et al., 2004). Examples of therapeutically valuable peptides that have been isolated from skin secretions of the extensively studied species X. laevis include caerulein, used to restore peristalsis in Cha intestinal paralysis after abdominal surgery and as a diagnostic aid in x-ray examination of the gallbladder and small bowel (Vincent et al., 1982), and xenopsin, shown to inhibit p ter 3 vascular leakage after tissue injury (Gao and Wei, 1993). Analysis of X. laevis skin secretions has led to the isolation and characterization of a range of peptides with growth inhibitory activity against bacterial and fungal pathogens. These include magainin-1 and -2, encoded by the same gene (Giovannini et al., 1987; Zasloff, 1987), and multiple antimicrobial peptides that are derived from the post- translational processing of the biosynthetic precursors of caerulein (Gibson et al., 1986; Richter et al., 1986), xenopsin (Gibson et al., 1986; Soravia et al., 1988), and peptide glycine-leucine-amide (Andreu et al., 1985). Although magainin-2 has only modest antimicrobial potency, a lysine-substituted C-terminally α-amidated analog, termed pexiganan, has shown potential as a topical anti-infective agent for the treatment of infected foot ulcers in diabetic patients and as a possible treatment for impetigo (Lipsky et al., 2008; Gottler and Ramamoorthy, 2009). Despite the fact that X. laevis was the first amphibian species in which cutaneous antimicrobial peptides were unambiguously identified (Zasloff, 1987), frogs of the Pipidae family have not been extensively studied as a source of potential anti-infective agents. Besides X. laevis, antimicrobial peptides have been isolated only from the diploid frog Silurana tropicalis (formerly Xenopus tropicalis) (Ali et al., 2001), the tetraploid frog X. borealis (Mechkarska et al., 2010), and the octoploid frog X. amieti (Conlon et al., 2010b). Consequently, the aim of the present study was to identify novel peptides with therapeutic potential in norepinephrine-stimulated skin secretions of the Eritrea clawed frog Xenopus clivii (also known as Peracca’s clawed frog). Structural characterization of these peptides allows insight into the molecular evolution of the diverse array of Xenopus antimicrobial peptides. X. clivii is a tetraploid frog (chromosome number 2n = 36) with a distribution centered on the Ethiopian plateau that is also found in northwestern Kenya and adjacent Sudan (Frost, 2010). It is a wholly aquatic species whose wide-ranging natural habitats are relatively high altitude (820 - 2745 m elevation), still and slow-flowing

67 permanent water bodies in montane forest and grassland, Acacia woodland and savannah. It is a resilient species and is currently listed as a species of least concern in the IUCN Red List of Threatened Species (Measey et al., 2004). Although still relatively abundant, populations have declined due to habitat loss. The nomenclature used for designating the antimicrobial peptides identified in this study is that used previously for peptides from X. laevis (James et al., 1994), X. amieti (Conlon et al., 2010b), and X. borealis (Mechkarska et al., 2010). The magainin, caerulein- precursor fragment (CPF), and xenopsin-precursor fragment (XPF) peptide families are recognized. The species origin is denoted by C and paralogs are differentiated by numerals e.g. CPF-C1 and CPF-C2. 3 r te 2. Experimental p Cha

2.1. Collection of skin secretions All procedures with live animals were approved by the Animal Research Ethics committee of U.A.E. University (Protocol No. A21-09) and were carried out by authorized investigators. Two specimens of X. clivii (male 41 g, female 15 g) were supplied by Xenopus Express Inc. (Brooksville, FL, USA). The animals were collected in Ethiopia (0902N, 3842E). Each animal was injected at two sites in the dorsal lymph sac with norepinephrine bitartrate (40 nmol/g body mass) and immersed in water (100 ml) for 15 min at room temperature. The solution containing the secretions was acidified with trifluoroacetic acid (TFA) (final concentration 1% v/v) and stored at -20 oC. The pooled

solution was passed at a flow rate of 2 mL/min through 6 Sep-Pak C-18 cartridges (Waters Associates, Milford, MA, USA) connected in series. The bound material was eluted with acetonitrile/water/TFA (70.0:29.9:0.1, v/v/v; 10 mL) and freeze-dried.

2.2. Antimicrobial and hemolytic assays Reference strains of Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25726), Klebsiella pneumoniae (ATCC 700603), and Pseudomonas aeruginosa (ATCC 27853) were purchased from the American Type Culture Collection (Rockville, MD, USA). The Euroclone I NM8 strain of Acinetobacter baumannii has been described previously (Conlon et al., 2010a). Purification of the peptides was monitored by incubating lyophilized aliquots of chromatographic effluent in Mueller-Hinton broth (50 L) with an inoculum (50 L of 106 colony forming units/mL) from a log-phase culture of reference strains of S. aureus and E. coli in 96-well microtiter cell-culture plates for 18 h at 37 oC in a humidified atmosphere of air. After incubation, the absorbance at 630 nm of each well was

68 determined using a microtiter plate reader. Minimum inhibitory concentration (MIC) of the peptides was measured in the concentration range of 3 - 100 μM by a standard double dilution method (Clinical and Laboratory Standards Institute, 2008) and was taken as the lowest concentration of peptide where no visible growth was observed. This value was confirmed by measurement of absorbance at 630 nm. In order to monitor the validity and reproducibility of the assays, incubations were carried out in parallel with increasing concentrations of antibiotics (ampicillin for S. aureus, E. coli, and A. baumannii, and ciprofloxacin for K. pneumoniae and P. aeruginosa) as previously described (Mechkarska et al., 2010). Hemolytic activity against human erythrocytes taken from a healthy donor was Cha measured as previously described (Conlon et al., 2010b). The LC50 value was taken as the mean concentration of peptide producing 50% hemolysis in two incubations. p ter 3

2.3. Peptide purification The skin secretions, after partial purification on Sep-Pak cartridges, were redissolved in 0.1% (v/v) TFA/water (4 mL) and injected onto a (2.2 x 25-cm) Vydac 218TP1022 (C-18) reversed-phase HPLC column (Grace, Deerfield, IL, USA) equilibrated with 0.1% (v/v) TFA/water at a flow rate of 6.0 mL/min. The concentration of acetonitrile in the eluting solvent was raised to 21% (v/v) over 10 min and to 63% (v/v) over 60 min using linear gradients. Absorbance was monitored at 214 nm and 280 nm and fractions (1 min) were collected. The abilities of freeze-dried aliquots (50 L) of the fractions to inhibit the growth of S. aureus and E. coli were determined as described in the previous section. Fractions associated with antimicrobial activity were successively chromatographed on (1 x 25-cm) Vydac 214TP510 (C-4) and (1 x 25-cm) Vydac 208TP510 (C-8) columns. The concentration of acetonitrile in the eluting solvent was raised from 21% to 56% over 50 min and the flow rate was 2.0 mL/min.

2.4. Structural characterization The primary structures of the peptides were determined by automated Edman degradation using an Applied Biosystems model 492 Procise sequenator (Foster City, CA, USA). MALDI-TOF mass spectrometry was carried out using a Voyager DE-PRO instrument (Applied Biosystems) that was operated in reflector mode with delayed extraction and the accelerating voltage in the ion source was 20 kV. The instrument was calibrated with peptides of known molecular mass in the 2 - 4 kDa range. The accuracy of mass determinations was  0.02%.

69 2.5. Secondary structure prediction Prediction of secondary structure and determination of helicity per residue for the peptides were performed using the AGADIR program (Muñoz and Serrano 1994). AGADIR is a prediction algorithm based on the helix/coil transition theory which predicts the helical behaviour of monomeric peptides. Calculations were performed at pH 7 and 298 K. A minimum percentage of 1% helicity/residue was considered to predict the presence of a helix.

3. Results 3 r te 3.1. Purification of the peptides p The pooled skin secretions from X. clivii, after partial purification on Sep-Pak C-18 Cha cartridges, were chromatographed on a Vydac C-18 preparative reversed-phase HPLC column (Fig. 1).

Fig. 1. Reversed-phase HPLC on a preparative Vydac C-18 column of skin secretions from X. clivii, after partial purification on Sep-Pak cartridges. The fractions designated by the horizontal bar inhibited the growth of E. coli and S.aureus and were purified further. The dashed line shows the concentration of acetonitrile in the eluting solvent.

70 Compared with chromatograms from the related species X. amieti (Conlon et al., 2010b) and X. borealis (Mechkarska et al., 2010) generated under the same conditions, the resolution was poor and relatively broad peaks were obtained. The fractions with retention times between 39 and 51 min (denoted by the bar in Fig. 1) contained peptides that inhibited the growth of both E. coli and S. aureus. These fractions were individually subjected to further purification on a semipreparative Vydac C-4 column and those components with growth-inhibitory activity were purified to near homogeneity, as assessed by a symmetrical peak shape and mass spectrometry, by a final chromatography on a semipreparative Vydac C-8 column. The methodology is illustrated by the purification of CPF-C1 (Fig. 2). The approximate final yields of purified peptides (nmol) were magainin- C1 330, magainin-C2 290, XPF-C1 195, CPF-C1 760, and CPF-C2 255. Cha p ter 3

Fig. 2. Purification to near homogeneity of CPF-C1 on semipreparative (A) Vydac C-4, and (B) Vydac C-8 columns. The arrowheads show where peak collection began and ended. The peak containing CPF-C1 is denoted by the symbol + and inhibited the growth of both E. coli and S. aureus.

3.2. Structural characterization The primary structures of the antimicrobial peptides isolated from X. clivii skin secretions were established by automated Edman degradation and are shown in Fig. 3. MALDI-TOF mass spectrometry was used to confirm the proposed structures and to demonstrate that CPF-C1 is C-terminally α-amidated.

71 M+Hobs M+Hcalc Magainin-C1 GVGKFLHSAKKFGQALASEIMKS 2433.4 2433.3 Magainin-C2 GVGKFLHSAKKFGQALVSEIMKS 2461.5 2461.4 XPF-C1 GWASKIGQALGKVAKVGLQQFIQPK 2651.7 2651.5

CPF-C1 GFGSLLGKALRLGANVL.NH2 1684.0 1684.0 CPF-C2 GLGSLLGKALKFGLKAAGKFMGGEPQQ 2702.5 2702.5

Fig. 3. Amino acid sequences, observed molecular masses (M+H obs), and calculated molecular masses (M+H calc) of the antimicrobial peptides isolated from skin secretions of X. clivii. 3 r

te 3.3. Antimicrobial activities p The abilities of the peptides in the X. clivii skin secretions to inhibit the growth of Cha reference strains of the Gram-positive bacterium S. aureus and the Gram-negative bacterium E. coli are compared in Table 1. Only CPF-C1 displayed appreciable hemolytic

activity against human erythrocytes (LC50 = 140 µM) at concentrations up to 200 µM (Table 1).

Table 1. Minimum inhibitory concentrations against microorganisms and hemolytic

activity against human erythrocytes (LC50) of the endogenous peptides isolated from skin secretions of X. clivii.

Peptide E. coli S. aureus LC50 Magainin-C1 100 >100 >200 Magainin-C2 50 >100 >200 XPF-C1 12.5 >100 ND CPF-C1 6 6 140 CPF-C2 25 25 >200

Data are expressed as µM. ND: not determined.

The MIC values for CPF-C1 and the orthologous peptides CPF-AM1 from X. amieti (Conlon et al., 2010b) and XT-7 from S. tropicalis (Ali et al., 2001) against reference strains of A. baumannii, K. pneumoniae, and P. aeruginosa are shown in Table 2.

72 Table 2. Minimum inhibitory concentrations of peptides belonging to the CPF family against reference strains of Gram-negative bacteria

Peptide K. pneumoniae P. aeruginosa A. baumannii

CPF-C1 25 25 3 CPF-AM1 25 25 6 Peptide XT-7 50 50 12.5

Data are expressed as µM. Cha p

4. Discussion ter 3 The present study has identified five peptides with varying degrees of antimicrobial activity in norepinephrine-stimulated skin secretions of the tetraploid frog X. clivii. As shown in Fig. 4, a comparison of their primary structures reveals that they represent orthologs of previously described peptides (magainin, CPF, and XPF) isolated from the diploid frog S. tropicalis (Ali et al., 2001), the tetraploid frogs X. laevis (Gibson et al., 1986; James et al., 1994) and X. borealis (Mechkarska et al., 2010), and the octoploid frog X. amieti (Conlon et al., 2010b). Application of the AGADIR algorithm to predict helical content of peptides indicates that magainin-C1 has only a weak propensity to adopt an α-helical conformation between residues 13 and 18 (% helicity/residue between 1.0 and 1.40) and the substitution Ala17  Val in magainin-C2 results in a further destabilization of the helix. Similarly, XPF-C1 is predicted to adopt a α-helical conformation between residues 6 and 16 but the value for the % helicity per residue is also low (between 1.0 and 1.6). It has been shown using model peptides that a stabilized amphipathic α-helical conformation is an absolute requirement for cytolytic activity against Gram-positive bacteria whereas the structural requirements for activity against Gram-negative bacteria are less stringent (Giangaspero et al., 2001; Powers and Hancock, 2003). Provided that cationicity and hydrophobicity are maintained, peptides with impaired helicity are still active. Consistent with this hypothesis, the magainin peptides and XPF-C1 were active against the Gram-negative E. coli but showed only very weak activity against the Gram-positive S. aureus.

73 Magainin-related peptides

X. clivii-C1 GVGKFLHSAKKFGQALASEIMKS X. clivii-C2 GVGKFLHSAKKFGQALVSEIMKS X. borealis-B1 G**KFLHSAGKFGKAFLGEVMIG X. borealis-B2 GIGKFLHSAGKFGKAFLGEVMKS X. amieti-AM1 GIKEFAHSLGKFGKAFVGGILNQ X. amieti-AM2 GVSKILHSAGKFGKAFLGEIMKS X. laevis-1 GIGKFLHSAGKFGKAFVGEIMKS X. laevis-2 GIGKFLHSAKKFGKAFVGEIMNS 3 r te p Procaerulein-derived peptides Cha

X. clivii-C1 GF*GSLLGKALRLGANVLa X. borealis-B4 GLLTNVLGFLKKAGKGVLSGLLPL

X. amieti-AM1 GL*GSVLGKALKIGANLL.NH2

X. amieti-AM2 GIGSALAKAAKLVAGIV.NH2 X. amieti-AM3 GL*GSVLGKILKMGANLLGGAPKGA X. amieti-AM4 GL*GSLVGNALRIGAKLLa S. tropicalis XT-6 GFLGSLLKTGLKVGSNLLa S. tropicalis XT-7 GLLGPLLKIAAKVGSNLLa

X. clivii-C2 GLGSLLGKAL*KFGLKAAGKFM*GGEPQQ X. borealis-B1 GLGSLLGKAF*KIGLKTVGKMMGGAPREQ X. borealis-B2 GLGSLLGKAF*KIGLKTVGKMMGGAPR X. borealis-B3 GLGSLLGSLF*KFIPK******LLPSIQQ X. laevis 1 GLASFLGKAL*KAGLK*IGAHLLGGAPQQ X. laevis 2 GFASFLGKAL*KAALK*IGANMLGGTPQQ X. laevis 3 GFGSFLGKAL*KAALK*IGANALGGSPQQ X. laevis 4 GLASLLGKAL*KAGLK*IGTHFLGGAPQQ S. tropicalis XT-1 GFLGPLLKLAAKGVAK**VIPHLIPSRQQ

74 Proxenopsin-derived peptides

X. clivii-C1 GWASKIGQALGKVAKVGLQQFIQPK X. borealis-B1 GFKQFVH*SMGKFGKAFVGEIINPK X. borealis-B2 GWASKIGTQLGKMAKVGLKEFVQS X. amieti-AM1 GWASKIAQTLGKMAKVGLQELIQPK X. laevis 1 GWASKIGQTLGKIAKVGLQGLMQPK X. laevis 2 GWASKIGQTLGKIAKVGLKELIQPK X. tropicalis XT-2 GVWSTVLGGLKKFAKGGLEAIVNPK S. tropicalis XT-3 GLASTLGSFLGKFAKGGAQAFLQPK S. tropicalis XT-4 GVFLDALKKF***AKGGMNAVLNPK Cha p ter 3 Fig. 4. A comparison of the primary structures of the antimicrobial peptides from X. clivii with orthologous peptides from the octoploid frog X. amieti, the tetraploid frogs X. laevis and X. borealis, and the diploid frog S. tropicalis. Conservation of amino acid residues is emphasized by the shading. In order to maximize sequence similarity, residue deletions denoted by (*) have been introduced in some sequences. (a) indicates that the peptide is C-terminally -amidated.

In contrast, the CPF family peptides showed broad-spectrum activity and the most abundant antimicrobial peptide in the X. clivii skin secretions, CPF-C1 was also the most potent in inhibiting the growth of bacteria (MIC = 6 µM against both E. coli and S. aureus) (Table 1). The AGADIR algorithm predicts that CPF-C1 has a high probability of adopting a stable α-helical conformation between residues 4 and 14 (% helicity/residue between 1.0 and 4.2). Similarly, CPF-C2 has a high probability of adopting a stable α-helical conformation between residues 2 and 18 (% helicity/residue between 1.0 and 5.8). The higher potency of the arginine-containing CPF-C1 is probably a consequence of its increased cationicity (isoelectric point of 11.49 compared with 10.55 for CPF-C2). Previous studies with a range of naturally occurring and model peptides (reviewed in Conlon et al., 2007) have shown that an increase in peptide cationicity promotes interaction with the negatively charged bacterial cell membrane and so increases antimicrobial potency. On the other hand, similar studies using naturally occurring (Dathe et al., 1997) and model peptides (Stark et al., 2002) have shown that increasing overall hydrophobicity increases hemolytic activity. The calculated mean hydrophobicities of the peptides based upon the hydrophobicity scales for amino acids of Kyte and Doolittle (1982) are +15.3 for CPF-C1 and +0.6 for CPF-C2. Thus, the increased hemolytic activity of CPF-C1 (LC50 = 140 µM) compared with CPF-C2 (LC50 > 200 µM) is consistent with its appreciably greater hydrophobicity.

75

The study supports an earlier conclusion (Mechkarska et al., 2010) that evolutionary pressure to conserve the primary structures of the antimicrobial peptides from the clawed frogs has not been strong (Fig. 4). In peptides belonging to the magainin family from the four species studied to-date, only 7 out of 23 amino acids are invariant and conservation of amino acid sequence in peptides of the CPF and XPF families is even less. A comparison of the primary structures of peptides of the CPF family peptides suggests that they divide naturally into two sub-groups but, even after deletions have been introduced into some sequences in order to maximize structural similarity, only the N-terminal glycine residue in the first group and 5 amino acids in the second group are invariant. Nevertheless, amino acid substitutions are almost always conservative and Schiffer-Edmundson (1967) helical wheel representations of the structures indicate that the amphipathic character of the

3 peptides has been maintained (data not shown). r te

p Antimicrobial peptides belonging to the peptide glycine-leucine-amide family have been isolated from skin secretions of X. laevis (PGLa), X. amieti (PGLa-AM1 and Cha PGLa-AM2), X. borealis (PGLa-B1 and PGLa-B2), and S. tropicalis (peptide XT-5) [reviewed in (Mechkarska et al., 2010)] but an ortholog was not identified in the X. clivii secretions. Similarly, as shown in Fig. 4, three XPF paralogs (XT-2, XT-3, and XT-4) were identified in skin secretions of the diploid frog S. tropicalis (Ali et al., 2001) whereas only one XPF peptide was identified in the X. clivii secretions. One possibility is that more peptides belonging to these families are present in the X. clivii secretions but their activity is so low that they have escaped detection. Alternatively, the gene(s) encoding the peptides have been deleted from the X. clivii genome during the course of evolution. This explanation is consistent with the conclusion of a previous study with the octoploid frog X. amieti that nonfunctionalization, involving deletion of a duplicated gene or its degeneration to a pseudogene, is the most common outcome following polyploidization events in the Xenopus/Silurana genera (Conlon et al., 2010b). Extended-spectrum β-lactamase (ESBL)-producing Gram-negative bacteria are becoming increasingly prevalent and their antibiotic resistance necessitates novel therapeutic intervention. Bacteria which express ESBL genes have the capacity to hydrolyse a broad spectrum of β-lactam antibiotics, including carbapenems (Bush, 2010). Treatment of patients with bacterial infections caused by such multi-resistant pathogens is challenging as antibiotic options are becoming increasingly limited. Although some effective new types of antibiotics against multidrug-resistant Gram-positive bacteria, such as methicillin-resistant S. aureus (MRSA), have been introduced or are in clinical trials, the situation regarding new treatment options for infections produced by emerging multidrug- resistant Gram-negative pathogens such as A. baumannii, K. pneumoniae, and P.aeruginosa is less encouraging (Norrby et al., 2005; Livermore, 2009). The present study has demonstrated CPF-C1 and other members of the CPF family [CPF-AM1 from X. amieti (Conlon et al., 2010b) and XT-7 from S. tropicalis (Ali et al., 2001)] show relatively potent

76 growth-inhibitory activities against reference strains of these Gram-negative bacteria. Future studies will investigate their effectiveness against ESBL-producing clinical isolates. Previous work from the laboratory has demonstrated the feasibility of using peptides against ESBL-producing pathogens (Eley et al., 2008). The frog skin peptide, ascaphin-8 and its lysine-substituted analogs were effective against a range of clinical isolates of ESBL- producing strains of E. coli and K. pneumoniae. Like ascaphin-8, CPF-C1 shows moderate hemolytic activity against human erythrocytes (LC50 = 140 µM) which limits its applications for systemic use but a strategy has been developed for design of analogs of naturally occurring frog skin antimicrobial peptides that retain activity against microorganisms but are non-hemolytic (Conlon et al., 2007). For example, the LC50 value against human erythrocytes for the potent analog [Lys10]ascaphin-8 was > 500 µM Cha compared with 55 µM for the native peptide (Eley et al., 2008). Future studies will p investigate structure-activity relationships for CPF-C1 with a view to the design of non- ter 3 toxic analogs with enhanced potency against microorganisms.

Acknowledgments This work was supported by a Faculty Support Grant (NP/10/05) from United Arab Emirates University.

77

REFERENCES Ali, M.F., Soto, A., Knoop, F.C., Conlon, J.M., 2001. Antimicrobial peptides isolated from skin secretions of the diploid frog, Xenopus tropicalis (Pipidae). Biochim. Biophys. Acta 1550, 81-89. Andreu, D., Aschauer, H., Kreil, G., Merrifield, R.B., 1985. Solid-phase synthesis of PYLa and isolation of its natural counterpart, PGLa [PYLa-(4-24)] from skin secretion of Xenopus laevis. Eur. J. Biochem. 149, 531-535. Bush, K., 2010. Alarming β-lactamase-mediated resistance in multidrug-resistant Enterobacteriaceae. Curr. Opin. Microbiol. 13, 558-564. Clarke, B.T., 1997. The natural history of amphibian skin secretions, their normal functioning and potential medical applications. Biol. Rev. Camb. Philos. Soc. 72, 365-379. 3 r Clinical Laboratory and Standards Institute, 2008. Methods for dilution antimicrobial susceptibility te p tests for bacteria that grow aerobically.Approved Standard M07-A8. CLSI, Wayne, PA.

Cha Conlon, J.M., Al-Ghaferi, N., Abraham, B., Leprince, J., 2007. Strategies for transformation of

naturally-occurring amphibian antimicrobial peptides into therapeutically valuable anti-infective agents. Methods 42, 349-357. Conlon, J,M., Ahmed, E., Pal, T., Sonnevend, A., 2010a. Potent and rapid bactericidal action of alyteserin-1c and its [E4K] analog against multidrug-resistant strains of Acinetobacter baumannii. Peptides 31,1806-1810. Conlon, J.M., Al-Ghaferi, N., Ahmed, E., Meetani, M.A., Leprince, J., Nielsen, P.F., 2010b. Orthologs of magainin, PGLa, procaerulein-derived, and proxenopsin-derived peptides from skin secretions of the octoploid frog Xenopus amieti (Pipidae). Peptides 31, 989-994. Dathe, M., Wieprecht, T., Nikolenko, H., Handel, L., Maloy, W.L., MacDonald, D.L., Beyermann, M., Bienert, M., 1997. Hydrophobicity, hydrophobic moment and angle subtended by charged residues modulate antibacterial and haemolytic activity of amphipathic helical peptides. FEBS Lett. 403, 208-212. Eley, A., Ibrahim, M., Kurdi, S.E., Conlon, J.M., 2008. Activities of the frog skin peptide, ascaphin-8 and its lysine-substituted analogs against clinical isolates of extended-spectrum beta-lactamase (ESBL) producing bacteria. Peptides 29, 25-30. Evans, B.J., Kelley, D.B., Tinsley, R.C., Melnick, D.J., Cannatella, D.C., 2004. A mitochondrial DNA phylogeny of African clawed frogs: phylogeography and implications for polyploid evolution. Mol. Phylogenet. Evol. 33, 197-213. Frost, D.R., 2010. Amphibian species of the world: an online reference. Version 5.4. American Museum of Natural History, New York, USA. Electronic database accessible at http://research.amnh.org/ herpetology/ amphibia /index.php. Gao, G.C., Wei, E.T., 1993. Xenopsin, neurotensin, neurotensin(8-13) and N-acetyl-neurotensin(8-13) inhibit vascular leakage in rats after tissue injury. Pharmacol. Exp.Ther. 265, 619-625.

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Giangaspero, A., Sandri, L., Tossi, A., 2001. Amphipathic -helical peptides. A systematic study of the effects of structural and physical properties on biological activity. Eur. J. Biochem. 268, 5589-5600. Gibson, B.W., Poulter, L., Williams, D.H., Maggio, J.E., 1986. Novel peptide fragments originating from PGLa and the caerulein and xenopsin precursors from Xenopus laevis. J. Biol. Chem. 261, 5341-5349. Giovannini, M.G., Poulter, L., Gibson, B.W., Williams, D.H., 1987. Biosynthesis and degradation of peptides derived from Xenopus laevis prohormones. Biochem. J. 243, 113-120. Gottler, L.M., Ramamoorthy, A., 2009. Structure, membrane orientation, mechanism, and function of pexiganan - a highly potent antimicrobial peptide designed from magainin. Biochim. Biophys. Cha Acta 1788, 1680-1686. p

James, S., Gibbs, B.F., Toney, K., Bennett, H.P., 1994. Purification of antimicrobial peptides from an ter 3 extract of the skin of Xenopus laevis using heparin-affinity HPLC: characterization by ion-spray mass spectrometry. Anal. Biochem. 217, 84-90. Kyte, J., Doolittle, D.F., 1982. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157, 105-132. Lipsky, B.A., Holroyd, K.J., Zasloff, M., 2008. Topical versus systemic antimicrobial therapy for treating mildly infected diabetic foot ulcers: a randomized, controlled, double-blinded, multicenter trial of pexiganan cream. Clin. Infect. Dis. 47, 1537-1545. Livermore, D.M., 2009. Has the era of untreatable infections arrived? J. Antimicrob. Chemother. 64 Suppl 1, i29-36. Measey, J., Tinsley, R., Largen, M., 2004. Xenopus clivii. In: IUCN 2010. IUCN Red List of Threatened Species. Version 2010.3. Electronic database accessible at www.iucnredlist.org. Mechkarska, M., Ahmed, E., Coquet, L., Leprince, J., Jouenne, T., Vaudry. H., King, J.D., Conlon, J.M. 2010, Antimicrobial peptides with therapeutic potential from skin secretions of the Marsabit clawed frog Xenopus borealis (Pipidae). Comp. Biochem. Physiol. C Toxicol. Pharmacol. 152, 467-472. Muñoz, V., Serrano, L., 1994. Elucidating the folding problem of helical peptides using empirical parameters. Nature Struct. Biol. 1, 399-409. Norrby, S.R., Nord, C.E., Finch, R., 2005. Lack of development of new antimicrobial drugs: a potential serious threat to public health. Lancet Infect. Dis. 5, 115-119. Powers, J.P., Hancock, R.E., 2003. The relationship between peptide structure and antibacterial activity. Peptides 24, 1681-1691. Richter, K., Egger, R., Kreil, G., 1986. Sequence of preprocaerulein cDNAs cloned from skin of Xenopus laevis. A small family of precursors containing one, three, or four copies of the final product. J. Biol. Chem. 261, 3676-3680.

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Schiffer, M., Edmundson, A.B., 1967. Use of helical wheels to represent the structures of proteins and to identify segments with helical potential. Biophys. J. 7, 121-135. Soravia, E., Martini, G., Zasloff, M., 1988. Antimicrobial properties of peptides from Xenopus granular gland secretions. FEBS Lett. 228, 337-340. Stark, M., Liu, L.P., Deber, C.M., 2002. Cationic hydrophobic peptides with antimicrobial activity. Antimicrob. Agents Chemother. 46, 3585-3590. Vincent, M.E., Wetzner, S.M., Robbins, A.H. 1982. Pharmacology, clinical uses, and adverse effects of ceruletide, a cholecystokinetic agent. Pharmacotherapy 2, 223-234. Zasloff, M., 1987. Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms and partial cDNA sequence of a precursor. Proc. Natl. Acad. Sci. USA 84, 5449-5453. 3 r

te p Cha

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Chapter 4

Genome duplications within the Xenopodinae do not increase the multiplicity of antimicrobial peptides in Silurana paratropicalis and Xenopus andrei skin secretions

M. Mechkarska, E. Ahmed, L. Coquet, J. Leprince, T. Jouenne, H. Vaudry, J.D. King, K. Takada, J.M. Conlon Comparative Biochemistry and Physiology (2011) 6:206-212. Chapter 4 A putative genome duplication event within the the within event genomeduplication A putative ABSTRACT stimulated skinsecretions of has produced the octoploid frog frog tetraploid aureus against activity withgrowth-inhibitory peptides Silurana the in events polyploidization following genes peptide antimicrobial duplicated fate of indicate, therefore, thatno frog from tetraploid the and -AN2), and one caerulein-precursor fragm XPF family fromand-A two (XPF-AN1 the components comprised two peptides frommagainin the family, (magainin-AN1 and -AN2), CPF peptides showed potent, broad-spectrum antimicrobial activity. The -SP2), and one peptide orthologous to peptide glycine-leucine-amide(PGLa-SP1). The - SP2 and -SP3), two peptides fromthe xenopsin-precursor fragment family (XPF-SP1 and comprised three peptides belongingto the caer antimicrobial peptides were previouslyisolated from diploidthe frog octoploid frog these peptides indicateacloseph . Structural characterization demonstrated thatthe Xenopus Xenopus and S. paratropicalis S. X. amieti lineages. lineages. X. borealis . Under the same experimental conditions, seven orthologous nfunctionalization (gene deletion) has been the most common S. paratropicalis and S. paratropicalis andasecond polyploidization within the , and five from the tetraploid frog . PeptidomicX. andrei analysisof norepinephrine- ylogenetic relationship between 84 N2), two from the PGLafamily fromthe N2), two (PGLa-AN1 ulein-precursor fragment family (CPF-SP1, ent (CPF-AN1). Theprim ent X. andrei Silurana Escherichia coli led to identification of multiple multiple of identification to led lineage has given rise to the S. paratropicalis and X. andrei X. clivii S. tropicalis, S. ary structures of Xenopus Staphylococcus components . Thedata X. andrei andthe lineage lineage nine

Chapter 4

, species Xenopus Silurana Xenopus whichare as separate as separate Hymenochirus ) (Evans et al., ) (Evans Xenopus Silurana X. amieti, X.andrei, victorianus + X. ruwenzoriensis and ruwenzoriensis X. X. wittei and and Silurana lineage has given X. laevis X. borealis,X. X. clivii, X. fraseri, (Evans, 2008; Frost, 2011; Evans Evans 2011; Frost, 2008; (Evans, reclassified in the genus

) 2n = 40 as well as at least two and number for pipid frogs of 2n = 20 was was 20 = 2n of frogs pipid for number t in skin secretions of many, although by by although many, of secretions skin in t within the family Pipidae (Frost, 2011). 2011). (Frost, Pipidae family the within with some some 85 ive drugs (Zhang and Falla, 2010) and for the the for and 2010) Falla, and (Zhang drugs ive species distributed in five genera genera five in distributed species laevis petersii ploidization eventin an ancestral

is controversial. Traditionally, the single diploid diploid single the Traditionally, controversial. is X. Silurana predates the polyploidization event that , and Xenopus, and oduced the dodecaploid species species oduced the dodecaploid from Silurana S. paratropicalis i, X. muelleri, X. pygmaeus 41 million years ago in a putative diploid (2n = 18) Silurana but the species has now been has species but the , Xenopus has a complex evolutionary history and its members have undergone at undergone have members its and history evolutionary a complex has chromosome number (36 and 40) in the two tetraploid lineages suggests tetraploid species (Tymowska, 1991; Tymowska and Fischberg, 1982). epitropicalis and (2n = 108) (Evans et al., 2005; Du Pasquier et al., 2009). Allopolyploidization, Allopolyploidization, 2009). al., et Du Pasquier 2005; al., et (Evans = 108) (2n Xenopus S. Xenopus tropicalis A putative genome duplication event within the Pseudhymenochirus , Introduction least 33 at frogs comprise The clawed The taxonomic status of The taxonomic Peptideswith antimicrobial activity are presen otential therapeutically valuable anti-infect (Cannatella and Trueb, 1988). Cladistic analysis based upon nucleotide sequences of of sequences nucleotide upon based analysis Cladistic 1988). Trueb, and (Cannatella Xenopus of monophyly the supports strongly genes mitochondrial insight that they provide into the evolutionary history of thespecies (Conlon, in press). species is unclear (Frost, 2011). Subsequent independent genome duplication events have have events duplication genome independent Subsequent 2011). (Frost, unclear is species 2n = 72 ( with species octoploid seven rise at to least given X. boumbaensis, X. itombwensis, X. lenduensis, X. vestitus, 2005). Two further independent polyploidizations involving the combination of tetraploid of tetraploid combination the involving polyploidizations independent further Two 2005). pr have and octoploid genomes X. longipes in which two species hybridize and the descendant inherits the complete genome of both both of genome complete the inherits descendant the and hybridize species two which in the in duplication of genome mode principal the be to considered is ancestors, et al., 2011). The taxonomic status of taxonomic et al., 2011). The 1. X. gilli, laevis, X. X. largen Pipa eight well ( characterized tetraploid species has generated The genus The genus least onepolyploidization event such speciesall that share either a tetraploid(2n36) = karyotype or euploid multiples (2n = 72 or 2n = 108) (Kobel and1991). Du Pasquier, et al., (Evans reported been ecies have sp additional, as yet unnamed, Although several 2004;2008), al., Evans a single et poly united inXenopodinae the (de Sá andHillis, 1990) but the use of twogenera "underscores termed termed species that has retained the ancestral chromo the retained has species that further unnamed occurred between 21 and ancestor (Kobel, 1996; Evans, 2007). The difference in that divergence of trenchant biological and historical differences between 2004). the two clades" (Evans et rise al., to genus (Kobel,1996). no means all, species of Anura (frogs and toads) and have excited interest as a source of of source as a interest excited have and toads) and (frogs Anura of species all, means no p Chapter 4 characterized than,the 2011). (Conlon et al., 2010), declined due toharvesting for human co stable. Its IUCN RedList status is‘Least swamps andsmall ponds in lowland forest. Thespecies is adaptable andits population shady are habitats natural Its 2011). (Frost, Congo of Republic the and Guinea Equatorial in occurs and probably Republic African Central western and Gabon northern Cameroon, their mating calls.Andre’s clawed frog referred to thespecies as S. paratropicalis S. S. paratropicalis extrem and Congo Democraticof Republic Both species occur in theCongoBasin antimicrobial peptidespresen studies with common ancestral geneby aseries of dupli N-terminal regions of the precursors suggesting that the peptides may have evolved froma and proxenopsin reveals significant structural similaritythe signal in peptide and sequences ofcDNAs encoding the biosynthetic described [reviewed in (Mechkarska et al., 2010)]. A comparison of the nucleotide precursors of caerulein, xenopsin, and peptid antimicrobal peptides derived from the post-translational processing of the biosynthetic multiple ofthe 1987), magainins (Giovanni Zasloff, discovery etal., initial 1987; Skin secretions of X. tropicalis from thepeptides consistency, maintain To magainin-AN2. and magainin-AN1 insight into the diversity and evolutionary history of MALDI-TOF massspectrometrycoupled withre amide (PGLa),caerulein-precur X. borealis, classified according to the terminology used previously for peptides from peptides from frogs of the Pipidae family andso the peptides described in this study are termed CPF-ST1, CPF-ST2, andCPF-ST-3 respectively, the peptides XT-2, XT-3, and ( SP by isdenoted origin species The recognized. (XPF) peptide familiesare peptides. There isnouniver S. paratropicalis The Gabon clawed frog The present study is part of an on-going program ofpeptidomic analysis using and X.clivii that were originally termed XT-1, XT-1, thatXT termed wereoriginally X. laevishave since been extended toinclude thecharacterization of the , wasfirst recognized astetraploid (2n = 40) by Tymowska (1991) who ) or AN ( , and epitropicalis S. X. laevis have proved to be arich source of such peptides. Following the X. borealis (Mechkarska etal.,2010),andX. borealis Cameroon clawed frog (Conlon et al., 2011). Xenopus Xenopus X. t in skin secretions of of secretions t skin in sally acceptednomenclature fo Silurana paratropicalis Silurana

andrei sor fragment (CPF),andxenopsin-precursor fragment nova VII. Although morphologically very similar, similar, very morphologically Although VII. nova ) and paralogs are differentiated by numeralse.g. nsumption (Tinsley S. tropicalis Xenopus amieti Xenopus 86 cation events (HuntandBarker,1988).These Concern’ butnumbers in certain areas have encompassing Gabon, Cameroon, western Cameroon, western Gabon, encompassing e glycine-leucine-amidee (PGLa) have been S. epitropicalis epitropicalis S. precursors ofprocaerulein,promagainin, e northeastern Angola The magainin, peptide glycine-leucine- peptide magainin, The -6, and XT-7 (Ali et al., 2001) are are now etal.,2001) (Ali and XT-7 -6, S. tropicalis versed-phase HLPC that aims to gain may be distinguished on the basisof the on distinguished maybe is closely related to, but lesswell but to, related isclosely Silurana is widely distributed in coastal in iswidely distributed and Measey, 2004). r designatingantimicrobial (Ali et al., 2001), (Alietal., and with which itissympatric which with X. clivii(Conlonetal., Xenopus (Frost, 2011). host-defense X. laevis, X. amieti .

Chapter 4

L of  S. aureus in 96-well in

(ATCC 700603), (ATCC 90028), and a through 6 Sep-Pak C-18 6 Sep-Pak through

(ATCC 25726) (ATCC C. The pooled solutions from from solutions pooled The C. o l) with an inoculum (50 inoculum an with l)  onnected in series. Bound material was material Bound in series. onnected (n = 2; male 15 g, female 22 g) were were 22 g) 15 g, female male (n = 2; Candida albicans Candida increasing concentrations of antibiotics A) c A21-09) and were carriedout by authorized C in a humidified of air. After atmosphere Escherichia coli Escherichia o 87 E). Both species were supplied by Xenopus Xenopus by were supplied species E). Both .9:0.1, v/v/v) and freeze-dried. freeze-dried. and .9:0.1, v/v/v) creasing concentrationsof ampicillin. Minimum (Euroclone I NM8 strain) were measured in the the in measured were strain) NM8 I (Euroclone peptides against reference strains of reference against peptides and 58 X. andrei

 (n = 2; male 8 g, female 18 g) were collected in in collected were 18 g) female 8 g, male 2; = (n M by standard double dilution methods (Clinical (Clinical methods dilution double by standard M μ XPF-ST2, and XPF-ST3 respectively, and XT-5 is now now is XT-5 and respectively, XPF-ST3 and XPF-ST2, N, 09  E). Adult 04  08  ater/ TFA (70.0:29 ater/ paratropicalis (ATCC 25923)

S. E. coli (ATCC 25726), Klebsiella pneumoniae N, 11 Acinetobacter baumannii 48  Materials and methods Collectionof skin secretions withAll experiments live animals were approved Researchby the Animal Ethics Antimicrobial assays assays Antimicrobial Purificationof the peptides was monitored by incubating lyophilized aliquotsof

colony forming units/mL) from a log-phase culture of reference strains of strains reference of culture a log-phase from units/mL) forming colony 6 Cameroon (02 10 (ATCC 25923), (ATCC 27853),and aeruginosa Pseudomonas incubation, the absorbance of630determinednm at was using each well a microtiter plate Inreader. order to the validity monitor reproducibility and of the assays, incubationswith were carried outbacteriaparallel with in in inhibitory concentrations of the (MIC) concentration range of 3 - 100 Laboratory Standards Institute, 2008a,b) and concentrationwere taken as the lowest of by measurement were confirmed The values was observed. growth no visible where peptide of absorbanceat 630order In nm. monitor to the validity andreproducibility of the assays, incubations were carried out in parallel with Staphylococcus aureus Staphylococcus investigators. Adult XT-4 are now termed XPF-ST1, now termed XT-4 are 2.1. committee of U.A.E. University (Protocol No. 2. termed PGLa-ST1. termed eluted with acetonitrile/w 2.2. (50 broth Mueller-Hinton in effluent chromatographic cartridges (Waters Associates, Milford, MA, US Milford, MA, (Waters Associates, cartridges each species were separately passed at a flow rate of 2 mL/min at a flow rate of 2 passed separately were each species clinical isolate of collected in Cameroon (03 collected in Cameroon Express(Brooksville, Inc. FL, USA). Each was injected animal at two sites in the dorsal in and immersed weight) body per g nmole (80 bitartrate norepinephrine sac with lymph trifluoroacetic with was acidified secretions the containing The solution min. 15 for water -20 at stored and v/v) 1% concentration (final (TFA) acid microtiter cell-cultureplates for 18 h at 37 Chapter 4 280 nm andfractions (1 min) were collect to 63%(v/v) over 60 min usinglinear gradients. Absorbancemonitored was at 214 nmand concentration of acetonitrileinthe elutingsolv TFA/ with IL, (v/v) 0.1% USA) equilibrated (2.2 x 25 cm) Vydac 218TP1022 (C-18) reversed-phase HPLC column (Grace, Deerfield, cartridges, were redissolved in 0.1% (v/v) TFA/water (4 mL) and separately injected onto a 2.3. for (ampicillin of the fractions to inhibit the growth of of the growth inhibit to the fractions of and amphotericin for in the previous section.Frac hydrophobicity scales for amino acids of Kyte and Doolittle (1982). of amino acids (1982). scalesfor Kyteand Doolittle hydrophobicity were basedupon the the peptides of mean hydrophobicities Calculated of a helix. presence 278 K. A minimum percentage of 1% helicity/residue wasconsidered to predict the the helical behaviour of monomeric peptides. Calculations were performed at pH 7 and the algorithmon based AGADIR isa prediction peptides were performed using the AGADIR program (Muñoz and Serrano 1994). 2.5. mass determinations was of accuracy The range. 2 mass- 4 kDa the molecular in ofknown with peptides calibrated extraction andtheaccelerating vo instrument (Applied Biosystems) that wasoperated in reflector mode with delayed USA). MALDI-TOF mass spectrometry wascarried out using a Voyager DE-PRO degradation using an Applied Biosystemsmodel 494 Procise sequenator (Foster City, CA, 2.4. raised from 21% to 56% over 50 min and the flow rate was 2.0 mL/min. Vydac 208TP510 (C-8)column. Theconcentratio cm) a(1 x25 and column (C-4) 214TP510 cm) Vydac a(1 x 25 on chromatographed

The lyophilized skin secretions from both sp purification Peptide Prediction of secondary structure and determination of helicity perresidue for the Secondary structure prediction The primary structures of the peptides were determined by automated Edman Edman The primary structures of automated were determined peptides by the Structural characterization S.

aureus C.

albicans and  0.02%. tions associated withantimicr E. coli; ciprofloxacin for K.pneumoniae ) as previously described (Mechkarska et al., 2010). et al., 2010). (Mechkarska described aspreviously ) ltage inthe ionsource was20kV.The instrumentwas S. aureus ed. The abilitiesoffreeze-dried aliquots(50 88 ent was raised to 21%(v/v) overminwas raised ent 10 and water at a flow rate of water ataflowrate mL/min.6.0 The of ecies, after partial purification on Sep-Pak Sep-Pak on ecies, after partial purification helix/coil transition theory which predicts predicts which theory transition helix/coil and n of acetonitrile intheeluting solvent was E. coli E. obial activitywere successively were determined asdescribed and P.aeruginosa; and  L) L)

Chapter 4 or or E. coli reversed-phase HPLC HPLC reversed-phase and were purified further. The and were purified further. The fractions designated by the bar the by designated The fractions . C-18 column of skin secretions from column of skin secretions from C-18 , after partial purification on Sep-Pak on Sep-Pak purification partial , after to further purification on a semipreparative on a semipreparative purification further to and S. aureus in the eluting solvent. Only absorbance at 214 nm at 214 eluting solvent. Only absorbance in the Vydac C-18 preparative Vydac C-18 preparative 89 ssed by a symmetrical peak shape and massssed by a symmetrical and peak shape on a semipreparative Vydac C-8 column. The The C-8 column. Vydac on a semipreparative E. coli S. paratropicalis after partial purification on Sep-Pak cartridges on Sep-Pak partial purification after

Results Purificationtheofpeptides paratropicalis from S. from secretions skin pooled The These fractions were individually subjected fractions were individually These The fractions with retention times between 44 and 57 min (denoted by the bar in in bar by the (denoted min 57 44 and between times retention with The fractions

Vydac C-4 column and those components with growth-inhibitory activity were purified to to purified were activity growth-inhibitory with components those and column C-4 Vydac as asse (> 98% purity), homogeneity near by a final chromatography spectrometry, the in peptide abundant most of the purification the by illustrated is methodology dashed line shows the concentration of acetonitrile the concentration dashed line shows is shown. S. paratropicalis, displayed growth inhibitoryactivityagainst Reversed-phase HPLC on a preparative Vydac Reversed-phase HPLC on a preparative Fig. 1. 3. 3.1 C-18 cartridges, were chromatographed on a column (Fig. 1). 1). (Fig. column S. aureus. Fig. 1) contained peptides that inhibited to varying degrees the growth of either either of growth the degrees varying to inhibited that peptides contained 1) Fig. Chapter 4 ended. The peak denotedby the cross inhibited the growthof Vydac C-4,and C-8columns. (B)Vydac Thearrowheads show wherepeakcollection began and Fig. 2. secretions, CPF-SP3 by sequen SP1 230. CPF-SP377 CPF-SP2235, 320, CPF-SP1 were and Vydac C-8 column (Fig. 2B).The approximate finalyields ofpurified peptides (nmol) Purification to near homogeneity ofCPF-SP3 from Purificationtonearhomogeneity tial chromatography on the on tial chromatography 90 5, XPF-SP1 155, XPF-SP2 190, and PGLa- and 190, 5, 155, XPF-SP2 XPF-SP1 S. paratropicalis E. coli E. and Vydac C-4 column (Fig. 2A) S. aureus onsemipreparative (A) . Chapter 4 , X. andrei peptides peptides (Conlon et al., al., et (Conlon splayed growth inhibitory X. clivii S. paratropicalis The approximate final yields of purified approximate The , after partial purification on Sep-Pak C-18

to further purification on a semipreparative a semipreparative purification on to further ical peak shape and mass spectrometry, by a spectrometry, mass and ical peak shape dac C-18 column of skin secretions from dac C-18 column 91 Vydac C-8 column. Chromatography on these these on Chromatography column. C-8 Vydac The fractions designated di . X. andrei s with skin secretions from s with skin and were purified further. The dashed line shows the The dashed and were purified further. secretions (Fig. 1), the resolution (Fig. secretions 1), the on this was column S. aureus and S. S. paratropicalis S. E. coli stent with previous studie previous with stent Reversed-phase HPLC on a preparative Vy HPLC on a preparative Reversed-phase Consi Purificationtheof peptides from X. andrei from secretions skin pooled The

(Fig. 3). 3). (Fig. Fig. 3. cartridges, were chromatographed on a Vydac C-18 preparative reversed-phase HPLC HPLC reversed-phase preparative C-18 Vydac a on chromatographed were cartridges, the of purification for used conditions same the under column purification on Sep-Pak cartridges after partial 3.2. 2011)with and the between times retention with fractions The obtained. were peaks broad relatively and poor activity. antimicrobial with peptides contained 3) Fig. bar in the by (denoted min 51 39 and subjected fractions were individually These to purified were activity growth-inhibitory with components those and column C-4 Vydac by a symmetr as assessed near homogeneity, on a semipreparative final chromatography peaks. well-defined in sharp, resulted columns peptides (nmol)were magainin-AN1 25, magainin-AN2 370, PGLa-AN1 20, PGLa-AN2 85, CPF-AN1 20, XPF-AN1 70, and XPF-AN2 15. concentration of acetonitrile in the eluting solvent. solvent. eluting the in acetonitrile of concentration activity against against activity Chapter 4 structures and todemonstrate thatCPF- shown in Fig. 4.MALDI-TOF mass spectrometryused wasto confirm the proposed assume that the peptides are C-terminally (M PGLa-AN2 areC-terminallyPGLa-AN2 α and Fig. 4. 3.3. X. GaS1GMATKAGTALGKVAKAVIGAAL.NH PGLa-SP1 P-P GLASTIGSLLGKFAKGGAQAFLQPK XPF-SP2 P-P GFWSSALEGLKKFAKGGLEALTNPK XPF-SP1 P-N GFASVLGKALKLGANLL.NH GMASKAGSVLGKLAKVAIGAL.NH CPF-AN1 PGLa-AN2 X. andrei GaA1GMASKAGSVLGKVAKVALKAAL.NH GWVSKIGQTLGKMAKVGLQELIQPK GWASKIGQTLGKMAKVGLQELIQPK PGLa-AN1 XPF-AN2 2404.92404.4 XPF-AN1 2417.32417.4 Magainin-AN2 GVSKILHSAGKFGKAFLGEIMKS Magainin-AN1 GIKEFAHSLGKFGKAFVGGILNQ P-P GFLGSLLKTGLKVGSNLL.NH GFLGPLLKLGLKGAAKLLPQLLPSRQQ CPF-SP3 GFLGPLLKLGLKGVAKVLPHLIPSRQQ2878.92878.8 CPF-SP2 CPF-SP1 S. paratropicalis

r andrei calc) of the antimicrobial peptides isolated from skin secretions of

The primary structures Structural characterization X. andrei Aminoacidsequences,obs .

The calculated masses of CPF-SP3, PGLa-SP1, CPF-AN1, PGLa-AN1 and PGLa-AN2 PGLa-AN2 and PGLa-AN1 CPF-AN1, PGLa-SP1, ofCPF-SP3, masses calculated The

skin secretions wereestablished by automated Edman degradation and are of theantimicrobial peptidesisolated from -amidated. erved molecular masses (M masses molecular erved α -amidated. -amidated. SP3, PGLa-SP1, CPF-AN 92 2 2 r obs), andcalculated molecular masses 2 2 2 1997.3 1997.2 2460.3 2649.7 2708.4 2680.6 1669.9 1670.0 1940.2 2068.2 1815.1 1815.4 2857.5 2856.8 S. paratropicalis M 1, PGLa-AN1 and 1, PGLa-AN1 r obs M S. paratropicalis 2459.4 2648.4 2708.5 2680.5 r calc and Chapter 4 25 6 25 12.5 12.5 25 P. aeruginosa C. albicans e skin secretions inhibit to 50 25 Silurana paratropicalis and neumonia p . K M) against microorganisms of the of the microorganisms against M) are compared in Table 1. In view of the of the 1. In view in Table are compared  6 93 12.5 peptides but limited data were obtained for the for the obtained were data limited but peptides X. andrei isolated from skin secretions of skin secretions from isolated coli coli S. aureus A. baumannii 50 6 50 25 6 50 25 6 50 >100 50 12.5 50 >50 50 ND ND 50 50 ND ND 25 100 ND ND ND ND ND ND 100 >100 25 100 >100 >50 25 ND 100 >100 >50 >50 ND ND 100 50 200 ND E. Minimum inhibitory concentrations ( concentrations inhibitory Minimum Antimicrobial activities The abilities of thepeptides purified in the S. paratropicalis

CPF-SP2 CPF-SP1 CPF-SP3 XPF-SP1 XPF-SP2 PGLa-SP1 Magainin-AN2 XPF-AN1 PGLa-AN2 low amounts of purified peptides obtained, it was not possible to determine the growth growth the determine to possible was not it obtained, peptides purified of amounts low of the all potencies inhibitory Data are expressed as µM. ND: not determined determined ND: not as µM. expressed are Data endogenous peptides Xenopus andrei Table 1. the growth of a range of clinically relevant bacteria more abundant magainin-AN2,XPF-AN1,PGLa-AN2. 3.4. Chapter 4 S. paratropicalis deletion. Procaerulein-derived peptides and al., 2010), S. paratropicalis Fig. 5. Proxenopsin-derived peptides S. tropicalis frogs the tetraploid and 2010) (magainin, PGLa, CPF, and XPF) isolated fromthe octoploid frog structures reveals that they represent orthologs of previously described dermal peptides the octoploid frog frog tetraploid the no in activity ofantimicrobial degrees varying 4. S. tropicalis S. paratropicalis S. tropicalis S. paratropicalis S. paratropicalis S. tropicalis PGLa-related peptides S. tropicalis S. tropicalis S. tropicalis S. paratropicalis S. paratropicalis This study has described the purification and characterization of six peptides with Discussion Acomparison oftheprimary structures of the antimicrobial peptides from thetetraploid frog, X. clivii withorthologous peptides fromthediploid frog S. paratropicalis PL-T GMATKAGTALGKVAKAVIGAAL.NH PGLa-ST1 XFS3 GVFLDALKKF***AKGGMNAVLNPK XPF-ST3 .X. andrei Asshown in Figs. 5 and 6, acomparison of their primary P-T GLLGPLLKIAAKVGSNLL.NH CPF-ST3 P-T GFLGSLLKTGLKVGSNLL.NH CPF-ST2 P-T GFLGPLLKLAAKGVAKVIPHLIPSRQQ CPF-ST1 P-T GLASTLGSFLGKFAKGGAQAFLQPK GVWSTVLGGLKKFAKGGLEAIVNPK XPF-ST2 XPF-ST1 (Conlon et al., 2011). etal.,2011). (Conlon PL-P GMATKAGTALGKVAKAVIGAAL.NH PGLa-SP1 CFS2 GFLGPLLKLGLKGAAKLLPQLLPSRQQ GFLGPLLKLGLKGVAKVLPHLIPSRQQ CPF-SP2 CPF-SP1 XFS2 GLASTIGSLLGKFAKGGAQAFLQPK GFWSSALEGLKKFAKGGLEALTNPK XPF-SP2 XPF-SP1 CPF-SP3 GFLGSLLKTGLKVGSNLL.NH

X. laevis(Gibson et al., 1986), and seven antimicrobial peptides in skin secretions of

94 repinephrine-stimulated skin secretions of S. tropicalis X. borealis X. amieti(Conlon et al., . (*) denotes aresidue (Mechkarska et 2 2 2

2 2

Chapter 4

2 2 2 2 2 95

GFGSL*GKALRLGANVL.NH GWASKIGQALGKVAKVGLQQFIQPK GFKQFVH*SMGKFGKAFVGEIINPK GWASKIGTQLGKMAKVGLKEFVQS GWASKIGQTLGKIAKVGLQGLMQPK GIKEFAHSLGKFGKAFVGGILNQ GIKEFAHSLGKFGKAFVGGILNQ GIKEFAHSLGKFGKAFVGGILNQ GVGKFLHSAKKFGQALASEIMKS GVGKFLHSAKKFGQALVSEIMKS G**KFLHSAGKFGKAFLGEVMIG GIGKFLHSAGKFGKAFLGEVMKS GIGKFLHSAGKFGKAFVGEIMKS GLGSVLGKALKIGANLL.NH GLGSVLGKILKMGANLLGGAPKGA GLGSLLGKALKFGLKAAGKFM*GGEPQQ GLGSLLGKAFKIGLKTVGKMMGGAPREQ GLGSLLGKAFKIGLKTVGKMMGGAPR GLGSLLGSLFKFIPK******LLPSIQQ GLLTNVLGFLKKAGKGVLSGLLPL GLASFLGKALKAGLK*IGAHLLGGAPQQ GIGSALAKAAKLVAGIV.NH GLGSLVGNALRIGAKLL.NH GWASKIGQTLGKMAKVGLQELIQPK GWVSKIGQTLGKMAKVGLQELIQPK GWASKIAQTLGKMAKVGLQELIQPK GFASVLGKALKLGANLL.NH GVSKILHSAGKFGKAFLGEIMKS GVSKILHSAGKFGKAFLGEIMKS -B1 -B2 -B1 -B2 -B1 -B2 -B3 -B4 1 2 GWASKIGQTLGKIAKVGLKELIQPK 1 2 3 4 GFASFLGKALKAALK*IGANMLGGTPQQ GFGSFLGKALKAALK*IGANALGGSPQQ GLASLLGKALKAGLK*IGTHFLGGAPQQ -AM2 -AM4 -C1 -AN1 -AN2 -AM1 -C1 -AN1 -AN1 -AN2 -AM1 -AM2 -C1 -C2 -1 -2 GIGKFLHSAKKFGKAFVGEIMNS -AM1 -AM3 -C2 X. amieti X. amieti X. clivii X. andrei X. borealis X. borealis X. laevis X. laevis X. andrei X. andrei Procaerulein-derived peptides Procaerulein-derived X. andrei X. amieti X. amieti X. clivii X. clivii X. borealis X. borealis X. laevis X. laevis X. amieti Proxenopsin-derived peptides X. clivii X. borealis X. borealis X. borealis X. borealis X. laevis X. laevis X. laevis X. laevis X. amieti Magainin-related peptides Magainin-related X. andrei X. amieti X. clivii Chapter 4 X. andrei X. andrei Fig. 6. X. amieti PGLa-related peptides X. The distribution of the antimicrobial peptides in the skin secretions of inthe were magainins detected not the to orthologous peptides X. laevis X. borealis X. borealis X. amieti X. andrei (Fig. 7).

borealis Co A comparison of the primary structures Acomparisonoftheprimary of theantimicrobial peptides fromtheoctoploid frog nsistent with previous work with the diploid frog frog diploid the work with previous with nsistent with orthologous peptides fromthe octoploid frog , X. clivii -AN1 -AM1 -AM2 -AN2 -B2 -B1 , and X. laevis . (*)denotes a residue deletion.

GMASKAGAIAGKIAKVALK*AL.NH GMASKAGSIVGKIAKIAL*GAL.NH GMASKAGTIAGKIAKTAIKLAL.NH GMASTAGSVLGKLAKAVAIGAL.NH GMASKAGSVLGKLAKVAI*GAL.NH GMASKAGSVLGKVAKVALKAAL.NH GMASKAGSVLGKVAKVALKAAL.NH 96 X. amieti S. tropicalis S. paratropicalis , and the tetraploid frogs, X. andrei (Ali et al., 2001), etal.,2001), (Ali 2 2 2 2 2 2 2

secretions. is atypical isatypical Most abundant Least abundant

S. tropicalis CPF-ST1 CPF-ST3 CPF-ST2 PGLa-ST1 XPF-ST3 XPF- XPF-ST2 ST1

S. paratropicalis CPF-SP3 CPF-SP1 CPF-SP2 PGLa-SP1 XPF-SP2 XPF- SP1

X. borealis CPF-B1 Magainin PGLa-B2 Magainin XPF-B2 CPF- CPF-B4 PGLa- XPF-B1 -B2 -B1 B3 B1 97

X. clivii CPF-C1 Magainin Magainin- CPF-C2 XPF-C1 -C1 C2

X. amieti Magainin- CPF-AM1 PGLa-AM1 CPF-AM2 PGLa- CPF- Magainin- XPF- CPF- AM2 AM2 AM4 AM1 AM1 AM3

X. andrei Magainin- PGLa-AN2 XPF-AN1 Magainin PGLa- CPF- XPF-AN2 AN2 -AN1 AN1 AN1

Fig. 7. Relative abundance of the antimicrobial peptides isolated from skin secretions of the diploid frog S. tropicalis, the tetraploid frogs S. paratropicalis, X. borealis and X. clivii, and the octoploid frogs X. amieti and X. andrei.

Chapter 4 Chapter 4 but the LC to determine accuratelythe hemolytic activity opportunistic yeast pathogen E. coli ≤ (MIC potency high relatively displaying agent, anti-infective valuable atherapeutically into for development varied appreciably (Table 1). CPF-SP1 represen independent polyploidization events. octoploid ancestor whereas the octoploid frogs, 2007). It was proposed that (Evans, gene RAG1 the and 2008) Evans, etal.,2004; (Evans DNA mitochondrial of sequences nucleotide of from with data comparisons isconsistent This conclusion identical. against areference strain lineage that produced Silurana the within event tetraplodization the that suggesting (Fig. 5) CPF-ST2 to identical X. amieti particularly close phylogenetic relationship between the octoploid frogs, the to-date, including thesecretionsforms in molecular of allother multiple speciesstudied andin abundance hydrophobicity, hydrophobicity, against microorganisms determined is by complex interactions among cationicity, with their physicochemical properties (Table 2). The antimicrobial potency of a peptide which the pathogens have not been exposed. (Livermore, 2009).There isclearlyanurgent n new treatment options for infections produced by these microorganisms isnot encouraging pathogens poses aparticularly serious threat to public health andthe situation regarding the PGLa peptides from species (Mechkarska etal.,2010 antimicrobial peptides have generally beenpoorly conserved both within and between evolutionary history ofclosely related species. The primary structures ofthe into the insight can provide secretion in frogskin peptides antimicrobial the agents, interaction with the negatively charged phospholipids in the bacterial cell membrane and and membrane cell bacterial the in phospholipids charged negatively the with interaction using model X. andrei The antimicrobial potencies andspecificities The observed potenciestheof As well as their potential for development in development for As potential well astheir The potent and broad spectrum CPF antimi , A. baumannii astheamino acidsequences ofthema 50 value was > 100 µM. The emergence of multidrug-resistant Gram-negative Gram-negative ofmultidrug-resistant emergence µM.The was>100 value secretions and this inlowyield. and secretions  -helical peptides have demonstrated th  -helicity and amphipathicity [reviewed in (Conlon et al. 2007)]. Studies Studies et al.2007)]. (Conlon in [reviewed amphipathicity and -helicity S. paratropicalis S. paratropicalis S. , 25 µM) againstreference strains K. pneumonia S. paratropicalis oftheGram-positive C. albicans X. amieti ; Conlon et al., 2011). However, the primary structures of , whereas onlyasingle CPF peptide was isolatedfrom S. paratropicalis , and P.aeruginosa , was relatively recent. The data in Fig. 6 indicate a a Fig. indicate in 6 The data recent. was relatively X. andrei (MIC =25µM). There was insufficient material and 98 eed for new types ofan ts the component with the greatest potential potential thegreatest with component ts the are the same and CPF-SP3 is arethesame tropicalis andCPF-SP3is S. of thepeptide against human erythrocytes S. aureus to therapeutically valuable anti-infective anti-infective valuable therapeutically to , and X. boumbaensis X. vestitus of the peptides isolated in this study study this in isolated peptides the of crobial peptidesar gainins andthePGLa-1 peptides are at an increase incationicity promotes peptides correlate moderately well moderately correlate peptides (MIC =6µM) andagainst the of theGram-negative bacteria . The peptide wasalsoactive and X. wittei timicrobial agents to agents timicrobial e presentin high shareacommon and X. andrei arosefrom Xenopus

Chapter 4 -helical  18-22 18-22 icity scales for amino -helical α domain 5-11 10-16, 10-16, 5-11 Non-helical 5-15,18-22 6-14 5-17 9-18 9-19 9-18 -helical domain between domain -helical

Charge at pH 7 +4 +3 +4 +3 +2 +3 +3 +2 +3 Non-helical +3 Non-helical +5 +4 +4 ith its lower charge (+2 at pH 7) and low low 7) and pH at (+2 charge lower its ith lical domains were made using the AGADIR tentthis with observation,and CPF-SP1 99 Xenopus andrei and Xenopus of +4 at pH 7 and a predicted  predicted a 7 and pH at +4 of ttle. Predictions of he -helical conformation. hydrophobicity hydrophobicity  Silurana paratropicalis Silurana Physicochemical Physicochemical propertiesthe antimicrobial of peptides isolated skin from e low activity ofe low activity XPF-SP1 is consistentw Th Peptide Mean CPF-SP3 +14.6 Magainin-AN1 +4.6 Magainin-AN2 +5.6 CPF-AM1XPF-AN2 +18.1 PGLa-AN1 -2.7 +18.4 CPF-SP1CPF-SP2 +10.5 +6.7 XPF-SP1XPF-SP2PGLa-SP1 -6.3 +7.9 +20.3 XPF-AN1 -5.1 PGLa-AN2 +20.3 Table 2. secretions of residues 5 - 11, are more potent than CPF-SP3 which has a charge of +3 and a low a low and of +3 has a charge which CPF-SP3 than potent more are 11, - 5 residues propensity to adopt a mean hydrophobicity. The reduced potency of PGLa-SP1 is more difficult to explain as it as it explain to difficult is more of PGLa-SP1 potency The reduced hydrophobicity. mean amphipathic stable, a can adopt and 7 pH at +4 of charge a possesses conformation. The peptide is strongly hydrophobic and it is possible that its activity is activity its that possible is it and hydrophobic strongly is The peptide conformation. program. acid residues of Kyte and Dooli acid residues Mean hydrophobicities (H) of the peptides are calculated using the hydrophob using calculated are the peptides of (H) Mean hydrophobicities CPF-SP2 with a molecular charge charge a molecular with CPF-SP2 thereby increasesantimicrobial activity. Consis Chapter 4 expressed antimicrobial gene products ascomponents present in low abundance and/or with X. of eachindividualpeptide. microorganisms in the environment than inferred from data obtained by measuring the MIC and It isprobable, therefore, against activity inhibitory X. PGLa from and Magainin-2 oligomerize. to atendency and lowsolubility by reduced incorporation of premature stop codons and frameshiftmutations (Evans et al., 2005). Xenopus (Sammut et al.,2002; Du Pasquier et al., 2009). Studies of duplicated RAG-1 genes in complex major among histocompatibility and 2007) (Evans, immunity adaptive RAG1 and RAG2, proteins crucial to the process of somatic rearrangement of DNA in silencing in octoploid and dodecaploid species wasseen in the case of genes encoding albumins instead of the expected six. A similar high level of gene deletion and/or gene instead ofthe expected four and the dodecaploid species to be greater. The serum ofthe octaploid species appears events polyploidization third and second the following loss gene duplicate rate of and Fischberg, 1986).However, inthe case Silurana/Xenopus 1993). Consistent with this estimate, electrophoretic analysis ofserum proteins from20 gene copiesin duplicate the 23% of that only estimated it et and hasbeen al., Chain 2008), 2006; Evans, per site)(Chain and substitutions nonsynonymous per sitethan substitutions synonymous copies of the duplicate gene are subject to st in tetraploid gene pairs duplicate of numbers large of Analysis 2004). Katju, and (Lynch role biological a new toadopt evolves gene duplicated the which in neofunctionalization and functionality; share genes daughter gene or its degeneration to a pseudogene; subfunctionalization in which the mother and gene has three possible evolutionary fates – 2000). A duplicated Whitton, and (Otto species predominate polyploid that Xenopodinae in the but itisonly among Anura species the individual in sporadically occurred have of the teleost lineage (Lundin et al., 2003). Subsequent genome duplications arebelieved to history of the chordates (2R hypothesis) and once in fish immediately before the radiation have occurred several times during vertebrate evolution –twice during the very early Xenopus andrei

laevis X. andrei It is not claimedthattheantimi Polyploidization events involving duplication of the complete genome, are believed to arebelieved genome, complete ofthe duplication involving events Polyploidization and and species displayed two albumin bands whereas act synergistically so that the peptides in combination show more potent growth potent more show combination in peptides the that so act synergistically skin secretions in this study necessarily represent the full complement of of fullcomplement the represent necessarily study this in secretions skin skin secretions provides greater prot Silurana have been lost following tetraploidization (Hughes and and Hughes, (Hughes tetraploidization following lost been X. laevis have species revealed that species have indicated that degeneration of paralogs occurs via that the mixture of antimicrobial peptides in the the in peptides antimicrobial of mixture that the

than either peptide tested alone (Westerhoff et al., 1995). etal.,1995). alone (Westerhoff tested peptide either than E. coli crobial peptides isolated from Xenopus S. paratropicalis 100 nonfunctionalization involving deletion of the of the octoploid and dodecaploid species, the rong purifying selection and and containedX. andrei only three albumins ection fortheanimal pathogenic against Silurana , S. X. S. epitropicalis S.

tropicalis ruwenzoriensis species has shown that both S. paratropicalis gave only one (Graf (a greater number of and all tetraploid alltetraploid and S. paratropicalis contained three

genes and

Chapter 4

X. clivii lineage and lineage Silurana skin secretions of the diploid species species diploid the of secretions skin (Mechkarska et al., 2010) and et al., 2010) and (Mechkarska the skin secretionstetraploid of the species 101 lineage. The existence of multiple biologically (Conlon et al., 2010) is not appreciably different different appreciably not is 2010) al., et (Conlon X. borealis X. amieti , it is tempting to speculate that they are evolving towards a and secretions appears to be a clear example of S. paratropicalisclear example be a to secretions appears X. andrei (Ali et al., 2001). Similarly, theSimilarly, 2001). multiplicity (Ali et al., ofpeptides the in secretionsof the S. paratropicalis

Acknowledgements Acknowledgements workThis was supported a Facultyby Support Grant (NP-10-11-103) from U.A.E. verygrowth-inhibitory weak activity may havebeen However, missed.to it is fair claim that the multiplicity of antimicrobialpeptides in the octoploid frogs frogs octoploid the S. that in the greater paratropicalis is not appreciably S. tropicalis species that in the tetraploid from is It protocol. experimental same the exactly using analyzed when 2011) al., et (Conlon antimicrobial of fate common most the is nonfunctionization that therefore, concluded, peptide genesfollowingputative the genome duplication event in the the second polyploidization in the Xenopus active CPF peptides in the PGLa and XPF the of potencies antimicrobial low very of the view In subfunctionalization. peptides in University. The authors thank Burley Lilley ofXenopus Expresshelp forwith sample collection. new, as yet undetermined function (neofunctionalization). (neofunctionalization). function undetermined yet as new, Chapter 4 Evans, B.J., 2007. Ancestry influences the fate Du Pasquier,L.,Wilson,M.,Sa de Sá,R.O., Hillis,D.M.,1990. Phylogene Conlon, J.M.,Mechkarska, M., Ahme Conlon, J.M.,Al-Ghaferi,N.,Ahmed, E.,Meetani, M.A., Leprince, Conlon, J.M., Al-Ghaferi, N.,Abraham,B., Leprince, J.,2007. Strategiesfor transformation of Conlon, J.M., in press.Structuraldiversity and species distribution ofhost-defense peptides in frog Cannatella, D.C., Trueb, L.,1988.E Ali, M.F.,Soto,A.,Knoop,F.C REFERENCES Chain, F.J.,Evans, B.J.,2006. Multiplemechanis Chain, F.J.,Ilieva,D., Evans, Clinical Laboratory and Standards Institute,2008a. Clinical Laboratory andStandards Institute,200 Evans, B.J., Kelley, D.B., Tinsley, R.C., D.B.,Tinsley, MelnickEvans, B.J.,Kelley, D.J.,Cannate Evans, B.J., 2008. Genome evolution and frog Purification and properties ofantimicrobial peptidesskin from secretions oftheEritrea clawed dodecaploid an integration of ribosomalDNAandmorphology. Mol.Biol. Evol. 7, 365-376. of theoctoploid frog of magainin,PGLa, procaerulein-derived, andpr agents. Methods 42, 349-357. am naturally-occurring skin secretions. Cell Mol. Life Sci. in press. 89. secretions ofthediploidfrog, duplicates inthetetraploidfrog Pipidaefrog family (Anura). Zool. J.LinnaeanSoc. 94, 1-38. polyploidization polyploidization of clawedfrogs ( susceptibility testingof yeast.Appr tests forbacteria that grow aerobically. vertebrate allopolyploidization. BMC Evol.Biol. 8, 43. Mol. Phylogenet. Evol. 33, 197-213. phylogeny ofAfrican clawed frogs: phylogeography Silurana). Front.Biosci.13, 4687-4706. Xenopus clivii Xenopus ruwenzoriensis (Pipidae).Comp. Biochem.Physiol. Xenopus amieti phibian antimicrobial peptides into therapeutically valuable anti-infective B.J., 2008.Duplicate gene evoluti ., Conlon, J.M., 2001. Antimicrobialpeptides isolatedfromskin mmut, B., 2009. The fate of duplicated immunity genes inthe mmut, B.,2009.Thefateofduplicatedimmunity Xenopus tropicalis volution ofpipoidfrogs:intergener Xenopus laevis d, E.,Leprince, J.,Vaudry, H. Xenopus oved Standard M27-A3. CLS1, Wayne, PA. oved Standard M27-A3.CLS1,Wayne, (Pipidae). Peptides 31, 989-994. . FrontBiosci.14, 177-191. tic relationshipsofthe pipidfrogs Approved Standard M07-A8. PA. CLSI, Wayne, speciation genetics ofclawed frogs ( ). Genetics 176,1119-1130. 102 8b. Reference method forbrothdilutionantifungal . PLoSGenet.2, e56. of duplicatedgenesmi Methods for dilution antimicrobial susceptibility oxenopsin-derived peptides ms theretainedexpressionofgene promote (Pipidae).Biochim. Biophys, Acta 1550,81- and implications forpolyploid evolution. C Toxicol. Pharmacol.153, 350-354. lla, D.C.,2004.Amitochondrial DNA on and expression inthe wakeof J., Nielsen, P.F , King,J.D., Takada, K.,2011. ic relationships oftheaquatic Xenopus llions ofafter years fromskinsecretions ., 2010.Orthologs Xenopus and Silurana: and Chapter 4 . Xenopus Xenopus Xenopus ) from the Xenopus J. Biol. Chem. 261, e fragments originating e fragments Version 5.5. American Version 5.5. American cates. Trends Genet. 20, cates. Trends Genet. timicrob. Chemother. 64 Chemother. timicrob. cratic Republic of the Congo: of the Congo: cratic Republic obel, H.R. (Eds) The Biology of The Biology (Eds) obel, H.R. hydropathic character of a protein. character of a protein. hydropathic three other prohormones from the three other prohormones groups of chromosomal regional Xenopus laevis. . Methods Cell Biol. 36, 9-34. . Methods Cell Biol. Biochem. J. 243, 113-120. 243, 113-120. J. . Biochem. USA. Electronic database accessible at at accessible database Electronic USA. ussion of the biogeography of African clawed clawed African of ussion of the biogeography fections arrived? J. An d: an online reference. reference. d: an online FEBS Lett. 233, 282-238. 282-238. FEBS Lett. 233, , J.E., 1986. Novel peptid , J.E., 1986. B., Hanner, R., Tinsley, A new species 2008. R., R.C., B., Hanner, ution in polyploid species of the genus ution 103 rsity Zootaxa conservation. 1780, 55-68. Xenopus laevis d frog species (Anura: Pipidae: d frog genome doublings at the root of the vertebrates. J. Struct. Funct. J. the vertebrates. root of the at genome doublings lationship of promagainin to ) from the Itombwe Massif, Demo ) from the Itombwe Massif, speciation. In: Tinsley, R.C., K R.C., Tinsley, In: speciation.

etology/ amphibia /index.php. etology/ amphibia /index.php. volution of RAG-1 in polyploid RAG-1 2005. Evolution of D.C., k, D.J., Cannatella, osynthesis and degradation of and degradation 1987. Biosynthesis D.H., Williams, bson, B.W., : a different perspective. perspective. : a different Xenopus laevis prohormones r, D., Hallböök, F., 2003. Numerous , L., 1991. Genetics of Genetics L., 1991. , , Tobias, M.L., Kelley,, Tobias, M.L., D. and the caerulein and xenopsin precursors from xenopsin precursors and and the caerulein a . Clarendon Press, Oxford pp 391-401. pp 391-401. Press, Oxford . Clarendon Xenopus laevis . Mol. Biol. Evol. 10, 360-369. 10, 360-369. Evol. . Mol. Biol. Xenopus 105-132. J. Mol. Biol. 157, i29-36. Suppl 1, skin of Biochem. Genet. 24, 821-837. Biochem. Genet. laevis from PGL 2011. Description of a new 2011. Description of octoploi implications for DNA barcodes and biodive implications for herp http://research.amnh.org/ Democratic Republic of the Congo, with a disc with Congo, the of Democratic Republic in press. Zool. J. Rift. Albertine frogs in the New York, of Natural History, Museum Genomics 3, 53-63. Genomics 3, 53-63. paralogies strongly indicate two strongly indicate paralogies 5341-5349. from derived peptides 544-549. clawed frogs. Mol. Biol. Evol. 22, 1193-1207. 1193-1207. 22, Evol. Mol. Biol. frogs. clawed of clawed frog (genus Xenopus of clawed frog (genus Kobel, H.R., Du Pasquier Du Kobel, H.R., in era of untreatable the Has 2009. Livermore, D.M., Larhamma Lundin, L.G., Graf, J.D., Fischberg, M., 1986. Albumin evol Graf, J.D., Fischberg, M., 1986. Allopolyploid 1996. Kobel, H.R., for displayingsimple method A 1982. the Kyte, Doolittle, D.F., J., Hunt, L.T., Barker, W.C., 1988. Re W.C., 1988. Hunt, L.T., Barker, Hughes, M.K., Hughes, A.L., 1993. Evolution of duplicate genes in a tetraploid animal, in a tetraploid of duplicate genes Evolution Hughes, M.K., Hughes, A.L., 1993. Evans, B.J., Greenbaum, E., Kusamba, C., Carter, T.F., Tobias, M.L., Mendel, S.A., Kelley, D.B., D.B., Kelley, S.A., Mendel, M.L., Tobias, T.F., Carter, C., Kusamba, E., Greenbaum, B.J., Evans, L., Williams, D.H., Maggio Gibson, B.W., Poulter, Frost, D.R., 2011. Amphibian species of the worl the species of Amphibian Frost, D.R., 2011. Lynch, M., Katju, V., 2004.The altered evolutionary trajectories of gene dupli of trajectories Lynch,altered evolutionary V., 2004.The M., Katju, Giovannini, M.G., Poulter, L., Gi L., M.G., Poulter, Giovannini, Evans, B.J., Kelley, Melnic Evans, B.J., D.B., Carter, T.F. Evans, B.J., Chapter 4 Westerhoff, H.V., Zasloff, M.,Ro Tymowska, J.,Fischberg,M.,1982.AcomparisonTymowska, of Tymowska, J., 1991. Polyploidy J.,1991. Polyploidy and cytoge Tymowska, Zasloff, M.,1987.Magainins,acla Tinsley, R., J.,2004. Measey, Tinsley, Sammut, B.,Marcuz,A.,Pasquier, L.D.,2002.Th Otto, S.P., Whitton,J.,2000. incidence Polyploid Zhang, L., Falla,T.J.,2010.Poten Muñoz, V.,Serrano, L.,1994. Elucid Mechkarska, M.,Ahmed, E., Coquet, P.M., Riethorst, A., Jureti 49-157. and Picardwith those ofXenopus tropicalis and nucleolar organizer regionsofthenewtetraploid species D,M., Sessions, S.K. (Eds magainin-2 in Species. Version 2010.3. Electronic databa characterization oftwoactive forms a Acad. Sci.USA84,5449-5453. Biol. 618,303-327 32, 2698-2709. complex classIagenes in a dodecaploid amphibian, parameters. NatureSt Pharmacol. 152, 467-472. Marsabitfrog clawed J.M., 2010.Antimicrobial peptid Escherichia coli ruct. Biol. 1, 399-409. Xenopus borealis ) Amphibian Cytogenetics and Evolutio ć Xenopus andrei . D., 1995.Functional synergism ofthemagainins PGLaand sner, J.L.,Hendler, R.W.,De Waal, A., Vaz Gomes, A.,Jongsma, tial therapeutic applicationofhost defensepeptides.Methods Mol. , tumorcells and liposomes. Eu L.,Leprince, J.,Jouenne, T., ating the folding problem ofhe ss ofantimicrobialpeptidesfrom es withtherapeutic potential from skinsecretionsofthe netic variation in frogs ofthegenus nd partialcDNAsequenceofaprecursor. se accessible at www.iucnredlist.org.se accessibleat (Pipidae). Comp. Biochem. Physiol. C Toxicol. Physiol. Comp. Biochem. (Pipidae). 104 . In: IUCN 2010. IUCN Red List ofThreatened IUCNRed . In:IUCN2010. Gray (Anura,Pipidae).Cytogenet. Cell Gray Genet. 34, and evolution. Annu. Rev. Genet. 34, 401-437. e fateof duplicated ma the karyotype, constitutive heterochromatin, Xenopus ruwenzoriensis r. J. Biochem. 228, 257-264. Xenopus epitropicalis Vaudry. H., King, J.D.,Conlon, n, Academic Press, San Diego. lical peptides using empirical lical peptidesusingempirical Xenopus jor histocompatibility Xenopus . Eur.J.Immunol. skin:isolation, . In:Green, Proc. Natl. Fischberg

Chapter 5

Peptidomic analysis of skin secretions demonstrates that the allopatric populations of Xenopus muelleri (Pipidae) are not conspecific

M. Mechkarska, E. Ahmed, L. Coquet, J. Leprince, T. Jouenne, H. Vaudry, J.D. King, K. Takada, J.M. Conlon

Peptides (2011) 32:1502-1508. Chapter 5 erythrocytes. reference strains of bacteria (MIC 3 - 25 a separate species. TheCPF peptides showed potent, broad-spectrum activity against X.borealis related to closely primary structures ofthese peptides and -MW2), and three CPF peptides (CPF-MW1, -MW2 and -MW3). Comparison of the (PGLa-MW1 glycine-leucineamide (XPF-MW1), peptides two XPFpeptide one MW1), Mueller’s clawed frog ABSTRACT motif wasalsoisolatedthatshows limited secretions from identified only in frogs of the Dicroglossidae family. Thecomponents identified in skin other caerulein-precursor fragments(C to the magainins (magainin-M1 and -M2), xenopsin-precursor fragments (XPF-M1) and offi identification the in resulted range eastern Peptidomic analysis ofnorepinephrine-stimulated skin secretions from unclear and ithas been proposed that the western population represents aseparate species. is two populations the between relationship westAfrica.Thephylogenetic eastand in Xenopus species. A cyclicpeptide(WCPPMIPLCSRF.NH X.

muelleri Xenopus muelleri Xenopus from the western range comprised one magainin (magainin- X. muelleri to X.muelleri than PF-M1 and-M2)previously fo suggest that western population of of population western that suggest (Peters 1844) occupies two non-contiguous ranges ranges two non-contiguous occupies 1844) (Peters

structural similarity to the tigerinins, previously previously tigerinins, the to similarity structural 108 µM), but were hemolytic against human µM), butwerehemolyticagainst ve antimicrobial peptides structurally related related structurally peptides antimicrobial ve consistent with its proposed designation as designation itsproposed with consistent 2 ) containing theRFamide und in skin secretions of X. muelleri X. X. muelleri fromthe is more ismore Chapter 5

ility and group group species Mueller's and fraseri and

group laevis group S. tropicalis muelleri Hymenochirus, X. borealis Xenopus which are found which are ; the ; the like , Pipa muelleri group includes X. includes group

a component of the system of system of the a component -helix in ofenvironment the a  X. , fraseri are united in the Xenopodinae [14] Xenopodinae united in the are the within the family Pipidae [15]. All are Pipidae [15]. the family within

ith females reaching up to 9 cm snout-to- 9 cm reaching up to ith females Peters, 1844 (also known as and antifungal activities andwith the ab it is rare that orthologouspeptides two from quence even when those species are closely species are those when even quence croorganisms [39]. With few exceptions, the the exceptions, few With [39]. croorganisms veral members of a particular peptide family. family. peptide particular of a members veral ; and 109 species distributed in five genera genera five in distributed species and Xenopus X. clivii , and Xenopus, and

and Silurana proposed that they constitute constitute they that proposed Xenopus muelleri X. largeni, X. petersii, and X. victorianus Silurana , , X. borealis

X. gilli

[14,16,35]. laevis, is a relatively large aquatic species w is a relatively large aquatic species X. X. muelleri, so streams and rivers in the dry season, preferring agricultural and other altered altered other and agricultural preferring season, dry the in and rivers streams so Pseudhymenochirus , Mueller’s clawed frog Introduction Peptides with broad-spectrum antibacterial The clawed frogs comprise at least 33 at frogs comprise The clawed permeabilize mammalian are found cells in a variety skin offrom secretions species of includes includes X. pygmaeus X. pygmaeus Platanna) ponds, permanent and temporary both occupies It smaller. 20% about males and length vent and al havebeen divided into three species groupsbasis on the of similarities in morphology, the of mitochondrial genes: calls, and/or nucleotide sequences advertisement includes 1. Anura (frogs and toads). It is toads). Anura (frogs and to environment the in pathogens by invasion against animal the defends that immunity innate [3]. Suchhavepeptidesclinicalpossible attracted applicationsfor their interest in treating infections producedby antibiotic-resistantmi aqueous in structure secondary stable lack and cationic are peptides antimicrobial skin frog solutions but have the propensity an to form amphipathic phospholipid vesicle or solventmembrane-mimetic in a such as 50% trifluoroethanol-water on the similaritiesgrouped be in families of limited [4,30].peptides basis The together may contain frequently species a single from secretions skin and in amino acid sequence se as as well families different from peptides and Their primary structures hypervariable are se acid amino same the have species different related phylogenetically. Consequently the antimicrobial peptides in skin secretionsmay serve as valuablegainingfor phylogenetic insight intomarkers the evolutionaryhistory of [6,7,9].frogthe families different retainingdiploid the status that is thoughtbe to related the ancestral to state existingprior to tetraploid 10 The [12-14,21,22]. duplications genome whole more or one habitats and is seldom found in forests. In certain regions certain In forests. in found seldom is and habitats Pipa genus of the members for except Sahara the of south Africa in found in South America. The genera The genera America. South in reticulating and bifurcating both by characterized history evolutionary a complex have and modes of speciationAllopolyploidization[11]. events,which in species two hybridize and to rise given have ancestors, of both genome complete the inherits descendant the species, a single only with species dodecaploid and octoploid, tetraploid, Chapter 5 investigators. Adult Adult investigators. No. (Protocol U.A.E. University of committee 2.1. 2. numerals e.g. magainin-M1 and magainin-M2. denotedfor M by fragment fam (XPF)peptide xenopsin-precursor magainin, peptide glycine-leucineamide (PGL according to theterminology used previously for peptides from theXenopodinae [17]. The from frogs of the Pipidae family and so the peptides described in this study are classified X. andrei characterization of antimicrobial and other peptides in skin secretions from the to led that has work previous complements investigation The agents. infective anti- valuable therapeutically into for development thepotential with peptides antimicrobial gaining insight into the evolutionary history of the Xenopodinae conspecific. The study is part ofa program ofinvestigation with the dual purpose of frogs from both ranges to determine whether the two populations should be regarded as with MALDI-TOF mass spectrometry) of norepinephrine-stimulated skin secretions from HLPCcoupled (reversed-phase useanalysis wasto peptidomic study the present aim of encompassing BurkinaFaso, Ca referred to as scientific current namethe western population represents aseparate specie to northeastern Democratic Republic of Congo [16,31]. It has been proposed that the region of Nambia [16,18]. In the western range, into northwestern border areas ofthe Republic ofSouth Africa, Botswana and the Caprivi found along the East African coastal belt from extreme southern Kenya through Tanzania are declining due to loss of habitat and harvesting for human consumption [25]. [25]. consumption for human harvesting of and toloss habitat due are declining IUCN(Red itscurrent List)status is habitats, complete [15,25]. In view of its very wide distribution and tolerance of abroad range of X. gilli

All experiments with live animals were approved by the Animal Research Ethics Ethics theAnimalby Research wereapproved animals live with All experiments secretions skin of Collection Experimental There isno universally accepted nomenclatu X. muelleri revealing X.laevis with that to hybridize , isknown reproductive isolation is not [28], X.borealis X. occupies two discontinuous ranges. In the eastern range,

muelleri X. muelleri X. muelleri West in this article but this is not a recognized species name. isnotarecognized The this articlebut this West in [27], and [27],and X. andMW for from the eastern range (n = 2; male 43 g, female 43 g) were muelleri meroon, Côte d'Ivoire, northern Gabon, Nigeria, eastward X. clivii [26]. For convenience, the western population is is the western population convenience, For [26]. X. [8]. 110 ‘LeastConcern’ but numbers incertainareas

muelleri a), caerulein-precursor A21-09) and were carried out by authorized s with only the eastern population retaining retaining eastern population the only s with ilies are recognized. The species origin is is origin Thespecies recognized. are ilies X. re for designating antimicrobialre fordesignating peptides

muelleri West. Paralogs aredifferentiated by is widely distributed in azone fragment (CPF),and

and identifying identifying and X. [5], [5], X. amieti

muelleri is Chapter 5

(ATCC (ATCC colony forming forming colony 6 water at a flow rate flow water at a l of 10 of l  freeze-dried. The material was The material freeze-dried. aining the secretions from each from aining the secretions Staphylococcus aureus Staphylococcus after partial purification on Sep-Pak purification on Sep-Pak partial after r plate reader. Fractions associatedwith in the eluting solvent was raised to 21%raised to was solvent eluting in the kV. The instrument was calibrated with was instrument kV. The connected in series. Bound material was material Bound connected in series. l 494 Procisel sequenator(Foster CA). City, weight) and placed in and body weight) nmol/g oride (40 in 96-well microtiter cell-culture plates for 2 - 4 kDa range. The accuracy of massof accuracy range. The 4 kDa 2 -

cm) Vydac 218TP510 (C-18) reversed-phase reversed-phase (C-18) 218TP510 Vydac cm) Jos Plateau. Jos Animals from bothgroupswere lvent was raised 21%lvent from 56%was 50 over to min 111 l) with an inoculum (50 (50 inoculum an with l)  the peptidesbydetermined automated were Edman FA (70.0:29.9:0.1, v/v/v) and FA (70.0:29.9:0.1, University. The solutions cont University. (ATCC 25726) Escherichia coli Escherichia C in a humidified atmosphere of air. After incubation, the absorbance at 630 nm at absorbance the After incubation, of air. atmosphere C in a humidified o and

Structural characterization characterization Structural structures of The primary Peptide purification each group, from secretions The pooled skin

degradation using an Applied Biosystems mode Biosystems Applied an using degradation instrument DE-PRO a Voyager using out was carried spectrometry mass MALDI-TOF (Applied Biosystems) that was operated in with reflector mode delayed extraction and the 20 was source voltage in the ion accelerating in the mass of known molecular peptides 2.3. of each was determined well using a microtite Vydac 25 cm) x cm a (1.0 on chromatographed were successively activity antimicrobial The column. (C-8) 208TP510 Vydac cm) x 25 cm a (1.0 and column (C-4) 214TP510 concentrationof acetonitrile in the eluting so and the flow rate was 2.0 ml/min. cartridges, were injected onto a (1.0 cm x 25 a (1.0 cm onto injected cartridges, were IL) equilibrated with 0.1% (v/v) Deerfield, (Grace, TFA/ HPLC column group were pooled and separately passed at a flow rate of 2 ml/min through 6 Sep-Pak C-18 Sep-Pak 6 through 2 ml/min of rate a flow passed at separately and pooled were group MA) Milford, Associates, cartridges (Waters eluted with acetonitrile/water/T of acetonitrile concentration The of 2.0 ml/min. collected in South Malawi. Adult individuals from the western range (n = 2; male 32 g, g, 32 male 2; (n = range western the from individuals Adult Malawi. South in collected near Nigeria, in collected were 32 g) female 2.2. suppliedby Xenopus Express Inc. (Brooksville,injected was Each FL). animal via the hydrochl norepinephrine sac with dorsal lymph a solution of (100 distilledml) water15 formin. The frog was removed and the collection solution was acidified by addition trifluoroacetic of acid (TFA) (1 ml) and immediately to U.A.E. for shipment frozen ml). (2 TFA/water (v/v) 0.1% in redissolved (v/v)over10min and to63% (v/v) over60 using min lineargradients. Absorbancewas the of Purification were collected. (1 min) and fractions nm, and 280 nm 214 at monitored peptidesmonitoredincubatingby was lyophilized aliquots of chromatographiceffluent (100in µl) Mueller-Hintonbroth (50 units/ml) fromunits/ml) a log-phase culture of reference strains of 18 h at 37 25923) Chapter 5 coli concentrations of antibiotics (ampicillin for (ampicillin antibiotics of concentrations assays,incubations the of reproducibility and were confirmed by measurement ofabsorbance at 630 nm. Inorder to monitor the validity lowestthe as concentration ofpeptide whereno visible growth wasobserved. Thevalues concentration rangeof- 200 3 peptides against reference strains of (MIC) ofthe purified concentrations MD).Minimum (Rockville, inhibitory Collection 2.4. Pr Center of University Medical Nebraska determinations was measured as previously described [27]. TheLC C. 2.5. spectrometry. rate was 6 ml/min. Thestructureofthe of acetonitrile in theelutingso lvent was raised from 21%to 56% over min60 andthe flow phase HPLC on a(2.2 cm25 cm) x Vydac 218TP1022 (C-18) column. Theconcentration Co., Shanghai, China and was purified to near homogeneity (>98% purity) by reversed- scales for amino acids scales for [23]. Kyte and of Doolittle thehydrophobicity upon were based peptides the of hydrophobicities mean Calculated ofahelix. presence the to predict wasconsidered helicity/residue 1% of percentage monomeric peptides. Calculations were performed at pH7and 278 K.Aminimum of the helical behaviour predicts which theory transition the helix/coil on algorithm based peptides were performed using the AGADIR program [29]. AGADIR is a prediction 2.6. concentrationpeptide of producing 50% hemolysistwoin incubations.

albicans (ATCC 25726), and and 25726), (ATCC

Tigerinin-M1 (WCPPMIPLCSRF.NH Peptide synthesis Hemolytic activityagainsthuman erythroc Reference strainsof microorganisms were activities hemolytic and Antimicrobial Prediction of secondary structure and determination ofhelicity per residue for the Secondary structure prediction ) as previously described [27].  0.02%. Amino acid composition analyses were performed by the Candida albicans μ M by standard microdilution methods [1,2] and were taken Staphylococcus aureus Staphylococcus otein Structure Core Facility (Omaha, NE). Core Facility(Omaha, Structure otein 2 ) was supplied in crude form by China Peptides Peptides form crude China in by ) was supplied 112 peptide was confirmed by electrospray mass S. were carried out in parallel with increasing increasing with parallel in out were carried purchased fromthe American TypeCulture

aureus (ATCC 90028) were measured in the in the measured were 90028) (ATCC ytes taken from ahealthy donor was 50 and E.coli; and and amphotericin for value wastaken asthe mean (ATCC 25923), (ATCC 25923), Escherichia Chapter 5 3 contained tigerinin-M1. The dashed The contained tigerinin-M1. 3 The peaks designated 1 - 2 and 4 - 6 . dac C-18 column of skin secretions from column of skin secretions from C-18 dac y, peak 2 was associated with the ability to the ability with was associated 2 y, peak , after partial purification on Sep-Pak C-18

113 X. muelleri onitrile in the eluting solvent. solvent. eluting in the onitrile only whereas peaks 1 and 4 - 6 inhibited the growth of both both of growth the - 6 inhibited 4 1 and peaks whereas only -aminobutyric acid] (Calbiochem) as substrate as described [24]. [24]. described as as substrate (Calbiochem) acid] -aminobutyric E. coli . S. aureus and Results Enzymatic assay The ability of tigerinin-M1inhibit(100 µM) to the activity ofporcinepancreatic The pooled skin secretions from from secretions skin The pooled

Reversed-phase HPLC on a semipreparative Vy on a semipreparative HPLC Reversed-phase Fig. 1. after partial purification on Sep-Pak cartridges purification partial after X. muelleri 2.7. line shows the concentration of acet of concentration shows the line displayed Peak further. antimicrobial activityand were purified cartridges, were chromatographed on a Vydac C-18 semipreparative reversed-phase HPLC HPLC reversed-phase semipreparative C-18 Vydac a on chromatographed were cartridges, to subjected and 1 - 6 collected were designated peaks prominent 1). The (Fig. column assa of conditions the Under purification. further of growth the inhibit 3.1. Purification of the peptides from X. muelleri X. from peptides the 3.1. of Purification 3. E. coli elastase (Calbiochem, San Diego, CA) was determined using succinyl-Ala-Ala-Pro-Abu-p- using determined was CA) San Diego, (Calbiochem, elastase α = L- [Abu nitroanilide Chapter 5 acetonitrile in the eluting solvent. antimicrobial activity andwere purified further. The dashed lineshows the concentration of X. Fig. 2. Reversed-phase HPLC onasemipreparative Vy 3.2. Purification of 3.2.the peptides from X. muelleri magainin-M2 20,tigerinin-M155,XPF-M150,CPF-M1115,CPF-M240. of yields final approximate Vydac semipreparative symmetrical 6: peak peak (Fig. 2). HPLC columnusing the same expe ona chromatographed were cartridges, C-18

muelleri usqet tutrl nlss eosrtd ht ek cnand magainin-M1, contained 1 peak that demonstrated analysis structural Subsequent The pooled skin secretions from from secretions skin pooled The 2: magainin-M2, peak 3: tigerinin-M1, peak 4: XPF-M1, peak 5: CPF-M1, and CPF-M1, 5: peak XPF-M1, 4: peak tigerinin-M1, 3: peak magainin-M2, 2: P-2 Te etds ee uiid o er ooeet, s sesd y a by assessed as homogeneity, near to purified were peptides The CPF-M2. tion on Sep-Pak purifica cartridges partial after West e crmtgah on chromatography further by spectrometry, mass and shape peak - ad ya C8 oun a peiul dsrbd The described. previously as columns C-8 Vydac and C-4 uiid etds no) ee aannM 182, magainin-M1 were (nmol) peptides purified rimentalconditions asforthe X. muelleri X. muelleri 114 Vydac C-18 semipreparative reversed-phase West West, dac C-18 columnofskinsecretionsfrom

after partial purification on Sep-Pak . The peaksdesignated 1 -6 displayed X. muelleri skinsecretions Chapter 5

E. coli contained magainin- contained trated that peak 1 trated that re collected and subjected to further and subjected re collected La-MW2 (peak 2) on a semipreparative Vydac Vydac (peak 2) on a semipreparative La-MW2 peptides (nmol) were magainin-MW1 420,peptides were magainin-MW1 (nmol) on began and ended and the dashed line shows the the dashed line shows the ended and and on began eaks 1 and 2 were associated with the ability ability with the were associated 2 eaks 1 and ity of PGLa-MW1 on a semipreparative Vydac C-8 on a semipreparative Vydac PGLa-MW1 ity of 115 ive Vydac C-4 and Vydac C-8 columns. The methodology The methodology C-8 columns. C-4 and Vydac ive Vydac onlywhereas peaks36 - inhibited the growthboth of E. coli . Subsequent structural analysis demons analysis structural Subsequent . (A) Separation of PGLa-MW1 (peak 1) and PG (A) Separation of PGLa-MW1 The approximate final yields of purified The approximate final yields of The prominent peaks designated 1 - 6 we - 1 peaks designated The prominent S. aureus XPF-MW1 50, PGLa-MW1 230, PGLa-MW2 50, CPF-MW1 220, CPF-MW2 140 and and 140 CPF-MW2 220, CPF-MW1 50, PGLa-MW2 230, PGLa-MW1 50, XPF-MW1 CPF-MW38. C-4 column and (B) purification to near homogene (B) purification to near C-4 column and collecti peak where show arrowheads The columns. solvent. eluting the in acetonitrile of concentration Fig. 3. Fig. 3. purification. Under the conditions of assay, p of assay, conditions the Under purification. to inhibit the growth of and CPF-MW1, 4: peak PGLa-MW2, and PGLa-MW1 3: peak XPF-MW1, 2: peak MW1, near to purified were peptides The CPF-MW3. 6: peak and CPF-MW2, 5: peak further by spectrometry, mass and peak shape a symmetrical by as assessed homogeneity, on semipreparat chromatography C-4 on a Vydac PGLa-MW2 and PGLa-MW1 of separation partial the by illustrated is further by PGLa-MW1 of homogeneity near to purification by followed 3A), (Fig. column 3B). (Fig. column C-8 Vydac a on chromatograpy Chapter 5 Xenopus muelleri Xenopus their completeprimary structures are shown in Fig. 4. X. muelleri P-2 GGLGAKGKVKMGPE 29. 2893.6 2893.6 2892.4 muelleri Xenopus 2892.6 GLGSLLGKAFKFGLKTVGKMMAGAPREE GLGSLLGKAFKFGLKTVGKMMAGAPREQ CPF-M2 CPF-M1 XPF-M1 GWASKIGQTLGKMAKVGLKDLIQA 2513.8 2513.4 (M PGLa-MW2 GMASKAGAIAGKIAKTAIKIAL.NH Fig. 4. 2518.6 PGLa-MW1 GMASKAGSVLGKITKIALGAL.NH 2517.4 XPF-MW1 GWASKIGQTLGKLAKVGLKEFAQS GIGKFLHSAGKFGKAFLGEVMKS Magainin-MW1 2680.9 2680.5 Tigerinin-M1 WCPPMIPLCSRF.NH Magainin-M2 GFKQFVHSLGKFGKAFVGEMIKPK GIGKFLHSAGKFGKAFIGEIMKS Magainin-M1 The primarystructures 3.3. Structural characterization West. West. CPF-MW3 GLGSLLGKAFKFGLKTVGKMMGGAPR 2621.3 CPF-M 2621.5 CPF-MW1 GLGSLLGKAFKFGLKTVGKMMGGAPREQ r calc) of the antimicrobial peptides isolated from skin secretions of Amino acid sequences, observed molecular masses (M masses molecular observed sequences, Aminoacid W2 GLGSLLGKAFKFGLKTVGKMMGGAPREE 2879.8 2879.6 West skin secretions were established by automated Edman degradation and

West

of the antimicrobial pe 2

116 ptides isolatedfrom r 2 obs), andcalculated molecular masses 2 2083.4 2083.4 2083.3 1985.4 1985.4 1985.2 2878.6 2878.6 2878.6 X. muelleri muelleri X. [M [M r r +H]obs [M +H]obs [M 2423.1 2423.1 2423.3 2409.1 2409.3 2409.3 2409.1 1446.6 1446.6 1446.6 X. muelleri and X. muelleri muelleri X. r r +H]calc +H]calc and Chapter 5

and

50 200 200 70 105 180 ND LC  >500  = 70 µM), = 70 µM), > 200 µM). > 200 µM). 50 X. muelleri 50 25 25 50 25 ND 100 ND , and the opportunisticE. coli, and the of reference strains of the Gram- the of strains reference of -amidated and tigerinin-M1 exists in = 180 µM) showed moderately high high moderately showed µM) = 180 α 50 M) against microorganisms and hemolytic and hemolytic microorganisms against M)  200 6 6 50 117  It is probable that CPF-MW3 is a productpost- of the

, the Gram-negative bacterium bacterium Gram-negative , the are compared in Table 1. CPF-MW1 (LC CPF-MW1 1. in Table are compared West. X. muelleri West. 6 3 50 12.5 12.5 6 12.5 12.5 100 12.5 E. coli coli E. aureus S. albicans C. >200 >200 >200 and albicans

skin secretions to inhibitgrowth the

= 105 µM), and magainin-M1 (LC and magainin-M1 = 105 µM), C. 50 X. muelleri West 2 Minimum inhibitory concentrations ( concentrations inhibitory Minimum As there was some ambiguity withtosome regard identification was As there of the aminoacids at the The abilities of abundant the most nmol) peptides (> 50 in the Table 1. Tigerinin-M1 did not display antimicrobial activity and did not inhibit the activity of of activity the inhibit not and did activity antimicrobial display not did Tigerinin-M1 100 µM. of elastase at a concentration activity against human erythrocytes of endogenous the peptides isolated skin from secretions of the cyclic (disulphide-bridged) form. very hydrophobic C-terminus of the PGLa-MW2, proposed sequence was confirmed by (7), 7.0 Ala (4), 3.5 Gly (1), 1.2 Ser (1), 1.2 Thr [Found: analysis composition acid amino the show parentheses in Figures peptide]. residues/mol (4) 3.9 Lys (1), 1.1 Leu (4), 3.5 Ile mass MALDI-TOF sequences. proposed the from predicted residues of number spectrometry was used to that tigerinin- the confirm proposed and to demonstrate structures C-terminally are PGLa-MW2, and PGLa-MW1 M1, hemolytic activity against human erythrocytes1) (Tablewhereas activities hemolytic the of magainin-MW1, tigerinin-M1very and low (LC were PGLa-MW1 CPF-MW2 (LC CPF-MW2 ND: not determined CPF-M1 Magainin-M1 yeast pathogen pathogen yeast 3.4. Antimicrobial and hemolytic activities translational processing of CPF-MW1 and/or CPF-MW2 arising from proteolytic cleavage cleavage proteolytic from arising CPF-MW2 and/or of CPF-MW1 processing translational gene. a separate of product the than rather residue arginine a single at CPF-MW1 Tigerinin-M1 PGLa-MW1 Magainin-MW1 CPF-MW positive bacterium S. aureus X. muelleri Chapter 5 rich inhibitor of elastase,guameri of frogs have been shown to contain protease inhibitors [33]. The region of the cysteine- peptides containing the RFamide motif [34] are unknown. Skin secretions of several species biological role oftigerinin-M1, if any, and its evolutionary relationship to other vertebrate and and their inability to adopt aα adopt to inability their and H. rugulosus H. from peptide in this study, the assayused of conditions the However, under [32,38]. andsevenX. muelleri antimicrobial peptides in skin secretions of norepinephrine-stimulat in activity antimicrobial 4. tetraploid frogs tetraploid of previously described dermal peptides magainin, PGLa, CPF, and XPF isolated from the Determination ofthe primarystructures ofthe Hoplobatrachus rugulosus rugulosus Hoplobatrachus Tigerinin peptides have previously been identified only in previously. reported been not has the Xenopodidae to species belonging of secretions skin tig designated isprovisionally so (Fig. 6)and family peptide tigerinin the of similaritypeptides to limitedstructural shows but activity isolated from the Additionally, the Additionally, peptide fromtwothe populations of proposal that thelattershould eventua X. muelleri peptides fromfrogs the of frogs peptide whereas the to the X. ne articleas“X. the in (described upon the nucleotid ortholog from compared withthe ortholog from magainin-B2 from X. muelleri X. muelleri that suggest data

muelleri This study has described the purification of five peptides with varying degree of Discussion A disulphide-bridged peptide containing the RFamide motif at its C-terminus was Fejervarya subfamily Dicroglossinae X. amieti[5] and [14]. and or was notactiveagainsteither X. muelleri X. clivii X. laevis[17], X. muelleri X. muelleri X. muelleri X. muelleri have been reportedto displaybr X. muelleri X. muelleri X. muelleri X. muelleri X. borealis X. borealis e sequences ofmitochondr X. muelleri X. muelleri .

In particular,magainin-MW1 from X. andrei (Fig. 5). This conclusion is supported by cladistic analysis based based analysis cladistic by issupported conclusion (Fig. 5).This (unpublished data), and secretions, like those from those like secretions, West arenotconspecificther skin secretions. The peptide lacks antimicrobial or hemolytic lackshemolytic or antimicrobial secretions. The peptide skin X. muelleri X. muelleri West -helical conformation, the tigerinins from tigerinins the conformation, -helical West secretionsWest contained two members ofthisfamily. The X. borealis and XPF-MW1 shows only two amino acid substitutions XPF-MW1and shows amino two substitutions only acid of the family Dicroglossidae. w tetraploid”) as sister group to to assistergroup w tetraploid”) [28].A comparison of theprimary ofthese structures n, first isolated from theleech

X. borealis is more closely related to to is more related closely lly be described asa separate species X. muelleri species group (Fig. 5) strongly suggests that that suggests strongly (Fig. 5) species group [27],and 118 E. coli peptides reveals that they represent orthologs erinin-M1. Thepresence but five substitutions compared with the with compared substitutions five but ed skin secretions from the tetraploid tetraploid frog from the secretions skin ed ial DNAthatplaces X. orS.aureus hasthe same amino acidsequence. Fejervarya cancrivora oad-spectrum antimicrobial activity X. clivii X. clivii X. muelleri X. muelleri Hoplobatrachus tigerinus eby providing support for the the for support providing eby

[8] andfrom the octoploid Despite their low cationicity their lowcationicity Despite [8], did not contain aPGLa X. borealis (unpublished data). The X. borealis Hirudo nipponia [20] West is identical to to West isidentical X. muelleri X. muelleri of such peptide in . Hoplobatrachus No orthologous

[38] belonging muelleri rather than to than rather than to either West West. [32], [32],

Chapter 5 , West M. μ X. muelleri X. muelleri -amidated. -amidated. (3) (1) a a a a West sequence. X. muelleri sequence. West 119 GWASKIGQTLGKLAKVGLKEFAQS GIGKFLHSAGKFGKAFLGEVMKS GIGKFLHSAGKFGKAFLGEVMKS GLGSLLGKAFKIGLKTVGKMMGGAPR (1) GWASKIGQALGKVAKVGLQQFIQPK (6) GWASKIGQALGKVAKVGLQQFIQPK GMASKAGSIVGKIAKIAL*GAL GMASKAGTIAGKIAKTAIKLAL (1) GLGSLLGKAFKFGLKTVGKMMAGAPREQ (1) (7) GLGSLLGKAFKIGLKTVGKMMGGAPREQ GLGSLLGKALKFGLKAAGKFMGGEP*QQ (1) GLGSLLGKAFKFGLKTVGKMMAGAPREE GVGKFLHSAKKFGQALASEIMKSGVGKFLHSAKKFGQALVSEIMKS (7) (7) of amino acids that differ from the from differ that acids of amino . Structural differences are emphasized by the shading and the and the shading the by emphasized are differences . Structural ) indicates that the peptide is C-terminally  C-terminally is peptide the that ) indicates a

) scissile bond. A Met-Ile bond is also foundbond alsobut A Met-Ile tigerinin-M1 is in ) scissile bond. X. clivii X. ΄

, and GWASKIGQTLGKMAKVGLKDLIQA (5) West-MW1 West-MW2 GMASKAGAIAGKIAKTAIKLAL West-MW1 GLGSLLGKAFKFGLKTVGKMMGGAPREQ West-MW1 GLGSLLGKAFKFGLKTVGKMMGGAPREQ West-MW1 West-MW1 GMASKAGSVLGKITKIAL*GAL West-MW2 GLGSLLGKAFKFGLKTVGKMMGGAPREE West-MW2 GLGSLLGKAFKFGLKTVGKMMGGAPREE West-MW3 GLGSLLGKAFKFGLKTVGKMMGGAPR tides -B2 -M2 -B1 -M1 -B1 -B2 -B2 GWASKIGQTLGKMAKVGLKEFVQS (2) -M2 -B1 GFKQFVHSLGKFGKAFVGEMIKPK-B2 G**KFLHSAGKFGKAFLGEVMIG GIGKFLHSAGKFGKAFLGEVMKS (10) (4) (0) -M1 GIGKFLHSAGKFGKAFIGEIMKS (2) -C2 -C1 -C1 -C2 X. borealis , A comparison of the primary structures of the orthologous peptides from from peptides of the orthologous the primary structures A comparison of xenopsin-derived peptides xenopsin-derived muelleri

X. muelleri X. muelleri peptides Procaerulein-derived X. muelleri X. muelleri X. muelleri values in parentheses show the number parentheses values in X. borealis Fig. 5. X. muelleri X. X. borealis X. clivii X. muelleri X. borealis PGLa-related pep X. borealis X. muelleri X. muelleri X. borealis X. clivii X. muelleri-M1 X. clivii X. clivii X. muelleri X. borealis X. borealis X. muelleri the peptide not did inhibitporcine at a concentration pancreatic elastase 100of contains a Met(P1)-Ile( P1 Met(P1)-Ile( contains a Pro Magainin-related peptides (*) Denotes a residue deletion and ( and deletion a residue (*) Denotes Chapter 5 hydrophobicity, hydrophobicity, mammalian cells are determined by complex interactions among cationicity, against cytotoxic microorganisms activities their against and peptides skin frog of activities α that the peptide is C-terminally Fig. 6 H. rugulosus F. cancrivora F. cancrivora H. tigerinus H. tigerinus H. tigerinus H. tigerinus Tigerinin-related peptides X. muelleri potency against α pathogen against theGram-negative the CPFpeptides showthe greatest antimicrobi reached. However, thetherapeutic is charge molecular optimal acertain until potency antimicrobial increased and cationicity synthetic model peptides (reviewed in [4]) have shown a correlation between increasing and antimicrobial peptides occurring naturally both involving studies structure-activity Structural similarity is empha tigerinins from decrease in cationicity associated with the amino acid substitution Gln substitution acid the amino with associated cationicity in decrease erythrocytes (LC human against activity their hemolytic islimitedby forsystemic use, particularly -helicity) of the peptides isolated in this study are compared in Table 2. The antimicrobial antimicrobial in Table The 2. arecompared study this in isolated the peptides of -helicity) -helical conformation in a membrane-mimetic solvent (Table 2). The 2-fold decrease in in decrease 2-fold (Table a The 2). in membrane-mimetic solvent conformation -helical Consistent with previous observations with observations with previous Consistent The . Acomparison oftheprimary structures ofthetigerinin-like peptide from physicochemical properties (mean hydrophobicity, cationicity, and degree of C. albicans Hoplobatrachus tigerinus,rugulosus WCPPMIPL**CSRF  E. coliof CPF-MW2 compared with CPF-MW1 may beexplained by the 50 4 RVC*YAIPLPIC 4 RVC*YAIPLPICY 3 RVC*FAIPLPICH FC*TMIPIPRCY 2 1 -helicity and amphipathicity [4,30]. amphipathicity [4,30]. and -helicity in the range 70 – 105 µM). RVC*MAIPLPLCH RVC*SAIPLPICH . This activity correlates with a high propensity to adopt an extended extended an adopt to a correlatespropensity with high . activity This

RVC*MAIPLPLCH , the Gram-positive S.aureus E. coli,the sized by thesized by shading.(*) Denot  -amidated. potentialofthe CPF peptid 120 a a a a a

a al potency (MIC intherange 3- 25 µM) peptides from related species [5,8,27,28], [5,8,27,28], species related from peptides es aresiduedeletion and( , and the opportunistic yeast yeast the opportunistic , and es asanti-infective agents, ,

and Fejervarya cancrivora Fejervarya 28 28 X. 

muelleri Glu. Several a ) indicates with the . Chapter 5 2 2 7 7 West peptide 14-2 14-2 14-1 14-17 14-1 , , 9 8 22 -helical domain 10-16 2-11 2-11 α X. muelleri +3 Non-helical +3 +4 +3 +5 2-11, +5 +3 8- +5 +3 Non-helical +3 +4 Non-helical +4 +4 12-1 +4 +3 10-1 +3 +2 Non-helical +2 +3 2-11, +3 +4 2-12, +4 pH 7 Ile in the West West cal domains are made using the AGADIRcal domains are made Charge at at Charge 

thanpeptide either tested[37]. alone 20 eater antimicrobial potency and hemolytic at the peptidesat the in combination show more culated using the hydrophobicity scales for aminoculated using 121 E. coli Ile and Val 0 .6 .6  2 0

- -2.1 -0.6 - +5.3 +1.8 +4.3 +1.6 +1.6 +6.4 +7. +18.0 +18.1 Mean Mean 17 hydrophobicity hydrophobicity Doolittle [23]. Predictions of heli Xenopus muelleri and Xenopus muelleri muelleri Xenopus and muelleri Xenopus ore difficultore to account for the gr 1 1 3 Physicochemical propertiesofthe antimicrobial peptides skin isolatedfrom have shown that increasing overall hydrophobicity enhanced activity against against activity enhanced hydrophobicity overall increasing that X. laevis have shown MW MW It is m La-MW2 La-MW1 result in an increase in mean hydrophobicity. Studiessynthetic with analogs of magainin-2 from increasing time, hemolyticwhile, bacteria activityGram-positive at the [10,36]. same Althoughpotency the known relatively that of magainin-MW1 is low, it is the magainin peptides act synergistically with PGLa so th potent growth inhibitory against activity acid residues of Kyte and acid residues of program [29]. Mean hydrophobicities (H) of the peptides are cal are (H) of the peptides Mean hydrophobicities secretions of secretions Table 2. activity of comparedmagainin-M1 withBoth magainin-MW1. peptidesbear a charge +3 at pH7 but the substitutions Leu CPF- CPF-MW2 XPF-M1 CPF- Magainin-M1 Magainin-M2 CPF-M1 CPF-M2 Magainin-MW1 PG PG Tigerinin-M1 XPF-MW Peptide Chapter 5 Center, Omaha, NE) for amino acidcomposition analysis. collection and LaureySteinke andMichele F sample with for help Express Xenopus Lilleyof Burley thank Theauthors University. Acknowledgments [9]. species separate not represent do occupying the Mogollon Rim of central Arizona suggest that the two populations probably occupying regions in southern Arizona and frommorphologically distinct specimens secretions fromspecimens of are separatespecies [7].Ontheotherhand, frog leopard lowland Lithobates frog leopard relict the that probably not conspecific [6]and demonstrate that thisarticleto in described species [19]. Previous investigations with ranid frogs have used the samemethodology to differentiate between sub-species and even different population clusters ofthe same Litoria, amongfrogs. skin The peptide profiles of Australian hylid frogsfrom the genera antimicrobial peptides in skin secretions have value in inferring evolutionary relationships PGLa-MW1 of PGLa-MW2activities and aswellthe CPF peptides. Consequently, in the the in Consequently, This work was supported by a Faculty Support Grant (NP-10-11/103) from U.A.E. U.A.E. from (NP-10-11/103) Grant Support byaFaculty supported was work This This study has given further support to the assertion that the amino acid sequences of and Uperoleia X. muelleri Lithobates yavapaiensis Lithobates have been used, together with morphological and cognatemethods, the Chiricahua leopard frog frog leopard Chiricahua the West secretions, magainin-MW1 may act to potentiate the the secretions, potentiate West magainin-MW1 to may act

122 and tagoi tagoi Rana , although closely related phylogenetically, closelyrelated phylogenetically, , although the presence of identical peptides in skin skin in peptides identical of presence the ontaine (University of Nebraska Medical Lithobates chiricahuensis Lithobates Rana tagoi okiensis okiensis tagoi Rana

onca andthe Crinia are are ,

Chapter 5 Xenopus and Rana tagoi and tic relationship between tic relationship (Ranidae). Comp Biochem Comp (Ranidae). J, Nielsen PF. Orthologs of rategies for transformation of for transformation rategies therapeutically valuable anti- valuable therapeutically subtended by charged residues modulate charged residues subtended by peptides to the system of innate immunity peptides to the in secretions from discrete populations of discrete populations from in secretions nopsin-derived peptides from from skin secretions nopsin-derived peptides thods for dilution antimicrobial susceptibility susceptibility antimicrobial for dilution thods ation. Howard, D, Berlocher S, editors. New Berlocher ation. Howard, D, amphipathic helical peptides. FEBS Lett peptides. helical amphipathic ouenne T, Leprince J, Vaudry H, et al. H, J, Vaudry ouenne T, Leprince conspecific, phylogene of species, species criteria, and the process of process and the criteria, species of species, Vaudry H, King JD. Primary structures of skin H, King JD. Vaudry ndel L, Maloy WL, MacDonald DL, et al. L, Maloy ndel J, Vaudry H, Iwamuro S. Evidence from the from the Evidence H, Iwamuro S. Vaudry J, ). Genetics 2007;176:1119-30. 2007;176:1119-30. ). Genetics 123 pproved Standard M07-A8. CLSI, Wayne, CLSI, PA, 2008. Standard M07-A8. pproved Lithobates yavapaiensis Standard M27-A3. CLS1, Wayne, M27-A3. Standard PA, 2008. (Pipidae). Peptides 2010;31:989-94. Peptides 2010;31:989-94. (Pipidae). subspecies. Toxicon 2010;55:430-5. subspecies. Toxicon 2010;55:430-5. and Xenopus med E, Leprince J, Vaudry H, King JD et al. Purification and med E, Leprince med E, Meetani MA, Leprince Leprince MA, E, Meetani med , Abraham, B., Leprince, J. St J. B., Leprince, Abraham, , antimicrobial peptides okiensis that tagoi Rana (Ranidae) from central and southern Arizona. Peptides (Ranidae) from central and southern Xenopus amieti Xenopus (Ranidae) are not conspecific are not (Ranidae) (Pipidae). Comp Biochem Physiol C Toxicol Pharmacol 2011;153:350-54. Pharmacol 2011;153:350-54. Toxicol Physiol C Comp Biochem (Pipidae). speciation. In: Endless forms: species and speci species forms: Endless In: speciation. polyploidization of clawed frogs ( of clawed frogs polyploidization 1997;403:208-12. 1997;403:208-12. York: Oxford Press, 1998. p.57-75. Physiol C Toxicol Pharmacol. 2010;151:313-7. Pharmacol. 2010;151:313-7. Toxicol Physiol C frog clawed Eritrea of the secretions skin from peptides antimicrobial of properties clivii in sk peptides of antimicrobial Characterization Hydrophobicity, hydrophobic moment and angle of activity haemolytic and antibacterial Lithobates chiricahuensis 2011;32:664-9. antimicrobial peptides indicate a close, but not close, but a indicate peptides antimicrobial tagoi frogs Lithobates onca the leopard susceptibility testing of yeast. Approved yeast. of testing susceptibility tests for bacteria that grow aerobically. A that grow tests for bacteria of dermal structures primary in anurans. Cell Tissue Res 2010;343:201-12. Res 2010;343:201-12. Tissue Cell in anurans. and proxe procaerulein-derived, PGLa, magainin, frog of the octoploid bian antimicrobial peptides into peptides naturally-occurring amphibian antimicrobial infective agents. Methods 2007;42,349-57. Methods 2007;42,349-57. infective agents. REFERENCES REFERENCES [11] concept lineage The general de Queiroz, K. [12] of after millions years genes of duplicated the fate Evans BJ. Ancestry influences [8] Conlon JM, Mechkarska M, Ah [9] L, J Conlon JM, Mechkarska M, Coquet [10] H, Ha T, Nikolenko Dathe M, Wieprecht [7] T, J, Jouenne L, Leprince Conlon JM, Coquet [2] antifungal broth dilution for method Institute. Reference Standards Laboratory and Clinical [3] skin antimicrobial of Conlon JM. The contribution [4] Al-Ghaferi, N. Conlon, J.M., [6] T, Leprince L, Jouenne Conlon JM, Coquet [1] Institute. Me and Standards Laboratory Clinical [5] Conlon JM, Al-Ghaferi N, Ah Chapter 5 1] GibsonBW, PoulterL, Williams DH,MaggioJE.Novelpeptide fragments originating from [17] species FrostDR.Amphibian of theworld: [16] Sympat A. Elepfandt WA, Koch WJ, Fischer [15] DB, EvansBJ,Kelley Tinsley RC,Meln [14] EvansBJ.Genomeevolutionand speciation genetics ofclawedfrogs ( [13] 2] JungHI,KimSI,HaKS,JoeCO, KangKW. [20] RJ,Pukala Jackway TL, DonnellanSC,Sher [19] GJ,Patrick HarperEB,DA, Menegon Measey [18] 2] MaloneJH,ChrzanowskiTH,Michalak P.St [25] LargmanC. Isolation and characterizati [24] J,DoolittleDF.Asimp Kyte [23] KobelHR, Loumont C,TinsleyRC. The exta [22] KobelHR.Allopolyploid speciation. In: Ti [21] [26] Measey J, Tinsley R, MinterL,RödelM-O. J, Tinsley Measey [26] PGL Histor ofNatural Museum database accessible at http://r Xenopus laevis laevis Mol Phylogenet Evol 2004;33:197-213. ofAfrican evolution. phylogeny clawedfrogs:andimplicationsforpolyploid phylogeography Front Biosci2008;13:4687-706. trends. Peptides 2011;32:161-72. cDNA profilingofAustraliananurans:genusa Camerapix PublishersInternational; 2010. the EasternArcMountainsandCoastalForest 1986;261:5341-49. Xenopus laevis 1983;22:3763-70. J. Mol.Biol.1982;157:105-32. The biology of Xenopus 84. new humanleukocyte elastase inhibitor from www.iucnredlist.org; 2004 List ofThreatened Species. Version 201 a andthe caerulein and xenopsin precursors from Xenopuslaevis. . Oxford: Clarendon Press;1996.p 391-401. and Xenopus X. muelleri and . Oxford: Clarendon Press;1996.p9-33. Xenopus muelleri y, New USA;2011. y, York, le themethod fordisplaying hydropathic character of aprotein. esearch.amnh.org/ herpetology/ . PLoS One2007;2:e781. 124 (Pipidae).J.Zool2000;252:99-107. ick DJ, Cannatella DC. Amitochondrial DNA on of ratpancreatic elastase.Biochemistry Hirudo nipponia.JBiolChem.1995;270:13879- nsley RC,Kobel HR,nsley editors. The biologyof man PJ, Tyler MJ, man PJ,Tyler Bowie JH.Skin peptide and ry and hybridization betweenthe clawedfrogs Xenopus muelleri erility andgene expression inhybrid males of nt species. In: Tinsley an onlinereference. Version5.5. Electronic M, VoneshJR.FieldGuide to Amphibians of Isolation andcharacteriz s ofTanzania Nairobi,and Kenya. Kenya: 0.4. Electronic data nd speciesidentificationandevolutionary amphibia /index.php.American . In: IUCN2010.Red RC, Kobel HR, editors. Xenopus base accessible at ation of guamerin,a ation JBiolChem andSilurana). Chapter 5 . Xenopus Xenopus (Amphibia: , tumor cells cells tumor , Xenopus Xenopus Fejervarya cancrivora e peptides. Methods Mol Methods e peptides. Escherichia coli Escherichia T, Vaudryal. Genome et H, Vaudry H, et al. Antimicrobial et al. Antimicrobial Vaudry H, rification, characterization characterization and rification, thic, antibacterial peptides by slight peptides thic, antibacterial Rana tigerina, J Biol Chem 2001;276:2701- and its receptor in vertebrates. Cell Tissue in vertebrates. and its receptor NP, Devi AS, et al. Tigerinins: R. Nagaraj een peptide structure and antibacterial activity. activity. antibacterial and structure peptide een l. I: Amphibians of the savanna. West African l. sui K. Molecular evolution and functional functional evolution and sui K. Molecular ng problem of helical peptides using empirical problem of helical peptides ng er B, Shaw C. HV-BBI - a novel amphibian skinamphibian Shaw C. HV-BBI - a novel er B, Hendler RW,al.A,Hendler De Waal A, Vaz Gomes et in secretions of the Marsabit clawed frog frog clawed the Marsabit of in secretions pplication of host defens 125 ment. FEBS Lett 1997;417:135-40. Lett 1997;417:135-40. ment. FEBS species and subspecies of the genus subspecies of the and species skin secretions. Comp Biochem Physiol Part D Physiol Part Biochem Comp Xenopus andrei skin secretions. Hailong Y, KeyunZ, Ren L. Pu and (Pipidae). Comp Biochem Physiol C Toxicol Pharmacol 2010;152:467-72. Pharmacol 2010;152:467-72. Toxicol Physiol C Comp Biochem (Pipidae). Silurana paratropicalis Biol 2010;618:303-27. Biol 2010;618:303-27. cloning of two novel tigerinin-like peptides from skin secretions of secretions skin from peptides tigerinin-like two novel cloning of and liposomes. Eur J Biochem 1995;228:257-64. 1995;228:257-64. Eur J Biochem and liposomes. Peptides 2009;7:1228-32. Bowman-Birk-like trypsin inhibitor. Biochem Biophys Res Commun 2008;372:191-6. Commun 2008;372:191-6. Biophys Res Biochem inhibitor. Bowman-Birk-like trypsin 26RFa peptide of the orexigenic characterization Res 2011;343:475-81. the hydrophobic mo modifications of in magainin-2 PGLa and of the magainins Functional synergism Modulation of membrane activity of amphipa Modulation of borealis peptides of antimicrobial increase the multiplicity do not the Xenopodinae duplications within in 1994;1:399-409. Biol Nature Struct parameters. frog the Indian from peptides novel antimicrobial 07. London 1979;188:103-22. Anura) J Zool, Genomics Proteomics 2011;6:206-12. 2011;6:206-12. Genomics Proteomics Peptides 2003;24:1681-91. Chimaira, 2000. Edition Frankfurt: peptides with therapeutic potential from sk from potential with therapeutic peptides [38] Yuzhu S, Yi L, Lijun W, [39] a TJ. Potential therapeutic Zhang L, Falla [34] Tsut J, Ukena K, VaudryH, Leprince [35] 12 mating calls of Vigny The C. [37] Westerhoff HV, ZasloffRosner M, JL, [28] J, Jouenne L, Leprince Ahmed E, Coquet Mechkarska M, [30] betw relationship The RE. Powers JP, Hancock [33] T, Walk M, Chen W, Chen Zhou Song G, [36] Bienert M. DL, MacDonald Maloy WL, M, Beyermann E, Krause Dathe M, T, Wieprecht [29] the foldi Elucidating L. Muñoz V, Serrano [31] of West Africa, Vo Rodel MO. Herpetofauna [32] Sai KP, Jagannadham MV, Vairamani M, Raju [27] T, J, Jouenne Ahmed E, Coquet L, Leprince Mechkarska M,

Chapter 6

The hymenochirins: a family of antimicrobial peptides from the Congo dwarf clawed frog Hymenochirus boettgeri (Pipidae)

M. Mechkarska, M. Prajeep, L. Coquet, J. Leprince, T. Jouenne, H. Vaudry, J.D. King, J.M. Conlon

Peptides (2012) 35:269-275. Chapter 6 Pseudhymenochirus dwarf clawed frog norepinephrine-stimulated skin secretions fromtwo distinct populations of the Congo AKGX (IKLSPETKDNLKKVLKGAIKGAIAVAKMV.NH structurally-related peptides with broad-spectrumantimicrobial activity. Hymenochirin-1B species from thesistertaxon family Pipidaearea rich source of antimicrobi ( Xenopodinae subfamily the from frogs of secretions Skin ABSTRACT Silurana tropicalis and tropicalis Silurana antimicrobial structural the similaritylow with inthe peptide mostGALTQ) was abundant the hymenochirins-2B-5B have the general structure XKIPX erythrocytes (LC human against activity hemolytic weak relatively display µM).The peptides (MIC =80 Staphylococcus aureus growth of Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, the Pipinae and Xenopodinae. Synthetic replicates of hymenochirin-1B - 4B inhibit the 2 SX 2 AGAX 50 in the range 160 to >300 >300 µM). to range 160 the in 3 Hymenochirus boettgeri .COOH. Hymenochirin-3B (IKIPAVVKDTLKKVAKGVLSAVA (IKIPAVVKDTLKKVAKGVLSAVA Hymenochirin-3B .COOH. +Pipa)have not been investigated. Peptidomic analysis of Xenopus laevis (MIC intherange 10 - 40 µM) and Hymenochirus consistent with the with prop consistent (Tornier, 1896) hasled to identification of five 128 in the subfamily Pipinae ( Pipinae subfamily in the peptides isolated from skin secretions of al peptides with therapeutic potential but but potential therapeutic with al peptides secretions. Thehymenochirins show very 2 ) is C-terminally α Xenopus osed ancientdivergence of +Silurana -amidated whereas Candida albicans Hymenochirus 2 VKDTLKKV VKDTLKKV ) within the the ) within and and +

Chapter 6

Pipa [30] [30] [29], has been been has H. boettgeri, butpeptides . The present study . The present study [22]. These include peptide peptide include These quatorial Africa [18]. [18]. Africa quatorial tides termed caerulein- tides termed Xenopus was laevis the Hymenochirus within the family Pipidae within family the [8], Xenopus andrei [8], Xenopus muelleri [22], Xenopus Silurana tropicalis forest zone of central Africa from zone of central Africa from forest , and Xenopus, and rized species distributed in five genera in five distributed rized species of its wide distribution. wide However, it is a of its tion and characterization of several other other of several characterization and tion Xenopus amieti Xenopus cturally-related pep cturally-related tides, the magainins with growth inhibitory host-defense peptides host-defense to the PGLa family (peptide XT-5), the from West Africa provisionally designated designated provisionally West Africa from anti-infective agents. ) that are found in e 129 Silurana , Xenopus pygmaeus and Xenopus [22], [9], Xenopus [9], lenduensis ecently, antimicrobial peptides peptides re not recently, antimicrobial [1]. identified More Xenopus clivii Xenopus petersii have proved to be a rich source of host-defense peptides with with peptides host-defense of source a rich be to proved have Pseudhymenochirus , [28], [28], HymenochirusBoulenger, 1896 comprises four species ( Pipa , ranges widely through the equatorial equatorial the through widely ranges is unusual in that it relies solely on inertial suction to captureprey [4]. It is that were probably captivebred in [33]. Florida + Silurana) Introduction 33 well-characte comprise frogs The clawed Up untilUp this no time, species belonging togenus the The genus The genus X. laevis skin secretions led has to the isola Xenopus Hymenochirus [17,18]. All are found in Africa south of the Sahara except for members of the genus genus of the for members Sahara except the of south Africa in are found All [17,18]. 1. have also declined because of habitat habitat of declined because have also numbers trade and pet for the popular species of a population in reported has been Chyridiomycosis [37]. deforestation from modification H. boettgeri listed as a Species of Least Concern byInternational the Union for Conservation of Nature view in Species of Threatened List (IUCN) Red H. boulengeri, H. curtipes, and H. feae H. boettgeri of region Mayombe the to Gabon through south Cameroon and Nigeria southeastern western Republic of Democratic Congo and species the has been introduced into found generally aquatic, is species The [38]. non-indigenously USA Florida, southeastern in still, shadedpools[36]. rainforest and water in by slow-flowing lowland in rivers H. boettgeri of cutaneous presence for the investigated which are found in South America. The skins of frogs from the subfamily Xenopodinae Xenopodinae subfamily the from of frogs The skins America. South in are found which ( characterized species incompletely and an [30], X. muelleri West first amphibian species in which cutaneous pep cutaneous which in species amphibian first analysis Subsequent [39]. identified unambiguously were fungi and bacteria against activity of antimicrobial and anti-inflammatory activities (reviewed[10]). in Xenopus borealis belonging to the magainin, PGLa, CPF, and XPF families have been isolated from from isolated been have families XPF and CPF, PGLa, magainin, the to belonging from secretions skin norepinephrine-stimulated peptides that show therapeutic potential as stru (PGLa) and multiple glycine-leucine-amide precursor fragment (CPF) and xenopsin-precursor fragment (XPF) that are derived from the the from derived are that (XPF) fragment xenopsin-precursor and (CPF) fragment precursor xenopsin and of caerulein precursors biosynthetic of the processing post-translational respectively [21,35]. Peptides that belong XT-3, XT-2, (peptides family XPF the and XT-7), and XT-6, XT-1, (peptides CPF family of secretions skin from isolated been have XT-4) and we the magainins to orthologous Chapter 6 Pak cartridges, were separately injected on 2.2. v/v/v) and freeze-dried. The material wasredissolved in 0.1% (v/v) TFA/water (2 ml). connected in series. Bound material waselut mlof 2 min containing the secretions from both groups were acid (TFA) (1 ml) andimmediatelyfrozen for sh frog was removed and the collection solution was acidified by addition of trifluoroacetic (40 nmol/g bodyweight) andplacedin asolutio ofCongo. ataRepublic the Democratic site in were originallycollected 0.69-0.74 g; sex not determined) were purchased at apet store in St Louis, MO, USA and 2(n= weights 4; from animals group Sub-adult Cameroon. the atasitein collected Aquarium, California Academy ofScience, SanFrancisco, CA and were originally 1 from animals group Adult investigators. Two populations of the Congo dwarf clawed frog Committee ofU.A.E. University (Protocol No. frog clawed dwarf Congo the of populations norepinephrine-stim from activity antimicrobial describes the isolation and the structural and biological characterization of peptides with with 0.1% (v/v) TFA/waterat 0.1%(v/v) with aflowrate ml6.0 of min 218TP1022 (C-18) 2.1. 2. Cameroon the the Democratic Congo. and the Republic of broth (50  by incubating lyophilized aliquots of chromatographic effluent (100 µl) in Mueller-Hinton weremonitored peptides ofthe activities biological and Purification min) (1 were collected. using linear gradients. Absorbance was monitored at 214 nm and280 nm, andfractions over min (v/v) 60 63% to min and over 10 (v/v) 21% to wasraised solvent eluting the in culture of reference strains of

The skin secretions from each group of of group from each secretions skin The purification Peptide Collection of skin secretions skin of Collection Experimental Each animal wasinjected via the dorsal lymph sacwith norepinephrine hydrochloride Ethics theAnimalby Research animalswereapproved live All experiments with l) with aninoculum (50 -1 through 4 Sep-Pak C-18 reversed-phase HPLCcolumn (Grace,

(n = 4; weights 3.0-5.0 g; 2 female) were kept at the Steinhart Staphylococcus aureus  l of10

cartridges (WatersAssociat 130 ed with acetonitrile/watered with H. boettgeri, H. boettgeri to a preparative (2.2 6 colony forming units/ml) from alog-phase A21-09) and were carried out by authorized authorized by out carried were and A21-09) n (40ml) ofdistilledwaterfor 15min. The separately pooled and passed at a flow rate ipment to ipment to U.A.E. solutions The University. ulated skin secretions of two distinct two distinct secretionsof skin ulated (ATCC 25923) (ATCC -1 . Theconcentration of acetonitrile (Tornier, 1896) that originated in Deerfield, IL, USA) equilibrated equilibrated Deerfield,IL,USA) after partial purification on Sep- on purification afterpartial H. boettgeri es, Milford, MA, USA) cm x 25 cm) Vydac cmcm) 25 Vydac x

and and /TFA (70.0:29.9:0.1, Escherichia coli were sampled.

Chapter 6 . -1 . The identity and C in a humidified o -1 e eluting solvent wase eluting solvent (ATCCE. coli 25726), of acetonitrile in th of acetonitrile of acetonitrile in the of acetonitrile in the concentration The mn. ssociatedwith antimicrobial activity were 25 cm) Vydac 214TP510 (C-4) column and a a and column (C-4) 214TP510 Vydac cm) 25 rbance at 630 nm of each well was determined determined each well was of 630 nm at rbance phase HPLC on a (2.2 cm x 25 cm) Vydac Vydac x 25 cm) cm on a (2.2 HPLC phase 131 (ATCC 27853), and and 27853), (ATCC aeruginosa Pseudomonas (ATCC 25923), ltage in the ion source was 20 kV. The instrument was instrument was 20 kV. The ion source ltage in the S. aureus S. 0.02%. 0.02%.  (ATCC 700603), (ATCC 90028) were measured inthe concentrationrange of 2.5 - 160 in 96-well microtiter cell-culture plates for 18 h at 37

Peptide synthesis Peptide synthesis Ltd. China Peptides by form crude in supplied were - 5B Hymenochirins-1B Structural characterization characterization Structural the by peptides determined were automatedof structuresprimary The Edman Antimicrobial andhemolytic activities Culture Type American the from purchased were microorganisms of strains Reference

M by standard double dilution methods [5,6] and were taken as the lowest concentration concentration lowest the as taken were and [5,6] methods dilution double standard by M raised from 28% to 56% over 60 min and the flow rate was 6 ml min 6 ml was rate flow and the 60 min over 56% to 28% from raised 218TP1022 (C-18) column. The concentration The concentration column. (C-18) 218TP1022 2.4. near to purified were and China) (Shanghai, Ltd. Biochem GL and China) (Shanghai, reversed- by (> 98% purity) homogeneity 2.3. CA, City, Foster Biosystems, (Applied sequenator Procise 494 model a using degradation DE-PRO Voyager a using out carried was spectrometry mass MALDI-TOF USA). delayed with mode reflector in was operated that Biosystems) (Applied instrument vo extraction and the accelerating calibratedwith peptides of known in molecular thekDamass - 4 2 range. The accuracyof mass determinationswas atmosphere of air. After incubation, of air. After the abso incubation, atmosphere plateusing reader. Fractions a a microtiter x cm (1.0 on a chromatographed successively colu Vydac 208TP510 (C-8) x 25 cm) (1.0 cm min was 2.0 ml and rate flow the 56% over 50 min 21% to from was raised solvent eluting Collection inhibitoryMinimum (Rockville, MD). concentrationsof synthetic (MIC) the against hymenochirins 2.5. purity of the peptides was confirmed by electrospray mass spectrometry. Hymenochirin-5B Hymenochirin-5B spectrometry. mass electrospray by was confirmed peptides the of purity solution. concentrated in gel a viscous form to observed was and yield low in obtained was pneumoniae Klebsiella μ by confirmed were values The observed. was growth visible no where of peptide were incubations independent of three A minimum nm. 630 at of absorbance measurement performed.order In tovalidity monitor the and reproducibility of the assays, incubations Candida albicans Candida (ATCC 25726)

were carried out in parallel with increasing concentrations of antibiotics (ampicillin for S. aureus and E. coli, ciprofloxacin for K. pneumoniae and P. aeruginosa, and amphotericin for C. albicans) as previously described [28-30]. Hemolytic activity against human erythrocytes taken from a healthy donor was measured as previously described [28-30]. Peptides were tested in the concentration range

37.5 - 300 µM. The LC50 value was taken as the mean concentration of peptide producing 50% hemolysis in three independent incubations.

2.6. Secondary structure prediction Prediction of secondary structure and determination of % helicity per residue for the peptides were performed using the AGADIR program [31]. AGADIR is a prediction algorithm based on the helix/coil transition theory which predicts the helical behaviour of monomeric peptides. Calculations were performed at pH 7 and 278K. A minimum percentage of 1% helicity/residue was considered to predict the presence of a helix. Calculated mean hydrophobicities of the peptides were based upon the hydrophobicity scales for amino acids of Kyte and Doolittle [23].

3. Results 3.1. Purification of the peptides The pooled skin secretions from the group 1 population of H. boettgeri (Cameroon), after partial purification on Sep-Pak C-18 cartridges, were chromatographed on a Vydac C-18 preparative reversed-phase HPLC column (Fig. 1). Under the conditions of assay, the prominent peaks designated 1 - 5 were associated with the ability to inhibit the growth of both E. coli and S. aureus. Subsequent structural analysis demonstrated that these peaks contained hymenochirins 1- 5. The peptides were purified to near homogeneity, as assessed ter 6 ter 6

p by a symmetrical peak shape and mass spectrometry, by further chromatography on semipreparative Vydac C-4 and Vydac C-8 columns. The methodology is illustrated by

Cha purification of hymenochirin-3B on C-4 (Fig. 2A) and C-8 (Fig. 2B) columns. The approximate final yields of purified peptides (nmol) were hymenochirin-1B (40), hymenochirin-2B (55), hymenochirin-3B (425), hymenochirin-4B (90) and hymenochirin-5B (65).

132

Chapter 6 Peaks 1-5 1-5 Peaks . C-18 column of skin secretions from column of skin secretions from C-18 rin-3B on semipreparative (A) Vydac C-4, and (A) semipreparative rin-3B on

133 and were purified further. The dashed line shows the line The dashed further. were purified and after partial purification on Sep-Pak cartridges

onitrile in the eluting solvent. solvent. eluting in the onitrile S. aureus and and E. coli E. the Cameroon (Group 1)

from Purification to near homogeneity of hymenochi Purification Reversed-phase HPLC on a preparative Vydac Reversed-phase HPLC on a preparative Fig. 1. H. boettgeri H. boettgeri concentration of acetonitrile in the eluting solvent. eluting the in acetonitrile of concentration inhibited growth of (B) Vydac C-8 columns. The arrowheads show where peak collection began and ended. The dashed The dashed ended. and began collection where peak show arrowheads The columns. C-8 (B) Vydac acet of concentration shows the line Fig. 2. Chapter 6 and analysed by MALDI- analysed and fromthe group 1population (Fig. 3). The prominent peaks designated 1 - 5werecollected preparative reversed-phase HPLC column under the same condition used for secretions partial purification on Sep-Pak C-18 cartridges, were chromatographed on a Vydac C-18 3. H. boettgerifrom Fig. 3. Reversed-phaseHPLConapreparativeVydac further. The dashed line shows theconcentrat Pak cartridges population (Cameroon) of and theiramino acidsequences are shown inFig. 4. 2.

The pooled skin secretions from the group 2 population of The primarystructures of Structural characterization . Peaks 1-5were analysed by MALDI-TOF mass spectrometry and werepurified

the DemocraticRepublic of theCongo(Group 2) TOF mass spectrometry. H. boettgeri the antimicrobial peptides peptides antimicrobial isolatedfromthe the group 1 were established by automated Edman degradation ion ofacetonitrile in the eluting solvent. 134 C-18 columnofskinsecretionsfrom

after partialpurificationon Sep- H. boettgeri (Congo), after after (Congo), Chapter 6 are

calc , E. coli albicans

Mr C. . obs Mr obs H. boettgeri Mr -amidated. The amino acid acid amino The -amidated. α 3061.8 3062.4 3061.8 3061.8 3062.4 3061.8 Cameroon Congo 2 obs), and calculated molecular masses masses molecular and calculated obs), r inhibitreference the growth of strains with their proposed structures and secretions of secretions , the Gram-negative bacteria bacteria Gram-negative , the r masses ofpeptides the determined by r masses 135 in the range 160 to > 300 µM). µM). 300 > to 160 range the in 50 S. aureus isolated from skin from isolated VAKGVLSTIAGALST VAKGVLSTIAGALST 2860.8 2860.5 2861.0 KVAKGIFSAVAGAMTPS 2973.7 2973.7 2973.5 KVAKGVLSAVAGALTQ 2817.7 2817.9 2817.6 KVAKGVISAVAGALTQ 2865.7 2865.5 2865.9 erved molecular masses (M in Mueller-Hinton broth and its tendency bility of the peptide in Mueller-Hinton broth , and the opportunistic yeast pathogen aeruginosa P. , and ere was some ambiguitysome was withtoere regard identification of amino acids at the the Amino acid sequences, obs Amino acid sequences, As th Antimicrobial andhemolytic activities The abilities of the synthetic hymenochirins to

calc) of the antimicrobial peptides peptides antimicrobial the of calc) r hydrophobic C-terminus of hymenochirin-1B, the proposed sequence was confirmed by by was confirmed sequence proposed the hymenochirin-1B, of C-terminus hydrophobic (1), 1.1 Ser (1), 1.1 Thr (1.0), Clx (2), 2.1 Asx [Found: analysis composition acid amino Pro (7), 6.7 Lys (3), 3.0 Leu (3), 2.7 Ile (1), 1.1 Met (3), 2.9 Val (4), 4.0 Ala (2), 2.1 Gly predicted residues of number the show parentheses in Figures peptide]. residues/mol (1) 1.0 The molecula sequences. the proposed from MALDI-TOF mass spectrometry are consistent are spectrometry MALDI-TOF mass 3.3. demonstrate that only hymenochirin-1B is C-terminally C-terminally is hymenochirin-1B only that demonstrate but determined not were (Congo) 2 population group the from hymenochirins of sequences onreversed-phasethe4) and close (Fig.retention times similarities in masses molecular probably are populations both from peptides the that 3) indicate 1 and (Figs. HPLC identical. of Gram-positivethe bacterium K. pneumoniae Fig. 4. Hymenochirin-1B IKLSPETKDNLKKVLKGAIKGAIAVAKMV.NHHymenochirin-1B Hymenochirin-2B LKIPGFVKDTLKHymenochirin-2B IKIPAVVKDTLKHymenochirin-3B IKIPAFVKDTLKHymenochirin-4B IKIPPIVKDTLKKHymenochirin-5B (M compared in Table 1. The MIC values for hymenochirin-5B may be artefactually high high be artefactually may hymenochirin-5B for values MIC The 1. Table in compared restricted solu because of the ato gel form in concentrated solution. All peptides displayed relatively low hemolytic activities against erythrocytes human (LC Chapter 6 ribosomal 18S and28S genes strongly supported the sister-group relationship between analyses based upon thecomparison of Hymenochirus morphological charactersconcluded that among the clades within the family arenot entirely clear. Astudy based upon the isolation of antimicrobial peptides from several species from the genera genera fromthe species several from peptides ofantimicrobial isolation the frogs originating in the Cameroon. The work complements previous studies that have led to chromatographic properties indicat molecular masses close in similarities the determined, and werenot Congo sequences ofthe peptides from thepopulation the hymenochirins and given the suffix B to denote the species. Although the amino acid Mexican burrowingtoad been investigated. and thesingle ) belongingspecies (P.merlini to the genus fr secretions in skin identified Xenopus genus two populations from different geographical locations. Skin secretions from afrogfromthe antimicrobial peptides from skin secretions of the the frog of secretions skin from peptides antimicrobial ND 160 160 80 4. 80 20 >300 80 ND >300 80 40 determined. 20 225 Data are expressed as µM andare derived from a 80 40 ND 40 Hymenochirin-5B 40 10 20 20 Hymenochirin-4B 20 40 20 Hymenochirin-3B 20 20 Hymenochirin-2B 20 40 Hymenochirin-1B 20 Peptide activity against human erythrocytes (LC 1. Table H.

boettgeri. boettgeri. Pipidae is sister-groupto The Discussion Hymenochirus within the family Pipidae (reviewed in family (reviewed the within Pipidae study has describedthe purification and characterization offivestructurally related Minimum inhibitory concentrations against microorganisms and hemolytic , Pipa E. coli P. aeruginosa K. pneu K. aeruginosa P. coli E. , and Pseudhymenochirus have not beeninvestigated previously. Thepeptides have been termed Rhinophrynus dorsalis Rhinophrynus om arepresentative ofthe genus the Rhinophrynidae (represented by asingle species,the e thattheyareprobably iden 50 Silurana Silurana ) of the peptides) of isolated 136 the nucleotide sequences of the DNA from minimum ofthreeindependent incubations.ND: not than to Xenopus to than originating in the Democratic of inthe Republic originating [10,22]). Antimicrobialpeptides werenot ) [17,18]) but phylogeneticrelationships was more closely related to the genera moniae S. aureus C. albicans albicans C. aureus S. moniae H.

boettgeri boettgeri Pseudhymenochirus [3]. However, subsequent Pipa, tical to the peptides from peptides the tical to from secretions of skin (Pipidae) belonging to belonging (Pipidae) notably P.pipa notably Silurana has not yet yet not has LC and and [7], 50 Chapter 6 ) species species in Africa from the

a a Xenopus Xenopus Pseudhymenochirus + Hymenochirus Pipa Pseudhymenochirus + + fragment peptides, the caerulein- fragment and a range of of and a range ce similarity (reviewed [10]).data in The y which occurred at the time of separation of separation time occurred at the y which Hymenochirus Hymenochirus 137 Hymenochirus Hymenochirus S. tropicalis GWASKIGQTLGKIAKVGLKELIQPK GWASKIGQTLGKIAKVGLQGLMQPK GIGKFLHSAGKFGKAFVGEIMKS GIGKFLHSAKKFGKAFVGEIMNS

[21]. Amino acid sequence comparisons of comparisons acid sequence [21]. Amino X. laevis

IKIPAFVKDTLKKVAKGVISAVAGALTQ IKIPPIVKDTLKKVAKGVLSTIAGALST IKLSPETKDNLKKVLKGAIKGAIAVAKMV LKIPGFVKDTLKKVAKGIFSAVAGAMTPS IKIPAVVKDTLKKVAKGVLSAVAGALTQ IKLSPETKDNLKKVLKGAIKGAIAVAKMV LKIPGFVKDTLKKVAKGIFSAVAGAMTPS IKIPAVVKDTLKKVAKGVLSAVAGALTQ IKIPAFVKDTLKKVAKGVISAVAGALTQ IKIPPIVKDTLKKVAKGVLSTIAGALST [1] and rather than the relationship suggested by morphology [14]. [14]. morphology by suggested relationship the than rather ) from the Pipinae ( Pipinae the from ) in South America and America in South clade. XT-2 XT-3 GVWSTVLGGLKKFAKGGLEAIVNPK GLASTLGSFLGKFAKGGAQAFLQPK Pipa [39] and the xenopsin-precursor X. laevis [39] and the S. tropicalis -1 -2 -1 -2 + Xenopus and Xenopus + Xenopus Silurana S. tropicalis X. laevis X. laevis Hymenochirin-4B Hymenochirin-5B S. tropicalis Hymemochirin-1B Hymenochirin-2B Hymenochirin-3B X. laevis X. laevis Hymenochirin-3B Hymenochirin-4B Hymenochirin-5B Hymemochirin-1B Hymenochirin-2B XPF Silurana [15,16] DNA mitochondrial of sequences the nucleotide of comparisons Subsequent provide support for conclusion this and suggest that divergence thethe of Xenopodinae ( precursor fragment peptides, and the PGLa-related peptides that were isolated from skin skin from isolated were that peptides PGLa-related and the peptides, fragment precursor secretions of from peptides antimicrobial orthologous predates the divergence of the Pipinae subfamil divergence the predates of Africa and South America (105 119- years million ago, [27].MYA) The origin of the the to led Gondwana of breakup and Pipidae is at MYA) least Late Jurassic (150 establishment of of sequen degree greater indicate an appreciably of divergence ancient proposed the with consistent are thus Silurana Magainin [2]. As shown in Fig. 5, the hymenochirins show only very low structural similarity to the to similarity structural low very only show hymenochirins the 5, Fig. in As shown [2]. magainins from Chapter 6 residues. Gaps, denoted by *, are inserted into that the peptide isC-terminally Fig. 5. X. laevis X. laevis X. laevis X. laevis S. tropicalis Hymenochirin-5B Hymenochirin-4B Hymenochirin-3B Hymenochirin-2B Hymemochirin-1B CPF X. laevis S. tropicalis Hymenochirin-5B Hymenochirin-4B Hymenochirin-3B Hymenochirin-2B Hymemochirin-1B PGLa and CPFfamilies from degree of of degree effective hydrophobicity of a of hydrophobicity effective Retention time onreversed-phase HPLC has been shown to providemeasure a of the times which may self-aggregate. times tendency to which totheir be related hymenochirin-4B and -5B areeluted from the column with anomalously high retention of the hymenochirins do not correlate ex Th

A comparison of the primary structures ofpe Acomparisonoftheprimary e physicochemical properties (grand average of hydrophobicity, cationicity, and and cationicity, of hydrophobicity, average (grand e properties physicochemical α -helicity) of the peptides isolated in this study are compared in Table 2. 2. in are compared Table study inthis isolated peptides the -helicity) of CF3 GFGSFLGKAL*KAALKIGANALGGSPQQ GFASFLGKAL*KAALKIGANMLGGTPQQ GLASFLGKAL*KAGLKIGAHLLGGAPQQ CPF-3 CPF-2 CPF-1 CPF-4 X- GFLGPLLKLAAKGVAKVIPHLI*PSRQQ XT-1 GMATKAGTALGKVAKAVIGAAL XT-5 Hymenochirus boettgeri, Silur α -helicalpeptide [36]. However,  -amidated. Shadingisused to IKIPPIVKDTLKKVAKGVLSTIAGALST IKIPAFVKDTLKKVAKGVISAVAGALTQ IKIPAVVKDTLKKVAKGVLSAVAGALTQ LKIPGFVKDTLKKVAKGIFSAVAGAMTPS IKLSPETKDNLKKVLKGAIKGAIAVAKMV IKIPPIVKDTLKKVAKGVLSTIAGALST IKIPAFVKDTLKKVAKGVISAVAGALTQ IKIPAVVKDTLKKVAKGVLSAVAGALTQ LKIPGFVKDTLKKVAKGIFSAVAGAMTPS IKLSPETKDNLKKVLKGAIKGAIAVAKMV GLASLLGKAL*KAGLKIGTHFLGGAPQQ

GMASKAGAIAGKIAK*VALKAL some sequences to maximize structural similarity. structural to maximize sequences some 138 actly with calculated hydrophobicity as hydrophobicity calculated actly with ptides belonging tothemagainin, XPF,PGLa, ana tropicalis, andXenopuslaevis. indicate conser the observed retention times times retention observed the a a

ved amino acidved amino a a a indicates

Chapter 6 -helical α -helicity 5-27 8-16 9-16 7-16 9-16 α e face of the helix helix of the face e -helical domain from from domain -helical using the hydrophobicity using α -helical conformationthe with +6 +4 +4 +4 +4 segregating on on segregating tral region of the molecule (maximum (maximum of the molecule region tral -helicity and amphipathicity [11,12]. 16  on residues the opposite face (Fig. 6). ared with +4 for hymenochirins -2B, -3B, -3B, -2B, hymenochirins +4 for ared with 15 e [23]. Predictions of helical domains are made are made domains Predictions of helical e [23]. peptides are calculated are peptides 139 , and Lys 13 , and Leu , and 14 , Lys 689 664 596 12 GRAVY GRAVY pH 7 at Charge 0.169 0.466 0. 0. 0. , Val 11 , Lys 9 , Leu 3 , Asp 8 , Lys 6 H. boettgeri. , Glu 2 -helical conformation in the conserved cen conserved in the -helical conformation Physicochemical Physicochemical theproperties antimicrobial of peptides isolated skin from α Analysis of the secondary structure of the peptides using the AGADIR program [31] [31] program AGADIR the using peptides of the structure secondary the of Analysis Hymenochirin-1B differs from the other structurally similar hymenochirins by its its by hymenochirins similar structurally other the from differs Hymenochirin-1B indicates that hymenochirins -2B, -3B, -4B, and - 5B have a relatively weak propensity to to propensity weak a relatively have and - 5B -4B, -3B, -2B, hymenochirins that indicates adopt a greater cationicity (charge of +6 at pH 7 comp of +6 at pH greater cationicity (charge -4B, and -5B) and by its strong propensity to form a stable and extended extended and stable a form to propensity strong its by and -5B) and -4B, conformation (Table 2). The antimicrobial activities of peptides against microorganisms and cytotoxic their activities against mammalian cells are determinedbycomplex hydrophobicity, cationicity, interactions among structure-activitySeveral studies involvingbothnaturally occurring antimicrobial peptides between a correlation shown have [12]) in (reviewed peptides model and synthetic increasing cationicity and increased antimicrobial potency until a certain optimal molecular reached. charge is scales for amino acid residues of Kyte and Doolittl acid residues of Kyte and scales for amino using the AGADIR program [31]. Grand average of hydropathicity (GRAVY) of of the Grand average Hymenochirin-2B Hymenochirin-1B Hymenochirin-3B Hymenochirin-4B Hymenochirin-5B Table 2. of secretions Peptide and the hydrophobic Leu hydrophobic the and percentage helicity/residue = 1.5) whereas hymenochrin-1B is predicted to form a more form to predicted is hymenochrin-1B whereas 1.5) = helicity/residue percentage percentagestable (maximum helicity/residue = 20) and extended residues 5 to 27. A Schiffer-Edmundsen wheel representation [34] of N-terminal region of region N-terminal of [34] representation wheel Schiffer-Edmundsen A 27. 5 to residues hymenochirin-1B illustrates the amphipathic nature of the α Lys polar Chapter 6 amphipathic less pronounced (Table 2). It has been shown using model peptides that astabilized increased cationicityon potency of hymenoch positive bacterium bacterium positive [32]. Although effective new ofantibio effective types [32]. Although with resistance to commonly used antibiotics constitutes a serious threat to public health correlation betweenhemoly Fig. 6. model Gram-negative bacteriaarelessstringent [20]. Studies withanalogs of theamphipathic against Gram-positive bacteria determine the determine the LC determined by retention time on reversed-phase HPLC. There was insufficient material to activity ofhymenochirin-4B correlateswith α increased propensitytoformastable activity of hymenochirin-1B compared with hymenochirin-2B and -3B is consistent with its demonstrating theamphipathic -helical peptide alsoincrea The i The emergence in all regionsfungi and ofthe bacteria world pathogenic of of strains Schiffer-Edmundson α -helical peptide KLALKLALKALKAAKLA.NH peptide -helical ncreased growth inhibitory potency of hymenochirin-1B against the Gram- α -helical conformation isanabsolute 50 value of hymenochirin-5Bagainst humanerythrocytes. S. aureus S. nature of the predicted tic activityanddegreeof wheel representation of is consistent with the results results the with is consistent ses with hydrophobicity [13,35]. The increased hemolytic hemolytic increased The [13,35]. hydrophobicity ses with whereas thestructuralrequi α -helix. Hemolytic activity of an amphipathic amphipathic an of Hemolytic-helix. activity 140 tics against multidrug-resistant Gram-positive Gram-positive multidrug-resistant tics against irin-1B against Gram-n against irin-1B itsgreater effectiv α -helical conformation. the (1-18) regionof hymenochirin-1B α requirement for cytotoxic activity -helicity [11]. The greater hemolytic of these studies. The effects of 2 rements for activity against against rements activity for have shown a positive e hydrophobicity as e hydrophobicity egative bacteria are

Chapter 6 Bacillus and aeruginosa P. propriate toxicological propriate toxicological

E. coli, ersity. The authors thank Freeland Dunker, Dunker, Freeland authors thank ersity. The (MRSA) have been introduced or are in in are or introduced been have (MRSA) robial agents, with ap -lactam antibiotics are particularly difficult to difficult antibiotics are particularly -lactam eady been described and increasing use of this this of use increasing and described been eady 141 S. aureus ) for amino) composition acid analysis. and Laurey Steinke and Michele Fontaine (University of of (University Fontaine Michele and Steinke Laurey and µM), have the advantage over over advantage have the 40 µM), 20 - range in the (MIC ative pathogens such as as such ative pathogens > 300 µM). Further structure-activity studies are warranted to to are warranted studies structure-activity Further > µM). 300 50 H. boettgeri is less encouraging [26]. Infections caused by these carbapenemase- these by caused Infections [26]. encouraging less is , is used, is as a “last resort” therapy in the of management infectionsdue to This workwas supported by a Faculty Support Grant (NP/12/03) and a University Nebraska Medical Center, Omaha, NE Nebraska Medical Center, Omaha, DVM, the Steinhart Aquarium and the California Academy of Science for help in obtaining obtaining in help for Science of Academy California and the Aquarium Steinhart the DVM, skin from secretions Research Grant (G00000900) U.A.E. from Univ Acknowledgments Acknowledgments developofpeptides these analogs with increased activity against drug-resistant pathogenic microorganismshemolytic activity. that low retain bacteria suchas methicillin-resistant clinical trials, the situation regarding new treatment options for infections produced by produced infections for options treatment new regarding situation the trials, clinical Gram-neg multidrug-resistant K. pneumoniae K. to all beta that are resistant bacteria producing treat [19]. Colistin, the cyclic antibiotic peptidepolymyxin E derived from polymyxa antibioticThis resistant strains [24]. situationofhas more to is likely emergence lead the to necessitatednovelforsearch types a of antimic have microorganisms pathogenic pan-resistant which to properties, pharmacokinetic and not beenexposed. Hymenochirins-2B and3B, although displaying only moderate potency bacteria against Gram-negative against cytotoxicity low very having of peptides antimicrobial occurring naturally many human erythrocytes (LC multidrug-resistant[25]. not bacteria However, Gram-negative only high nephrotoxicity limits itsuse but resistance to colistin has alr

REFERENCES [1] Ali MF, Soto A, Knoop FC, Conlon JM. Antimicrobial peptides isolated from skin secretions of the diploid frog, Xenopus tropicalis (Pipidae). Biochim Biophys Acta 2001;1550:81-9. [2] Báez AM. The fossil record of the Pipidae. In: Tinsley RC, Kobel HR, editors. The biology of Xenopus. Oxford: Clarendon Press; 1996. p. 329-47. [3] Cannatella DC, Trueb L. Evolution of pipoid frogs: intergeneric relationships of the aquatic frog family Pipidae (Anura). Zool J Linnean Soc 1988;94:1-38. [4] Carreño CA, Nishikawa KC. Aquatic feeding in pipid frogs: the use of suction for prey capture. J Exp Biol 2010;213:2001-8. [5] Clinical Laboratory and Standards Institute. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved Standard M07-A8. CLSI, Wayne, PA, 2008. [6] Clinical Laboratory and Standards Institute. Reference method for broth dilution antifungal susceptibility testing of yeast. Approved Standard M27-A3. CLS1, Wayne, PA, 2008. [7] Conlon JM. Structural diversity and species distribution of host-defense peptides in frog skin secretions. Cell Mol Life Sci. 2011;68:2303-15. [8] Conlon JM, Al-Ghaferi N, Ahmed E, Meetani MA, Leprince J, Nielsen PF. Orthologs of magainin, PGLa, procaerulein-derived, and proxenopsin-derived peptides from skin secretions of the octoploid frog Xenopus amieti (Pipidae). Peptides 2010;31:989-94. [9] Conlon JM, Mechkarska M, Ahmed E, Leprince J, Vaudry H, King JD et al. Purification and properties of antimicrobial peptides from skin secretions of the Eritrea clawed frog Xenopus clivii (Pipidae). Comp Biochem Physiol C Toxicol Pharmacol 2011;153:350-4. [10] Conlon JM, Mechkarska M, King JD. Host-defense peptides in skin secretions of African clawed frogs (Xenopodinae, Pipidae). Gen Comp Endocrinol 2012; in press. [11] Dathe M, Schümann M, Wieprecht T, Winkler A, Beyermann M, Krause E, et al. Peptide helicity and membrane surface charge modulate the balance of electrostatic and hydrophobic ter 6 ter 6 interactions with lipid bilayers and biological membranes. Biochemistry 1996;35:12612-22. p [12] Dathe M, Wieprecht T. Structural features of helical antimicrobial peptides: their potential to

Cha modulate activity on model membranes and biological cells. Biochim Biophys Acta 1999;1462:71-87. [13] Dathe M, Wieprecht T, Nikolenko H, Handel L, Maloy WL, MacDonald DL, et al. Hydrophobicity, hydrophobic moment and angle subtended by charged residues modulate antibacterial and haemolytic activity of amphipathic helical peptides. FEBS Lett. 1997;403:208-12. [14] de Sa RO, Hillis DM. Phylogenetic relationships of the pipid frogs Xenopus and Silurana: an integration of ribosomal DNA and morphology. Mol Biol Evol 1990;7:365-76.

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L, Tossi A. Amphipathic L, Tossi A. Amphipathic Xenopus lenduensis (Pipidae). Comp Biochem Physiol C Toxicol Pharmacol 2010;152:467-72. 2010;152:467-72. Pharmacol Toxicol Physiol C Comp Biochem (Pipidae). and the caerulein and xenopsin precursors from and xenopsin precursors and the caerulein a and the octoploid frog octoploid and the effects of structural and physical prope effects of structural and physical 2001;268:5589-600. PGL treatment options? Drugs options? treatment database accessible at http://re at accessible database Museum of Natural Histor Front Biosci 2008;13:4687-706. 2008;13:4687-706. Front Biosci phylogeography frogs: clawed phylogeny of African Evol 2004;33:197-213. Mol Phylogenet peptides from skin secretions from peptides 1986;261:5341-49. 1986;261:5341-49. peptides with therapeutic potential from skin secretions of the Marsabit clawed frog Xenopus frog clawed of the Marsabit secretions skin from potential with therapeutic peptides borealis increase not do Xenopodinae the duplications within J Mol Biol 1982;157:105-32. t of resistance, review a colistin: colistin in multidrug-resistant colistin in multidrug-resistant 2006;50:2946-50. 2010;30:1279-91. 2009;64 Suppl 1:i29-i36. J Bot 2001;49:271-300. provincialism. Aust [21] L, Will Gibson BW, Poulter [19] the what are infections: Gram-negative Multidrug-resistant Poulakou G. H, Giamarellou [20] Giangaspero A, Sandri [16] Evans BJ, Kelley Tinsley RC, Me DB, [17] DC. The major cl Ford LS, Cannatella [18] DR. Amphibian Frost species of the world: [22] King JD, Mechkarska M, Coqu [29] Mechkarska M, Ahmed E, [24] RL, Owen RJ, Spelma Nation Li J, Rayner CR, [23] of a protein. Kyte J, Doolittle DF. A character method for displayingsimple the hydropathic [25] of Resurgence A 3rd, et al. L, Jarkowski Macander D, Yang JC, Anderson Ly N, LM, Lim [26] the era of untreatable Has DM. Livermore [27] The breakup history of Gondw McLoughlin S. [28] Leprince L, Ahmed E, Coquet Mechkarska M, [15] ( frogs of clawed genetics speciation and evolution BJ. Genome Evans

in Silurana paratropicalis and Xenopus andrei skin secretions. Comp Biochem Physiol D Genomics Proteomics 2011;6:206-12. [30] Mechkarska M, Ahmed E, Coquet L, Leprince J, Jouenne T, Vaudry H, et al. Peptidomic analysis of skin secretions demonstrates that the allopatric populations of Xenopus muelleri (Pipidae) are not conspecific. Peptides 2011;32:1502-8. [31] Muñoz V, Serrano L. Elucidating the folding problem of helical peptides using empirical parameters. Nature Struct Biol 1994;1:399-409. [32] Norrby SR, Nord CE, Finch R. Lack of development of new antimicrobial drugs: a potential serious threat to public health. Lancet Infect Dis 2005;5:115-9. [33] Raverty S, Reynolds T. Cutaneous chytridiomycosis in dwarf aquatic frogs (Hymenochirus boettgeri) originating from southeast Asia and in a western toad (Bufo boreas) from northeastern British Columbia. Can Vet J 2001;42;385-6. [34] Schiffer M, Edmundson AB. Use of helical wheels to represent the structures of proteins and to identify segments with helical potential. Biophys J 1967;7:121-35. [35] Soravia E, Martini G, Zasloff M. Antimicrobial properties of peptides from Xenopus granular gland secretions. FEBS Lett 1988;228:337-40. [36] Tachi T, Epand RF, Epand RM, Matsuzaki K. Position dependent hydrophobicity of the antimicrobial magainin peptide affects the mode of peptide-lipid interactions and selective toxicity. Biochemistry 2002;41:10723-31. [37] Tinsley R, Measey J, Burger M. Hymenochirus boettgeri. In: IUCN 2011. IUCN Red List of Threatened Species. Version 2011.2. . [38] United States Geological Survey. Hymenochirus boettgeri. USGS Nonindigenous Aquatic Species Database, Gainesville, FL. Electronic database accessible at http://nas.er.usgs.gov/queries/FactSheet.aspx?speciesID=66; 2012. [39] Zasloff M. Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms and partial cDNA sequence of a precursor. Proc Natl

ter 6 ter 6 Acad Sci USA 1987;84:5449-53. p

Cha

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Hybridization between the tetraploid African clawed frogs Xenopus laevis and Xenopus muelleri (Pipidae) increases the multiplicity of antimicrobial peptides in the skin secretions of female offspring

M. Mechkarska, M. Meetani, P. Michalak, Z. Vaksman, K. Takada, J.M. Conlon

Comparative Biochemistry and Physiology (2012) 7:285-291. Chapter 7 magainin-2 from magainin-2 not detected insecretions from X.laevis either PGLa-LM1, and CPF-LM1) were purified from thesecretions of the hybrid frogs that were werenotX. muelleri detected. Three previously undescribed peptides (magainin-LM1, previously found in skin secretions of X. laevis. Four peptides (magainin-M1, XPF-M1, CPF-M1, and tigerinin-M1) were CPF-7, XPF-1,XPF-2, and PGLa) wereidentified in secretions of both the hybrids and frogs. Eleven peptides (magainin-1, magainin-2, CPF-1, CPF-3, CPF-4, CPF-5, CPF-6, secretions from hybrid in the wereidentified peptides 18 of Atotal species. parent the muelleri Xenopus the common clawedfrog norepinephrine-stimulated skin secretions fromlaboratory-generated female F1hybrids of Peptidomic analysis wasused to compare the distribution of host-defense peptides in ABSTRACT adaptive strategy among secretions. skin As the female F1 hybrid in peptides host-defense skin of multiplicity the increases hybridization that indicate data The is and hemolytic. activity broad-spectrum potent, antimicrobial CPF-LM1 shows species. PGLa-LM1 and CPF-LM1 differ appreciably instructure from orthologs intheparent microorganisms in the environment. (Peters, 1844) with the corresponding distribution in skin secretions from by a single amino acid substitution (Gly acid substitution amino asingle X. laevisby Xenopus Xenopus laevis Xenopus species to increase protection against pathogenic pathogenic against protection increase to species X. muelleri 148 (Daudin, 1802) and Mueller’s clawed frog s are fertile, hybridization may represent an an may represent are fertile,hybridization s or X.muelleri but magainin-M2 and CPF-M2 from . Magainin-LM1. differs from 13  Ala) but Chapter 7

rise and

, and , X. largeni, andrei

, X. X. borealis (Mechkarska et (Mechkarska

constitute a third X. gilli

(Kobel et al., 1996; 1996; al., et (Kobel ptide glycine-leucine- , X. amieti (Conlon et al., 2011), 2011), al., et (Conlon X. andrei laevis, X. wittei X. muelleri, X. and X. clivii X. pygmaeus pygmaeus X. West (Mechkarska et al., 2011a), . and skin more than 25 years ago, ago, years 25 than more X. laevis skin have proved to be a rich source of host- of source rich be a to proved have d peptides that are derived from the post- species have been divided into three species species three into divided been have species (King et al., 2012). and an incompletely characterized species characterized species incompletely and an 10). These include pe include 10). These otherpeptides that show potential therapeutic group includes includes group group includes within the family Pipidae family within the currently comp fraseri X. muelleri X. itombwensis

149 oploid, and dodecaploid species with no extant with no extant species dodecaploid and oploid, phology, advertisement calls, and/or nucleotide nucleotide calls, and/or advertisement phology, X. (Conlon et al., 2010), 2010), al., et (Conlon Xenopus laevis Xenopus mieti muelleri X. ruwenzoriensis X. Xenopus X. muelleri X. pygmaeus X. a and ; the zations within the second group have given rise to thegiven the second group have zations within et al., 1988). More recently, antimicrobial peptides belonging share a common tetraploid ancestor; ancestor; tetraploid a common share (Mechkarska et al., 2010), 2010), al., et (Mechkarska of the biosynthetic precursors of caerulein [termed caerulein- [termed of caerulein precursors of the biosynthetic X. longipes fraseri group includes form a second form group; and X. borealis the (King et al., 2012),(King et al., and X. vestitus

(King et al., 2012), (King and et al., species retaining species the diploid status that is thought to berelated to the ancestral ; and The skinsfrogs offrom the genus Introduction genus of the frogs The clawed well characterized species although several unnamed species have been reported (Kobel (Kobel reported been have species unnamed several although species characterized well et al., 1996; Frost, 2011). All are found in Africa south of the Sahara. The genus has a has The genus Sahara. of the south Africa in are found All 2011). Frost, 1996; al., et of modes reticulating and bifurcating both involving history evolutionary complex speciation (Kobel,1996; Evans2004; Evans, et al., 2008). Allopolyploidization events, in both of genome complete the inherits descendant the and hybridize species two which oct tetraploid, rise to have given ancestors, 19 Xenopus X. petersii, and X. victorianus amide (PGLa) and structurally-relate multiple translational processing (XPF)] fragment xenopsin-precursor [termed xenopsin and (CPF)] fragment precursor 1986; Soravia al., et (Gibson state existing prior to one or more whole genome duplications (Kobel and Du Pasquier, Pasquier, Du and (Kobel duplications genome whole more or one to prior existing state 1991). thetime, ten At this tetraploid to the magainin, have PGLa, CPF, andbeen XPF families isolated norepinephrine-from skin secretions from stimulated groups on the basis of similarities in mor of similarities basis on the groups sequences of mitochondrial genes: the defense withpeptides antimicrobial and anti-inflammatory activities [reviewed in (Conlon ins were isolated from magain The et al., 2012)]. (Giovannini et al., 1987; Zasloff, 1987)analysis and of skin secretions this from species led to the isolation and characterization several of as anti-infective agents(Zhang and Falla, 20 1. X. clivii X. lenduensis designated provisionally West Africa from X. petersii X. boumbaensis allopolyploidi group. Further species dodecaploid Evans et al., 2004). It has been proposed that the seven extant octoploid species arose from from arose species octoploid seven extant the that proposed has been It 2004). al., et Evans Thus, 2011). al., et Evans 2008; (Evans, events allopolyploidization distinct three X. lenduensis al., 2011b) , Chapter 7 of Swaziland. Female hybrids (n=3; 5-6 years old; weights 43 – 48 g) wereinjected via of South Africa and the male parents were (Malone etal.,2007).The female parents were investigators. No. (Protocol U.A.E.University of committee classified according to the terminology used previously for peptides from the corresponding peptides fromthe parent species. Thepeptides described in this studyare 2.1. 2. are differentiated by numerals e.g. CPF-1 and CPF-2. by LM. Paralogs are denoted inthe hybrids identified peptides M the novel and designated tigerinus frogHoplobatrachus Asian the refers to termthe The tigerinin are recognized. (Gibson et al., 1986, Zasloff, 1987). The magainin, PGLa, CPF, and XPF peptide families stimulated skin secretions from female electrospray massspectrometry) present study wasto use peptidomicanalysis (reversed-phase HLPC coupled with 2008). These animals have been made availa males (Malone etal., 2007) and inthe ovary the testes hybrid in of expression gene tostudy used been has analysis microarray and greater dysfunctional effects of hybridization (Malone et al., 2007). that rule to Haldane’s an exception represents genus this that out pointed been ithas homogametic, males are and heterogametic consistently sterile and the females are fully or partially fertile. As partially females or the arefully and sterile consistently In etal.,1996). (Kobel to be absent found aregenerally incompatibilities gametic and laboratory inthe can be hybridized species etal., many calls(Fischer contrast, advertisement 2000). In distinctive their and natural hybrids were characterized both biochemically and morphologically and from species werefound in the same bodies of water in Mpumalanga province ofSouth Africa between sympatry of Areas 1996). (Yager, alsobeen described Kenya have X. victorianus western in seriously endangered endangered seriously for only described has been interbreeding the Extensive in wild. species

All experiments with live animals wereap live All experiments with secretions skin of Collection Materials and methods Materials and The generationof There haverelativelybeen few reports ofnaturalhybridization between Xenopus X. laevis X. laevis and X. muelleri X. laevis x X. gilli X. gilli X.muelleri x are few which mitigates against interbreeding but the the two but interbreeding mitigates arefew against which to compare the host-defense peptides in norepinephrine- X. muelleri X. (Picker, 1985). Hybrids between between Hybrids 1985). (Picker, F1 hybrids wereproducedas previously described (Sai etal.,2001).Orthologsfrom X. laevis X. 150

hybrids in the laboratory has been described muelleri ble to the investigators and the aim the of and the investigators to ble the heterogametic sex typically suffers the the suffers typically sex heterogametic the of hybrid females (Maloneand Michalak, A21-09) and were carried out by authorized such interspecies crosses, the males are family of cyclic peptides first identified in first identified cyclicpeptides family of X. laevis originating fromthe Cape region proved bytheAnimalResearch Ethics x originating fromthe Nkambeni area X.muelleri F1 hybrids with the the with F1 hybrids Xenopus X. borealis and the the X. laevisand X. muelleri femalesare X. laevis Xenopus and are

Chapter 7 (ATCC has been colony forming forming colony 6 muelleri ound material oundwas material

X. L of 10  the eluting solvent was raised raised was solvent the eluting 0:29.9:0.1, v/v/v) and freeze- v/v/v) and 0:29.9:0.1, Staphylococcus aureus Staphylococcus the secretions from each group each group from the secretions r plate reader. Fractions associatedwith in the vivarium at U.A.E. University. Skin at U.A.E. University. the vivarium in (weight 263 g) was supplied by Xenopus Xenopus by was g) supplied 263 (weight in 96-well microtiter cell-culture plates for quilibrated with 0.1%at (v/v) TFA/water a ent was raised from 21% to 56% over 50 min from was raised ent matographed on a (1.0 cm x 25 cm) Vydac Vydac 25 cm) x cm a (1.0 on matographed ffer (50 mM sodium chloride-25 mM sodium sodium mM chloride-25 sodium (50 mM ffer

fied on Sep-Pak cartridges using the same same using the cartridges on Sep-Pak fied cm) Vydac 218TP1022 (C-18) reversed-phase 151 X. laevis L) with an inoculum (50 an inoculum L) with  concentration of acetonitrile in of acetonitrile concentration /trifluoroacetic acid (TFA) (70. /trifluoroacetic (ATCC 25726) iversity. The solutions containing solutions iversity. The Escherichia coli Escherichia C in a humidified atmosphere of air. After incubation, the absorbance at 630 nm nm 630 at absorbance the incubation, After air. of atmosphere a humidified in C o and

A single adult female specimen of of specimen female adult A single Peptide purification Sep-Pak on purification partial after frogs, hybrid the from secretions skin pooled The

25923) 18 h at 37 h at 18 units/mL) from a log-phase culture of reference strains of strains reference culture of a log-phase from units/mL) of each well was determined using a microtite antimicrobial activity were successively chro activity were successively antimicrobial 214TP510 (C-4) column and a (1.0 x 25 Vydac cm) cm 208TP510(C-8) column.The in the eluting solv of acetonitrile concentration and the flow rate was 2.0 mL/min. eluted with acetonitrile/water mL). (2 TFA/water (v/v) 0.1% in redissolved was material The dried. acetate, pH 7.0) for 15 min. The frogs were removed and the collection solution was was solution collection and the removed were frogs The for 15 min. 7.0) acetate, pH for frozen and immediately mL) (1 acid hydrochloric concentrated of addition by acidified to U.A.E. Un shipment were pooled and separatelypassed at a flow rate2mL/min of through 6 Sep-PakC-18 in series. B connected Milford, MA, USA) Associates, cartridges (Waters the dorsal lymph sac with norepinephrine hydrochloride (40 nmol/g body weight) and and weight) body nmol/g (40 hydrochloride norepinephrine sac with lymph dorsal the bu of collecting (100 mL) solution a placed in to 21% (v/v) over 10 min and to 63% (v/v) over 60 min using linear gradients. Absorbance Absorbance gradients. linear using min 60 over (v/v) 63% to and min 10 over (v/v) 21% to of Purification were collected. (1 min) and fractions nm, and 280 214 nm at was monitored the peptides was monitored by incubating lyophilized aliquots of chromatographic effluent (50 broth Mueller-Hinton µL) in (100 Express(Brooksville, Inc. housed USA) and FL, secretions were obtainedsecretionspartially and puri from secretions of skin The collection hybrids. the for used protocol HPLC column (Grace, Deerfield, IL, USA) e HPLC column The 6.0 mL/min. of flow rate cartridges, were injected onto a (2.2 cm x 25 a (2.2 cm onto were injected cartridges, 2.2. described previously (Mechkarska et al., 2011a). 2011a). al., et (Mechkarska previously described Chapter 7 carried out in parallel with increasing concentrations of antibiotics (ampicillin for S. for (ampicillin of antibiotics concentrations increasing with parallel in carried out were assays,incubations the of andreproducibility nm. the validity 630 In tomonitor order visiblegrowth wasobserved. Thevalueswere where no ofpeptide concentration as were taken lowest and the 2008b) 2008a; Institute, 200 3 - of range and 25726), (ATCC PGLa-LM1 and CPF-LM1 against reference strainsof MD, inhib USA). Minimum (Rockville, Collection 2.5. spectrometry. rate was6mL/min. Thestructuresofthepe acetonitrile in the eluting solv of concentration The column. (C-18) a cm 218TP1022 on (2.2 HPLC cm) x 25 Vydac (Shanghai, China) and were purified to near homogeneity (> 98% purity) by reversed-phase 2.4. City,CA,USA). tems, Foster Biosys (Applied sequenator Procise novel peptides weredetermined by automated Edman degradation using amodel 492 350 °C. The accuracy of mass determinations to 10l/min, the nebulizer gas pressure was set to 70psi, and the drying temperature was skimmer voltage was 36.3 V and the trap drive was 74.2 V. The flow of drying gas was set with maximum accumulation time of300ms. Th m/z 2200 was fromto range 200 scan the and mass the positive spectrometer wassetto of Series iontrap mass spectrometeras previously 2.3. described previously (Mechkarska et al., 2011a). frogs. The purification of antimicrobial peptides from the same chromatographic procedures used for purification ofthe peptides from the hybrid

Reference strains of microorganisms werepurchased from the American Type Culture activities hemolytic and Antimicrobial PGLa-LM1 and CPF-LM1 were supplied in crude form by GL Biochem. Ltd. Peptide synthesis Electrospray-ionization mass spectrometry wascarried out using an Agilent 6310 Structural characterization Skin secretionsfrom Skin μ M by standard microdilution methods (Clinical Laboratory Standards Candida albicans X. laevis(20% of the total amount collected) were subjected to ent was raised from 21%to56% over 60 min andthe flow (ATCC 90028) were measur 152 ptides were confirmed byelectrospraymass confirmedmeasurement by of absorbance at was ± 0.05%. The primary structures of the the of structures primary ±0.05%. was The described (Zahid et al., 2011). The polarity polarity The etal., (Zahid 2011). described e capillaryvoltage was itory concentrations (MIC) of synthetic (MIC) ofsynthetic itory concentrations X. S. aureus

muelleri skin secretions has been (ATCC 25923), E.coli ed in theconcentration set to-3500V, the

aureus aureus Chapter 7 and were and value as was taken The peaks designated The peaks designated . 50 reversed-phase HPLC HPLC reversed-phase

and S. aureus coli E. X. muelleri hybrids x ) as previously described (Mechkarska et al., al., et (Mechkarska described ) as previously tration of acetonitrile in the eluting solvent. solvent. eluting in the acetonitrile of tration Vydac C-18 preparative Vydac C-18 preparative 153 albicans

C. peptide producing 50% hemolysis in three independent after partial purification on Sep-Pak cartridges after partial purification

hybrids ; and amphotericin for for and amphotericin ; X. muelleri x X. muelleri Reversed-phase HPLC on a semipreparative Vydac C-18 column of skin secretions from F1 from secretions of skin column C-18 Vydac on a semipreparative HPLC Reversed-phase Hemolytic activity against human erythrocytes taken from a healthy donor was was donor a healthy from taken erythrocytes human against activity Hemolytic Results Purificationtheofpeptides from X. laevis Sep-Pak on purification partial after frogs, hybrid the from secretions skin The pooled E. coli

purified further. The dashed line shows the concen line The dashed further. purified antimicrobial activity against against activity of antimicrobial degrees varying 1 - 13 displayed Fig. 1. X. laevis and measuredpreviously as described (Mechkarska2011a). et al., The LC 2011a). 3.1. 3. the mean concentration of concentration the mean incubations. C-18 cartridges, were chromatographed on a were chromatographed C-18 cartridges, column (Fig. 1). 1). (Fig. column Chapter 7 frogs (Fig. 2). 2). (Fig. frogs column using the same experimental conditions cartridges, were chromatographed on aVyd 3.2. columns. C-8 C-4 and Vydac Vydac semipreparative on chromatography homogeneity, asassessed by asymmetrical peak shape and mass spectrometry, by further of growth inhibited 2, and 8-10 3, peaks from material from peaks 1 - 11 inhibited to varying degrees the growth of ofassay, conditions the CPF-7.Under 13 peak CPF-LM1, peak 12 CPF-1, 11 peak CPF-6, peak 7 tigerinin-M1 +XPF-2, peak 8CPF-M1, peak 9 CPF-4 +CPF-5, peak 10 CPF-3 + PGLa-LM1,PGLa,4 peak peak 5magainin-LM1,peak 6XPF-1+ XPF-M1, revealed that peak 1 contained magainin-2, peak 2 magainin-1, peakmagainin-M1 3 + components present in the peaks are shown in Table 1. Subsequent structural analysis line showsthe concentration of acet degrees of antimicrobial activity against X. laevis Fig. 2. Reversed-phase HPLC onasemipreparative Vy

The pooled skin secretions fromThe pooledskin X.laevis from peptides of the Purification The prominentpeaks designatedwe 1 -13 after partialpurification on Sep-Pakcartridges onitrile inthe elutingsolvent. E. coli X. laevis, and 154

S. aureus ac C-18preparativereversed-phase HPLC S. aureus

after partialpurification onSep-PakC-18 re collected and themasses of the major as forthe skin secretions from the hybrid . The peaksdesignated 1- 9 varying displayed dac C-18 columnofskinsecretionsfrom . The peptides were purified to near near to purified were peptides . The and werepurified further. The dashed E. coli and material material and Chapter 7 . The . The , and the , and the S. aureus X. muelleri in-2, peak 2 magainin-1, peak X. laevis, has been described previously previously has been described re collected and subjected to further subjected and re collected rometry, by further chromatography on on chromatography further by rometry, 155 that peak 1 contained magain contained 1 peak that tigerinin-M1) from X. muelleri F1 hybrids are shown in Table 1. 1. Table in shown are hybrids F1 x X. muelleri The prominent 1 peaks designated The - 9 we prominent Purification of antimicrobial peptides (magainin-M1, magainin-M2, XPF-M1, XPF-M1, magainin-M2, (magainin-M1, peptides antimicrobial of Purification Structural characterization characterization Structural from peptides of the purified masses The molecular

purification. Under the conditions ofall peaksmaterial from assay, inhibited theof growth of growth the inhibited 6-9 and 3, 2, peaks and partially least E. coli at + CPF-6, CPF-3 7 peak CPF-5, + 6 CPF-4 peak 5 XPF-2, peak 4 XPF-1, peak 3 PGLa, as assessed homogeneity, near to purified were peptides The 9 CPF-7. peak 8 CPF-1, peak by a symmetricalspect mass and peak shape columns. C-8 Vydac C-4 and Vydac semipreparative CPF-M1, CPF-M2, and masses of the major components present in the peaks are shown in Table 1. Subsequent Subsequent 1. Table in shown are peaks the in present components major the of masses demonstrated structural analysis 3.3. (Mechkarska et al., 2011a). 2011a). al., et (Mechkarska X. laevis Chapter 7 Xenopus laevis Xenopus 1. Table X. laevisand list includes both peptides that have been previously identifiedandcharacterized in hybrid frogs that are notpresenthat hybrid frogs that magainin-1, magainin-2, CPF-1, CPF-3,CPF-4,CPF-5,CPF-6, CPF-1, magainin-2, magainin-1, that The prim Tigerinin XPF XPF XPF CPF CPF CPF CPF CPF CPF CPF CPF CPF PGLa PGLa Magainin Magainin Magainin Peptide Magainin Magainin Molecular masses ofthehost-defense pep ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ M1 2 1 LM1 M2 1 3 4 5 6 7 M1 ‐

LM1 ary structures of the andX. muelleri newly discovered peptides pr , Xenopus muelleri

‐

‐ ‐ ‐ ‐ ‐ M1 LM1 M2 M1 2 1 t in skin secretions ofthe parent species. The dataindicate 2664.6 2610.6 2618.6 2602.4 2647.3 2632.3 2586.8 2660.2 1968.8 2467.3 2410.3 X. , and

laevis peptides from the F1 hybrids are shown in Fig. 3. This This 3. inFig. areshown hybrids F1 the from peptides X. laevis

156 xX.muelleri 5512514.5 2515.1 46614 1446.6 2895.5 8452894.1 2894.5 2682.3 4492424.4 2424.9 muelleri X.

tides isolated from skinsecretions of

esent in skin secretions of the the of secretions skin in esent F1 hybrids. 2791.0 2664.8 2610.3 1948.0 2617.5 2602.6 2647.4 2632.3 2586.5 2659.6 2480.4 1969.0 2467.4 2410.3 F1 46.7 hybrid PF-7, XPF-1, XPF-2,

Chapter 7

X. laevis +H]calc , were not not , were r 2665.3 2515.0 2789.6 2424.9 1446.7 2410.9 2467.9 2480.3 . Magainin-M1, X. muelleri X. laevis obs), and calculated average average calculated and obs), r +H]obs [M r 2424.4 2410.3 2467.4 2480.4 1948.0 1948.0 1948.4 1446.7 [M

1969.0 1969.0 1969.5 2 2 t were not detected eithernot secretions were in from t NALGGSPQQ 2602.6 2603.1 157 ANMLGGAPQQ 2659.6 2659.4 VGKMMAGAPREQ 2894.1 2894.5 GTHFLGGAPQQ 2647.4 2648.2 GAHLLGGAPQQ 2617.5 2618.2 GANALGGAPQQ 2586.5 2585.5 GANALGGSPQQ 2632.3 2633.1 VGLQGLMQPK 2610.3 VGLQGLMQPK 2610.3 2611.2

IIPKLLPSIQQ 2791.0 2 KAFVGEIMKS KAFVGEIMNS KAFIGEIMKS KAFVGEIMNS . calc) of the antimicrobial peptides isolated from skin secretions of the of the secretions skin from isolated peptides antimicrobial calc) of the r GWASKIGQTLGKIAKVGLKELIQPK 2664.8 GWASKIGQTLGKIAKVGLKELIQPK hybrids. hybrids. X. muelleri . Magainin-M2 and CPF-M2, present. Magainin-M2 and CPF-M2, skin secretions in of or Amino acid sequences, observed average molecular masses (M masses molecular average observed sequences, acid Amino X. muelleri muelleri X. x PGLa-LM1 GMASKAGSVAGKIAKFALGAL.NH Magainin-2 GIGKFLHSAKKFG Magainin-2 GIGKFLHSAGKFG Magainin-M1 Magainin-1 GIGKFLHSAGKFG Magainin-1 and PGLa are present in skin secretions of both the hybrids and and hybrids the both of secretions skin in present are PGLa and XPF-M1, CPF-M1, and tigerinin-M1 are present in skin secretions of both the hybrids and and hybrids the both of secretions skin in present are tigerinin-M1 and CPF-M1, XPF-M1, X. muelleri GIGKFLHSAKKFA Magainin-LM1 PGLa GMASKAGAIAGKIAKVALKAL.NH XPF-1 XPF-2 GWASKIGQTLGKIAK XPF-M1 WCPPMIPLCSRF.NH Tigerinin-M1 2514.5 GWASKIGQTLGKMAKVGLKDLIQA CPF-1 CPF-3 GLASFLGKALKAGLKI CPF-4 GFGSFLGKALKAALKIGA CPF-5 GLASLLGKALKAGLKI CPF-6 GFGSFLGKALKTALKI CPF-7 GFASFLGKALKAALKIG CPF-M1 GFGSFLGKALKAALKI CPF-LM1 GFGSFLGSLFKTGLK GLGSLLGKAFKFGLKT molecular masses (M Fig. 3. detected in secretions from the hybrids. Magainin-LM1, PGLa-LM1, and CPF-LM1 are are and CPF-LM1 PGLa-LM1, Magainin-LM1, hybrids. the from secretions in detected present in the secretions of the hybrid frogs bu X. laevis Chapter 7 secretionsthe of hybrid frogsthatwere undescribed peptides (magainin-LM1, PGLa-LM1, and CPF-LM1) were purified from the peptides (magainin-M2 and CPF-M2) were not detected. In addition, three previously of secretions skin the in secretionsof the skin in peptides antimicrobial hybrid is expressing genes derived from both parent species sothat the multiplicity ofthe and subsequentlyin the Asian frog in tigerinin, firstidentified Zasloff, and 1987) PGLa,CPF, magainin, peptides degradation reveals that they represent orthologs of the previously described host-defense X. muelleri X. muelleri X. muelleri secretions are also presen the in identified peptides antimicrobial allthe 1, Table in Asshown species. 4. activity againsthuman erythrocytes ( 2. Table human erythrocytes,areshown inTable 2. yeastpathogen opportunistic Gram-positive bacteriumthe 3.4. antimicrobial activity in norepinephrine-stimulated skin secretions from female from secretions skin norepinephrine-stimulated in activity antimicrobial CPF-LM1 from CPF PGLa

The MICvalues forsynthetic PGLa-LM1 a activity Antimicrobial Thi Discussion ‐ LM1 ‐ LM1 s study has described the purification of 18 peptides with varying degree of Minimum inhibitory concentrations (

F1hybrids. Characterization ofthe (Fig. 4). 4). (Fig.

X. laevis 20 80 E. X. muelleri

coli

xX.muelleri t in the secretions of the hybrid. Four out of six peptides identified identified ofsix theout peptides secretions Four t in hybrid. the of X. muelleri

C. S. aureus

and XPF, first identified in XPF, and first identified (Mechkarska et al., 2011a). Thedata indicate that the albicans 80 S. 5 were found in the secretions of the hybrid but two but the hybrid of the secretions in found were F1 hybrids. F1 hybrids.

aureus

, theGram-negative bacterium not detectedin secretions from either M) ofsynthetic replicatesofPGLa-LM1 and ,

together with their hemolytic activities against together against their activities with hemolytic 158

 the hybrid is greater peptides by mass spectrometry and Edman peptides massby spectrometryandEdman M) againstmicroorganisms andhemolytic nd CPF-LM1 againstreference strains of 160 C. 40 albicans

X. laevis (Gibson et al., 1986; H. tigerinus H. tigerinus

than in either ineither parent than E. coli (Sai etal., (Sai 2001) >500 HC 30 50

X. laevis or X.

, andthe X. laevis

laevis x Chapter 7 and X. laevis

2

2

rences. Residue deletions (denoted by *) (denoted by Residue deletions rences. the novel peptides magainin-LM1, PGLa-LM1, and magainin-LM1, the novel peptides 159 the hybrid with orthologous peptides from peptides with orthologous the hybrid es to maximize structural similarity. similarity. structural maximize to es LGKALKAGLKI*GTHFLGGAPQQ

GLASFLGKALKAGLKI*GAHLLGGAPQQ GFASFLGKALKAALKI*GANMLGGTPQQ GFGSFLGKALKAALKI*GANALGGSPQQ GLASL GFGSFLGKALKTALKI*GANALGGSPQQ GFASFLGKALKAALKI*GANMLGGAPQQ GFGSFLGKALKAALKI*GANALGGAPQQ GLGSLLGKAFKFGLKTVGKMMAGAPREQ GLGSLLGKAFKFGLKTVGKMMAGAPREE GFGSFLGSLFKTGLKI*IPKLL*PSIQQ GIGKFLHSAKKFAKAFVGEIMNS GIGKFLHSAGKFGKAFVGEIMKS GIGKFLHSAKKFGKAFVGEIMNS GIGKFLHSAGKFGKAFIGEIMKS GFKQFVHSLGKFGKAFVGEMIKPK GMASKAGSVAGKIAKFALGAL.NH GMASKAGAIAGKIAKVALKAL.NH

M1 M2 M1 M2 ‐ ‐ ‐ ‐ . The shaded residues indicate amino acid diffe indicate . The shaded residues

A comparison of the primary structures of A comparison of the primary structures

muelleri muelleri laevis‐4 laevis‐5 laevis‐6 laevis‐7 laevis‐1 laevis‐2 laevis‐3 muelleri laevis‐1 laevis‐2 muelleri laevis

Fig. 4. X. X. X. X. of skin secretions CPF-LM1 identified in X. X. X. X. Hybrid‐LM1 X. CPF X. muelleri PGLa Hybrid‐LM1 X. X. X. Hybrid‐LM1 X. Magainin have been introduced into some sequenc into introduced have been X. Chapter 7 magainin-2 from magainin-2 treefrogs producedin captivity from female investigated the distribution of host-defense peptides in the skin secretions of F1 hybrid alternative explanation that PGLa-LM1 results from the expression of a mutated duplicated duplicated amutated of expression resultsfromthe PGLa-LM1 that explanation alternative However, PGLa was detected along with PGLa-L may havearisen from upregulation of the PGLa-M1 gene inherited from However, if the gene is presen peptides are present ins magainin-2 sequence and one copy of the magainin-1 sequence (Terry et al., 1988). Both X. laevis (Zasloff, 1987) but thededuced precurso magainin-LM1 shows only one amino acid substitution (Gly substitution acid amino one shows only magainin-LM1 the secretions of the hybrids. Their origins are amatter for speculation. As shown in Fig. 4, genome was preferentially silenced. et al.,2007). It wassugges whereas approximately 35%genes inherited from that approximately 1.2% of genes inherited from , apatternX. muelleri of differential gene expression was also observed and it was found relative a to X. muelleri hybrids. In malesrelated sterile F1 study of hybrid of in upregulated genes 4349 and X.muelleri relative to hybrids the in were genes upregulated and 777 genes upregulated in pattern of gene expression with 839 of genes upregulated in the hybrids relative to paternal with crossing by maternal femaleproduced offertileF1 hybrids ovary in the expression assertion is provided by acomprehensive analysis usingmicroarray technology of gene of altered regulation of expression in the hybrid genetic background”. Firm support for this The authors speculatedthatthe 2.6, 2.7, and 5.1) found only in secretions of the hybrid but not in either parental species. together with peptides synthesitogether withpeptides (Pukala et al., 2006). The skin secretions contained peptides common to only one parent hybrid was missed.hybrid was (Mechkarska et al., 2011a) so that it is possibl X.muelleri in than magainin-M1 9-fold) (approximately concentration ofthe muchhybrid. Magainin-M2lower secretions in in ispresent wasdetected M2, magainin- not but Magainin-M1, magainin-2. encoding domain the in mutation to subject arises from the expression of a duplicate magainin gene derived from that magainin-LM1 therefore, concentration as expected(Figs.1and 2). Itisprobable, The data complement, and are consistent with, the results of an earlier study which which earlierstudy an results the of complement,The with, data are consistent and Three novel peptides (magainin-LM1, PGLa-LM1 and CPF-LM1) were identified in PGLa was not detected in skin secretions of secretions skin in detected PGLa wasnot X. muelleri X. laevis. Magainin-1 and magainin-2 are encoded by the same gene in (Malone andMichalak, 2008). Th ecretionsthe hybrid of and ted, therefore, that the t but expressed at a very low level, ata lowlevel, the PGLa-LM1in hybrid expressed very t but zed by bothparentalspecies an X. leavis appearance of these novel peptides isa result of“some form relative to the hybrids. Similarly, a total of 2930 2930 of Similarly, atotal hybrids. the to relative 160 Litoria splendida Litoria e thatitspresencein thesecretionsof X. laevis were misexpressed in the hybrids (MechkarskaX. muelleri et al., 2011a). X. muelleri X. muelleri M1 in the hybrid secretions so that the the that so secretions M1 inthe hybrid r sequence contains five copies of the X. muelleri with magainin-2 in higher in higher X. laevismagainin-2 with e hybrids showed adifferential d fourpeptides (caerins 1.10, were misexpressed (Malone andmale 13 component ofthe hybrid

 Ala) compared with with compared Ala) X. laevis Litoria caerulea thathas been X. laevisand X. muelleri secretions X. laevis X. laevis .

Chapter 7 (Fig. 4). The amino amino The with the closest closest with the laevis.

X. muelleri X. X. laevis secretions (CPF-1, X. laevis secretions (CPF-1, -3, -5, -6, -4, 1986) but peptidethe was not detected in 161 ybrids suggesting that CPF-LM1 have may arisen CPF-M1 and CPF-M2 from er structural similarity to the CPF peptides identified in is another possibility. TheX. laevis is another originpossibility. of is CPF-LM1 or the hybrids. The proposed origin of the host-defense host-defense the of origin proposed The hybrids. X. laevis or the secretions compared with compared X. laevis secretions PGLa gene inherited from from inherited PGLa gene unclear. The peptide shows a great peptide shows a The unclear. the However,degree the of sequence identity is low (12 amino acid substitutionsoneand from peptide the CPF-5, with compared deletion acid amino All six CPF peptides resemblance). present in the h of the the secretions found in -7) are and from inherited gene CPF duplicate of a mutated expression the from acid sequence of CPF-2 is predicted from the nucleotide sequence of a cDNA of a cDNA sequence nucleotide the from predicted is CPF-2 of sequence acid al., et Richter 1985; et al., (Wakabayashi secretions from either peptideshybrid in the secretionsrepresented is schematically in Fig. 5. Chapter 7 the gene encoding the peptide is inherited from are foundinthehybridtype, Th skinsecretionsonly. indicated by X. Three novel peptides (magainin- X. muelleri Fig. 5. XPF-2 XPF CPF-7 CPF-6 CPF-5 CPF-4 CPF-3 CPF-1 Magainin-2 Mag ♀ PGLa 11 X.laevis peptides -1 ainin-1 Proposed originof the host-defense peptides

hybrids. Magainin-M2 andCPF-M2from

? ?

? X. laevis Magainin-LM1 PGLa-LM1 Magainin-M1 Magainin-M1 Ti ♀ Magainin-2 Magainin-2 Magainin-1 18 peptides CPF

gerinin-M1 XPF-M1 CPF-M1 F1 Hybrid Hybrid F1 XPF-2 CPF-6 CPF-5 CPF-7 CPF-4 CPF-3 CPF-1 XPF 1 PGLa x X. laevis X X -LM1 LM1, PGLa-LM1, and CPF-LM1), indicated by LM1, PGLa-LM1,andCPF-LM1),indicatedby bold X.muelleri 162 presentinskinsecretionsof female F1 e dashed line indicatesth X. muelleri

or X. muelleri were notdetected inthe hybrids as . X X at itisunclear whether Magainin-M2 Magainin-M2 Magainin-M1 Tigerinin-M1 ♂ ? ?

X. muelleri 6 peptides CPF-M2 x laevis X. XPF-M1 CPF-M1

Chapter 7 are are X. laevis x X. laevis studies ranging ranging studies from widely widely X. laevis from lineage (Conlon et al., et (Conlon lineage species (Mechkarska et species individuals studied by by studied individuals etic effects, such as DNA as such etic effects, Xenopus Xenopus X. muelleri ity. One may speculate, therefore, that the cts on expression of antimicrobial peptide peptide antimicrobial of expression on cts male parents of the hybridsoriginated from ybridization, even without polyploidization, polyploidization, without even ybridization, 163 ession often take place in addition to genome genome to place in addition take often ession the parent species. One such peptide, CPF-LM1 CPF-LM1 peptide, such One species. parent the muelleri

X. All of the inheritableAll of the epigen skin secretion from populations of populations from secretion skin easing body of evidence coming from from coming of evidence body easing of tetraploid and octoploid octoploid and of tetraploid e tetraploid (2n = 36) (Koroma et al., 36) (Koroma (2n = species, are tetraploid like the parent hybrids, These It shouldbe pointed out that the The study permits a comparison of the effe permits a comparison of the The study During the process of interspecies h of interspecies process During the muelleri.

separated regions of Africa contain antimicrobial peptides with the same primary structures structures primary same the with peptides antimicrobial contain Africa of regions separated (M. Mechkarska and J.M. Conlon,unpublished data). the Nkambeni area of Swaziland whereas the whereas of Swaziland area the Nkambeni populations two the that is It possible Malawi. South from were (2011a) al. et Mechkarska are synthesizingdifferentvariants molecular so the that novel three peptides identified in unlikely, is This parent. the male from be derived could hybrids of the secretions the that shown has been as it however, genes of inter-species hybridization in the laboratory with ancient allopolyploidization allopolyploidization ancient with laboratory the in hybridization inter-species of genes events in lineage. the Previous investigations analysing multiplicity the of antimicrobial peptidesin the skin secretions octoploid the from of paralogs numbers the that have shown 2012) al., et King 2011b; al., s from the tetraploid number e corresponding th than greater significantly not were frogs frogs.It was concluded, therefore, that nonfunctionalization (gene silencing) (Lynchand genes peptide antimicrobial duplicated of fate common most the been has 2004) Katju, followingputative the allopolyploidizationevents the in from genes peptide of antimicrobial complement full the In contrast, 2012). from genes peptide antimicrobial of six out as four as well F1 hybrids the in expressed X. hybrid, with a greater complement of host-defense peptides, is better protected against against protected better is peptides, host-defense of complement greater a with hybrid, firm but species parent the than the environment in microorganisms pathogenic by invasion evidence is required. support to this claim shows potent, broad spectrum antimicrobial activ antimicrobial spectrum broad potent, shows 2011). Additionally, hybridization has resulted in expression of three novel genes encoding of secretions skin in detected not peptides across biological systems from yeast and plants to humans that demonstrate that epigenetic that epigenetic that demonstrate humans to from yeast and plants systems biological across future by be inherited may stress environmental or hybridization by induced changes in [reviewed environments novel to adaptation to contribute may therefore and generations (Whitelaw and Whitelaw, 2006)]. methylation, histone small modification,interfering microRNA, RNA and spatial location haveofDNA, been shown to trigger changes in gene expression and ultimately phenotype Raz, and Jablonka 2002; (Finnegan, genotype in variation without generation, next the in 2009). Cytosine methylationof is one most the important epigenetic mechanisms operating of fragments of methylated analysis and genomes vertebrate and plant in patterns methylation distinct four revealed 2011) al., et (Koroma hybrids X. muelleri F1 epigenetic changes that control gene expr gene that control changes epigenetic is an incr There reconfigurations. Chapter 7 X.laevis. Arab Emirates United University Pharmacology, Grant (NP/12/03) from U.A.E. University. The authors thank Dr Murat Oz, Department of Acknowledgments the increased complement of host-defense peptides. willinherit species parental ofthe either the with hybrids of mating from offspring the the ar the effects.Since female hybrids epigenetic hybrids the in identified peptides novel the whether be determined to It remains to interspecies hybridization. statesdue methylation methylated fragments (20.9% of the markers disrupti observed previously with consistent of methylatedfragments withthe compared pare (70.6%) proportion higher asignificantly by characterized were females.The hybrids and clusters with thetwo parental species This work was supported by a Research Grant (G00000900) and a Faculty Support Support aFaculty and (G00000900) Grant byaResearch supported was This work X. laevis and 164 ons of hybrid transcriptomes. Hybrid-specific analyzed) wereidentifie e fertile, it is important to ascertain whether ascertain whether to e fertile,itisimportant in this study arise from stably inherited inherited arisefromstably study this in ntal species (64.5%), and this difference is for help in obtaining secretions from secretions obtaining in forhelp X. muelleri separate from hybrid males d indicative ofnovel d Chapter 7 Silurana). ) from the and ., 2010. Orthologs Xenopus J. Biol. Chem. 261, J. Biol. Chem. 261, r polyploid evolution. evolution. r polyploid Xenopus in skin secretions of African osynthesis and degradation of and degradation osynthesis Biol. stress. Curr. Opin. Plant l peptide fragments originating fragments l peptide J., Nielsen, P.F H., King, J.D., Takada, K., 2011. K., H., King, J.D., Takada, Xenopus laevis. a, D.C., 2004. A mitochondrial DNA C Toxicol. Pharmacol. 153, 350-354. 350-354. 153, Pharmacol. Toxicol. C hy and implications fo and implications hy Biochem. J. 243, 113-120. 243, 113-120. J. . Biochem. (Pipidae). J. Zool. 252, 99-107. 99-107. 252, J. Zool. (Pipidae). ussion of the biogeography of African clawed clawed African of ussion of the biogeography oxenopsin-derived peptides from skin secretions secretions from skin peptides oxenopsin-derived Methods for dilution antimicrobial susceptibility susceptibility antimicrobial dilution Methods for Reference method for broth dilution antifungal antifungal dilution for broth method Reference Sympatry and hybridization between the clawed between the clawed hybridization and Sympatry 165 Approved Standard M07-A8. CLSI, Wayne, CLSI, PA. Standard M07-A8. Approved Gen. Comp. Endocrinol. 176: 513–518. 513–518. 176: Comp. Endocrinol. Gen. speciation genetics of clawed frogs ( clawed frogs of speciation genetics (Pipidae). Peptides 31, 989-994. 989-994. Peptides 31, (Pipidae). oved Standard M27-A3. CLS1, Wayne, oved Standard PA. J.D., 2012. Host-defense peptides J.D., 2012. Host-defense peptides Xenopus muelleri d, E., Meetani, M.A., Leprince, Leprince, M.A., d, E., Meetani, med, E., Leprince, J., Vaudry, E., Leprince, J., Vaudry, med, etology/ amphibia /index.php. etology/ amphibia /index.php. ource of random variation in times of ms, D.H., Maggio, J.E., 1986. Nove J.E., 1986. ms, D.H., Maggio, bson, B.W., Williams, D.H., 1987. Bi D.H., Williams, bson, B.W., y, R.C., Melnick D.J., Cannatell y, Melnick R.C., Xenopus laevis prohormones Xenopus amieti Xenopus (Pipidae). Comp. Biochem. Physiol. (Pipidae). Comp. and the caerulein and xenopsin precursors from xenopsin precursors and and the caerulein a and laevis and Xenopus laevis Xenopus clivii Xenopus Front. Biosci. 13, 4687-4706. 4687-4706. Front. Biosci. 13, phylogeograp of African clawed frogs: phylogeny Mol. Phylogenet. Evol. 33, 197-213. 33, 197-213. Evol. Mol. Phylogenet. Pipidae: species (Anura: frog octoploid of a new 2011. Description a disc with the Congo, of Democratic Republic 276-290. 283, Zool. Rift. J. Albertine frogs in the from PGL New York, USA. Electronic database accessible at accessible database Museum of Natural Electronic History, USA. New York, herp http://research.amnh.org/ clawed frogs (Xenopodinae, Pipidae). frogs (Xenopodinae, clawed 5, 101-106. 5, 101-106. frogs 5341-5349. peptides derived from derived peptides tests for bacteria that grow aerobically. aerobically. grow that for bacteria tests susceptibility testing of yeast. Appr yeast. of testing susceptibility clawed Eritrea of the secretions skin from peptides antimicrobial of properties and Purification frog of magainin, PGLa, procaerulein-derived, and pr procaerulein-derived, PGLa, of magainin, frog of the octoploid Evans, B.J., Kelley, D.B., Tinsle Evans, B.J., Greenbaum, E., Kusamba, C., Carter, T.F., Tobias, M.L., Mendel, S.A., Kelley, D.B., D.B., Kelley, S.A., Mendel, M.L., Tobias, T.F., Carter, C., Kusamba, E., Greenbaum, B.J., Evans, Epialleles - s Finnegan, E.J., 2002. Frost, D.R., 2011. Amphibian species of the world: an online reference. Version 5.5. American 5.5. American Version reference. world: an online species of the Amphibian Frost, D.R., 2011. L., Willia Gibson, B.W., Poulter, Evans, B.J., 2008. Genome evolution and evolution Genome 2008. Evans, B.J., Conlon, J. M., Mechkarska, M., King, Conlon, J. M., Fischer, W.J., Koch, W.A., Elepfandt, A., 2000. 2000. A., Elepfandt, W.A., Koch, W.J., Fischer, Giovannini, M.G., Poulter, L., Gi L., M.G., Poulter, Giovannini, Clinical Laboratory and Standards Institute, 2008a. Institute, 2008a. and Standards Clinical Laboratory REFERENCES REFERENCES Clinical Laboratory and Standards Institute, 2008b. Standards and Laboratory Clinical Conlon, J.M., Al-Ghaferi, N., Ahme Conlon, J.M., Al-Ghaferi, Mechkarska, M., Ah Conlon, J.M., Chapter 7 King, J.D.,Mechkarska,M.,Coquet, Jablonka, E.,Raz.G.,2009. Transgen Kobel, H.R., 1996. Allopolyploid speciation. R.C.,In: Tinsley, Kobel, H.R. (Eds)TheBiology of Koroma, A.P.,Jones,R.,Michalak Kobel, H.R., Loumont, C., Tinsley, Kobel, H.R., Du Pasquier Malone, J.H., Michalak, P.,2008. Gene expression Lynch, M.,Katju,V.,2004.The altered evolutionaryLynch, trajectories of geneduplic Malone, J.H.,Chrzanowski, T.H., Mi Mechkarska, M.,Ahmed, E., Coquet, Pukala, T.L., Bertozzi,T.,Donnell Picker, M.D.,1985.Hybridization andhabitatselectionin Mechkarska, M.,Ahmed, E., Coquet, Mechkarska, M.,Ahmed, E., Coquet, J.M., 2012.Host-defensepeptidesskin from implications for the study of heredity Litoria caerulea hybridization in Xenopus 43. and (Eds.), TheBiology of Xenopus laevis 544-549. of Xenopus laevis secretions. Comp Biochem Physiol Part J.M., 2011a. Peptidomic an Pharmacol. 152, 467-472. Marsabit clawed frog J.M., 2010.Antimicrobial peptides south-western Cape Province. Copeia 1985,574-580. multiplicity of antimicrobial peptides in K., Conlon, J.M., 2011b. Genome duplications secretions ofinterspecific hybrid tree frogs and their parents, female R.J., Doyle, J.R., M.J., L.E.,Tyler, Llewellyn, Xenopus muelleri Xenopus pygmaeus . Clarendon Press,Oxford pp.391-401. and and . FEBSJ.273, 3511-3519. Xenopus X. muelleri X. muelleri (Pipidae) are not conspeci , L., 1991. Genetics of Xenopus , and theoctoploid frog Xenopus borealis . Physiol. Genomics43, 1276-1280. alysis ofskinsecretialysis , P., 2011. Snapshot of DNA methylation , P.,2011.methylation changes associated SnapshotofDNA with an, S.C., Bowie, J.H., Surinya-J . BMC Evol.Biol.8, 82. . PLoS One2:e781. R.C., 1996.The extant species. chalak, P., and gene 2007. Sterility . Clarendon Press,Oxford,pp. 9-33. L., Leprince, J., Jouenne, T., Vaudry, H., Takada, K., Conlon, L., Leprince, J.,Jouenne, T., Vaudry, H., King, J.D., Takada, L.,Leprince, J.,Jouenne, Vaudry, H., T., King, J.D.,Conlon, L.,Leprince, J.,Jouenne, Vaudry, H., T., King, J.D.,Conlon, erational epigenetic inheritanc erational epigenetic and evolution. Q.Rev. Biol, 84, 131 withtherapeuticsecretionsofthe potentialfromskin DGenomics Proteomics6,206-212. (Pipidae). Comp. Biochem. Physiol. CToxicol. Xenopus laevis 166 Silurana paratropicalis secretions ofthetetraploidfrogs secretions fic. Peptides 32,1502-1508. ons demonstratesthatth 2006. Host-defence peptide profilesoftheskin Xenopus lenduensis within the Xenopodinaedonot increase the analysis analysis ofof theovary hybridfemalesof Xenopus gilli . MethodsCellBiol. 36,9-34. ohnson, K.H., Liu, Y.,Jackway, e: Prevalence,mechanisms,and In: TinsleyR.C.,Kobel,H.R. expression inhybrid malesof and (Pipidae). Peptides 33, 35- andXenopus Litoria splendida e allopatricpopulations − ates. TrendsGenet. 20, Xenopus andreiskin 176. Xenopus petersii

laevis and inthe male

Chapter 7

Xenopus bson, B.W., bson, B.W., In: Tinsley In: Tinsley skin: isolation, skin: isolation, Proc. Natl. Acad. Acad. Natl. Proc. J. Biol. Chem. 276, J. Biol. Chem. 276, borealis.

Meetani, M.A., Conlon, M.A., Conlon, Meetani, (Pipidae). Gen. Comp. Gen. Comp. (Pipidae). Xenopus oore, C.H., Gi Xenopus t defense peptides. Methods Mol. Rana tigerina. Rana one, three, or four copies of the final copies of the final or four one, three, (prepro-magainins) and aspects of the and aspects of the (prepro-magainins) Xenopus amieti with insulin-releasing activities from skin from activities insulin-releasing with and . Clarendon Press, Oxford, pp. 121-141. 121-141. Oxford, pp. Press, . Clarendon hab, Y.H., Flatt. P.R., ape epigenotype within and across generations. generations. across and within ape epigenotype , N.P., Devi, A.S., Nagaraj, R., Sitaram, N., 2001. N., R., Sitaram, A.S., Nagaraj, Devi, , N.P., J.C., Giovannini, M.G., M J.C., Giovannini, 167 Antimicrobial properties of peptides from of peptides properties Antimicrobial Xenopus nd partial cDNA of a precursor. sequence skin: the mRNA contains an unusual repetitive structure. an unusual repetitive contains skin: the mRNA ss of antimicrobial peptides from Xenopus borealis tial therapeutic application of hos tial therapeutic Xenopus . FEBS Lett. 228, 337-340. 337-340. Lett. 228, . FEBS M.V., Vairamani, M., Raju . A small family of precursors containing containing familyof precursors A small . Biol. 618, 303-327. Biol. 618, 303-327. 2701-2707. Xenopus laevis product. J. Biol. Chem. 261, 3676-3680. 3676-3680. Chem. 261, Biol. product. J. Tigerinins: novel antimicrobial peptides from the Indian frog from peptides novel antimicrobial Tigerinins: R.C., Kobel, H.R. (Eds.), The Biology of of The Biology (Eds.), R.C., Kobel, H.R. 172, 314-320. Endocrinol. processing of this prepro-polypeptide. J. Biol. Chem. 263, 5745-5751. 263, 5745-5751. J. Biol. Chem. this prepro-polypeptide. processing of from precursor caerulein 1817-1828. 13, Nucleic Acids Res. R131-R137. 15, Hum. Mol. Genet. granular gland secretions granular for prepro-PGS coding 1988. The cDNA sequence J.M., 2011. Caerulein-and xenopsin-related peptides xenopsin-related Caerulein-and J.M., 2011. frogs, clawed of the secretions of two active forms a characterization Sci. USA 84, 5449-5453. Zhang, L., Falla, T.J., 2010. Poten Soravia, E., Martini, G., Zasloff, M., 1988. E., Martini, G., Zasloff, Soravia, G., 1986. Sequence of preprocaerulein cDNAs cloned from skin of cDNAs cloned from Sequence of preprocaerulein G., 1986. Kreil, R., Egger, Richter, K., Sai, K.P., Jagannadham, Sai, K.P., Jagannadham, Zasloff, M., 1987. Magainins, a cla Wakabayashi, T., Kato, H., Tachibana. S., 1985. Complete nucleotide sequence of mRNA for of mRNA sequence nucleotide Complete 1985. S., H., Tachibana. Kato, T., Wakabayashi, sh How lifetimes 2006. E., Whitelaw NC, Whitelaw, in communication acoustic and Sound production Yager, D.D., 1996. Terry, A.S., Poulter, L., Williams, D.H., Nutkins, Nutkins, D.H., Williams, L., Poulter, A.S., Terry, Zahid, O.K., Mechkarska, M., Ojo, O.O., Abdel-Wa O.O., Ojo, M., Mechkarska, O.K., Zahid,

Chapter 8

General discussion

Milena Mechkarska

Part of the discussion is published in the review: Host-defense peptides in skin secretions of African clawed frogs (Xenopodinae, Pipidae). J.M. Conlon, M. Mechkarska, J.D. King. Gen Comp Endocrinol (2012a) 176:513-518. Chapter 8 by was attempt an addition, In X. members seven 1987) al., tropicalis S. had Xenopodinae those oftheparents(Chapter7). characterized incompletely Pipidae the to belonging frogs of secretions skin from pathogens multidrug-resistant against potential eerh more research cationic concentration (MIC) concentration S. aureus and the hymenochirins-1B, -2B and -3B (from Pi CPF-B1,CPF-C1 agents: anti-infective into from skin secretions of frogs of the Pipidae family (Chapters in Table 1 and their antimicrobial and hemolytic activities are compared in compared Table 2. are and hemolytic activities antimicrobial 1and their Table in with MIC with MRSA MDRAB and areactive against very the CPF peptides addition, In erythrocytes. following viewpoints: (HC hemolytic activity -mean concentration of peptide producing 50% hemolysis low very advantage of the have they activity, and antifungal moderate antimicrobial studying the AMP profile of profile AMP the studying in parallel with parallel in andrei 50 is the isolation of AMPs with therapeutic with AMPs of isolation the is thesis this in described work the of aim The Seven peptides have been identified that show therapeutic potential for development AMPs AMPs, all newlyidentified skin the frog of majority the vast with common In Therapeuticp   The CPF peptides are potent againstthe  The importance ofthe AMPs isolated from Pipidae frogs is discussed from the ) ≥ α 225 225 and mode of inheritance of duplicated AMP genes. th on hybridization interspecies and the effect polyploidization Analysis of of evolutionary history. Clarification of taxonomic relationships between frog species and study of their resistant pathogens. Development ofnoveltherapeutically -helical peptides (Chapter , and ≤ family. At the beginning of 2010 only three species belonging to the subfamily the to belonging species three only 2010 of beginning the At family. C. albicans

12.5 μ 12.5 (Ali et al., 2001), the tetraploid frog tetraploid the 2001), al., et (Ali μ M. than h octoploid the f the of o M (Chapters M (Chapters tential of AMPs fromthePipidae frogsof family d o te rsne f Ms te ili frog diploid the - AMPs of presence the for analyzed been 0 Ms ee uiid rm h si scein o a additional an of secretions skin the from purified were AMPs 50 ≤ , 25 25 aiy Pipidae: family K. pneumonia . new tetraploid 1” (Chapter 1” tetraploid new “S. X. muelleri X. made to understand the mode of inheritance of AMP genes AMP of inheritance of mode the understand to made re f this of course the During 2010a). al., et (Conlon amieti X. μ M bu F1 1 , 1 hybrid frogs hybrid ). 2 t are only moderately cytotoxic against human human against cytotoxic moderately t areonly

and and West (Chapter West and (Chapter borealis X. , CPF-MW1 and CPF-SP1 (from Xenopodinae) Xenopodinae) (from CPF-SP1 and , CPF-MW1 170 4 ). Although the hymenochirins display only clinically relevant microorganisms relevant clinically P. aeruginosa pinae). Their primary X. laevis X. activecompoundsmultidrug- against and (Zasloff, 1987; Giovannini et Giovannini 1987; (Zasloff, laevis X. 5), x 4), and comparing it to it comparing and muelleri X. together with the with together muelleri X. withminimum inhibitory 2 - (Chapter boettgeri H. 2), 6 ) belong tothe class of X. structures are shown (Chapter clivii

E. coli e fate 3), 6). , Chapter 8

2 50 HC - >200 - - 70 - 6 >100 6 25 140 25

2 - - 25 25 K. pneumoniae P. aeruginosa GKAFKFGLKTVGKMMGGAPREQ tic activity against human erythrocytes erythrocytes tic activity against human ed by four different parameters that are that are parameters four different ed by

tive agents for use against drug resistant 1).In addition, there is no obvious common 171 Primary structure structure Primary GFGSLLGKALRLGANVL.NH GFLGPLLKLGLKGVAKVLPHLIPSRQQ GLGSLLGKAFKIGLKTVGKMMGGAPREQ IKLSPETKDNLKKVLKGAIKGAIAVAKMV.NH LKIPGFVKDTLKKVAKGIFSAVAGAMTPS IKIPAVVKDTLKKVAKGVLSAVAGALTQ C. albicans ) (Powers andHancock,2003), it is difficult tothe predict 1 S.aureus

li Hymenochirin-1B Hymenochirin-1B Hymenochirin-2B Hymenochirin-3B CPF-B1 CPF-C1 5 5 25 5 5 6 6 - 6 6 3 6 25 6 3 25 6 25 6 25 E. co potency of the AMPs is determin AMPs is of the potency West CPF-MW1 GLGSLL MIC against microorganisms and hemoly against microorganisms MIC ) of the peptides isolated from skin secretions of Pipidae frogs. frogs. Pipidae of secretions skin from isolated peptides the of ) Because the 50 ble 2. S. new tetraploid 1” CPF-SP1 Hymenochirin-1B 20 Hymenochirin-1B 10 20 Hymenochirin-2B 80 40 20 Hymenochirin-3B 80 40 20 80 20 40 40 225 20 >300 40 >300 H. boettgeri CPF-SP1 CPF-B1 CPF-C1 X. borealis X. borealis Frog species Frog species “ AMP X. clivii CPF-MW1 X. muelleri Data are expressed in µM. in are expressed Data Ta (HC Table 1. skin Naturally-occuring AMPs from secretions of Pipidae frogs with potential for anti-infec non-toxic, potent into development microorganisms. microorganisms. strongly interrelated (Chapter (Chapter interrelated strongly amino its from peptide a given of of action and mode selectivity, activity, antimicrobial mparison Co ofprimary the sequences properties. of the physicochemical or sequence acid sequence acid conserved amino ere is no th that reveals hymenochirins the and CPFs associatedwith antimicrobial activity (Table Chapter 8 amphipathic character of Edmundsonwheel representationand (Schiffer an Table 3usingthe AGADIR program(Munoz an Giangaspero etal.,2001).Analysis oftheseco etal., (Dathe the bacteria1997; isless stringent Gram-negative regarding situation the bacteria, while Gram-positive towards the activity requirement antimicrobial for (prokaryotic) ones. microorganisms,In terms effecton absolute isan of helicity charged negatively towards the action for seems important membranes, but less (eukaryotic) neutral activity forthe against featureisessential This et al.,1999). for the binding of peptides to membranes is the formation of observed for hymenochirin-2B and-3B with acharge of +4. Another strong driving force issimilar that +6 to with a hymenochirin-1B of charge activity Similarly, the +4. of issimi charge a+2 CPF-C1 with of activity the secretions ofPipidaefrogs. 3. Table 3). (Table a activity of peptide given the determine which parameters set of physicochemical yeohrn3 1. + 1.1 9-16 10.71 8-16 10.71 +4 +4 Hymenochirin-3B +19.5 Hymenochirin-2B +13.5 Hymenochirin-1B +4.9 CPF-MW1 CPF-C1 CPF-B1 CPF-SP1 Peptide α There i -helical conformation overat leastpart Physicochemical properties of the CPFs and the hymenochirins, isolated from skin s no simple correlation between peptide charge and membrane activity. Thus, Hydrophobicity Hydrophobicity Charge +15.3 +10.5 -0.6 -1.1 the 1). the helices (Fig. 6 06 5-27 10.65 +6 pH 7 +2 +4 +4 +4 172 of their structures. A Schiffer-Edmundson lar to that exerted by peptides with a charge acharge by with peptides exerted that lar to ndary structure of the peptides included in d Serrano, 1994) indicate thatallcanadopt , 1967) of the structures illustrates the the , 1967)of at α 11.49 5-14 11.73 5-11 IAlpha-helical pI 10.98 2-12, 14-17 10.98 2-12, -helical structures(Wieprecht domain 14-17 Chapter 8 3B potent antimicrobial activity,are CPF-C1 CPF-C1 CPF-SP1 CPF-SP1 Hymenochirin Hymenochirin -helical conformation. The polar Lys, Glu, Lys, polar The conformation. -helical the most active CPFs and hymenochirins the most active CPFs and hymenochirins α 173 s in the peptides, essential for s in the peptides, essential for nature of the predicted nature of the predicted 2B CPF-B1 CPF-B1 CPF-MW1 CPF-MW1 Hymenochirin Hymenochirin -Edmundson wheel representation of -Edmundson wheel representation Schiffer Fig. 1. Fig. 1. demonstrating the amphipathic Asp and Arg segregate on one face of the helix with the hydrophobic Leu, Ile, Pro and Val residues Ile, Leu, with the hydrophobic Asp and Arg segregate on one face of the helix on the opposite face. The basic residue shown in bold type. type. shown in bold Chapter 8 such aserythrocytes.Ifthetherapeutic inde investigated as these peptides were isolated and CPF-MW3 (from CPF-B2,CPF-B3 CPF-B4 thesis: (fromthis and towards towards Chapter (HC colistin to are resistant antibiotic-resistant of isolates clinical multiple against MICvalues with activity (Conlon et epitropicalis al.,S. 2012b). Thus, CPF-AM1 shows broad spectrum bactericidal (Conlon et al., 2010a), X.pygmaeus agents comesfrom otherworkcarried ( temporin-DRa erythraea brevinin-2-relatedpeptide (B2RP) ( ascaphin-8 ( for hemolysis of human erythrocytes) of the promising peptides is found to be too low for low too be to isfound peptides thepromising of erythrocytes) human of hemolysis for most hydrophobic but are the least hemolytic of the peptides in Tables 2 and and 3. Tables 2 in peptides ofthe arethe least hemolytic but most hydrophobic withthis consistent are fully thesis not this in presented The findings et al., Dathe 1997). etal.,1996; Skerlavaj etal.,1996; (Javadpour potency the hemolytic of increase an by followed often is in hydrophobicity increase charged membranes (Kiyota et al., 1996; Wiep (Wieprecht et al., 1997a) but plays asecondary role in their interaction with negatively bacterium CPF-PG1 from MRSA but are hemolytic (Conlon of of isolates arange clinical against -5µM) (MIC=2.5 activity growth-inhibitory potent (unpublished data). Two peptides -CPF-SE2 and CPF-SE3 -from Gram-negative bacteria,aswell and Gram-positive multidrug-resistant against activity exerting peptides, by following different frog species have been recently reviewed (Conlon and Sonnevend, 2011). The therapeutic potential asanti-infec potential therapeutic 50 The use of peptide-based anti-infective agents is limited by their toxicities to host cells cells host to toxicities their by islimited agents anti-infective peptide-based use of The The wheel representation of the amphipathic structure of hymenochirin-1B is shown in Further support for the CPF peptides as candidates for development into therapeutic therapeutic into for development ascandidates the CPF for support peptides Further Hydrophobicity correlates with the higher a the higher with correlates Hydrophobicity Possible clinicalapplications of = 145 µM) (King et al., 2012). Morememb S. aureus 6 ); kassinatuerin-1 ( . Therefore, it is difficult to explain why the hymenochirins have reduced activity S. aureus Aschapus truei Aschapus Rana draytonii X. pygmaeus or are less hemolytic when co when hemolytic less are or (MIC = 6 µM) and shows moderate hemolytic activity moderate activity hemolytic shows µM)and (MIC =6 X. muelleri ≤ (Conlon et al., 2012c), and ); brevinin-1BYa ( 25μ Kassina senegalensis ). has relatively high potency against the Gram-positive the Gram-positive against potency high relatively has M against reference strains of tive agents: alyteserin-1c(fromtive agents: West). However, their antimicrobial potential was not wasnot potential antimicrobial their However, West). , X. petersii the opportunistic yeast pathogen C. albicans pathogen yeast opportunistic the et al.,2012b).Similarly, a syntheticreplicateof Lithobates septentrionalis Lithobates α -helical AMPsisolated from skinsecretionsof out in the laboratory on AMPs from on X.amieti laboratory the out in from skin secretions in very low yields. x (ratio of MIC against microorganismsHC to MICagainst (ratio of x 174 rule. For example, the hymenochirins are the Rana boylii Rana recht etal.,1997b; Kwon etal.,1998).An mpared to the CPFpeptides. and ffinity of peptides for zwitterionic lipids lipids zwitterionic for of ffinity peptides X. borealis ); pseudin-2 ( ers oftheCPF family aredescribed in X.lenduensis A. baumannii, ); brevinin-2PRa ( Stenotrophomonas maltophilia Stenotrophomonas ), CPF-AN1 (from E. coli and ); B2RP-ERa ( Pseudis paradoxa); and (King et al., 2012), including strains that show epitropicalis S. Alytes obstetricans Alytes S. aureus Rana pirica Rana

X. andrei Hylarana , show and

and 50 ); ); )

Chapter 8

acne vulgaris (Melo acne vulgaris is important is in the (Banemann et al., 1998; et al., (Banemann P. acnes This mechanism can lead to mechanism This in vitro in lications during the course of the disease of the lications during the course possess the desired dual action: directly dual action: directly the desired possess agents for treatment of agents for treatment seases (Lewis, 2010; Berditsch et al., 2012). 2010; Berditsch seases (Lewis, ecord (Gould,1977; Hennig, 1979). In the aris is a disease affecting the pilosebaceous pilosebaceous the affecting is a disease aris including behavioural characteristics such as with cross-resistance to human-neutrophil- to cross-resistance with y of animal species was based entirely upon entirely based was species y of animal as impetigo in infants and children (Geria and (Geria children and infants in as impetigo ion may stillion may be For skin feasible. example, 175 S. aureus n of surface lesions such as the foot ulcers of diabetic n of surface lesions centuries, the recognition of taxa, such as genera, species species as genera, of taxa, such recognition centuries, the th th has been demonstrated has been demonstrated tion (Berditsch et al., 2012). tion (Berditsch et al., and suppress the inflammatory immune response (Popovic et al., al., et (Popovic response immune inflammatory the suppress and within(Bojar the and PSU Holland, 2004; et al., Kurokawa and early 20 and early th P. acnes P. Taxonomy and evolutionary history of Pipidae frogs frogs Pipidae of history evolutionary and Taxonomy 19 Back in the Natural resistanceAMPs to AMPs have attracted interest as therapeutic as therapeutic attracted interest AMPs have and subspecies and establishing the phylogen the and establishing and subspecies r fossil and the characteristics morphological 1950s definitionthe of taxa was facilitated by and of biochemical development the Following attributes. and other calls advertisement generated experimentally provides provides experimentally generated bacterial persistence responsible for chronic di for chronic bacterial persistence responsible pexiganan to resistance evolved that reported (2012) Brockhurst and Habets Moreover, infections. to response immune innate the in role key a plays which defensin-1 Bishop and 2006). Finlay, Development of inducedresistance will also occur et (Groisman of are orders that rates at but 2008) al., et Llobet 2006; Otto, 1996; Nikaido, 1992; al., magnitude lower than those observed for conventional antibiotics (Ge et al., 1999; Perron et cell the damage not do which concentrations AMP sub-toxic However, 2006). al., membranes bacterial can populations stimulate to switch a metabolically to dormant state forma biofilm associated with (Mouser et al., 2003;(Mouser Sugisaki et al., 2009).Frogal., et skin peptides showpotential for they because topical agent into a development of growth inhibit systemic use, alternative routes of applicat use, alternative systemic 2012). infections in otherwise healthy young individuals are a common clinical manifestation of of manifestation clinical are a common individuals young healthy otherwise in infections community-acquired problem inseriousinfections2012). (Skov MRSA is a MRSA et al., infections of the superficial such epidermis, colonizatio in Schwartz, 2010) and patients (Bowling et al., 2009). Consequently, the peptidesfind may application as topical promote to regimes therapeutic in and infections skin combating in options treatment wound healing. et al., 2006; Popovic2012). et al., vulg Acne withunit both(PSU), bacterial and inflammatory et al., 2009; components(Bhambri by proliferation is lesion acne an of development the in role A major 2010). al., et Grange acnes Propionibacterium comp the to contributes acne and of pathogenesis 2009). The inflammatory immune response of to host the Chapter 8 and species (Conlon etal.,2004; Appon inta of are frogs valuable dermal peptides sequences of individual peptides from one sp peptide family (Tennessen and Blouin, 2007). Since there is virtually no overlap in the Skin secretions from singlespeciesfrequen a acommonfamilies evolutionary thatshare orig similarities in their amino acid sequences, frog skin AMPs may begrouped together in the peptide families (Duda et al.,2002; Te diversity of the extraordinary explaining selection, via positive rapidly evolve and encoded and for study ofevolutionary history of the different frog families. AMPs aregenetically possess the necessary features tobeconsidered isolated to-date from skin secretions of species belonging to Xenopodinae (Chapters AMPs The latterview. the for support Pipinae provide and from Xenopodinae isolated Coll 2008). etEvans, Evans al., 2004; 1990; Pseudhymenochirus (Yanai et al., 2011). The hymenochirins duplicated genesmaintained are in S. tropicalis from secretions skin in families PGLa,CPF, identified -magainin, peptide XPF and - initially Conlon et al., 2010a; Conlon et al, 2012b; King et al., 2012) aregrouped in the samefour ( Xenopodinae nucleotidethe sequence of mitochondrialgenes providedsupport for divergence of the of comparisons upon based analyses subsequent However, 1988). Trueb, and (Cannatella genera the to related closely comparison of molecular featur molecular biology techniques, the study of animal evolution hasincreasingly relied upon divergence of the Pipinae from the Xenopodinae (Chapter (Chapter the Pipinae of Xenopodinae fromthe divergence withanearlier consistent from the Xenopodinae peptides similarity to lowstructural very On the basismorphologicalof char There isanincreasingbody ofevidence that X. laevis and X.laevis are and 90% identical within coding regions and roughly half of the 2-4). (Figs. Silurana ) before the divergence of the Pipinae subfamily (de Sa and Hillis, Pipinae Sa and ) ofthe subfamilyHillis, the (de before divergence + This finding is not unexpected as the genomic DNA sequences of Hymenochirus Xenopus es (Guttman, 1973; Graur and Li, 2000; Carroll et al., 2001). etal., Carroll 2001). Li, and 2000; Graur 1973; es (Guttman, after the putative tetraploidization took place place took tetraploidization X. laevis putative afterthe ) fromthePipinae( acters alone,itwasconcluded that yi et al., 2004; Zheng etal., 2005). Zheng yi etal., 2004; isolated from skinsecretions of , 176 xonomic evaluations of closely related genera genera closelyrelated of evaluations xonomic Pseudhymenochirus Pipa, and Pseudhymenochirus ectively, the amino acidsequences of AMPs tly containseveralmembers ofaparticular ecies toanother (Conlon etal., 2004), the nnessen, 2005). On the basis of limited limited of the basis On 2005). nnessen, in (Goraya etal.,2000 asareliablemarker AMPspresent infrogskinsecretions 6 ). Hymenochirus for anuran taxonomy taxonomy anuran for ; Duda et al., 2002). etal., ; 2002). Duda H. Silurana Silurana than to to than

show show boettgeri + Xenopus Pipa + is more 2 - 5 ;

Chapter 8 GVGKFLHAAGKFGKALMGEMMKS GVSQFLHSASKFGKALMGEIMKS GVGKFLHSAKKFGQALASEIMKS GVGKFLHSAKKFGQALVSEIMKS GLKEVAHSAKKFAKGFISGLTGS GLKEVAHSAKKFAKGFISGLTGS GLKEVLHSTKKFAKGFITGLTGQ GIGKFLHSAKKFGKAFVGEVMKS GISQFLHSAKKFGKAFAGEIMKS GIKEFAHSLGKFGKAFVGGILNQ GVSKILHSAGKFGKAFLGEIMKS GIGQFLHSAKKFGKAFVGEIMKS G**KFLHSAGKFGKAFLGEVMIG GIGKFLHSAGKFGKAFLGEVMKS GIGKFLHSAGKFGKAFIGEIMKS GFKQFVHSLGKFGKAFVGEMIKPK GIGKFLHSAGKFGKAFLGEVMKS GIKEFAHSLGKFGKAFVGGILNQ GVSKILHSAGKFGKAFLGEIMKS GIGKFLHSAGKFGKAFVGEIMKS GIGKFLHSAKKFGKAFVGEIMNS GIGKFLHSAGKFGKAFVGEIMKS 177 LM1 GIGKFLHSAKKFAKAFVGEIMNS

SE1 L1 L2 ST1 PG1 PG2 P2 B1 B2 M1 M2 West MW1 P1 P1 AN1 AN2 C1 C2 x X. muelleri AM1 AM2 1 1 2 X. andrei X. andrei X. lenduensis X. amieti X. amieti X. pygmaeus X. pygmaeus X. lenduensis X. laevis X. clivii X. clivii X. muelleri X. muelleri X. muelleri X. petersii X. borealis X. borealis X. laevis X. petersii S. tropicalis S. epitropicalis X. laevis Magainin-related peptides Chapter 8 introduced in to some sequences to in tosome introduced belonging to the taxonXenopodinae. Aminoacid Fig. 2. X. andrei X. andrei X. amieti X. amieti X. lenduensis X. lenduensis X. lenduensis X. lenduensis X. pygmaeus X. laevis X. muelleri X. muelleri X. borealis X. borealis X. petersii X. laevis S. epitropicalis GMATKAGTALGKVAKAVIGAAL S. epitropicalis “S. newtetraploid1”SP1 S. tropicalis PGLa-related peptides Conservation of amino acid residues is emphasized by the shading. Acomparison oftheprimary structures ofmagainin and PGLa-related peptidesspecies from AN2 AN1 AM2 AM1 xX.muelleri 1 WestMW2 PG1 WestMW1 B2 B1 P1 L GMASTVGSIFGKLAKTAL*GAL GMASTAGSVLGKLAKVAL*GAL GMASTAGSVLGKLAKVAIKAAL GMASTAGSIFGKLAKTAL*GAL L4 L3 L2 L1 ST1

SE2 SE1 maximizestructur L1 GMASKAGSVAGKIAKFAL*GAL LM1 178 GMASKAGSVLGKLAKVAI*GAL GMASKAGSVLGKVAKVALKAAL GMASTAGSVLGKLAKAVAIGAL GMASKAGSVLGKVAKVALKAAL GMASKAGAIAGKIAKVALK*AL GMASKAGTIVGKIAKVAL*NAL GMASKAGAIAGKIAKTAIKLAL GMASKAGSVLGKITKIAL*GAL GMASKAGSIVGKIAKIAL*GAL GMASKAGTIAGKIAKTAIKLAL GMASTAGSIAGKIAKVALK*AL GMATKAGTALGKVAKAVIGAAL GMATAAGTTLGKLAKFVI*GAV GMATKAGTALGKVAKAVIGAAL al similarity. al similarity. residue deletions denoted by * have been a denotesC-terminal a a a a a a a a a a a a a a a a a a a a

α -amidation. Chapter 8

a a a a a a a a a a a a -amidation. Conservation of amino of -amidation. Conservation α GLGSLLGKALKFGL*KAAGKFMGGEP*QQ GFLGSLLKTGLKV****GSNLL GFGSLLGK*ALRL****GANVL GFLGPLLKLGLKGVAKVIPHLI**PSRQQ GFLGPLLKLGLKGVAKVIPHLI**PSRQQ GFLGPLLKLGLKGAAKLLPQLL**PSRQQ GLGSLLGKAFKIGL*KTVGKMMGGAPREQ GLGSLLGSLFKFIP*K****LL**PSIQQ GLGSLLGKAFKFGL*KTVGKMMAGAPREQ GLGSLLGKAFKFGL*KTVGKMMAGAPREE GLGSLLGKAFKFGL*KTVGKMMGGAPREQ GLGSLLGKAFKFGL*KTVGKMMGGAPREE GFGSFLGKALKAGL*KLGANLLGGAP*QQ GFGSLLGKALKAGL*KLGANLLGGAP*QQ GFLGSLLKTGLKV****GSNLL GLLGPLLKIAAKV****GSNLL GLGSVLGK*ALKI****GANLL GIGSALAK*AAKL****VAGIV GLGSLVGN*ALRI****GAKLL GFASVLGK*ALKL****GANLL GFGSFLGKALKAAL*KIGANALGGAP*QQ GLASFLGKALKAGL*KIGSHLLGGAP*QQ GFGSFLGKALKAAL*KIGANVLGGAP*QQ GFGSFLGKALKAAL*KIGANVLGGAP*EQ GFGSFLGKALKAAL*KIGADVLGGAP*QQ GFLGPLLKLAAKGVAKVIPHLI**PSRQQ GFLGPLLKLAAKGVAKVIPHLI**PSRQQ GLASFLGKALKAGL*KIGAHLLGGAP*QQ GFASFLGKALKAAL*KIGANMLGGTP*QQ GFGSFLGKALKAAL*KIGANALGGSP*QQ GLASLLGKALKAGL*KIGTHFLGGAP*QQ GFGSFLGKALKTAL*KIGANALGGSP*QQ GFASFLGKALKAAL*KIGANMLGGAP*QQ GFGSFLGKALKAAL*KIGANALGGAP*QQ 179 denotes C-terminal C-terminal denotes a LM1 GFGSFLGSLFKTGL*KIIPKLL**PSIQQ LM1 GFGSFLGSLFKTGL*KIIPKLL**PSIQQ SE3 SE1 SE2

ST2 ST3 L1 L2 L3 GIGSLLAK*AAKL****GANLL GIGSALAK*AAKL****VAGIV GLGTFLGN*ALKTGLKIGANLL ST1 M2 West MW1 West MW2 PG2 PG3 B1 B2 M1 P1 P2 P3 P4 P5 AM2 AM4 AN1 C1 AM1 C2 6 7 1 1 2 3 4 5 A comparison of the primary structures of CPF-related peptides from species belonging to the CPF-related peptides from species belonging to of A comparison of the primary structures acid residues is emphasized by the shading. shading. the by emphasized is residues acid taxon Xenopodinae. Amino acid residue deletions denoted by * have been introduced in to sometaxon Xenopodinae. Amino acid residue * have been introduced deletions denoted by sequences to maximize structural similarity. Fig. 3. X. amieti X. amieti X. andrei X. lenduensis X. lenduensis X. amieti “S. new tetraploid 1” SP3 S. epitropicalis X. clivii GFLGSLLKTGLKV****GSNLL X. lenduensis S. tropicalis S. tropicalis X. laevis x X. muelleri X. clivii X. pygmaeus X. pygmaeus X. muelleri X. muelleri X. muelleri X. borealis X. borealis X. muelleri X. petersii X. petersii X. petersii X. petersii X. laevis X. laevis X. petersii X. laevis X. laevis X. laevis S. epitropicalis S. epitropicalis X. laevis X. laevis S. tropicalis 1” SP1 “S. new tetraploid 1” SP2 “S. new tetraploid GFLGPLLKLGLKGVAKVLPHLI**PSRQQ GFLGPLLKLGLKGAAKLLPQLL**PSRQQ CPF-related peptides Chapter 8 ructural similarity. sequences tomaximizestructuralsimilarity. deletions denotedby taxon Xenopodinae.Aminoacidresidue*havebeenintroducedintosome Fig. 4. X. andrei X. andrei X. amieti X. clivii X. muelleri X. muelleri X. borealis X. borealis X. laevis X. laevis S. epitropicalis S. epitropicalis S. epitropicalis S. epitropicalis S. tropicalis GLASTIGSLLGKFAKGGAQAFLQPK S. tropicalis GFWSSALEGLKKFAKGGLEALTNPK “S. newtetraploid1”SP2 “S. newtetraploid1”SP1 S. tropicalis XPF-related peptides acid residues is emphasized by the shading. Acomparisonoftheprimary structures of XPF AN2 AN1 AM1 C1 2 1 WestMW1 M1 B2 B1 ST3 ST2 ST1

SE1 SE4 SE3 SE2 a denotes C-terminal 180 GWVSKIGQTLGKMAKVGLQELIQPK GWASKIGQTLGKMAKVGLQELIQPK GWASKIAQTLGKMAKVGLQELIQPK GWASKIGQTLGKIAKVGLKELIQPK GWASKIGQTLGKIAKVGLQGLMQPK GWASKIGQTLGKLAKVGLKEFAQS* GWASKIGQTLGKMAKVGLKDLIQA* GWASKIGTQLGKMAKVGLKEFVQS* GFKQFVHSM*GKFGKAFVGEIINPK GVFLDA***LKKFAKGGMNAVLNPK GVWSTVLGGLKKFAKGGLEAIVNPK GLASTLGSFLGKFAKGGAQAFLQPK GLFLDT***LKKFAKAGMEAVINPK GVWTTILGGLKKFAKGGLEALTNPK GFWTTAAEGLKKFAKAGLASILNPK GLASTIGSLLGKFAKGGAQAFLQPK GWASKIGQALGKVAKVGLQQFIQPK -related peptides from species belonging to the α -amidation. Conservation of amino Chapter 8

in -helical lineage α S. tropicalis (Evans et al., (Evans Xenopus . There are several are several . There S. paratropicalis of the XPF family timicrobial peptides peptides timicrobial 4 (Mechkarska et al., (Mechkarska 4 new tetraploidnew 1” (Evans lackburn and Beier,2011). ; King et al., 2012; Conlon et Conlon 2012; al., et King ; X. itombwensis -amidated amino acid residue residue acid amino -amidated 5 es, it was pointed out that the the that out was pointed es, it al., 2004; Evans et al., 2005; 2005; al., et Evans 2004; al., - α 2 Silurana re detected in skin secretions from nova VII” (Graf and and (Graf VII” species nova Xenopus ddressed in this thesis with the aim ofddressed in this thesis with aim the y structures of the an y structures analysed in Chapter analysed in Chapter has been expanded to include two new two include to expanded has been nopodinae for which further confirmation is no acids are invariant in the first group and group first the in invariant are no acids The amino acid sequences acid The amino according to the terminology of Flajnik et al.according to the terminology of Flajnik 181 ictions indicate that the amphipathic Xenopus and therefore invalid (B invalid and therefore Silurana However, a peptide belonging to the magainin family new tetraploid1”used is instead of . ized species status (Evans et status (Evans ized species recognized octoploid species – species octoploid recognized has been maintained(Chapters

, analysis of mitochondrialDNA suggests that tetraploidization . nomen nudum S. paratropicalis S. paratropicalis (Evans et al., 2011). At the same time, there are several there are several time, At the same et al., 2011). (Evans Silurana Silurana ) 8 is a Silurana skin (Ali et al., secretions 2001) we new tetraploidnew 1”2-4). (Figs. The magainins were not detected in either species S. paratropicalis The first exampleThe first is the tetraploid During the last 4 years, the genus 4 years, the During the last In the genus the In Evolutionary pressure to conserve the primar the conserve to pressure Evolutionary Silurana character of all the peptides peptides the of all character a been have members such Two 2007). Evans, providing evidencetheir to confirm putativedesignation. species entriesstatus with of a fully al., 2012a). al., 2012a). needed in order to gain recogn gain to order in needed incompletelyofXe characterized the members X. lenduensis X. and 2008) studies involvingfirst describedfrog this as “ Fishberg, 1986; Tymowska, 1991) and subsequently as “ Inet al., 2004). the publication arising from the work in Chapter as it was referred 2011), (1993). However,as it is not a separate speci recognized as “ name the Consequently, (Chapter Discussion the term term (Fig. 3). Even after deletions(Fig. 3). Even after been have introduced sequences some order in into to 3 ami only the structural similarity, maximize only glycineN-terminal the residue is invariant inthe groupof amidated the CPFs. bothNevertheless,XPF and in CPF peptides, the acid amino substitutions generally are structure pred secondary conservative and and sowas speculated it the that gene encoding the magainins arose in the “ after its divergence from from Xenopodinaefrom has not been uniform (Figs. 2-4). Peptides belonging the to PGLa family magainin the by followed invariant) acids amino 22 of out (7 conserved strongly are most invariant). acids 23 amino of out (4 family invariant). acids amino of 25 3 out (only conserved poorly been have peptides divide they that suggests CPF-peptides of the structures primary of the A comparison naturally intogroups two with and without a C-terminally occurred by allopolyploidization. Hence another diploid (2n=20) ancestral species is is species ancestral (2n=20) diploid another Hence allopolyploidization. by occurred predictedhave to existed in addition tothe the ancestor diploid of species (Evans2005; et al., Evans,2007). AMP families All -XPF PGLa,found - and CPF in S. tropicalis Chapter 8 given the working name Chapter Chapter “ expression of the tetraploid genomesmore sothat AMP genes have beensilenced in inthe event the polyploidization that into two subgroups (Chapter 2011). sub-species andeven different population clus have been used, together with morphological andcognate methods, to differentiate between peptide profiles of Australian hylid frogs from the genera populations probably do not represent separate species (Conlon et al., 2011). The skin specimens occupying the Mogollon Rim ofcentral Arizona suggest that the two calls, this population is referred to as to isreferred population calls, this reports involving molecular genetics studies and analysis ofthe evolution of advertisement the westernpopulation represents aseparate that proposed has been It 2011). Frost, etal.,2010; Harper 2000; (Rodel, Africa west and “ in peptide(s) magainin of the presence that thatitispossible so 2012b) of activity the reported antimicrobial However, expressed, three amino acid differences from the gene encoding magainin-relateda peptide (referred to asmagainin-ST1) that would bear, if search ofthe from tetraploid the purified was recently closely related to X.borealis to related closely X.muelleri the in (b) twoPGLa members aredetectedin not conspecific because(a) noorthologous peptide hasthe same amino acidsequence, and 2011). Comparison of the AMP profiles of the two frog populations suggests that they are chiricahuensis from secretions sp skin in peptides are separatespecies (Conlon etal., 2010c). frog leopard lowland conspecific (Conlon etal.,2010b) tagoi tagoi Rana that to demonstrate thesis this for the separate species designation of S. S. S. epitropicalis S. in - than magainin the gene for the 1” - including new tetraploid new tetraploid 1”secretions may havebeen The second example ofan incompletely ch The ei Previous investigations of ranid frogs have usedthe samemethodology described in 5 . Two discontinuous (allopatric) populations populations (allopatric) discontinuous . Two ght octoploid species have complex taxonomic relationships and can bedivided S. tropicalis

occupying regions in southern Arizona and frommorphologically distinct secretions. On the basis ofthe AMP sequences, Lithobates yavapaiensis Lithobates genome database (Hellsten et al., X. muelleri

than to to than 1 , Fig.11). According to this proposed evolutionary scenario, and that the relict leopard Lithobatesfrog relictleopard that the and X. muelleri. X. muelleri. ecimens ofthe Chiricahua leopard frog Silurana X. new tetraploid West. West. X. muelleri (Measeyetal.,2004 West X. muelleri S. epitropicalis epitropicalis S. 182 On the other hand, the presence of identical lineage gave rise to species with differential withdifferential riseto species gave lineage , although closely related phylogenetically, closelyrelated phylogenetically, , although species (Measeyetal.,2011). In previous missed. An alternative explanation may be magainin-SE1 is very weak (Conlon et al., etal., is (Conlon very weak magainin-SE1 ters oftheal., same species(Jackway et and Therefore, the data provide strong support support strong provide data the Therefore, S. epitropicalis S. aracterized species isthe frog which is West secretions while PGLa is lacking PGLaislacking while secretions West Rana tagoi okiensis okiensis Rana tagoi 1 (Evans etal.,2004;Tobias etal., X. muelleri (Conlon et al., 2012b). A BLAST 2010) reveals the presence ofa Crinia ortholog (magainin-SE1). X. muelleri X. muelleri , occupy ranges in east east in ranges occupy Litoria, ) that is analysed in ) thatisanalysedin S. tropicalis are probably not and

West onca Lithobates Lithobates Uperoleia . and the the and

is more andin Chapter 8

are ” subgroup) ” subgroup) X. lenduensis X. andrei (Conlon et al., (Conlon et vestitus-wittei X. amieti X. amieti and ) and 4 anisms thatanisms lead to retention, followed by secretions confirm thesecretions confirm particularlyclose of duplicated genes suggesting evolutionary evolutionary suggesting genes of duplicated (Evans et al., 2008; Evans et al., 2011). The 2011). al., et Evans 2008; al., et (Evans three evolutionary fates for a duplicated gene three evolutionary 183 (which belongs to the “ the to belongs (which imary structures between AMPs from from AMPs structures between imary (Chapter X. andrei (Chapter A priori Xenopus per se will not lead to an increase the in complexity of suggest that there have been at least three independent independent three least at been have there that suggest nd hybridization in Xenopodinae Xenopodinae in nd hybridization ” subgroup. More detailedthe analysis mitochondrial of X. lenduensis X. X. itombwensis vestitus-wittei share a common octoploid ancestor that differs from the octoploid octoploid the from differs that ancestor octoploid a common X. andrei share and Effect of polyploidization a polyploidization Effect of carry large numbers All extant vertebrates Gene or genome duplications duplications Gene or genome allooctopolyploidization events in ancestor of the “ of ancestor X. amieti and DNA and gene sequences of RAG1 and RAG2 of the newly identified octoploids octoploids identified newly the of RAG2 and of RAG1 sequences gene and DNA X. lenduensis pr in differences 2010a). observed The species the sister-pair from orthologs and 2012) al., et (King consistent with the fact that results from ourworkresults on AMPs in from skin between relationship phylogenetic originated from a different octoploid ancestor. However, further analysis is necessary to necessary is analysis further However, ancestor. octoploid a different from originated species. octoploid different the between relations the clarify adoption of new function, versus loss of duplicated genes have broader implications at both both at implications broader have genes duplicated of loss versus function, new of adoption level. evolutionary an and a genetic are proposed (Lynch and Katju, 2004): (a) nonfunctionalization which involves deletion of deletion involves which (a) nonfunctionalization 2004): Katju, and (Lynch are proposed which in subfunctionalization (b) a psuedogene; into degeneration its or gene daughter the (c) and role, biological same the share and retained are genes daughter and mother the neofunctionalization in which the mother anddaughter genes are retained but the daughter massive by followed are events WGD Usually, role. biological a new adopt to gene evolves mech the However, et al., 2002). (Gu gene loss conservation of genes that arise through local, regional or global DNA duplication events events duplication DNA global or regional local, through arise that genes of conservation (Lynch and2000). Conery, Thehypothesis 2R – influenced initially by studiesonHox the occurred (WGD) duplications genome whole of rounds two that postulates - gene clusters in relatively rapid succession, perhaps within 10 million years, sometime very early in MYA 550-600 approximately of amphioxus divergence after but evolution vertebrate (Escriva et al., 2002; Furlong andHolland, 2002; Lundin2003).et al., furtherA roundof WGD in the main branch of the vertebrate lineage that produced all the teleost fishes (approximately 320-350 MYA)(Robinson-Rechavi et al., 2001). In additionWGD to the postulatedhypothesis,2R the have in there been duplication events involving multiple large of duplications segmental genes, individual of translocations and duplications 2001; Martin, 1999; (Hughes, chromosomes whole perhaps DNA and of stretches 2001). et al., Robinson-Rechavi life forms but they are an important evolutionary strategy by which organisms acquire organisms which by strategy evolutionary are an important they but forms life phenotypic diversity (Ohno,1970). Chapter 8 to its unduplicated relative relative itsunduplicated to 1991). (Tymowska, Martino and Sinsch, 2002) and ploidy reaches one of its highest levels -dodecaploidy family dopolyploids comprise the majority ofthe species (Duellman and Trueb, 1994; a diploid background (severalmillion years) (Lynch and Conery, 2000). Only in the Pipidae in genes duplicated of lifetime typical the than same isolder Atthe time,it duplication. 2R recent than the teleost-speci XPF families is no greater in the tetraploids tetraploids greaterinthe no XPF familiesis Conlon et al., 2012b). The multiplicity ofthe peptides belonging to the PGLa, CPF, and to datearecompared inTable4(Chapters events. polyploidization more one or genes following duplicated of systemin excellent an constitutes Xenopodinae tetraploid tetraploid octoploid in the greater significantly XPF is not families in the diploid Evans etal., 2005). occurs via incorporation of premature stop codons and frameshiftmutations (Evans, 2007; paralogs of RAG1/RAG2 latterexample,degeneration the immunity. In DNA inadaptive encoding RAG1 and RAG2 proteins crucial to theprocess ofsomatic rearrangement of case ingenes the of also and etal., 2009) Pasquier Du etal.,2002; (Sammut genes dodecaploid mostcommon fateof duplicated AMP genes following polyploidizationevents. The duplication of the genome in the ancestor of the Xenopodinae is significantly more more issignificantly ancestor the Xenopodinae of the in ofthegenome The duplication The distribution AMPs of The distribution secretions in skin A similar high level of gene deletion and/or gene silencing in octoploid and Xenopus Xenopus S. tropicalis S. species. The datasuggest, therefore, X. laevis species is seen among major histocompatibility complex (MHC) complex among isseen major species histocompatibility . Similarly, the multiplicity of the magainin, PGLa, CPF, and PGLa,CPF, magainin, and the of . multiplicity the Similarly, has retained ~50% of the duplicated gene loci when compared S. tropicalis S. fic duplication and the ancient vertebrate specific . Because the taxonisso well defined, the 184 S. epitropicalis S. 2 - 5 from all species from Xenopodinae studied studied from Xenopodinae fromallspecies ; Conlon et al., 2010a; King et al., 2012; which to study the evolutionary trajectory trajectory evolutionary the to study which that nonfunctionalization is the isthe nonfunctionalization that and“ Xenopus S. new tetraploid 1” than than 1” new tetraploid speciesthanin the Chapter 8

6 9 9 5 9 X. amieti and the the and tions in these studies S. aureus 1 6 2 11 2 6 1 0 3 1 0 3 4 2 4 2 4 2 0 2 1 2 0 2 4 1 4 2 ated from skin secre ated ry weak growth-inhibitoryactivity may have 185 2 2 2 2 2 2 Magainin PGLa CPF XPF Total ), and the two PGLa peptides (PGLa-B1 and PGLa-B2) PGLa-B2) and (PGLa-B1 peptides PGLa two and the ), 4 727 2 72 1 2 2 4010 4 36 3 368 2 1 0 5 1 2 36 365 1 2 0 2 72 207 3 3 1 0 406 2 3 1 0 36 36 el (2n) ) have moderately well conserved primary structures and show show and structures primary conserved well have moderately ) 2 Ploidy lev

(Chapter (Chapter West 36 1 2 3 1 7 ensis Multiplicity of the AMPs present in skin secretions of diploid, tetraploid, and borealis borealis alis alis tidomic analysistidomic does allow not discrimination betweendeletionduplicated of the

maeus maeus elleri lleri X. mieti Pep The Xenopodinae provide examples of subfunctionalization among the AMPs. As AMPs. the among of subfunctionalization examples provide Xenopodinae The new tetraploidnew 1” (Chapter X. lendu X. andrei X. andrei X. laevis X. laevis X. petersii X. pyg S. epitropicalis X. bore X. mu X. a X. clivii X. mu S. tropicalis S. “S. new tetraploid1”“S. Table 4. octoploid frogs of the Xenopodinae. necessarily represent the full complement of expressed AMP gene products as components as components products gene AMP of expressed complement full the represent necessarily ve with and/or abundance low very in present been missed. from and CPF-AM4) (CPF-AM1 peptides CPF two 5, the Table in shown from CPF-SP3) and (CPF-SP1 peptides CPF two the 2010a), al., et (Conlon “ from comparable potencies against the Gram-positive bacterium gene from the frog genome and silencing at the level of transcription or translation. translation. or transcription of level the at silencing and genome frog the from gene it isSimilarly, not claimed thatAMPs isol the Chapter 8 Th from Xenopodinae. 5. Table the organism against environmental pathogens. Gram-negative bacterium CPF-SP1 PGLa-AM2 CPF-AM4 CPF-AM1 Peptide PGLa-AM1 PGLa-AM1 PGLa-B1 CPF-SP3 PGLa-B2 PGLa-B2 e basicresiduesinthepeptides,ssentialforpotentantimicrobialactivity,are showninboldtype. Examples ofsubfunctionalization and possible neofunctionalization among AMPs GMASTAGSVLGKLAAVAIGAL. GMAS GMAS GMAS GFLGSLLKTGL GFLGPLLKLGLGVAVLPHLIPSRQQ GLGSLVGNAL GLGSVLG Amino acidsequence KVAVAL KAGSVLG KAGSIVG KAGTIAG . They appear to be sharing the responsibility of protecting of the responsibility sharing to be appear E. coli.They K AL R K K KIA IGA KIA IGANLL.NH VGSNLL.NH KLL.NH K KTAI IALGAL.NH 186 KLAL.NH K 2 2 2 AAL.NH NH 2 2 2 2 >200 256 12.5 E. coli MIC 12.5 12.5 25 550 25 25 50 25

>200 12.5 S. aureus MIC 25 25 6 Chapter 8 ) was ) was 7 Ile have have → Ile 19 were present in were present are interpreted as are interpreted ) without increasing increasing ) without 1 ong the AMP genes in genes in ong the AMP muelleri

ophobicity, stability of the ophobicity, X. may have been an important important an been have may X. vestitus → Thr andLys female frogs (Chapter (Chapter frogs female

5 ) to study the mode of inheritance of inheritance mode the study ) to and 5 Xenopus ) is determined interactions bycomplex 1 species (Table 3) suggests the conclusion the conclusion 3) suggests (Table species le 5). This suggestsle the possibility of ast to the Mesozoic (Late Jurassic around (Late Jurassic to the Mesozoic ast pathicity promotes interactionbetween the membrane of prokaryotes, and so generally generally so and prokaryotes, of membrane these hybrids, like the parent species, are are parent species, like the hybrids, these een species (reviewed in Evans, 2008). Skin 2008). Evans, in (reviewed een species t in the hybridshavepresumed to and are ., 2007a). However, in the case of the PGLa PGLa of the case the in 2007a). However, ., (Chapter (Chapter hybridsgenerated in the laboratory represent a neofunctionalization am neofunctionalization e, comparing the total number of AMPs in the the in AMPs of number total the e, comparing 187 Xenopus X. muelleri x X. laevis ). In addition, three novel AMPs, not detected skinin 7 X. muelleri ity (net positive charge), hydr charge), ity (net positive ongst representatives of ion and AMP multiplicity ion and AMP multiplicity of these frogs. Thus, X. wittei frogs. Thus, of these X. laevis and X. , the aminoX. amieti acid substitutions, the Lys X. as four laevis as well out of AMPs six from In order to appreciate the possibility of the possibility appreciate order to In Interspecies hybridizat Interspecies Polyploidization in clawed frogs occurs through mainly allopolyploidization – genome The origin of the Pipidae dates back at le dates the Pipidae of origin The -helix,degree andof amphipathicity (Conlon et al., 2007a; Powers and Hancock,2003). among four parameters: cationic four parameters: among subfamily Xenopodinae,subfamily necessarydiscuss to it is structure-activity relationships the among (Chapter AMP of an The potency products. gene α Increasing cationicity while maintaining amphi cell charged negatively more the and peptide et al (Conlon potency antimicrobial increases from peptides compared to that of compared the total amountofgenetic information as tetraploid= 36)(2n (Koroma2011). et al., It was confirmed that the full of complement AMPs from of AMPs. It is possible that the interspecies interspecies that the possible It is AMPs. of model of ancient allopolyploidization events in the lineage (Chapter neofunctionalization with PGLa-AM2 having adopted a new biological role but the nature nature but the role biological new a adopted having PGLa-AM2 with neofunctionalization ofnew this function is currently unknown. duplication associated with hybridization betw secretionAMPs profile from hybrid resulted in a selectiveresulted decrease a in in cationicity which turnhas in resulted complete in disappearance of antimicrobial activity (Tab secretions of the hybrid (Chapter presen were species, parent secretions of the Therefor of hybridization. arisen as a result hybrids, the tetraploid from with those factor in the evolution that interspecies hybridization leads to increased multiplicity of in AMPs skin the secretions. San 2010; Mauro, San 2007; al., et Roelants 2005; Bossuyt, and (Roelants MYA) 150 Mauro et al., 2005). The extensive fossil record that spansabout 90 million the(from years of Pipidae population movements large scale that et al., 2005) indicates Cretaceous) (Trueb, of periods different during highlands African Central the in occurred probably have species observed am Sympatry time. Chapter 8 occurring AMPs has been established in the laboratory (Conlon et al., 2007a). It involves hemolytic. Asuccessful strategy for structuralmodification of promising naturally analogues with improved antimicrobial potency against MDR pathogens that are non- be the subject of extensive structure-activity relationships studies aimed at generating They forwill drug templates design. targeted good so represent and activity antimicrobial -2B and -3B realized. AMPs yetto be skin has offrog potential therapeutic the Consequently, practice. clinical pathogenic fungi, no anti-infective peptide based upon their structures has been adopted in certain against resistant bacteria and ofantibiotic strains against activity potent displaying threat addressthe growing will help therapeutics research: future for directions following from the Pipidae frogs family suggest County Calaveras p’ints about that frog that’s any better’n any other frog” in evolution of the Xenopus the in role important an may play fertilefemale hybrids partially fertile.The or fully In interspecies crosses themales areconsiste the greatersex typicallysuffers heterogametic that the to Haldane’s rule exception an represents genus this that out pointed As 1996). etal., (Kobel absent to be found aregenerally incompatibilities gametic and laboratory the in the the wild, in ). etal.,2000 Fischer 1996; Yager, there have been relatively few reports of conclusive evidence islacking. allopolyploids, the products of interspecies hybridization (Tinsley and Jackson, 1998) but The CPF-B1, CPF-C1, CPF-MW1, CPF-SP1 peptides, aswell ashymenochirin-1B, Development of frog AMP-based anti-infectives asa platform for next-generation development intoth Design of analogues of the CPFs an polyploid of secretions AMPs on skin from work fromthe gained newinsights The More than 150 years have passed since Mark Twain penned the words: “I don’t see no Future perspectives Many present day representatives of Xenopus mainly amongst members of the

that are (1867). Now we actually do know why some frogs are better than others. females are heterogametic and males are homogametic, it has been been males ithas arehomogametic, and femalesare heterogametic

erapeutic agents highlighted in the Discussion (Table 1) exert broad spectrum frogs. frogs. dysfunctional effects of hybridization (Malone et al., 2007). In contrast,many Xenopus naturally occurring hybrids in the genus genus in the hybrids occurring naturally 188 laevis andthe ntly found to be sterile and the to the befemales sterileand are found ntly d the hymenochirins with potential for d thehymenochirinswithpotential of multidrug-resistant infections. Despite Despite infections. multidrug-resistant of and Xenopus Silurana The Celebrated Jumping Frog of muelleri species can behybridized in live in sympatry. in However, live group ( Picker, 1985; Xenopus

Chapter 8 models of of models and using in vivo using and in vitro provides stability againstproteases. d properties of the best analogues of the of analogues best of the properties lly or totally resistant to proteolysis without of new therapiesof new to counter the increasing rnative strategy is to select unnatural amino sed by tumours becoming unresponsive to unresponsive becoming by tumours sed ons associated with increased susceptibility severely limitsseverely their usefulness for systemic oteases, at substitutions acids D-amino by with decrease in helicity andhydrophobicity -helicity, alsobe tend than less hemolytic to α lls compared withnon-neoplastic cells. Anti- 189 -dimethylated aminoacid and its incorporation into a α , α -aminoisobutyric acid (Aib) (Conlon et al., 2007b) and ornithine (Strom et (Strom ornithine and 2007b) al., et (Conlon (Aib) acid -aminoisobutyric α Cancer chemotherapy faces challenges po challenges faces Cancer chemotherapy Another worthwhile avenue for future exploitation is the evaluation of CPF peptides peptides CPF of evaluation the is exploitation future for avenue worthwhile Another A major obstacle to the development of effective peptide-based anti-infective drugs is The conjugation a fatty acid at of moiety the N-terminus of peptide scaffolds hasbeen todue infections to a compromised immune and/or system impaired woundhealing, the combined antimicrobial andantitumor or insulin-releasing activity of these peptidesmay prove particularly helpful in the development health burden of diseases. the andhymenochirins, theanalogues,well as their for antitumour as and insulin-releasing potential. Since cancerdiabetes and are conditi peptide introduces conformational constraints. As a result, Aib substitution leads to stabilization of an existinghelical conformation an selective increase in cationicity concominant in the transformationnaturally of the occurring intoAMPs non-toxic therapeutic agents. pharmacokinetic efficacy and the Subsequently, naturallyoccurring peptides will be investigated both for need urgent an is There 2011). Tiwari, and (Chen agents used anti-cancer commonly exert that profiles toxicological and pharmacokinetic appropriate with drugs of types new preferential cytotoxicity towards tumour ce cancer activityhas of beenAMPs studiedin much less detail but it has been shown that diseases caused by MDR pathogens. pathogens. MDR by caused diseases their half-lives short in the circulation which applications.Peptides lacking stable secondary structures are susceptibleproteolysis to by a been have approaches al., 2002). Several et (Schmidtchen proteases range of microbial developed increase stability to the of peptide analogues whichproteolytic enzymes towards involve couplingpeptidesof the to fatty acids and substitutions of L-amino acidsby et Tossi 2007b; al., et Conlon 2006; al., et (Chen acids amino unnatural or acids D-amino al., 2012). demonstrated to improve significantly antimicrobial activity (Shalev2006; et al., Jerala, 2007) andknown is alsocompensate to for a loss of hydrophobicity withinpeptidic the chainbased on amino residue acid substitutions (Malina and2005). Shai,only natural As prof the residues acidL-amino are targets susceptible sites wouldrenderpeptide the partia lossof antimicrobial activity (Chen et al., 2006; Conlon and Sonnevend, 2011; Conlon, et by decreasing D-stereoisomers, al., 2012d). al., 2003).Aib is a non-chiral, their L-counterparts (Papo et al., 2002). An alte An al., 2002). (Papo et their L-counterparts as acids, such Chapter 8 and previo ofthe those with compared willbe they structures areknown, their primary willbe Once determined. species X. itombwensis sister group with approach usedinChapters in silenced of the electrophoretic patterns ofthe albumins reveals thatmore globin genes have been events are suggested (Evans et al., Based onmolecular analysis ofthe mtDNA, at Two other tetraploids similar to similarto tetraploids other Two for th patterns similarelectrophoretic display and species with the other members of the Xenopodinae. AMP secretory profile of The incomplete. isstill species oftheir status (EvansCharacterization etal.,2004). 2 and 1 X. fraseri of Xenopodinae being more closely related to related closely more being Xenopodinae of monophyletic group with tetraploid may show increased potency ther and potency increased may show (Burki and Fischberg, 1985). the rest of the species, may haveevolved as aresult of an independent genome duplication genus in tetraploids the expression of ofglobin evolution the of studies comparative detailed X. boumbaensis X. borealis concentrations aslow as 3 x 10 particularly high potency, producing a significan diabetes The CPF peptides 2013). Consequently, these compounds show pote BRIN-BD11 etal.,2012e). cells (Conlon tumour target selectively that analogues more potent design to template agood represent alyteserin-2a, isolatedfr The taxonomic relationship between the octoploids Based on advertisement calls and/or molecular advertisement callsand/or characters, Based on Further studies to elucidate taxonomic relationships among frogs from the Pipidae Similarly,many frog skin peptides have been shown to stimulateinsulin-release from X. lendu Xenopus and , X. vestitus X. clivii

clonal ensis S. tropicalis suggest that that suggest and , and the dodecaploid X. lenduensis (Chapter , X. wittei -cells at low concentrations that are not toxic to the cells (Ojo etal., thecells(Ojo to toxic are that not concentrations β-cells atlow X. muelleri than in X.wittei than usly characterized ort om skin secretions fromom skin X. fraseri X. fraseri are morphologically similar, e.g. possess equal number of claws, 2 X. fraseri X. , and the , other half with from skin secretions of - X. fraseri 4 5 which shares common ancestry of half of their genome with ; Conlon et al., 2010a; King King etal.,2010a; ; Conlon , the full complement of of AMPs inthese skin secretions of complement full , the X. fraseri West and “ -11 will provide insight into the taxonomic relations of this of relations taxonomic the into insight will provide 2011). According tothescenario, X. pygmaeus efore greatertherapeutic potential. , despite sharing the the same chromosome sharing with , number despite (Burki and Fischberg, 1985). By applying the same may occupy an anomalous position in the evolution evolution the in position anomalous an occupy may M (Srinivasan et al., 2013). CPF peptides from X. ruwenzoriensis have been identified and referred as 190 S. hologous peptides from new tetraploid 1”, described Chapters in leastthreeindependent Silurana eir albumins (Burki and Fischberg, 1985). t increase in the rate of insulin release at release at the rate in of insulin t increase ntial for treatment of patients with Type Type 2 with patients treatment of for ntial , the octoploids amieti X. (ConlonA. obstetricans et al., 2009a), X. amietiand X. laevis and X. vestitus and than the other tetraploids. Both Both tetraploids. the other than (Kobel, 1996).However, more et al., 2012). Anexpected X. andrei S. epitropicalis S. X. fraseri X. andrei, X. amieti allooctoploidization X. wittei X. vestitus , and X. andrei . Comparison X. is complex. iscomplex. cf. fraseri cf. formsa forms a show 2 - 5 , ,

Chapter 8 increases the the increases . Moreover, the the . Moreover, . Since this genus is and X. ruwenzoriensis X. muelleri extend the study on effect of and, unexpectedly, AMPs are AMPs unexpectedly, and, Hymenochirus outlines the importance of 1 and are dodecaploid (the highest level of level highest (the dodecaploid are Pseudhymenochirus (Evans et al., 2004; Bewick et al., 2012), it et al., 2004; Bewick (Evans to address ambiguities regardingorigin the of proposed to analyze the skin secretions from secretions the skin analyze to proposed 191 X. longipes common to all the members of the genus, other other of the genus, to all the members common and Pseudhymenochirus Pseudhymenochirus ith different chromosome sets. Consequently,differentchromosome anith alternative Pipa studied to-dateP. pipa is ), they represent a good model to to model a good represent they ), belonging to the genus and the other in An genera Pipidae alternative family. explanation is Xenopus on AMPs multiplicityon in Inofsecretions. the skin light presented findings in evolved independently and are considered to be products of such such of products be to considered are and independently evolved

, it is expected that theof numberwill not AMPs the be substantially different 4 . This includes female dorsal skin brooding and AMPs may be toxic to the eggs. eggs. the to be toxic may AMPs and brooding skin dorsal female includes This . Insight into the mode of inheritance of AMP genes in interspecies hybrids in interspecies genes of Insightof inheritance the mode AMP into X. laevis and male female between hybridization Interspecies Investigations polyploidizationof the link between multiplicityand of AMPs The evolutionarydescribed scenarioChapter in Relationships between different taxa in the subfamily Pipinae have been much less less much been have Pipinae subfamily the in taxa different between Relationships Pipa ploidy observed in in observed ploidy taxonomically closely related to Hymenochirus of thecomparison A of P. merlini will containsecretions AMPs. the skin is expected that ofstructures putative peptides these with hymenochirins the will provide insightinto the between relationship taxonomic from thegenes number will havefrom tetraploidsbeen AMP in and as many octoploids, interspecies of as a model considered are dodecaploids the However, silenced. hybridizationparents between w Chapter gene dosage gene (although outcome less likely) is a possibility: anAMPs which increasednumber of include components. novel with together parents both with common peptides X. longipes X. ruwenzoriensis Since these species. multiplicity of theleads AMPs and to expressionofnovel peptides in skin the secretionsof allopolyploidization events between different octoploid and tetraploid ancestors (Evans et (Evans ancestors tetraploid and octoploid different between events allopolyploidization al., 2004). Furtherinvestigations are required allopolyploidization for the speciation of Xenopodinae. Both Both Xenopodinae. of speciation the for allopolyploidization absent from skin secretions (Conlon et al., 2009b). In order to answer the question whether whether question the answer to order In 2009b). al., et (Conlon secretions skin from absent or is species-specific the lack of AMPs is is temptingwill be analyzed.species to It speculate that the AMP genes have evolved after of Pipa divergence the behavior reproductive unusual the by necessitated genes AMP the of nonfunctionalization of only representative of genus of genus representative only the single species P. merlini the single species outcome of this research be will a better understandingofthe evolutionary history of these frogs differentiation andpermit may between the alternative hypotheses. studied than thoseXenopodinae. in the It is Chapter 8 which the mother is either mother iseither the which Usin 2007). etal., Evans, 2005; Evans al., 2004; the this finding might be species that the but possibility increase expected An in is peptides. AMP multiplicity the novel of maternal and paternal genomes onthe mode ofinheritance ofthe AMP genes; and 3) origin the of 2) influence frogs; hybrid of secretions skin AMPsin the of multiplicity 1) to: complement of AMPs is stably transferred into the the complement next AMPsisstably of into generation. transferred is the to whether be answered novel main question The (F2 willbe generation) undertaken. and X. muelleri the frommating the offspring AMPs the are of in fertile, analysis hybrids female the addressed.Since be to needs peptides the novel AMPsincluding of complement investigation. further needs also species parent the than environment the in microorganisms pathogenic by invasion against protected better will be peptides, active novel AMPs,including complement of The secretions. the skin in peptides novel parent present in the hybrid environment. factors ence transcription from toof other re-activated the the pres expression will be due after genome duplicationstakeplace(Chapter and Michalak, 2008; Koroma, et in the cytosine methylation pattern and histone modifications (Chain et al., 2008; Malone Xenopus X.laevisxborealis female as such hybrids female F1frogs (Chapter muelleri Dramatic genetic andepigenetic reconfigur The issueofwhether epigenetic changes will pl background such as: mis-expression and novel gene expression pattern, or changes changes or pattern, gene expression novel and as:mis-expression such background lineage and are thought to beevolutionary equidistant from X. laevisx 7 X. laevis ). It is proposed to extend this research by studying other species -specific is notexcluded. -specific X. borealis al.,2011). As many AMPgenesina species aresilenced or X. borealis hybrids with either of their parental species parental oftheir with either hybrids 192 This mechanismmay explainthe presence of 4 . Both ), it seems reasonable to speculate that their that speculate to ), itseemsreasonable hypothesis that a hybrid with a greater a agreater that hybrid with hypothesis ations are known to occur in the hybrid , would provide more information relating g both directions of interspecies cross, in ay a role in the inheritance of the full the full of inheritance the in ay arole X. borealis and X. muelleri X. laevis(Evans et X. laevisx belong to to belong Chapter 8

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Summary

The emergence of pathogenic bacteria and fungi resistant to commonly used antibiotics poses a serious threat to public health and necessitates novel treatment approaches in order to control infections. Antimicrobial peptides (AMPs) are one of the central components of the system of innate immunity and due to their non-specific and highly destructive mechanism of killing, pathogens will develop resistance at lower rates than conventional antibiotics. Skin secretions of frogs from the family Pipidae are a rich source of AMPs which show potential for development into therapeutic agents. Until recently, the only representatives of the Pipidae family frogs from which dermal AMPs had been identified were the diploid frog Silurana tropicalis, the tetraploid frog Xenopus laevis and the octoploid frog Xenopus amieti. Therefore, this program of research was undertaken with the aim to isolate, purify and characterize AMPs with therapeutic potential from skin secretions of other polyploid species of African clawed frogs of the Pipidae family. Emphasis is given to the application of the AMPs as markers to elucidate the taxonomic relationships and evolutionary history of the frogs. The study also investigates the effects which polyploidization and interspecies hybridization have had on the multiplicity of AMPs in frog skin secretions. Chapter 2 and Chapter 3 present data from the peptidomic analysis of norepinephrine- stimulated skin secretions of two well-characterized and closely related tetraploid Xenopus species – X. borealis and X. clivii. Multiple peptides with varying degrees of antimicrobial activity were isolated. Structural characterization demonstrated that they were orthologous to magainins, peptide glycine-leucine-amide (PGLa), caerulein-precursor fragments (CPFs) and xenopsin-precursor fragments (XPFs), previously isolated from S. tropicalis, X. laevis and X. amieti. CPF-B1 and CPF-C1 were the most abundant AMPs in the skin secretions of X. borealis and X. clivii respectively. CPF-B1 (GLGSLLGKAFKIGLKTV GKMMGGAPREQ) was active against clinical isolates of the hospital-associated pathogens, methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Acinetobacter baumannii (MDRAB) with MIC = 5 µM and showed low hemolytic activity against human erythrocytes (HC50 >200 µM). CPF-C1 (GFGSLLGKALRLGANVL.NH2) also showed potent activity against a range of Gram-negative bacteria. CPF peptides, therefore, represent promising candidates for development into therapeutic agents for use against these emerging antibiotic-resistant pathogens. The genera Silurana and Xenopus are united in the subfamily Xenopodinae and have a complex evolutionary history. Chapter 4 includes data from the peptidomic analysis of skin secretions from an incompletely characterized tetraploid species termed “S. new tetraploid 1” with chromosome number 2n=40 and the octoploid species X. andrei (2n=72). The species represent model systems in which to study of the fate of duplicated AMP genes following putative allopolyploidization events. Multiple peptides belonging to the PGLa, XPF, and CPF familes were identified. The primary structures of the AMPs from X. andrei indicate a

205 close phylogenetic relationship between this species and the previously studied X. amieti. Three CPF peptides from “S. new tetraploid 1” showed potent, broad-spectrum antimicrobial activity and are present in high abundance. In contrast, only a single CPF peptide was isolated in low yield from the X. andrei secretions. There is no increase in the multiplicity of the AMPs in skin secretions of “S. new tetraploid 1” and the octoploid X. andrei when compared to the diploid S. tropicalis (2n=20) and the tetraploid X. laevis (2n=36). It is concluded that nonfunctionalization (gene silencing) has been the most common fate of duplicated AMP genes following polyploidization in the Silurana and Xenopus lineages. AMPs constitute a characteristic ‘‘fingerprint’’ of a particular frog species that may be used for an unequivocal taxonomic classification. Two populations of the tetraploid X. muelleri, occupying separate non-contiguous ranges in east and west Africa, are studied in Chapter 5. Their taxonomic relationship is unclear and it has been proposed that the western population represents a separate species referred to as X. muelleri West while the eastern population retains the original name X. muelleri. A comparison of the primary sequences of AMPs in skin secretions reveals that no orthologous peptide from the two populations of X. muelleri has the same amino acid sequence. Additionally, the X. muelleri secretions, like those from X. clivii, did not contain a PGLa peptide whereas the X. muelleri West secretions contained two members of this family. The data indicate that X. muelleri West is more closely related to X. borealis than to X. muelleri and so provide strong support for the proposal that X. muelleri and X. muelleri West should be described as separate species. In contrast to species in the subfamily Xenopodinae, frogs from the subfamily Pipinae have not been investigated as a source of AMPs. The AMP profile in skin secretions from Hymenochirus boettgeri as a representative of genus Hymenochirus (subfamily Pipinae) is described in Chapter 6. A novel family of five structurally-related peptides, designated as hymenochirins, was identified. Hymenochirin-1B (IKLSPETKDNLKKVLKGAIKGAIAV AKMV.NH2) is C-terminally α-amidated whereas hymenochirins-2B - 5B have the general structure XKIPX2VKDTLKKVAKG X2SX2 AGAX3.COOH. The most abundant peptide in the secretions was hymenochirin-3B (IKIPAVVKDTLKKVAKGVLSAVAGALTQ). Synthetic replicates of hymenochirin-1B - 4B possess broad-spectrum antimicrobial activity and relatively weak hemolytic activity and so represent potential candidates for development into therapeutically valuable agents against drug-resistant pathogens. The hymenochirins show very low structural similarity with the antimicrobial peptides isolated from skin secretions of S. tropicalis and X. laevis consistent with the proposed ancient divergence of the Pipinae and Xenopodinae. The F1 hybrid frogs X. laevis x X. muelleri represent a model of interspecies hybridization in the Pipidae family that does not result in an increase in ploidy. They are

206 studied in Chapter 7 and the data obtained provide an insight into the mode of inheritance of AMPs. A total of 18 different AMPs were isolated from skin secretions of the female hybrids. In addition to the complement of AMPs from the parent species, three previously undescribed peptides (magainin-LM1, PGLa-LM1, and CPF-LM1) were purified from the secretions of the hybrid frogs that were not detected in secretions from either X. laevis or X. muelleri. Magainin-LM1 differs from magainin 2 from X. laevis by a single amino acid substitution (Gly13  Ala) but PGLa-LM1 and CPF-LM1 differ appreciably in structure from orthologs in the parent species. CPF-LM1 shows potent, broad-spectrum antimicrobial activity and is hemolytic. The data indicate that hybridization increases the multiplicity of host-defense peptides in skin secretions. As the female F1 hybrids are fertile, hybridization may represent an adaptive strategy among Xenopus species to increase protection against pathogenic microorganisms in the environment. The thesis is completed by a general discussion in Chapter 8 of the results and conclusions in Chapters 2-7. The potential of AMPs from skin secretions of frogs belonging to the Pipidae family is reviewed from three different aspects: promising candidates for development into therapeutic valuable anti-infective agents; reliable taxonomic and phylogenetic markers; and tools to study the fate of duplicated genes in Xenopus and Silurana. The interspecies Xenopus hybrids are proposed as a suitable model to perform future studies on the mode of inheritance of skin AMPs.

207

Samenvatting

De opkomst van pathogene bacteriën en schimmels, die resistent zijn tegen conventionele antibiotica vormen een serieus gevaar voor de volksgezondheid en vereist een nieuwe aanpak om infecties in bedwang te houden. Antimicrobiële peptiden (AMPs) vormen een van de centrale componenten in het aangeboren immuun system en door hun niet specifieke en hoogst vernietigende mechanismen, zullen pathogenen langzamer resistentie opbouwen dan bij gebruik van conventionele antibiotica. De huidsecreties van kikkers van de Pipidae familie vormen een rijke bron van AMPs die het ontwikkelen van therapeutische middelen mogelijk zou kunnen maken. Tot voor kort waren alleen AMPs- geïdentificeerd van de kikkerhuid van de diploid kikker Silurana tropicalis, van de tetraploide kikker Xenopus laevis en van de octoploide kikker Xenopus amieti, die allen behoren tot de Pipidae familie. Om die reden werd dit onderzoeksprogramma uitgevoerd met het doel om AMPs met therapeutisch potentiaal te isoleren, te zuiveren en te karakteriseren ten opzichte van de huidsecreties van andere polyploide soorten van de Afrikaanse klauwkikker van de Pipidae familie. De nadruk ligt op het gebruik van AMPs als markers om de taxonomische relaties en de evolutionaire geschiedenis van de kikkers in kaart te brengen. Deze studie onderzoekt de effecten die polyploidizatie en interspecies hybridisatie gehad hebben op het multipliceren van AMPs in de huidsecretie van deze kikkers. Hoofdstukken 2 en 3 presenteren de data van de peptidomische analyse van norepinephrine-gestimuleerde huidsecreties van twee goed gekarakteriseerde en nauw verwante tetraploide Xenopus soorten – X. borealis en X. clivii. Meerdere peptiden met verscheidene antimicrobiële activiteiten werden geïsoleerd. Structuurkarakterisatie toonde aan dat deze ortholoog waren tot magainins, peptide-leucine-amide (PGLa), caerulein- precursor fragmenten (CPFs) en xenopsin-precursor fragmenten (XPFs), die al eerder waren geïsoleerd van S. tropicalis, X. laevis en X. amieti. CPF-B1 en CPF-C1 waren de meest voorkomende AMPs in de huidsecreties van respectievelijk X. borealis en X. clivii. CPF-B1 (GLGSLLGKAFKIGLKTVGKMMGGAPREQ) was actief bij de klinische isolatie van de ziekenhuis-gerelateerde pathogene, methicillin-resistente Staphylococcus aureus (MRSA) en de meervoudig-resistente Acinetobacter baumannii (MDRAB) met een MIC = 5 µM en een lage hemolytische activiteit tegen menselijke erythrocyten

(HC50 > 200 µM). CPF-C1 (GFGSLLGKALRLGANVL.NH2) was ook sterk actief tegen verschillenden Gram-negatieve bacteriën. CPF peptiden representeren daarom een groep van veelbelovende kandidaten voor de ontwikkeling van therapeutische middelen tegen het verschijnen van antibiotica resistente pathogenen. De genera Silurana en Xenopus zijn verenigd in de subfamilie Xenopodinae en hebben een complexe evolutionaire geschiedenis. Hoofdstuk 4 bevat data van de peptidomische analyse van huidsecretie van een onvolledig gekarakteriseerde tetraploide species genaamd “S. new tetraploid 1” met chromosoom nummer 2n=40 en de octoploid species X. andrei

211 (2n=72). Deze soorten vertegenwoordigen een model waarin het van gedupliceerde AMP genen bestudeerd kunnen worden, na een putatieve allopolyploidizatie gebeurtenis. Verscheidene peptiden behorende tot de PGLa, de XPF, en de CPF families werden geïdentificeerd. De primaire structuren van de AMPs van X. andrei wijzen op een nauwe phylogenetische verwantschap tussen deze species en de eerder bestudeerde X. amieti. Drie CPF peptiden van de “S. new tetraploid 1” vertoonden een sterke, breed-spectrum antimicrobiële activiteit en zijn in overmaat aanwezig. Daarentegen werd slechts een enkel CPF peptide en in lage kwantiteit geïsoleerd uit de secreties van de X. andrei. Er is geen toename in de multipliciteit van de AMPs in huidsecreties van de “S. new tetraploid 1” en de octoploid X. andrei ten opzichte van de diploid S. tropicalis (2n=20) en de tetraploid X. laevis (2n=36). De conclusie is dat de meest voorkomende uitkomst van gedupliceerde polyploidisatie in de Silurana en Xenopus geslacht is niet-functionalisatie (gene silencing). AMPs vormen een karakteristieke “vingerafdruk” van een specifiek kikkersoort die gebruikt zou kunnen worden voor een eenduidige taxonomische classificatie. Twee populaties van de tetraploid X. muelleri, die apart voorkomen in twee afzonderlijke en niet aan elkaar verbonden gebieden in oost en west Afrika zijn bestudeerd in hoofdstuk 5. Hun taxonomische relatie is niet duidelijk en er is voorgesteld dat de westerse populatie een aparte soorten vertegenwoordigt, genaamd X. muelleri West, terwijl de oostelijke populatie zijn oorspronkelijke naam X. muelleri behoudt. Een vergelijking van de primaire sequenties van de AMPs in de huidsecreties maakt duidelijk dat geen orthologe peptide van de twee X. muelleri populaties dezelfde serie aminozuren vertoont. Bovendien, bevatten de huidsecreties van X. muelleri, net als die van de X. clivii, niet een PGLa peptide terwijl de X. muelleri West secreties twee leden van deze familie bevatten. De resultaten tonen aan dat X. muelleri West meer gelieerd is aan X. borealis dan aan X. muelleri. Dit het voorstel ondersteunt dat X. muelleri en X. muelleri West het best als twee verschillende soorten kunen beschreven worden. In tegenstelling tot de species in de subfamilie Xenopodinae, zijn de kikkers van de subfamilie Pipinae nog niet onderzocht als een potentiele bron van AMPs. Het AMP profiel in de huidsecreties van Hymenochirus boettgeri als vertegenwoordiger van de genus Hymenochirus (subfamily Pipinae) is beschreven in hoofdstuk 6. Een nieuwe familie van vijf structuur-gerelateerde peptiden, hymenochirins genoemd, is geïdentificeerd.

Hymenochirin-1B (IKLSPETKDNLKKVLKGAIKGAIAVAKMV.NH2) is C-terminally

α-amidated terwijl hymenochirins-2B - 5B de algemene structuur XKIPX2VKDTLKKV

AKGX2SX2 AGAX3.COOH hebben. De meest voorkomende peptide in de secreties was hymenochirin-3B (IKIPAVVKDTLKKVAKGVLSAVAGALTQ). Synthetische kopieën van hymenochirin-1B - 4B bevatten breed-spectum antimicrobiële activiteit en een relatief zwakke hemolytische activiteit en vertegenwoordigen dus potentiele kandidaten voor de ontwikkeling van therapeutisch waardevolle middelen tegen medicijnen-resistente pathogene. De hymenochirins vertonen een zeer geringe structurele gelijkenis met de

212 antimicrobiële peptiden die geïsoleerd waren van huidsecreties van S. tropicalis en X. laevis wat weer consistent is met de voorgestelde oude afscheiding van de Pipinae en de Xenopodinae. De F1 hybride kikkers X. laevis x X. muelleri vormen een interspecies hybridizatie model in de Pipidae familie die geen toename in ploidy initieert. Deze zijn bestudeerd in hoofdstuk 7 en de verkregen resultaten geven een inzicht in de overerving van AMPs. In totaal werden 18 AMPs geïsoleerd van de huidsecretie van de vrouwelijke hybriden. Naast het aantal AMPs, afkomstig van de ouderlijke soorten, werden drie niet eerder beschreven peptiden (magainin-LM1, PGLa-LM1, en CPF-LM1) gezuiverd uit de secreties van de hybride kikkers die niet ontdekt waren in secreties afkomstig van of X. laevis of van X. muelleri. Magainin-LM1 verschilt van magainin 2 van de X. laevis met een enkele aminozuur substitutie (Gly13  Ala), maar PGLa-LM1 en CPF-LM1 verschillen aanzienlijk in structuur vergeleken met hun orthologen in de ouderlijke speciën. CPF-LM1 vertoont een sterke breedspectrum antimicrobiële activiteit en is hemolytisch. De data suggereren dat hybridisatie de multipliciteit van gastheer-verdedigende peptiden in huidsecreties doet toenemen. Aangezien de vrouwelijke F1 hybriden vruchtbaar zijn, zou hybridisatie een aanpassingsstrategie voor de Xenopus soorten kunnen zijn om zich beter te verdedigen tegen pathogene micro-organismen in de hun omgeving. Het proefschrift wordt in hoofdstuk 8 afgesloten met een algemene discussie over de resultaten en de conclusies uit hoofdstukken 2-7. Het potentieel van AMPs afkomstig van huidsecreties van de Pipidae kikkerfamilie wordt beschreven vanuit drie verschillende aspecten: als potentiele kandidaten voor de ontwikkeling van therapeutisch interessante anti-infectie middelen; als betrouwbare taxonomische en phylogenetische markers; en als gereedschap om het lot van gedupliceerde genen te bestuderen in Xenopus en Silurana. De interspecies Xenopus hybride wordt voorgesteld als een geschikt model voor verdere studie in de soort van vererving van dermaal afkomstige AMPs.

213

Acknowledgements

“Ancora imparo. [I am still learning.]” -Michelangelo, at age 87 in 1562

I wish to thank my colleagues, family and friends who have contributed in various ways to the realization of this thesis. Particularly, I am grateful to: Professor J. Michael Conlon, my supervisor, for his: time and effort in explaining the fascinating world of frog antimicrobial peptides; thought provoking discussions of the papers and the thesis; and introduction to the collaborators. I deeply appreciate the constant support provided to me and the experience I have obtained under his guidance in a new research direction. Professor Jerry M. Wells, my promotor, for responding positively to my enquiry and understanding my special circumstances, thereby enabling me the opportunity to defend my thesis. The outstanding collaborators and co-authors: Dr Jay King and Dr Pawel Michalak (USA); Dr Mohammed Meetani (UAE); Dr Koji Takada (Japan); Professor Hubert Vaudry, Professor Thierry Jouene, Dr Laurent Coquet and Dr Jerome Leprince (France) for creating the “butterfly effect” internationally and for fine-tuning the text of the published articles. Professor Wim Lammers for translating – together with his wife Marijke – the Summary into Dutch and for the stimulating talks prior to the defence. Dr Agnes Sonnevend for sharing the ATCC microbial strains, multidrug-resistant clinical isolates, and two of the figures in the Introduction. Ivanna Lizarriturri, a talented graphic designer and a reliable friend in a time of need, for creating the cover page of this dissertation and the invitations for the public defence. Loes Bugter, Trudy Janssen, and Denise Magendans for extending excellent administrative support with a personalized touch. Dr Marjolein Meijerink, who kindly accepted the role of a paranymph with such enthusiasm. My colleagues Eman, Azim, Wasila, Kholoud, Hama, Manju, Akela, Mohammed, Marwa, Annie, Ghada, Waqar, Mahendra, Mansoor, Abdulqadir, Anne and Basheer for creating a friendly atmosphere, making things work as good as possible, and for enlightenment during difficult moments. My exceptional friend Pamelchi, for standing beside me during the happy moments to celebrate and the hard moments to motivate, accepting to be my paranymph, and continuing to be all what I could ask for in a friend; even being predictably irrational. My supporting friends outside science – you know who you are! And finally, my very special appreciation goes to my mom, dad and my wonderful son Ivo for the trust, love and encouragement – you are just the best! Thank you for believing in me! Ivcho, the quote under the acknowledgements is to inspire you. Dear Past, I am grateful for the lessons! Dear Future, I am ready!

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Personalia

Curriculum vitae List of publications

Curriculum Vitae

Milena Mechkarska was born on 9th of October 1969 in Sofia, Bulgaria. She graduated with a gold medal from the National School of Mathematics and Natural Sciences (NSMNS), class Biotechnology in Sofia in 1987 and immediately continued her education in Genetic and Cellular Engineering at Sofia University “St. Kliment Ohridski” (Sofia, Bulgaria). During the course of the study she participated in the research of the Laboratory of Virology under the supervision of the late Prof. Tatiana Varadinova and wrote a thesis entitled “Zinc-containing complexes as a novel therapy for herpes simplex virus type 1 infection”. After receiving her MSc degree in 1992 she specialized further in the field of Virology and Immunology at the Faculty of Biology, Sofia University (Sofia, Bulgaria), the Bulgarian Academy of Sciences (Sofia, Bulgaria) and the International Centre for Genetic Engineering and Biotechnology (New Delhi, India). She gained extensive experience in the field of host-pathogen interactions working on herpes simplex and hepatitis E viral models. She obtained a second MSc in Ecology and Environment from Sikkim Manipal Academy (New Delhi, India) with a thesis entitled “Interfaces between ecology and virology: the SARS model” with Prof. Norbert Nowotny (Department of Microbiology and Immunology, United Arab Emirates University) as a supervisor. Subsequently, she spent four years in the laboratory of Prof. Basel Al-Ramadi (Department of Microbiology and Immunology, United Arab Emirates University) on a project studying the differential activation of murine macrophages by cytokine-expressing Salmonella strains. At the beginning of 2010 she joined the laboratory of Prof. J. Michael Conlon at the Department of Biochemistry (United Arab Emirates University). Her main research focus since then has been isolation and characterization of biologically active peptides from frog skin secretions with particular emphasis on antimicrobial peptides from skin secretions of polyploid frogs from the Pipidae family as the PhD topic. The PhD research, under the supervision of Prof. J. Michael Conlon and Prof. Jerry M. Wells, was completed in 2012 and is described in this thesis. Currently, she is focusing on finding answers to some of the questions proposed in this thesis for future research.

221 List of publications

Directly related to the PhD topic: Mechkarska, M., Ahmed, E., Coquet, L., Leprince, J., Jouenne, T., Vaudry, H., King, J.D., Conlon., J.M., 2010. Antimicrobial peptides with therapeutic potential from frog skin secretions of the Marsabit clawed frog Xenopus borealis (Pipidae). Comp Biochem Physiol 4:467-472. Conlon, J.M., Mechkarska, M., Ahmed, E., Leprince, J., Vaudry, H., King, J.D., Takada, K., 2011. Purification and properties of antimicrobial peptides from skin secretions of the Eritrea clawed frog Xenopus clivii (Pipidae). Comp Biochem Physiol 153:350-354. Mechkarska, M., Ahmed, E., Coquet, L., Leprince, J., Jouenne, T., Vaudry, H., King, J.D., Takada, K., Conlon, J.M., 2011. Genome duplications within the Xenopodinae do not increase the multiplicity of antimicrobial peptides in Silurana paratropicalis and Xenopus andrei skin secretions. Comp Biochem Physiol 6:206-212. Mechkarska, M., Ahmed, E., Coquet, L., Leprince, J., Jouenne, T., Vaudry, H., King, J.D., Conlon, J.M., 2011. Peptidomic analysis of skin secretions demonstrates that the allopatric populations of Xenopus muelleri (Pipidae) are not conspecific. Peptides 32:1502-1508. Mechkarska, M., Prajeep, M., Coquet, L., Leprince, J., Jouenne, T., Vaudry, H., King, J.D., Conlon, J.M., 2012. The hymenochirins: a family of antimicrobial peptides from the Congo dwarf clawed frog Hymenochirus boettgeri (Pipidae). Peptides 35:269-275. Mechkarska, M., Meetani, M., Michalak, P., Vaksman, Z., Takada, K., Conlon, J.M., 2012. Hybridization between the tetraploid African clawed frogs Xenopus laevis and Xenopus muelleri (Pipidae) increases the multiplicity of antimicrobial peptides in the skin secretions of female offspring. Comp Biochem Physiol 7:285-291.

Related to the general research field: Mechkarska, M., Ojo, O.O., Meetani, M.A., Coquet, L., Jouenne, T., Abdel-Wahab, Y.H.A., Flatt, P.R., King, J.D., Conlon, J.M., 2011. Peptidomic analysis of skin secretions from the bullfrog Lithobates catesbeianus (Ranidae) identifies multiple peptides with potent insulin-releasing activity. Peptides 2:203-208. Conlon, J.M., Mechkarska, M., Coquet, L., Jouenne, T., Leprince, J., Vaudry, H., Kolodziejek, J., Nowotny, N., King, J.D., 2011. Characterization of antimicrobial peptides in skin secretions from discrete populations of Lithobates chiricahuensis (Ranidae) from central and southern Arizona. Peptides 32:664-669.

222 Conlon, J.M., Mechkarska, M., Ahmed, E., Coquet, L., Jouenne, T., Leprince, J., Vaudry, H., Hayes, M.P., Padgett-Flohr, G., 2011. Host defense peptides in skin secretions of the Oregon spotted frog Rana pretiosa: Implications for species resistance to chytridiomycosis. Devel Comp Immun 35:644-649. Ojo, O.O., Abdel-Wahab, Y.H.A., Flatt, P.R., Mechkarska, M., Conlon, J.M., 2011. Tigerinin-1R: a potent, non-toxic insulin-releasing peptide isolated from the skin of the Asian frog, Hoplobatrachus rugulosus. Diabetes Obes Metab 13:1114-1122. Zahid, O., Mechkarska, M., Ojo, O.O., Abdel-Wahab, Y.H.A., Flatt, P.R., Meetani, M.A., Conlon, J.M., 2011. Caerulein-and xenopsin-related peptides with insulin-releasing activities from skin secretions of the clawed frogs, Xenopus borealis and Xenopus amieti (Pipidae). Gen Comp Endocrin 172:314-320. King, J.D., Mechkarska, M., Coquet, L., Leprince, J., Jouenne, T., Vaudry, H., Takada, K., Conlon, J.M., 2012. Host-defense peptides from skin secretions of the tetraploid frogs Xenopus petersii and Xenopus pygmaeus, and the octoploid frog Xenopus lenduensis (Pipidae). Peptides 33:35-43. Conlon, J.M., Mechkarska, M., Arafat, K., Attoub, S., Sonnevend, A., 2012. Analogues of the frog skin peptide alyteserin-2a with enhanced antimicrobial activities against Gram- negative bacteria. J Pep Sci 18:270-275. Conlon, J.M., Mechkarska, M., King, J.D., 2012. Host-defense peptides in skin secretions of African clawed frogs (Xenopodinae, Pipidae). A review. Gen Comp Endocrin 176:513- 518. Conlon, J.M., Mechkarska, M., Prajeep,, M., Sonnevend, A., Coquet, L., Leprince, J., Jouenne, T., Vaudry, H., King, J.D., 2012. Host-defense peptides in skin secretions of the tetraploid frog Silurana epitropicalis with potent activity against methicillin-resistant Staphylococcus aureus (MRSA). Peptides 37:113-119. Conlon, J.M., Mechkarska, M., Prajeep, M., Arafat, K., Zaric, M., Lukic, M., Attoub, S., 2012. Transformation of the naturally occurring frog skin peptide, alyteserin-2a into a potent, non-toxic anti-cancer agent. Amino Acids - in press. Attoub, S., Mechkarska, M., Sonnevend, A., Radosavljevic, G., Jovanovic, I., Lukic, M.L., Conlon, J.M., 2012. Esculentin-2CHa: a host-defense peptide with differential cytotoxicity against bacteria, erythrocytes and tumor cells. Peptides - in press. Srinivasan, D., Mechkarska, M., Abdel-Wahab, Y.H.A., Flatt, P.R., Conlon, J.M., 2012. Caerulein precursor fragment (CPF) peptides from the skin secretions of Xenopus laevis and Silurana epitropicalis are potent insulin-releasing agents. Biochimie - in press.

223 Relevant to the research field of host-pathogen interactions: Varadinova, T., Bontchev, P., Nachev, C., Shishkov, S., Strahilov, D., Paskalev, Z., Toutekova, A., Panteva, M., 1993. Mode of Action of Zn-complexes on Herpes Simplex Virus type 1 Infection in vitro. J Chemother 1:3-9. Varadinova, T., Shishkov, S., Panteva, M., Bontchev, P., 1996. Effect of complexes of cobalt with aminoacids on the replication of Herpes simplex virus type1 (HSV-1). Metal Based Drugs 3:149-154. Panteva, M., Varadinova, T., Shishkov, S., Angelova, A., Ivanovska, N., Popova, P., 1996. Immunomodulating effect of zinc complexes in vitro and in vivo. Infectology (bulg.) 3:81- 84. Shishkov, S., Varadinova, T., Panteva, M., Bontchev, P., 1997. Effect of complexes of Zn(II), Co(II) and Cu(II) with D-aminosugars on the replication of Herpes simplex Virus type 1(HSV-1). Metal Based Drugs 1:35-38. Panteva, M., Varadinova, T., Turel, I., 1998. Effect of copper acyclovir complexes on Herpes simplex virus types 1 and 2 (HSV-1, HSV-2) infection in cultured cells. Metal Based Drugs 1:24-28. Panteva, M., Korkaya, H., Jameel, S., 2003. Hepatitis viruses and the MAPK pathway: is this a survival strategy? A review. Virus Res 92:131-140. Moin, S., Panteva, M., Jameel, S., 2007. The Hepatitis E Virus (HEV) ORF3 Protein Increases Expression and Oligomerization of the Mitochondrial Voltage Dependent Anion Channel (VDAC) and Protects Cells from Mitochondrial Depolarization and Death. J Biol Chem 29:21124-21133. Fernandez-Cabezudo, M.J., Azimullah, S., Nurulain, S.M., Mechkarska, M., Lorke, D.E., Hasan, M.Y., Petroianu G.A., al-Ramadi, B.K., 2008. The organophosphate Paraoxon has no demonstrable effects on the murine immune system following subchronic low dose exposure. Int J Immunopath Pharmac 4:891-901. Fernandez-Cabezudo, M.J., Mechkarska, M., Azimullah, S., al-Ramadi, B.K., 2009. Modulation of macrophage proinflammatory functions by cytokine-expressing Salmonella vectors. Clin Immunol 1:51-60. Fernandez-Cabezudo, M.J., Lorke, D.E., Azimullah, S., Mechkarska, M., Hasan, M.Y., Petroianu, G.A., al-Ramadi, B.K., 2010. Cholinergic stimulation of the immune system protects against lethal infection by Salmonella enterica serovar Typhimurium. Immunology 3:388-398.

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Cover photo: Xenopus laevis, kindly provided by Wernher Krutein / photovalet.com Cover design: Ivanna Lizarriturri Design and layout: Milena Mechkarska

Printed by: GVO drukkers & vormgevers B.V. Ponsen & Looijen