Antimicrobial peptides and the interplay between microbes and host Towards preventing porcine infections with Streptococcus suis Rogier A. Gaiser Thesis committee Promotor Prof. Dr Jerry M. Wells Professor of Host-Microbe Interactomics Wageningen University Co-promotor Dr Peter van Baarlen Assistant professor, Host-Microbe Interactomics Wageningen University Other members Prof. Elizabeth Wellington, University of Warwick, UK Dr Stefano Donadio, NAICONS Srl / Ktedogen Srl, Italy Dr Paul Ross, University College Cork, Ireland Prof. Dr Hauke Smidt, Wageningen University This research was conducted under the auspices of the Graduate School of Wageningen Institute of Animal Sciences (WIAS) Antimicrobial peptides and the interplay between microbes and host Towards preventing porcine infections with Streptococcus suis Rogier A. Gaiser Thesis submitted in fulfilment of the requirement for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. Dr A.P.J. Mol in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Friday 7 October 2016 at 1.30 p.m. in the Aula. Rogier A. Gaiser Antimicrobial peptides and the interplay between microbes and host: Towards preventing porcine infections with Streptococcus suis 240 pages. PhD thesis, Wageningen University, Wageningen, NL (2016) With references, with summaries in English and Dutch ISBN: 978-94-6257-891-3 DOI : 10.18174/387767 Voor diegenen die ik liefheb TABLE OF CONTENTS Chapter 1 General Introduction 9 Chapter 2 Frog skin host-defense peptides as potential candidates 45 for treatment of topical bacterial infections Chapter 3 Transcriptome analysis of the generic and adaptive 67 responses by Streptococcus suis to sub-lethal concentrations of LL-37 Chapter 4 Compositional Profiling of Porcine Microbiota to Identify 95 Bacteria with Strong Positive or Negative Associations with Streptococcus suis Abundance Chapter 5 Narrow-spectrum Inhibition of Streptococcus suis by 121 Delta-Lysins of Commensal Staphylococcus pasteuri Isolated from Porcine Tonsil Microbiota Chapter 6 Modulation of mucosal barrier function and immunity by 163 valinomycin, an antibiotic ionophore produced by the mammalian commensal Rothia nasimurium Chapter 7 General Discussion 203 Appendices Summary 229 Samenvatting Acknowledgement Curriculum Vitae Training Activities 1 CHAPTER 1 General Introduction CHAPTER 1 10 GENERAL INTRODUCTION Antibiotic discovery and the emergence of antibiotic resistance Antibacterial chemotherapy is an important component of modern medicine and it has drastically improved human health and life expectancy worldwide, bringing benefits to the individual and society as a whole [1]. Use of antibiotics in livestock and poultry, initially mainly for growth promotion but also to control endemic disease among increasingly larger groups of animals, has also revolutionized animal production agriculture [2]. This started with the discovery and development of sulphonamides and penicillin during the first half of the twentieth century, followed by the so called Golden Era of drug discovery, which Chapter 1 Chapter accelerated the discovery of many novel classes of antibiotics [3] (Figure 1). However, extensive (over- and mis-) use of antibacterial drugs over the past decades have favoured the selection and spread of resistant bacteria [4]. The high level of antibiotics utilized in the livestock and poultry industry is considered to have contributed to the global spread of antibiotic resistance [5]. As a consequence, there are plans to further reduce antibiotic use in livestock across Europe [6]. Nevertheless, resistance of clinically relevant bacteria to commonly used antibiotics combined with the current void in discovery and market approval of successful novel antibacterial drugs remains a global health emergency [7, 8]. The rising prevalence of antibiotic resistance in bacteria is a leading healthcare priority worldwide, with drug resistant pathogens causing increased morbidity, mortality and overall healthcare costs in both human and veterinary medicine [8, 9]. Annually, 25.000 Europeans and 23.000 Americans are estimated to die as a direct result of infections with multidrug- resistant bacteria [8, 10]. Although it is difficult to estimate, bacterial antimicrobial resistance has been reported to cost the European Union economy approximately €1.5 billion and the US health-care system between $21 billion to $34 billion per year [7, 11]. Yet, it is not so much the current impact of antibiotic resistance but the potential future implications that are worrisome. Antibiotic resistance is expected to lead to increased morbidity and mortality, with an associated exorbitant increase in health-care costs and indirect economic impacts worldwide [7, 9, 12]. Compounds that were found in the Golden Era of antibiotic discovery (1940s to 1960s) or derivatives of these compounds encompass practically all currently used antibiotics and despite the enormous high-tech discovery efforts which started in the 1990s only two new classes of antimicrobials have been approved in the last 20 years [3, 13] (Figure 1). History has shown that the introduction of every new antibiotic was rapidly followed by the emergence of resistance in the target microorganism, and the required steady flow of novel classes of antibiotics and derivatives has been outpaced by the emergence and spread of antibiotic resistance [13, 14]. Although there are hundreds of essential proteins in bacteria, the number of targets of currently used antibiotics is relatively small [13]. The most successful antibiotics hit only three targets or pathways: the ribosome (affecting protein synthesis), DNA gyrase or DNA topoisomerase (affecting DNA synthesis), or cell-wall synthesis. Other antibiotics target RNA synthesis, folic acid metabolism pathways and the bacterial cell membrane (Figure 2). 11 CHAPTER 1 Trimethoprim Streptogramins Quinolones Lincosamides Chloramphenicol Tetracyclines Macrolides Glycopeptides Aminoglycosides Beta-lactams Lipopeptides Sulfonamides Oxazolidinones 1930s 1940s 1950s 1960s 1970s 1980s 1990s 2000s 2010s The Golden Era Emperical whole-cell antibacterial screens Target-based HTS SBDD: FBDD + VHTS Figure 1. History of antimicrobial drug discovery, showing the “Golden Era” of antimicrobial drug discovery from ~1945 – 1976. Market approval of different classes of antibiotics per decade are indicated above the timeline. The principal strategies employed in antimicrobial drug discovery are displayed below the timeline (HTS: High- Throughput Screening, SBDD: Structure-Based Drug Discovery, FBDD: Fraction-Based Drug Discovery, VHTS: Virtual High-Throughput Screening). Figure is adapted from [15] and [14]. Antibiotic resistance is a normal ecological phenomenon which is widespread in the environment. Over billions of years bacteria have evolved to resist the action of naturally occurring antibacterial compounds [16]. It has been shown that antibiotic resistance in bacteria already existed before the modern selective pressure caused by human use of antibiotics was introduced [17, 18]. It is this dramatic increase of selective pressure caused by the extensive use of antibiotics over the last 70 years that has accelerated the evolution and spread of bacteria that are resistant to antibiotics. Although antibiotic resistance is a complex phenomenon, its mechanisms can be classified in three categories, namely intrinsic, acquired and adaptive resistance. Some common antibiotic targets and mechanisms associated antibiotic resistance are shown in Figure 2. 12 GENERAL INTRODUCTION Antibiotic Targets Antibiotic Resistance Penicillins Cephalosporins Glycopeptides Carpabenems decreased penetration Monobactams cell wall synthesis Quinolones increased efflux DNA synthesis Rifampicin target modification RNA synthesis P enzymatic modification Chapter 1 Chapter Lipopeptides cell membrane or inactivation target overproduction folic acid metabolism Sulphonamides A + B AB Trimethoprim protein synthesis bypass pathways target Oxazolidinones Macrolides antibiotic Chloramphenicol Clindamycin phosphate Aminoglycosides P Tetracyclines Figure 2. Antibiotic targets and mechanisms of resistance. Figure adapted from Wright (2010)[19] and Lewis (2013) [13]. Alternatives to conventional antibiotics The alarming problem of antibiotic resistance has prompted renewed interests in alternative strategies to combat pathogenic microbes, such as the use of bacteriophages [20], synthetic antimicrobial peptides and peptoids [21-24], and naturally occurring bacteriocins [25- 27]. Renewed optimism about natural product discovery and the overwhelming number of uncharacterized biosynthetic gene clusters (BGCs) has also accelerated efforts to find novel antimicrobial secondary metabolites from microbes [28-30]. Vaccination, often being relatively cheap and highly effective, is another historically successful strategy against bacteria causing infectious disease. Although new tools and vaccination strategies are continuously being developed, still a myriad of challenges remain, such as persistent, highly variable and/ or novel pathogens, complex (e.g. polymicrobial) infections, and target populations that are difficult to reach [31]. The work in this thesis will focus on antimicrobial peptides as an alternative strategy to reduce the risk of infections by pathogenic bacteria. Antimicrobial peptides As mentioned above, the use of antimicrobial peptides (AMPs) is one of the alternatives to conventional antibiotics