DOTTORATO DI RICERCA IN BIOCHIMICA CICLO XXVI (A.A. 2010-2013) Membrane-active derivatives of the frog skin peptide Esculentin-1 against relevant human pathogens Docente guida Coordinatore Prof.ssa Maria Luisa MANGONI Prof. Francesco MALATESTA Prof.ssa Donatella BARRA Dottorando Vincenzo LUCA Dicembre 2013 DOTTORATO DI RICERCA IN BIOCHIMICA CICLO XXVI (A.A. 2010-2013) Membrane-active derivatives of the frog skin peptide Esculentin-1 against relevant human pathogens Dottorando Vincenzo LUCA Docente guida Coordinatore Prof.ssa Maria Luisa MANGONI Prof. Francesco MALATESTA Prof.ssa Donatella BARRA Dicembre 2013 To my brother INDEX INDEX List of papers relevant for this thesis 1 List of other papers 1 1. INTRODUCTION 3 1.1 Antimicrobial peptides and innate immunity 3 1.2 AMPs: structural features and mechanism of action 6 1.3 AMPs and resistance 13 1.4 Amphibian AMPs 16 1.5 The esculentin family 17 1.6 The 1-18 fragment of esculentin-1b, Esc(1-18) 19 1.7 The 1-21 fragment of esculentin-1a, Esc(1-21) 20 1.8 Candida albicans pathogen 21 1.9 Pseudomonas aeruginosa pathogen 23 1.10 Caenorhabditis elegans: a simple infection model 24 2. AIM OF THE STUDY 27 3. MATERIALS AND METHODS 28 3.1 Materials 28 3.2 Microorganisms and nematode strains 28 3.3 Circular dichroism (CD) analysis 29 3.4 Hemolytic activity 29 I INDEX 3.5 Determination of the minimal peptide concentration inhibiting the growth of free-living microorganisms 30 3.6 Anti-biofilm activity 31 3.7 Time-killing of free-living microorganisms (yeasts and bacteria) 33 3.8 Peptide’s effect on the viability of hyphae 34 3.9 Inhibition of hyphal development 35 3.10 Membrane permeabilization of C. albicans and P. aeruginosa 35 3.11 Scanning electron microscopy (SEM) on planktonic and biofilm cells 38 3.12 Measurement of reactive oxygen species (ROS) in C. albicans 39 3.13 Apoptosis assay on C. albicans 41 3.14 Staining protocols: fluorescent probes for microscopic assessment of peptide’s distribution and fungal membrane integrity 42 3.15 C. elegans infection 43 3.16 Worm survival assay 44 3.17 Peptide’s effect on the number of yeast cells and their switch to the hyphal form, within the nematode intestine 44 3.18 Statistical analysis 45 4. RESULTS 46 4.1 Structural properties of the Esc(1-18) and Esc(1-21) fragments 46 4.2 Anti-Candida activity of Esc(1-18) 48 4.2.1 In vitro activity 48 4.2.1.1 Anti-Candida activity of all-L/all-D Esc(1-18) enantiomers 48 4.2.1.2 Membrane perturbation of both yeast and hyphal forms 50 II INDEX 4.2.1.3 Fluorescence studies 54 4.2.1.4 Inhibition of hyphal development 56 4.2.2 In vivo activity 57 4.2.2.1 Peptide effect on C. elegans survival 57 4.2.2.2 In vivo inhibition of hyphal development and peptide localization 59 4.3 Anti-pseudomonas activity of Esc(1-21) 60 4.3.1 Antimicrobial activity on planktonic cells 60 4.3.2 Anti-Biofilm activity 62 4.3.3 Mode of action 62 4.3.3.1 Membrane perturbation assays and SEM 62 4.4 Hemolytic activity 68 5. DISCUSSION 69 6. REFERENCES 73 ACKNOWLEDGMENTS 91 APPENDIX - REPRINT OF PAPERS 92 III List of papers relevant for this thesis 1. Luca V, Olivi M, Di Grazia A, Palleschi C, Uccelletti D, Mangoni ML (2013a) Anti-Candida activity of 1-18 fragment of the frog skin peptide esculentin-1b: in vitro and in vivo studies in a Caenorhabditis elegans infection model. Cell Mol Life Sci. IN PRESS. 2. Luca V, Stringaro A, Colone M, Pini A, Mangoni ML (2013b) Esculentin(1-21), an amphibian skin membrane-active peptide with potent activity on both planktonic and biofilm cells of the bacterial pathogen Pseudomonas aeruginosa. Cell Mol Life Sci 70, 2773-2786. List of other papers 1. Coccia C, Rinaldi AC, Luca V, Barra D, Bozzi A, Di Giulio A, Veerman ECI, Mangoni ML (2011) Membrane interaction and antibacterial properties of two mildly cationic peptide diastereomers, bombinins H2 and H4, isolated from Bombina skin. Eur Biophys J 40:577-88. 2. Grieco P, Luca V, Auriemma L, Carotenuto A, Saviello MR, Campiglia P, Barra D, Novellino E, Mangoni ML (2011) Alanine scanning analysis and structure-function relationships of the frog- skin antimicrobial peptide temporin-1Ta. J Pept Sci 17:358-65. 1 3. Grieco P, Carotenuto A, Auriemma L, Saviello MR, Campiglia P, Gomez-Monterrey IM, Marcellini L, Luca V, Barra D, Novellino E, Mangoni ML (2012) The effect of D-amino acid substitution on the selectivity of temporin L towards target cells: identification of a potent anti-Candida peptide. Biochim Biophys Acta 1828:652-60. 4. Falciani C, Lozzi L, Pollini S, Luca V, Carnicelli V, Brunetti J, Lelli B, Bindi S, Scali S, Di Giulio A, Rossolini GM, Mangoni ML, Bracci L, Pini A (2012) Isomerization of an antimicrobial peptide broadens antimicrobial spectrum to Gram-positive bacterial pathogens. PLos One 7:e46259. 5. Grieco P, Carotenuto A, Auriemma L, Limatola A, Di Maro S, Merlino F, Mangoni ML, Luca V, Gatti S, Campiglia P, Gomez-Monterrey I, Novellino E, Catania A (2013) Novel α-MSH peptide analogues with broad spectrum antimicrobial activity. PLos One 8:e61614. 2 1. INTRODUCTION 1. INTRODUCTION 1.1 Antimicrobial peptides and innate immunity Antimicrobial peptides (AMPs) are important components of the non- specific innate immunity and form a first line of defense against infection by pathogenic microorganisms. They are widespread in nature, being produced at all levels of the evolutionary scale: in bacteria and fungi, as well as in higher eukaryotes as reported below [Brown and Hancock, 2006]. Schematic representation of some living organisms producing AMPs In animals, AMPs are located at those anatomical sites that easily come into contact with microorganisms, such as the skin and the mucosal or epithelial surfaces of the respiratory, genitourinary and gastrointestinal tracts [Bals, 2000]. They are also mostly found in circulating granulocytic leukocytes. 3 1. INTRODUCTION AMPs are promptly synthesized at low metabolic cost, stored in large amounts into granules for extracellular secretion. Peptide antibiotics differ from the classical antibiotics, which are synthesized by microorganisms and made stepwise, with different enzymes at each step [Boman, 1995]. Indeed, AMPs are encoded by genes and ribosomally synthesized as pre-propeptides. The synthesis of AMPs can be constitutive or inducible by microbial components. However, it depends on the cell type and differentiation state. In animals, including humans, normal flora of bacteria exists on the skin, in the mouth, in the intestine, in the reproductive organs and in the air ways. It is likely that the control of the natural flora is due to a continuous production of peptide antibiotics. The main advantage of AMPs as factors of the innate immunity is that they can function without high specificity and memory. The innate immune system can respond in a matter of hours before the adaptive immunity is activated. The time needed to synthesize and process a pre-propeptide with 100-150 residues is certainly shorter than the time required to produce two heavy and two light immunoglobulin chains, the production of which also requires the differentiation of a memory cell to a plasma cell. This is a very important aspect considering the difference in the growth rate between a bacterium (generation time 20-30 min) and a lymphocyte (24-48 hours of doubling time). Thus, a primary bacterial infection can never be eliminated by a clonally-based mechanism, because this response is not fast enough. Even in a secondary microbial infection (when memory cells are present), it would likely take too long time to mobilize antibodies and/or specific effector 4 1. INTRODUCTION cells to curb a bacterial infection without the help of innate immune components [Boman, 1995]. Overstimulation of innate immunity, however, leads to a syndrome termed sepsis. Innate immunity is triggered when bacterial molecules (for example lipopolysaccharide, LPS) interact with host pattern recognition receptors including Toll-like receptors (TLRs). After TLRs binding, effector mechanisms that assist the prevention or resolution of infections are stimulated. Stimulation of innate immunity by natural mechanisms, for example using TLR agonists, creates the risk of inducing or exacerbating potentially harmful pro-inflammatory responses [Hancock and Sahl, 2006]. AMPs are known to play an important role in both innate and adaptive immunity [Zasloff, 2002]. AMPs from higher eukaryotes also act as intracellular signaling molecules and coordinate the innate and adaptive host defense responses, influencing processes like cytokine release, cell proliferation, angiogenesis, wound healing, chemotaxis, control and resolution of the inflammatory response (Fig. 1.1). Some of these properties would be considered pro-inflammatory, but AMPs actually suppress TLR signaling responses, by blocking the LPS-stimulated production of cytokines, like tumor necrosis factor-alpha (TNF-α), and therefore, the emergence of septic shock syndrome in animal models [Hancock and Sahl, 2006]. Note that the LPS-binding properties of many AMPs are believed to play an important role in detoxifying LPS during infection (Rosenfeld et al, 2006b). In addition, some AMPs, like human defensins and LL-37, are chemoattractive peptides for leukocytes. Specifically, they can have a dual role: (i) killing microorganisms, at high concentrations, and (ii) attracting leukocytes to the 5 1. INTRODUCTION site of infection, at low concentrations [Dürr and Peschel, 2002], thus providing a link between the innate and adaptive immunity. Figure 1.1 Biological roles of host defense peptides. Both direct antimicrobial killing and innate immune modulation occur for the majority. Although certain peptides have one or the other activity, preferentially.