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 Worldwide spread of infections caused by multidrug resistant bacteria 1 r A global problem is rapidly evolving and expanding to the point of posing a serious te p threat to public health. This is the emergence of pathogenic bacteria and fungi resistant to commonly used antibiotics, which cause increased morbidity and mortality, and impact Cha heavily on healthcare costs (Livermore, 2009). Antibiotic resistance is a type of drug resistance in which a microorganism is able to survive exposure to an antibiotic. Antibiotics have been considered to be the single most significant discovery in medicine. However, even at the early stage after their discovery, antibiotic resistance had already begun to emerge (Rammelcamp and Maxon, 1942). Bacteria are either non-responsive or resistant to the action of antibiotics by a range of different mechanisms (Fig. 1). Fig. 1. Bacterial resistance mechanisms to antibiotics. (1) Enzymatic degradation as a result of production of proteases; (2) Target alteration such as composition of the membrane, LPS or intracellular molecules; (3) Reduction of permeability by changing trans-membrane potential or modification of membrane fluidity; (4) Over-expression of efflux pumps causing immediate export; (5) Protection of intracellular target; (6) Overproduction of target; and (7) Bypassing the action on target. Key: enzyme (proteases) E ; antibiotic ; intracellular target ; protection ; alteration of target . Kindly provided by Prof. T. Pal (2012). 10 Resistance can arise either by mutations of bacterial genes, or even more frequently by Cha horizontal gene transfer. Antibiotic resistance genes may be mobilized between the same or p even different species by plasmids or transposons or acquired by natural competence for ter 1 transformation by exogenous DNA. Thus, a gene for antibiotic resistance that evolves via natural selection may be shared in or across the population(s). The resistant bacteria are classified into multidrug-resistant (MDR), extensively drug-resistant (XDR) and pandrug- resistant (PDR) categories (Magiorakos et al., 2012). MDR is defined as acquired non- susceptibility to at least one agent in three or more antimicrobial categories, XDR is defined as non-susceptibility to at least one agent in all but two or fewer antimicrobial categories (i.e. bacterial isolates remain susceptible to only one or two categories) and PDR is defined as non-susceptibility to all agents in all antimicrobial categories (Magiorakos et al., 2012). The genes for resistance to antibiotics, like the antibiotics themselves, are ancient (Donadio et al., 2010). Although there were low levels of pre-existing antibiotic- resistant bacteria (Nelson, 2009; Caldwell and Lindberg, 2011), the major factor in development and selection of MDR strains and in sharing of resistance between bacterial species is the widespread use of antibiotics (Hawkey and Jones, 2009). Examples of the misuse of antibiotics include their sales over the counter without a prescription; inappropriate prescription by doctors; addition to livestock feed; and unsound practices in the pharmaceutical manufacturing industry involving release of new antibiotics despite the fact that resistant strains have already been documented. The problem is made worse by the increasing trend for regional and international travel among human populations (Cohen, 1992; Tomasz, 1994; Swartz, 1997). This makes the emergence of antibiotic resistance “the most eloquent example of Darwin’s principle of evolution” (Livermore, 2009). One of the major drug-resistant human pathogens is a strain of the Gram-positive aerobic, non-motile coccus: Staphylococcus aureus. This microorganism is carried by approximately one third of the human population. It causes local infections by colonizing mucous membranes and skin which can, in later stages, develop into systemic infections, such as bacteremia and sepsis in both adults and children. The second group of infections is toxin- and superantigen-mediated diseases (Kurlenda and Grinholc, 2012; and references therein). S. aureus possesses a wide spectrum of virulence factors (Feng et al., 2008; Plata et al., 2009) that enable it to bypass the barriers of the host defence system and it can survive hostile environmental conditions due to its extraordinary versatility and adaptability to antibiotic pressure (Lowy, 1998; Lowy, 2003). The development of antibiotic resistance by S. aureus is summarized in Fig. 2. S. aureus was one of the first bacteria in which penicillin resistance was found (Rammelcamp and Maxon, 1942) and the specific role of penicillinase was subsequently identified (Kirby, 1944; Bondi and Dietz, 1945). This necessitated the clinical use of penicillinase-resistant derivatives such as methicillin as the antibiotics of choice. However, reports describing the emergence of methicillin-resistant S. aureus (MRSA) quickly 11 followed (Jevons, 1961). Since then, MRSA has become endemic in many countries and 1 poses a significant problem worldwide (Kreiswirth et al., 1993). The subsequent usage of r te newer classes of antibiotics also quickly caused appearance of resistant S. aureus strains p (Bozdogan et al., 2003). Thus, apart from being resistant to all β-lactam antibiotics, MRSA Cha may not be responsive to macrolides, clindamycin, fluoroquinolones, tetracycline and gentamicin (Feng et al., 2008). CA-MRSA causes healthcare associated infections First CA-MRSA infections CA-MRSA Several reported from infections new classes Australia and reported from Penicillin Methicillin antibiotics USA each continent 1940’s 1960 1980 1990’s 2012 1940 - 1960 1961 1980’s 1990 - 2000 Spread of First Epidemic MRSA HA-MRSA plasmid- MRSA strains reported infections borne reported from hospitals widespread in penicillinase in the UK many countries Fig. 2. Timeline for the introduction of antibiotics into medical practice. The history of the emergence of methicillin-resistant S. aureus (MRSA) with the subsequent change in epidemiology of MRSA infections is presented in parallel. CA – community-acquired; HA – hospital-acquired. Kindly provided by Dr. A. Sonnevend (2012). The epidemiology of infections caused by MRSA is rapidly changing. Originally, MRSA was associated predominantly with hospital-acquired infections (HA-MRSA) (Chambers and Deleo, 2009; Rosenthal et al., 2010); however, in the past 10 years, community-acquired MRSA (CA-MRSA) has become prevalent. CA-MRSA infections occur in otherwise healthy people who have not been recently hospitalized or had a medical procedure (e.g. dialysis, surgery, catheter) and usually involve skin infections, such as abscesses, boils, and other pus-filled lesions. Frequently though, CA-MRSA