The Pennsylvania State University. The Graduate School. Department of Biochemistry and Molecular Biology VIRULENCE AND INFECTION: INTERACTIONS BETWEEN

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1 The Pennsylvania State University The Graduate School Department of Biochemistry and Molecular Biology VIRULENCE AND INFECTION: INTERACTIONS BETWEEN BORDETELLA BRONCHISEPTICA AND THE IMMUNE SYSTEM A Dissertation in Biochemistry, Microbiology, and Molecular Biology by Sarah J. Muse 2014 Sarah J. Muse Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2014

2 The dissertation of Sarah J. Muse was reviewed and approved* by the following: Eric T. Harvill Professor of Microbiology and Infectious Disease Dissertation Advisor Chair of Committee Andrea Mastro Professor of Microbiology and Cell Biology Robert F. Paulson Professor of Veterinary and Biomedical Sciences K. Sandeep Prabhu Professor of Immunology and Molecular Toxicology Scott B. Selleck Professor and Head, Department of Biochemistry and Molecular Biology *Signatures are on file in the Graduate School

3 iii ABSTRACT Infectious diseases are a source of serious problems for humanity. By understanding how pathogenic bacteria utilize virulence factors to interact with their host, researchers can increase their knowledge of the mechanisms underlying infections and develop improved preventative measures and treatments to combat disease. In these studies, the respiratory pathogen B. bronchiseptica was used to study different aspects of virulence. B. bronchiseptica arnt, which encodes a protein that modifies the lipid A by adding glucosamine, was shown to be required for resistance to cationic antimicrobial peptides. Although arnt does not alter TLR4 agonist activity, the presence of arnt was necessary for transmission of B. bronchiseptica between hosts in the mouse model. These data indicate a novel role for lipid A modification in influencing between host infection dynamics. In a second series of experiments, we investigated how a pathogen could overcome early innate immune responses by manipulating host cell signaling. B. bronchiseptica infection of cultured macrophages revealed that clpv, a component of the Type VI secretion system (T6SS), is involved in decreasing levels of caspase-1, an important proinflammatory signaling molecule and inflammasome component. The decrease in caspase-1 is mediated through induction of autophagy. This mechanism suggests a novel interplay between a recently characterized bacterial secretion system and inflammasome signaling and hints at a bacterial virulence strategy to manipulate potentially damaging immune responses. Finally, Hcp, a core component of the T6SS, was investigated and was found to be necessary for bacterial processes, such as cell division and growth.

4 iv TABLE OF CONTENTS List of Figures... vi List of Tables... viii Acknowledgements... ix Abbreviations... xi Chapter 1 Introduction Host-pathogen interactions... 2 Bordetella as a model... 6 Bordetella Virulence Factors... 7 Preface References Chapter 2 Enzymatic Modification of the Lipid A by ArnT protects B. bronchiseptica against Cationic Peptides and Is Required for Transmission Abstract Introduction Materials and Methods Results Discussion Author Contributions References Chapter 3 B. bronchiseptica and the Caspase-1 Inflammasome Abstract Introduction Materials and Methods Results Discussion Author Contributions References Chapter 4 Hcp is necessary for normal B. bronchiseptica growth and morphology Abstract Introduction Materials and Methods Results Discussion Author Contributions

5 v References Chapter 5 Summary and Significance Synopsis Enzymatic Modification of the Lipid A by an ArnT Protects B. bronchiseptica Against Cationic Peptides and Is Required for Transmission Summary and Implications Future Directions Bordetella bronchiseptica and the Inflammasome Summary and Implications Future Directions Hcp is necessary for normal B. bronchiseptica growth and morphology Summary and Implications Future Directions Conclusion References Appendix A Parameters of B. bronchiseptica Infection in Relation to Caspase

6 vi LIST OF FIGURES Figure 1.1. Bacterial-Host Interaction Model Figure 1.2. Inflammasome Signaling Pathway Figure 1.3. BvgAS System Figure 1.4. Pertussis Toxin and Adenylate Cyclase Toxin Figure 1.5. Bordetella Adhesins Figure 1.6. Schematic of the basic structure of lipopolysaccharide Figure 1.7. Type III Secretion System (T3SS) Figure 1.8. Type VI Secretion System (T6SS) Figure 2.1. Ions corresponding to glucosamine additions in RB50 are not seen in RB50ΔarnT Figure 2.2. ArnT was not required for induction of TNF TNFα in murine macrophages, complement resistance, or adherence to the lung epithelial cells Figure 2.3. ArnT was required for resistance to killing by cationic antimicrobial peptides Figure 2.4. ArnT did not contribute to growth or resistance in the respiratory tract in a high dose model of respiratory infection Figure 2.5. ArnT decreased the mean infectious dose of B. bronchiseptica Figure 2.6. ArnT was required for transmission of B. bronchiseptica between mice Figure 2.7. ArnT did not affect neutrophil recruitment or shedding from the host Figure 3.1. IL-1 and the pro-inflammatory immune response Figure 3.2. clpv is required for mortality in B. bronchiseptica infected IL-1R -/- mice Figure 3.3. IL-1R is required for control of B. bronchiseptica respiratory colonization Figure 3.4. The Type VI Secretion System (T6SS) Figure 3.5. Deletion of clpv does not affect induction of IL-1β in murine lungs during infection Figure 3.6. Caspase-1 Inflammasome Complex Figure 3.7. Pathogen Interaction with the Inflammasome

7 vii Figure 3.8. Autophagy and the Inflammasome Figure 3.9. Effect of clpv and Caspase-1 during B. bronchiseptica Colonization in the Mouse Model Figure Model of T6SS affecting the inflammasome in macrophages Figure clpv was required for reduction of capase-1 protein in cultured macrophages Figure clpv is required for IL-1β production Figure clpv did not affect Casp1 mrna levels Figure clpv is necessary for in decreased levels of Nlrp3 protein Figure clpv is required for induction of autophagy and degradation of caspase Figure 4.1. The Type VI Secretion System (T6SS) Figure 4.2. Hcp protein Figure 4.3. Construction and confirmation of RB50Δhcp Figure 4.4. hcp contributed to bacterial growth Figure 4.5. hcp is required for polymyxin B resistance Figure 4.6. RB50Δhcp optical density did not correspond to colony forming units Figure 4.7. hcp is required for normal morphology in B. bronchiseptica Figure 4.8. Model of hcp in B. bronchiseptica growth Figure A.1. Measuring T6SS dependent caspase-1 activity Figure A.2. The Type III Secretion System is required for decreased levels of caspase-1 protein

8 viii LIST OF TABLES Table 4.1. Primer Sequences

9 ix ACKNOWLEDGEMENTS Chapter 2 of this dissertation Enzymatic modification of lipid A by ArnT protects Bordetella bronchiseptica against cationic peptides and is required for transmission, has been published in Infection and Immunity; 2014 Feb;82(2): All permissions have been obtained regarding the reproduction of text and figures of this manuscript within the dissertation as well as for diagrams in Chapters 1, 3, and 4. First, I would like to thank my advisor Dr. Eric Harvill for his help and guidance throughout my research and my committee members Dr. Kenneth Keiler, Dr. Andrea Mastro, Dr. Robert Paulson, and Dr. K. Sandeep Prabhu for their input and advice. I would also like to thank current members of the Harvill lab, Dr. Laura Goodfield (for heroic efforts in template formatting), Dr. William Smallridge, Liron Bendor, Dr. Yury Ivanov, Tyler Malys, Kai Hu, and Dr. Bodo Lintz, as well as past lab members Dr. Olivier Rolin, David Place, Dr. Sara Hester, Dr. Xuqing Zhang, Dr. Laura Weyrich, Dr. Jihye Park, Dr. Alexia Karanikas, Heather Feaga, Nathan Jacobs, Chetan Safi, and Melissa Augustino for their invaluable support and patience over the years. In addition, I would like to acknowledge our collaborators Dr. Andrew Preston at the University of Bath, UK, Dr. Robert Ernst and Lauren Hittle at the University of Maryland School of Dentistry, and Dr. Shokrollah Elahi Dr. Volker Gerdts at the University of Saskatchewan, Canada, and Dr. Sarah Barchinger, formerly from the Ades lab. I would like to extend my thanks to Dr. Mary Kennett, Dr. Pamela Hankey, and the National Institutes of Health (NIH) for providing me training and funding during my

10 x research through the Animal Models of Inflammation Training Grant. I would also like to thank Dr. Girish Kirimanjeswara and his lab for assistance with the caspase-1 project. Special thanks are given to Rachel Markley for help with the bone marrow derived macrophages and Dr. Kalyan Dewan for technical suggestions. In addition, I would like to acknowledge the excellent staff members at the Penn State Core Facilities for help with my work. I would like to thank Missy Hazen, John Cantolina, Dr. Ruth Nissly, and Dr. Greg Ning from the Microscopy and Cytometry Facility for all their hours of training and help. I would also like to thank Dr. Tatiana Laremore and Dr. Philip Smith from the Proteomics and Mass Spectrometry Core Facility for help with 2D gel proteomics and bacterial protein identification. Finally, I would like to thank my parents and friends for their constant support and understanding. Thank you to Matthew Moreau for editorial input for the Hcp project. Special thanks to Nick, Anand, Greg, Sheila, and Jan for helping me with this process. In the words of Julian of Norwich, All shall be well, and all shall be well and all manner of things shall be well.

11 xi ABBREVIATIONS 3MA: 3-methyl adenine ACT: Adenylate cyclase toxin BG: Bordet-Gengou agar BMDM: Bone marrow derived macrophage CAMP: Cationic antimicrobial peptide CDC: Center for Disease Control CFU: Colony forming unit DMEM: Dulbecco s modified Eagle s medium DNT: Dermonecrotic toxin ELISA: Enzyme Linked Immunosorbent Assay FBS: Fetal bovine serum FHA: Filamentous hemagglutinin Fim: Fimbriae GlcN: Glucosamine Hcp: haemolysin coregulated protein HRP: Horseradish Peroxidase ID50: Dose to infect 50% of individuals IL: Interleukin LB: Luria Bertani broth LBP: lipopolysaccharide binding protein LPS: lipopolysaccharide

12 xii MALDI-TOF: matrix-assisted laser desorption ionization time of flight mbd3: mouse β-defensin 3 MOI: Multiplicity of infection NLR: Nod-like receptor protein PAMP: Pathogen associated molecular pattern pbd1: Porcine β-defensin 1 PBS: Phosphobuffered saline Prn: Pertactin PRR: Pattern recognition receptor PT: Pertussis toxin SDS: Sodium dodecyl sulfate T3SS: Type III secretion system T6SS: Type VI secretion system TLR: Toll-like receptor TNF: Tumor necrosis factor USDA: United States Department of Agriculture

13 Chapter 1 Introduction.

14 2 Infectious diseases, which occur when transmissible pathogens interact with their hosts, are a source of major problems for humanity. According to the World Health Organization, infectious diseases are responsible for 16.2% of all deaths worldwide, account for four of the top ten causes of death, and are the predominant cause of death for people from low-income countries (2). Besides the staggering contribution to mortality, infectious diseases also cause disruption on an emotional, social, and economic level for the population (41). As estimated by the CDC, the annual cost of infectious diseases, both direct and indirect, is approximately $120 billion (78). In addition to directly affecting global human health, infectious diseases that afflict animals are also a burden on agriculture. The USDA reports that infectious diseases are responsible for billions of dollars lost by livestock producers each year (99). The incidence of infectious disease is an eminent and pervasive threat to human health and well-being (through human restricted pathogens and the rise of zoonoses (60)) as well as the food supply and world economy (5). To combat this critical dilemma, scientists are working to improve current protective measures as well as develop novel strategies though increased understanding of how pathogens cause disease. Host-pathogen interactions Pathogens are organisms that have characteristics that enable them to cause damage to their hosts. Types of pathogens include viruses, fungi, other eukaryotic microorganisms, and bacteria. Pathogenic bacteria, the focus of this dissertation, are classified thusly due to their ability to cause tissue damage and/or cell death in the host.

15 3 However, successful infectious pathogens have multiple key traits that enable the optimal utilization of their host environment : the capacity to overcome initial barriers and colonize a specific microenvironment; the ability to compete with commensal microbiota within a niche; the capability to survive, evade, and possibly manipulate host immune defenses; and the ability to transmit to a new host (28). The first objective for pathogenic bacteria is to prevail against the challenges imposed by the host microenvironment and establish an infection (Figure 1.1). The bacteria must navigate physical impediments, such as the skin or the mucosa at preferred points of entry (35). Then, the bacteria must contend with the mechanical perturbation of the mucocilliary interface (88) as well as innate immune defenses such as lysozymes (26), antimicrobial peptides (104), and complement (59). The bacteria must produce appropriate adherence factors (17) and have the ability to provide sufficient protection to survive and colonize the target environmental niche (80). Another important aspect of pathogenicity, which was not widely considered until recently, is the competition of invading microbes with commensal microbiota (34). Because of the adaptations of the commensal microbe species, they create a barrier against invasive species (45). Pathogens that are competing for the same space or pool of resources need methods to interfere with homeostasis and gain an advantage. The competition between the pathogen and commensals could involve direct killing (49), the disruption of biofilms (70), or differences in ability to withstand bacteriophage (10), or manipulation of an immune response (13). The host immune response is a complex system that is structured to create multiple layers of defenses against invading pathogens. A cursory overview of the

16 4 immune processes illustrates potential problems for the pathogenic bacteria in question. In addition to the previously mentioned difficulties, pathogens must also contend with other aspects of innate immunity (34). The bacteria would also encounter resident immune cells or even epithelia that respond to bacterial pathogen associated molecular patterns (PAMPs) (54). The immune cells would take advantage of highly conserved pattern recognition receptors (PRRs) (71) such as the transmembrane TLR (Toll-like receptor) or the cytosolic NLR (Nod-like receptor protein) (Figure 1.2) (42). Downstream signaling events, such as TLR4 activation of MyD88 and NF-kB (63) or even inflammasome complex formation between a Nod-like receptor, caspase-1, and Asc (92), result in induction of pro-inflammatory cytokines (36) as well as cell recruitment to the site of infection (50). Another obstacle would be to avoid or survive engulfment by phagocytic cells (12) as well as prevent antigen presentation and recruitment of the adaptive immune response (8). That process would influence the recruitment and differentiation of T cells (91), B cells (93), and ultimately the humoral immune response (37). To avoid detection or favorably alter the type of immune response elicited, pathogens rely on the production and delivery of specific virulence factors that manipulate host defense cellular properties (15), ensuring bacterial survival. After successfully replicating in the host environment, the infectious pathogenic bacteria must then attempt to transmit. Virulence factors provide a selective advantage in enabling the bacteria to move to another host without causing the death of the current host (102). Experiments with the mouse infection model indicate that bacterial transmission correlates with the generation of localized inflammation (74). The presence

17 5 of virulence factors such as the Type III secretion system and adenylate cyclase toxin is required for increased recruitment of polymorphonuclear leukocytes (PMN) and increased levels of bacterial transmission from the murine nasal cavity (89). The presence of PMNs could contribute to transmission by increasing bacterial shedding levels by promoting a coughing or sneezing response (14) and augmenting the flow of mucus out of the site of infection (6). These findings indicate that virulence factor pathogenicity could aid in the spread of infection. To accomplish all of these objectives, successful bacterial pathogens rely on the production of virulence factors. These molecules or proteins have specific functions that enable the bacteria to subvert host defenses, acquire resources, as well as facilitate transmission. Virulence factor properties are numerous and varied, but all abilities work to promote bacterial survival in the various conditions encountered as the infection progresses. However, caveats exist concerning the appropriate use and regulation of virulence factors. The ability of many virulence factors to promote pathogenicity are highly dependent on the ecological niche of the bacteria (25). Virulence factors are not necessarily multipurpose, nor are they effective for all stages of the pathogen s life cycle. For example, the B. bronchiseptica Type III secretion system and Bvg regulated virulence factors cause disease and allow for persistence in swine but do not necessarily contribute to transmission (76, 77). The successful utilization of virulence factors is constrained by functional, temporal, and environmental considerations. Therefore, understanding the molecular mechanisms of virulence factors requires the use of a model system for both host and pathogen.

18 6 To investigate virulence strategies utilized by bacteria and the effects that those traits have on host organs and immune system, the respiratory pathogen Bordetella bronchiseptica was used. Bordetella as a model B. bronchiseptica is one of the most commonly studied species of the Bordetella genus and is classified as a member of the classical bordetellae (67). The other members are B. pertussis, the causative agent of whooping couch in humans (19), and B. parapertussis (61), strains of which have been isolated from patients with whooping cough symptoms but has also been shown to cause disease in sheep (21). B. bronchiseptica, B. pertussis, and B. parapertussis strains are considered subspecies of a single species adapted to various hosts (67) because of similarities in DNA sequence (100), insertion sequence (IS) element polymorphisms (95), multilocus enzyme electrophoresis (MLEE) typing (75), and metabolic characteristics (81). B. bronchiseptica is considered a progenitor-like subspecies in relation to the other members of the classical bordetellae based on measurements of genetic diversity (82) and has a broad host range (38). Notably, B. bronchiseptica has been shown to cause a form of atrophic rhinitis, a swine respiratory disease (1) that results in facial distortions that lead to difficulties in feeding and the development of respiratory distress. Atrophic rhinitis has been detected in over twenty percent of farms surveyed and results in costly herd losses every year (1, 16). Further contributing to agricultural difficulties, B. bronchiseptica infection increases swine susceptibility to other respiratory diseases,

19 7 such as Haemophilus parasuis (22) and Pasteurella multocida (24), as well as the severity of co-infection with porcine respiratory coronavirus (23). In addition to infecting swine, B. bronchiseptica has been shown to causes disease in a wide range of mammals, including dogs (57), rabbits (62), guinea pigs (43), koalas (69), sea otters (94), and even humans (82). It is especially noteworthy that B. bronchiseptica also infects mice (67). This enables the use of a murine infection model to study the natural interaction between host and pathogen while enabling investigators to take advantage of the extensive body of literature and tools available for the study of mice. Using the mouse model, as well as through experimentation with cell culture and molecular biology techniques, researchers are able to mechanistically delineate the immunomodulatory and damage-inducing phenomena that occur during host-pathogen interactions by identifying and characterizing virulence factors in B. bronchiseptica. Bordetella Virulence Factors The major virulence regulatory system in Bordetella is the BvgAS two component system (31). BvgS acts as a sensor kinase and, in response to environmental stimuli, acts through a phosphorelay system to phosphorylate the BvgA regulator, resulting in dimerization and activation and repression of transcription (Figure 1.3A) (32, 98). This system serves as a master regulator for Bordetella virulence and controls the upregulation and downregulation of virulence gene sets in the various Bvg phases (Figure 1.3B) (72). In the Bvg + phase, virulence associated genes such as toxins and adherence

20 8 factors are upregulated (73), but they are downregulated in the Bvg - phase in favor of expression of metabolic and motility genes (3, 4). The genes expressed in this phase are necessary for successful B. bronchiseptica colonization of the host. The Bvg- phase may be required for survival outside of the host and in nutrient limiting conditions. An intermediate Bvg i phase also exists in which adherence factors and metabolic genes are upregulated but the other gene sets from both phases are not (97, 103). Changes in Bvg phase directly influence the virulence factors produced in response to the environment. A major class of virulence factors is bacterial toxins. The most famous toxin associated with Bordetella is pertussis toxin (PT) (85). PT is Bvg regulated (27) and functions by causing ADP-ribosylation of host proteins and altering G protein signaling (Figure 1.4 A,B) (56). Although the genome of B. bronchiseptica contains the coding regions necessary to produce PT, expression of the locus has not been observed (7). Adenylate cyclase toxin (ACT) is a calmodulin-sensitive adenylate cyclase/hemolysin that is expressed in Bvg + phase (67). However, non-bvg regulated factors, such as CO2 (47), have also been shown to influence its expression. ACT induces the overproduction of camp (Figure 1.4C,D), resulting in impaired phagocytosis (30) and induction of apoptosis in macrophages (44). B. bronchiseptica produces adenylate cyclase toxin and dermonecrotic toxin which have been shown to interact with host cell pathways. Dermonecrotic toxin (DNT) is highly conserved among the bordetellae and was discovered when B. pertussis extracts caused necrosis on the skin of treated mice (33). Properties of DNT include activation of Rho GTPase to inhibit cell differentiation and proliferation (52), disruption of the host cell cycle (58), and inhibition of antibody

21 9 response in mice (96). DNT is also a key virulence factor in causing the aforementioned atrophic rhinitis in swine (51). Another important type of virulence factor in B. bronchiseptica is adhesins. Fimbriae (Fim2/3), filamentous hemagglutinin (FHA), and pertactin (Prn) (Figure 1.5 A, B, C) enable B. bronchiseptica to adhere to respiratory tract cells (39) and also serve as components of the acellular pertussis vaccine. Fim and FHA are both necessary for tracheal colonization and are important for induction of an antibody response (53, 68). The membrane component lipopolysaccharide (LPS) (Figure 1.6) can also influence the ability of bacteria to survive and evade the host immune response. LPS consists of 3 main regions: the lipid A, which serves to anchor the LPS into the outer membrane of Gram negative bacteria (65); the core oligosaccharide, which commonly linked to the lipid A through 2-keto-3-deoxyoctulosonic acid; and the O antigen, which attaches to the core and consists of repeating polysaccharide chains (86). LPS can protect B. bronchiseptica from antimicrobial peptides (40), but the lipid A portion also interacts with TLR4 (66), resulting in the generation of a proinflammatory response. The addition of modifications to the lipid A can, in turn, influence changes in the host immune response. Previous work has shown that PagP palmitoyl transferase is necessary for colonization (87) and resistance to complement (84). The O-antigen component of LPS can also influence the bacterial interaction with the host. The presence of O-antigen is necessary for B. bronchiseptica colonization of the respiratory tract (46) and acts to protect bacteria from antimicrobial peptides and complement deposition (9). The bordetellae have 3 horizontally acquired O-antigen

22 10 types (48), and these polysaccharides have different levels of immungenicity, affecting resistance to antibody mediated killing. Secretion systems function to export proteins required for virulence. In B. bronchiseptica, two secretion systems are associated with pathogenesis: the Type III secretion system (T3SS) and the recently characterized Type VI secretion system (T6SS). The T3SS consists of a needle-like injection apparatus powered by a system designated ATPase (bscn) that transports effector proteins externally or into host cells (Figure 1.7) (79). The T3SS is required for caspase-1 dependent necrosis (96) in culture as well as inhibition of NF-kB signaling (105). In the mouse model, the T3SS is necessary for colonization in the lungs and inhibits the proinflammatory interferon (IFN)-γ that is required for antibody mediated clearance (83). The B. bronchiseptica T6SS was recently identified (20) and an initial characterization was performed (101). The T6SS consists of 13 core components as well as 13 Bordetella specific genes (Figure 1.8A). These proteins form a T4 phage tail-like secretion apparatus which has been shown to be contact dependent in other systems. Important conserved system components include Hcp, which has been shown to form nanotubule structures through which effectors can pass (Figure 1.8B) (18); VgrG, the tip of the secretion apparatus which interacts with membrane components present in target cells (90); IcmF, a conserved inner membrane protein that has been shown to contribute to ATP hydrolysis and binding (64); and ClpV, a AAA+ ATPase which serves as the main power source for the system and enables contraction of the apparatus (55). The T6SS is thought to assemble by polymerization of the Hcp-based tubule in an ATP dependent manner after the base-plate complex of proteins that span the inner and

23 11 outer membranes are brought together (29). Then, the VipA/VipB sheath is hypothesizes to form in the uncontracted position around the Hcp tubule, which is capped with the VgrG protein. After the system is stimulated, the VipA/VipB sheath contracts, enabling effector proteins, Hcp, and VgrG to be delivered into the target cell or the extracellular milieu (Figure 1.8C) (11). clpv is required for colonization in the mouse model, contributes to lung pathology (101), inhibits antibody mediated clearance by suppression of proinflammatory cytokines (L. S. Weyrich, unpublished), and is required for B. bronchiseptica mediated displacement of the nasal cavity microbiota (L.S. Weyrich, unpublished data). Additonally, it has been shown to affect IL-6, IL-1β, IL-17, and IL-10 cytokine responses in vitro and IFN-γ in vivo (101). Besides Hcp, none of the effector molecules secreted by the B. bronchiseptica T6SS have been identified. However, potential candidates include the proteins encoded by loci BB0797 (tssd) and BB0798 (tsse), which form a putative toxin-antitoxin system. Additional work has also been performed analyzing the role of BB0804 (tssj), which encodes a gp25-like lysosome and is hypothesized to be part of the base-plate of the apparatus or possibly secreted. Preliminary results have shown that TssJ does not affect cytotoxicity in culture and is not required for colonization of the respiratory tract in the mouse (H. Feaga, unpublished). However, during infection, the presence of TssJ has been shown to be required for the expression of the pro-inflammatory cytokines IL-6 and IL-1β as well as the chemokines KC and MIP1-α. B. bronchiseptica is capable of producing a wide array of virulence factors to facilitate its survival, replication, and transmission to a new host. By performing

24 12 experiments to characterize the interface of virulence strategies with the immune system, we hope to create a more detailed understanding of how virulence factors function in order to counter their effects and prevent disease. Preface The focus of this dissertation is investigating how the respiratory pathogen B. bronchiseptica utilizes virulence strategies to interact with the host and survive. The work presented in Chapter 2 features arnt, which encodes an enzyme that adds a glucosamine modification on the B. bronchiseptica lipid A. This virulence factor provides resistance to antimicrobial peptides and influences the ability of the bacteria to initially colonize the nasal cavity as well as its ability to affect leukocyte recruitment and facilitate transmission. The material in Chapter 2 was previously published in Infection and Immunity. In Chapter 3, an examination of a novel mechanism of virulence factor mediated manipulation of host immune cells is presented. In cultured macrophage cells, the presence of clpv, which encodes the major power source of the T6SS, is required for a decrease in caspase-1, a central inflammatory signaling protein. This decrease is facilitated through the clpv dependent induction of autophagy, implicating the involvement of the T6SS in a host cell degradation pathway to decrease inflammatory signaling. The experiments described in Chapter 4 concerned the investigation of Hcp, a core component of the T6SS (a system previously implicated in virulence in culture and

25 13 the mouse model), which is required for essential bacterial properties such as membrane stability and normal cell division. The overall results are summarized in Chapter 5, and the significance of the findings and virulence strategies on host-pathogen interactions is discussed.

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31 90. Sha, J., J. A. Rosenzweig, E. V. Kozlova, S. Wang, T. E. Erova, M. L. Kirtley, C. J. van Lier, and A. K. Chopra. Evaluation of the roles played by Hcp and VgrG type 6 secretion system effectors in Aeromonas hydrophila SSU pathogenesis. Microbiology 159: Shaler, C. R., C. Horvath, R. Lai, and Z. Xing. Understanding delayed T-cell priming, lung recruitment, and airway luminal T-cell responses in host defense against pulmonary tuberculosis. Clin Dev Immunol 2012: Sollberger, G., G. E. Strittmatter, M. Garstkiewicz, J. Sand, and H. D. Beer. Caspase-1: the inflammasome and beyond. Innate Immun 20: Spera, J. M., C. K. Herrmann, M. S. Roset, D. J. Comerci, and J. E. Ugalde. A Brucella virulence factor targets macrophages to trigger B-cell proliferation. J Biol Chem 288: Staveley, C. M., K. B. Register, M. A. Miller, S. L. Brockmeier, D. A. Jessup, and S. Jang Molecular and antigenic characterization of Bordetella bronchiseptica isolated from a wild southern sea otter (Enhydra lutris nereis) with severe suppurative bronchopneumonia. J Vet Diagn Invest 15: Stibitz, S., and M. S. Yang Genomic plasticity in natural populations of Bordetella pertussis. J Bacteriol 181: Stockbauer, K. E., A. K. Foreman-Wykert, and J. F. Miller Bordetella type III secretion induces caspase 1-independent necrosis. Cell Microbiol 5: Sukumar, N., M. Mishra, G. P. Sloan, T. Ogi, and R. Deora Differential Bvg phase-dependent regulation and combinatorial role in pathogenesis of two Bordetella paralogs, BipA and BcfA. J Bacteriol 189: Uhl, M. A., and J. F. Miller Autophosphorylation and phosphotransfer in the Bordetella pertussis BvgAS signal transduction cascade. Proc Natl Acad Sci U S A 91: United States Department of Agriculture, R. E. E. I. S INFECTIOUS DISEASE PATHOGENESIS STUDY AND PREVENTION DEVELOPMENT van der Zee, A., H. Groenendijk, M. Peeters, and F. R. Mooi The differentiation of Bordetella parapertussis and Bordetella bronchiseptica from humans and animals as determined by DNA polymorphism mediated by two different insertion sequence elements suggests their phylogenetic relationship. Int J Syst Bacteriol 46: Weyrich, L. S., O. Y. Rolin, S. J. Muse, J. Park, N. Spidale, M. J. Kennett, S. E. Hester, C. Chen, E. G. Dudley, and E. T. Harvill. A Type VI secretion system encoding locus is required for Bordetella bronchiseptica immunomodulation and persistence in vivo. PLoS One 7:e Wickham, M. E., N. F. Brown, E. C. Boyle, B. K. Coombes, and B. B. Finlay Virulence is positively selected by transmission success between mammalian hosts. Curr Biol 17: Williams, C. L., P. E. Boucher, S. Stibitz, and P. A. Cotter BvgA functions as both an activator and a repressor to control Bvg phase expression of bipa in Bordetella pertussis. Mol Microbiol 56: Yang, D., O. Chertov, and J. J. Oppenheim The role of mammalian antimicrobial peptides and proteins in awakening of innate host defenses and adaptive immunity. Cell Mol Life Sci 58: Yuk, M. H., E. T. Harvill, P. A. Cotter, and J. F. Miller Modulation of host immune responses, induction of apoptosis and inhibition of NF-kappaB activation by the Bordetella type III secretion system. Mol Microbiol 35:

32 20 Figure 1.1. Bacterial-Host Interaction Model. Bacterial pathogens must overcome multiple challenges over the course of infection. Initial obstacles include overcoming physical barriers to colonization such as mucus or the beating of cilia, withstanding antimicrobial peptides, complement, and lysozymes, outcompeting the commensal microflora, and prevention of engulfment by resident phagocytes. After the establishment of the pathogen, pro-inflammatory cytokine and chemokine responses initiated by the epithelium and resident immune cells and antigen presentation lead to the recruitment of leukocytes as well as T and B lymphocytes to the site of infection and the initiation of a humoral immune response. Modification of figure created by O. Rolin.

33 21 Figure 1.2. Inflammasome Signaling Pathway. Maturation and secretion of IL-1β requires 2 signals: the priming signal leads to synthesis of pro-il-1β, pro-il-18, and other components of the inflammasome, such as NLRP3, and the second signal results in assembly of the inflammasome, activation of caspase-1, and release of mature cytokines IL-1β and IL-18 into the extracellular milieu. Currently, the nature of the second signal is debated. The 3 proposed models of activation are shown: 1) extracellular ATP, which activates the purinergic P2X 7 receptor and causes subsequent recruitment of pannexin-1 hemichannel to the plasma membrane and K + efflux; 2) lysosomal rupture after engulfment of crystalline or particulate agonists; and 3) reactive oxygen species (ROS), which upregulate NLRP3 expression and activate the inflammasome. PAMP, pathogen-associated molecular pattern; DAMP, danger-associated molecular pattern; TLR, Toll-like receptor; dsdna, double-stranded DNA. Reprinted with permission from the American Physiological Society, Am J Physiol Lung Cell Mol Physiol Oct 15;303(8):L

34 22 Figure 1.3. BvgAS System. A) BvgS is a polydomain histidine sensor kinase that contains (from the amino to the carboxyl terminus) two periplasmically located venus flytrap domains (VFT1 and VFT2), a transmembrane domain, a PAS domain, a histidine kinase domain (HK), a receiver domain (Rec) and a histidine phosphoryl transfer domain (Hpt). BvgA is a response regulator protein that becomes autophosphorylated, and the phosphoryl group is then transferred to the Rec domain, followed by the Hpt and finally to the Rec domain of BvgA. Phosphorylated BvgA dimerizes and activates the expression of virulence-associated genes. B) Bvg Phase Gene Expression Profiles. The Bvg + phase occurs when BvgAS is fully active and is characterized by maximal expression of genes that encode adhesins and toxins and minimal expression of motility genes and bipa. The Bvg phase occurs when BvgAS is inactive and is characterized by maximal expression of bvgr and motility genes and minimal expression of toxins, adhesins and bipa. The Bvg i phase occurs when BvgAS is partially active and is characterized by the maximal expression of adhesins and bipa (expressed exclusively in this phase) and minimal expression of toxins and motility genes. Reprinted with permission from Nature Publishing Group, Nat Rev Microbiol Apr;12(4):

35 23 Figure 1.4. Pertussis Toxin and Adenylate Cyclase Toxin. A) Structure of Pertussis Toxin (PT) indicating the A subunit (catalytic subunit) and the B pentamer (transport subunits). B) Model of PT ADP-ribosylation. PT binds to a sialoglycoprotein host cell receptor, is endocytosed and trafficked to the endoplasmic reticulum (ER) where the B pentamer dissociates from the A subunit. The A subunit then traffics on exosomes to the cytoplasmic membrane, where it ADP-ribosylates the α-subunit of heterotrimeric G proteins, altering the ability of the G proteins to regulate multiple enzymes and pathways, including their ability to inhibit cyclic AMP (camp) formation. C) Structure of Adenylate Cyclase Toxin (ACT) indicating the adenylate cyclase domain connect via a hydrophobic segment to the RTX domain. D) Model of ACT function. The RTX domain of ACT interacts with complement receptor 3 (CR3). The hydrophobic segments of the linker region form pores in the membrane, enabling the passage of cations and the adenylate cyclase domain is translocated into the cytoplasm. Adenylate cyclase activity is stimulated by binding to calmodulin in the host cell, leading to an increase in camp production. Reprinted with permission from Nature Publishing Group, Nat Rev Microbiol Apr;12(4):

36 24 Figure 1.5. Bordetella Adhesins.. A) Filamentous haemagglutinin (FHA) is a TpsA exoprotein that is translocated across the outer membrane through its cognate TpsB pore protein, FhaC. This translocation occurs via the two-partner secretion pathway. Processing during translocation removes the carboxyterminal prodomain (yellow) from the full-length FhaB protein to produce the mature ~250 kda FHA protein. B) Bordetella fimbriae are type I pili. Fim2 and Fim3 are the major pilin subunits and FimD is likely to be the fimbrial tip protein. Both Fims and FHA are required for adherence to ciliated epithelial cells and modulating the host immune system. C) Pertactin is a classical autotransporter which is thought to play a role in cell adherence. The C-terminal ~30 kda domain (orange) forms a channel in the outer membrane, which is required for the translocation of the ~70 kda β-helical passenger domain (blue) to the cell surface. Reprinted with permission from Nature Publishing Group, Nat Rev Microbiol Apr;12(4):274-88

37 25 Figure 1.6. Schematic of the basic structure of lipopolysaccharide. LPS consists of three regions: from the bottom, lipid A (chair structure indicates di-glucosamine head group, red circles indicate phosphate groups, wavy lines indicate acyl chains),core sugars, and O-antigen, which consists of repeating units (denoted in brackets, with an n )of oligosaccharides. Reprinted via the Creative Commons Attribution (CC BY) license, Front Cell Infect Microbiol Feb 12;3:3

38 26 Figure 1.7. Type III Secretion System (T3SS). Schematic representation of components of T3S S, indicting conserved cytoplasmic, membrane spanning, and extracellular components of the apparatus. Reprinted with permission from the American Society of Microbiology, Microbiol Mol Biol Rev Jun;76(2):

39 27 Figure 1.8. Type VI Secretion System (T6SS). A) T6SS locus from B. bronchiseptica strain RB50. Each arrow corresponds to relative gene length. Homologues of T6SS core components are labeled. B) Model of assembled T6SS, indicating central tube and conserved components. C) Model of T6SS Apparatus Action. Reprinted with permission from Nature Publishing Group, Nature Feb 26;483(7388):182-6 and Creative Commons Attribution (CC BY) license, Front Microbiol Jul 18;2:155

40 28 Chapter 2 Enzymatic Modification of the Lipid A by ArnT protects B. bronchiseptica against Cationic Peptides and Is Required for Transmission.

41 29 Abstract Pathogen transmission cycles require many steps: initial colonization, growth and persistence, shedding, and transmission to new hosts. Alterations in the membrane components of the bacteria, including lipid A, the membrane anchor of lipopolysaccharide, could affect any of these steps via its structural role in protecting bacteria from host innate immune defenses, including antimicrobial peptides and signaling through TLR4. To date, lipid A has only been shown to affect the withinhost dynamics of infection rather than the between-host dynamics of transmission. We investigated the effects of lipid A modification in a mouse infection and transmission model. Disruption of the Bordetella bronchiseptica locus (BB4268) revealed that ArnT is required for addition of glucosamine (GlcN) to B. bronchiseptica lipid A. ArnT modification of lipid A did not change its TLR4 agonist activity in J774 cells, but deleting arnt decreased resistance to killing by cationic antimicrobial peptides, such as polymyxin B and β-defensins. In the standard infection model, mutation of arnt did not affect B. bronchiseptica colonization, growth, persistence throughout respiratory tract, recruitment of neutrophils to the nasal cavity, or shedding of the pathogen. However, the number of bacteria necessary to colonize a host (ID50) was five-fold higher for the arnt mutant. Furthermore, the arnt mutant was defective in transmission between hosts. These results revealed novel functions of the ArnT lipid A modification and highlight the sensitivity of low dose infections and transmission experiments for illuminating aspects of infectious diseases between hosts. Factors such as ArnT can have important effects on

42 30 the burden of disease and are potential targets for interventions that can interrupt transmission.

43 31 Introduction Lipopolysaccharide (LPS), the major component of the outer membrane of Gramnegative bacteria, is known to affect interactions with the host in a variety of ways that have been illuminated using host infection models. Upon initial contact with the host mucosa, LPS can protect pathogens from innate host defenses, such as complement and cationic antimicrobial peptides (CAMPs) (26). LPS is extracted from bacterial membranes by LPS binding protein (LBP), which then transfers the LPS to the CD14, a glycosyl-phosphatidylinositol-anchored protein (22). Then CD14 presents the LPS to the Toll-like receptor 4 (TLR4)-MD-2 complex, which recognizes the lipid A through interaction with hydrophobic regions in the binding pocket of MD-2 (16). The TLR4- MD-2 complex dimerizes and acts as a scaffold for MyD88 or TRIF (14), creating a signaling cascade that results in mobilization of the transcription factor NF-κB (16), induction of the expression of proinflammatory cytokines, such as TNF-α and IL-6 (33), as well as chemokines in cells of the innate immune system (13). TLR4 signaling also facilitates the recruitment of adaptive immune responses, particularly through the activation of dendritic cells (DC), which are induced by LPS to migrate to regional lymph nodes and present antigens to T cells (12). Lipid A-TLR4 interactions are therefore central to host-pathogen dynamics during infections by Gram-negative bacteria. Consequently, it is not surprising that pathogens regulate their lipid A structure through a number of covalent modifications which can affect interactions with host immunity (21, 26).

44 32 Bordetella bronchiseptica is a Gram-negative coccobacillus, closely related to B. pertussis and B. parapertussis and the causative agents of whooping cough in humans. B. bronchiseptica is highly infectious in mice, providing a model system in which the role of specific Bordetella virulence factors during infection can be probed in the context of a natural host infection (19). Adhesins, toxins, and other factors that enable B. bronchiseptica to thrive within the host are chiefly controlled by the two-component regulatory system, BvgAS (1, 20). These virulence-associated genes are expressed maximally in the Bvg + phase and transcriptionally repressed in the Bvg phase (4). Modifications of the lipid A of B. bronchiseptica are regulated by BvgAS (15, 17, 18). B. bronchiseptica lipid A consists of a glucosamine disaccharide backbone anchored to the bacterial outer membrane by a series of acyl groups (15) (Figure 2.1 C, D, E structure of the lipid A). Normally, B. bronchiseptica lipid A is penta-acylated with 3- OH C14 acyl groups at the 2 and 2 positions and a 3-OH C10 at the 3 position. The 3 position is empty due to the deacylase activity of the outer membrane enzyme PagL (15). Secondary or piggyback acylations at the 2 position are either a 2OH-C12 or C12 with the presence of 2OH-C12 dependent on the lipid A dioxygenase, LpxO (15). PagP is a Bvg-regulated lipid A palmitoyl transferase that adds palmitate as a secondary acylation at the 3 position, generating a hexa-acylated structure (24, 25). Finally, the major lipid A species contains a single phosphate at the C-4, although some molecules are also phosphorylated at the C-1 position (17). In monophosphorylated lipid A species, the C-4 phosphate is decorated with a GlcN molecule; however, in lipid A molecules that possess two phosphate groups, only one GlcN modification is observed at either the C-1 or the C-4 position. Orthologues of the lipid A modification enzyme, ArnT, which

45 33 decorates the lipid A phosphates of Salmonella typhimurium with aminoarabinose are conserved among Bordetella species (34). The modification of phosphate groups with aminoarabinose decreases the net negative charge on LPS and renders S. typhimurium resistant to the antimicrobial cationic peptide, polymyxin B (11). In B. pertussis, the ArnT activity of the homologue LgmB is induced in the Bvg + phase and mediates the addition of GlcN to both terminal phosphate groups of the lipid A, which is associated with increased stimulation of TLR4 activity in the HEK-Blue assay and upon infection of human macrophages (17, 18, 30). Deletion of arnt did not affect resistance to killing by polymyxin B (17). ArnT-mediated addition of GlcN has also been reported for B. parapertussis (9) and B. bronchiseptica strain 4650 (18). In this story, we characterized the function of the B. bronchiseptica arnt homologue, BB4268, by the construction and analysis of an arnt mutant in B. bronchiseptica strain RB50, henceforth referred to as RB50ΔarnT. Similar to its function in B. pertussis, we found that B. bronchiseptica arnt was required for Bvg + phasedependent addition of GlcN to the lipid A. No change in the TLR4 agonist activity of the mutant strain was observed; however, loss of resistance against polymyxin B and β- defensin mediated killing was detected. Loss of arnt had no effect on bacterial growth or persistence when bacteria were seeded throughout the respiratory tract by standard high dose inoculation; however, the arnt mutant was not transmitted between mice, even though the mutant was shed from index cases at the same level as wild type. Furthermore, RB50ΔarnT required approximately a 5-fold increase in mean inoculation dose compared to wild type to initiate infections. Together these results showed that deleting arnt had no observable effects in standard virulence and pathogenesis assays but

46 did affect LPS modification, which had a major impact on shedding and transmission of B. bronchiseptica. 34 Materials and Methods Bacterial strains and growth. Bordetella bronchiseptica strains RB50 (4), RB50Δwbm (2) and RB50ΔarnT were maintained on Bordet-Gengou agar (Difco) supplemented with 10% defibrinated sheep blood (Hema Resources) and 200 μg/ml streptomycin (Sigma-Aldrich) and cultured in Stainer-Scholte broth (3) at 37 C until grown to mid-log phase approximate OD600 of 0.5. For the mbd3 killing assay, E. coli K12 bacteria were grown in Luria Bertani broth at 37 C until mid-log phase. Mutation of BB4268. The genomic DNA template was made by resuspending several colonies of plategrown bacteria in 0.5 ml of water, boiling in a water bath for 5 min, spinning at top speed in a bench- top microcentrifuge for 2 minutes and taking 0.2 ml of supernatant. 1 μl of supernatant was used per PCR reaction. Each PCR reaction comprised genomic DNA template, buffer as directed by the manufacturer, dntps (25 mm each), 20 ng of each primer, 5% (v/v) DMSO, 5 mm MgCl2 and 2.5 units of TAQ DNA polymerase (Promega). Primers used to amplify an approximately 1 kb section of B. bronchiseptica 4268 were 5 ATGTAGCCGACCAGCTTG 3 and 5 ATCCATGCAACCCCATGC 3, corresponding to bases and respectively, of the B. bronchiseptica RB50 genome sequence, Genbank accession number BX (23).

47 35 PCR reactions were incubated at 94 C for 5 min followed by 30 cycles of 94 C for 75 s, 60 C for 75 s and 72 C for 90 s, followed by a final step of 72 C for 7 min. The PCR product was cloned into pgem-t Easy (Promega) according to the manufacturer s instructions. A non-polar kanamycin resistance cassette was ligated into a unique StuI site residing in the middle of the cloned BB4268 region. The resulting BB4268-kan region was subcloned into pex100t (29) and the resulting construct was moved into the conjugation donor strain SM10 (31) by transformation. Bacterial conjugations were performed as described previously (2). The expected chromosomal rearrangements in conjugants were confirmed by Southern hybridization analyses. B. bronchiseptica arnt was amplified by PCR using primers incorporating NdeI and HindIII restriction endonuclease recognition sites at the 5 and 3 ends of the amplicon respectively. Following digestion of the PCR product with NdeI and HindIII, this fragment was cloned behind the B. bronchiseptica pagp promoter into pbbrkanpagp (25), replacing the pagp CDS in this construct. The arnt containing construct was moved into wild type B. bronchiseptica and the B. bronchiseptica arnt mutant by conjugation as described (25). Lipid A purification. LPS was purified from 1 liter, overnight B. bronchiseptica cultures as described previously (35). Briefly, the bacteria were harvested, lyophilized, and treated with DNase I and proteinase. The bacteria were then boiled, and LPS was extracted by collecting the phenol fraction after using the hot phenol-water method. Further treatment of LPS with RNase A, DNase I, and proteinase K ensured removal of contaminating

48 36 nucleic acids and proteins (7). Hydrolysis of LPS to isolate lipid A was accomplished with 1% sodium dodecyl sulfate (SDS) at ph 4.5 as described (3). Confirmation of lipid A structures. The lipid A structures were confirmed by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. LPS was isolated using a rapid small-scale isolation method (36). Cell culture pellets (1 10 ml of an overnight culture) were resuspended in a 1.0 ml aliquot of TriReagent (Molecular Research Center; Cincinnati, OH, USA) and incubated at room temperature for 15 min. Chloroform (200 ml) was added and the samples were vortexed and incubated at room temperature for 15 min. Samples were centrifuged for 10 min at 13,400xg and the aqueous layers were collected. Water (500 ml) was added to the lower layer and vortexed. After 30 min, the samples were centrifuged as above and the aqueous layers were pooled. Two more aliquots of water were added to each sample for a total of four extractions. The combined aqueous layers were frozen and lyophilized. The LPS was then hydrolyzed to lipid A. Lyophilized LPS was resuspended in 0y.5 ml 1% sodium dodecyl sulfate (SDS) in 10 mm sodium acetate buffer, ph 4.5 (3). Samples were incubated at 100C for 1 h, frozen, and lyophilized. The dried pellets were washed in 0.1 ml of water and 1 ml of acidified ethanol (100 ml 4 N HCl in 20 ml 95% EtOH). Samples were centrifuged at 2300xg for 5 min and the supernatant discarded. The lipid A pellet was further washed (twice for a total of three washes) in 1 ml of 95% EtOH. The entire series of washes was repeated twice. A final wash step was carried out in 100% ethanol. Lipid A was extracted in a mixture of chloroform, methanol, and water (3:1:0.25, vol/vol/vol). One microliter of this extract was then spotted onto a MALDI

49 37 target plate followed by 1 ul of Norharmane matrix and air dried. Samples were analyzed on a Bruker AutoFlex Speed (Bruker Daltonics, Billerica, MA) mass spectrometer, which was calibrated using Agilent Tuning Mix (Agilent Technologies, Foster City, CA). In vitro Macrophage Stimulation and TNFα Detection. J774 murine macrophages were cultured in Dulbecco s modified Eagle s medium (DMEM, Difco) supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, 1% nonessential amino acids, and 1% sodium pyruvate weight/volume. The cells were grown to approximately 85% confluency in a 96 well plate. In order to compare TNFα secretion, J774 cells were inoculated with RB50 or RB50ΔarnT at an MOI of 0.001, 0.01, 0.1 or 1. Medium was harvested 2 hours post-inoculation, and an ELISA to detect TNFα concentrations was performed according to the manufacturer s instructions (R&D Systems). These assays were performed two times in quadruplicate. Adherence Assay. Rat epithelial (L2) cells were grown to 80% confluence in 96 well plates using Dulbecco s modified Eagle medium (DMEM, Difco)-F-12 medium supplemented with 10% fetal bovine serum. These cells were inoculated with 10 4 CFU of RB50 or RB50ΔarnT (MOI 0.2). Plates were then centrifuged for 5 min at 250 g, followed by incubation at 37 C with 5% CO2 for 40 min. Wells were then washed four times with 1 ml of the growth medium to remove non-adherent bacteria. L2 cells were then treated with 0.5 ml of 0.125% trypsin (Sigma Aldrich), followed by incubation for 10 min at 37 C. The total volume of each well was brought up to 1 ml with growth medium and homogenized by pipetting. Dilutions were plated on BG plates containing 40 μg of

50 38 streptomycin/ml to determine CFU counts, which were then used to calculate the proportion of adherent bacteria, expressed as a percentage of the original inoculum. Serum Killing Assay. Approximately 10 3 CFU RB50, RB50Δwbm (O-antigen mutant), or RB50ΔarnT in 50 µl of PBS were incubated with serum from mice naïve to Bordetella, at concentrations ranging from 0 15% solution by volume. After 1 h incubation at 37 C followed by 5 min incubation on ice, the entire 50 μl sample was plated onto Bordet- Gengou blood agar containing streptomycin (20 μg/ml). Colonies were enumerated after 2-days of incubation at 37 C. This assay was performed two times in quadruplicate. β-defensin Killing Assay CFU RB50 and RB50ΔarnT were incubated with 0, 5, and 10 μg/ml of synthetic pbd-1 (6) and mbd3 (R&D Systems) in 100 µl of PBS at 37 C for 2 hours after which the reaction mixture was plated on BG agar and incubated at 37 C for 2 days to calculate CFU. For the mbd3 assay, E. coli K12 bacteria were used as a positive control. Polymyxin B Susceptibility Assay. Cultures of RB50 or RB50ΔarnT were diluted to 10 6 CFU/mL into a final 1mL volume of PBS, PBS containing 10mg/mL polymyxin B, or PBS containing 100 mg/ml of polymyxin B. Suspensions were incubated for 2 hours at 37 C, following which, the number of organisms remaining in each sample was determined by quantitative culture on BG agar plates.

51 39 Colonization studies. C57BL/6 (wild type), and C3H/HEJ (TLR4-deficient) mice were obtained from Jackson Laboratories and bred in our Bordetella-free, specific pathogen-free facilities at The Pennsylvania State University. Bacteria grown overnight to an optical density at 600 nm of approximately 0.3 in liquid culture were diluted in PBS to approximately 2 x 10 6 CFU/mL. For a high dose/ high volume inoculation, 50 µl of the inoculum (10 4 CFU) was pipetted onto the external nares of 4 6 week old mice that had been lightly sedated with 5% isoflurane in oxygen. For low dose/low volume inoculations, bacterial cultures were further diluted in PBS to concentrations of 10 3 CFU/mL and for high dose/low volume to 4 x 10 4 CFU/mL. Mice were inoculated with doses of 5 CFU and 200 CFU respectively in 5μL using the previously described procedure. Groups of three or four animals were sacrificed on days 3, 7, 14, and 28 post-inoculation or as indicated, and the nasal cavity, trachea, and lungs were excised. Bacterial numbers in the respiratory tract were quantified by homogenization of each tissue in PBS followed by plating onto Bordet-Gengou blood agar containing streptomycin (20 μg/ml). Colonies were enumerated after 2 days of growth at 37 C. All protocols were reviewed and approved by The Pennsylvania State University Institutional Animal Care and Use Committee (IACUC), and all animals were handled in accordance with institutional guidelines. Shedding Analysis. Shedding was assessed by lightly swabbing the external nares for 10 seconds using a Dacron-polyester tipped swab. Swab tips were cut off and placed into 1 ml of PBS. Samples were vortexed vigorously and cultured on Bordet Gengou agar (Himedia).

52 40 Analysis of Leukocyte Recruitment. Prior to dissection, ml of PBS was perfused through the left ventricle of mice while venous runoff was collected from the orbit. Nasal bones were dissected and placed in 1 ml of DMEM containing 5% FBS and 1 mg/ml collagenase D. Samples were incubated for 45 min at 37 o C and subsequently disaggregated into a single cell suspension by mechanical disruption over a 70 µm mesh screen. Subsequently, 2x10 6 cells per well were then added to 96 well plates. Samples were resuspended in FC blocking buffer (200:1 anti-cd16/32 BD Biosciences in PBS + 2% FBS) and incubated on ice for 20 min. Following wash, cell surface markers were labeled with the following antibodies in PBS + 2% FBS: anti-cd45 APC-cy7 400:1 (BD Biosciences), anti-cd11b Horizon V450 (BD Biosciences), anti-ly6g APC (E Bioscience). Statistical Analysis. Data analysis between groups was performed using a One-Way Analysis of Variance test to evaluate statistical significance with p values <0.05 considered significant. The Reed Muench calculation was used for determination of ID50 (27). Results ArnT Is Required for Modification of Lipid A with Glucosamine. To investigate the role of ArnT enzymatic activity in GlcN modification of lipid A, the B. bronchiseptica RB50 arnt homologue, previously designated BB4268, was mutated to generate strain RB50ΔarnT. Purified lipid A isolated from RB50 or RB50ΔarnT was analyzed by matrix-assisted laser desorption ionization time of flight

53 41 (MALDI-TOF) mass spectrometry in the negative ion mode. Bordetella bronchiseptica lipid A had previously been characterized by Preston et al. (25). Peaks indicating a glucosamine addition were present at m/z 1651 and 1667, representing the monophosphorylated penta-acylated lipid A with a two acyl-oxo-acyl C12 or 2OH-C12 respectively, and m/z 1731, corresponding to the 1651 species with diphosphate additions (Figure 2.1A). The presence of the palmitate in acyl-oxo-acyl linkage at the C3 position was previously characterized and shown to be facilitated by the B. bronchiseptica PagP palmitoyl transferase (25). The lipid A form containing the phosphate addition was previously confirmed to be heterogeneously present in the bacterial population (15). Both of these peaks were entirely absent in the RB50ΔarnT spectrum (Figure 2.1B). Complementation with the arnt gene restored the phenotype (data not shown), demonstrating that ArnT was required for addition of glucosamine to Bordetella bronchiseptica lipid A. All other characteristic peaks were observed for both wild type and RB50ΔarnT. TLR4 Stimulation in Murine Macrophages, Serum Resistance and Adhesion Were Not Affected by arnt Mutation. ArnT-mediated modification of lipid A has been shown to enhance stimulation of TLR4 by B. pertussis LPS (17). To determine if the increased TLR4 agonist activity of B. bronchiseptica LPS was dependent on GlnN addition, cultures of murine macrophages (J774) were inoculated with either wild type strain RB50 or the arnt mutant. Release of the pro-inflammatory cytokine TNFα into the medium was used as a surrogate measurement of TLR4 receptor activity. Cultures were inoculated with MOIs ranging from to 1 and the concentration of TNFα in the medium was determined 2 hours

54 42 after inoculation by quantitative ELISA (Figure 2.2A). The amount of TNFα detected in the supernatant of cells exposed to B. bronchiseptica increased in a dose dependent manner; however, at each MOI, the amount of TNFα released by macrophages was similar between RB50 and RB50ΔarnT inoculated cell cultures. These data suggest that agonist activity of lipid A for TLR4 was not affected by ArnT-mediated addition of glucosamine. B. bronchiseptica is protected from the antimicrobial activities of serum complement by its LPS; strains lacking either O-antigen, such as (2), or the outer core oligosaccharide and O-antigen portions of LPS are highly susceptible to killing by serum complement. The B. bronchiseptica RB50 mutant with a deletion of wbm, a locus necessary for the assembly of the O antigen, lacks this structure and has the aforementioned defect in resistance to complement-mediated killing (2). To determine whether mutation of arnt affected serum resistance, approximately 10 3 CFU of RB50, RB50Δwbm, or RB50ΔarnT were incubated with concentrations of serum ranging from 0-15% (by volume). Whereas RB50Δwbm was killed by 5% serum, serum concentrations of up to 15% had no effect on either wild type or arnt mutant bacteria, demonstrating that mutation of arnt did not affect serum resistance (Figure 2.2B). Loss of the GlcN substitution might alter the structure of the outer membrane and destabilize interactions that facilitate bacterial adherence to respiratory epithelial cells. To test this, L2 rat lung epithelial cells were inoculated with between 5 1,000 CFU of the wild type strain or RB50ΔarnT. No significant difference was observed in the number of wild type or mutant bacteria recovered from the trypsin treated epithelial cells

55 43 (Figure 2.2C). This result suggests that binding of RB50 to the respiratory epithelium was not affected by deletion of arnt. Glucosamine Additions to Lipid A Contribute to Resistance against Cationic Antimicrobial Peptides. Host epithelial cells and leukocytes produce small cationic peptides, defensins, and cathelicidins that exhibit antimicrobial activity against numerous bacterial species (5, 8, 32). Defensins are amphipathic molecules proposed to intercalate into the bacterial cell membrane and use like-charge repulsion to disrupt membrane integrity (8). Disruption of the arnt locus rendered S. typhimurium susceptible to killing by cationic peptides. To test whether arnt provided a similar adaptation for B. bronchiseptica, we compared the ability of RB50 and RB50ΔarnT to survive in the presence of varying concentrations of antimicrobial peptides. RB50 was resistant to killing by polymyxin B at concentrations up to 100 µg/ml. However, greater than 90% of RB50ΔarnT were killed by 10 µg/ml, and 99.99% of the mutant bacteria were killed by 100 μg/ml of polymyxin B (Figure 2.3A). In addition, wild type RB50 survived a concentration of 10 µg/ml of the porcine β-defensin 1 (pbd1), whereas more than 99% of RB50ΔarnT were killed by 5 µg/ml of pbd1. Surprisingly, RB50 and the arnt mutant showed no significant difference in susceptibility to killing by mouse β-defensin 3, which shares homology with pbd1 (Figure 2.3C). Our results suggested that modification of lipid A by GlcN rendered RB50 more resistant to killing by some, but not all, antimicrobial cationic peptides.

56 44 B. bronchiseptica arnt Is Not Necessary for Infection of the Mouse Respiratory Tract. To determine whether decreased resistance to CAMPs would result in reduced fitness within a host, we compared the ability of the RB50 and the arnt mutant to grow and persist during experimental infections of mice. Following inoculation of C57BL/6 mice with 10 4 CFU of either RB50 or RB50ΔarnT in 50 µl of PBS, mice were dissected after 3, 7, 14 and 28 days. At each time point the number of CFU recovered from the respiratory tract of mice inoculated with RB50ΔarnT were similar to that recovered from RB50 inoculated mice (Figure 2.4). This result suggested that addition of GlcN to the lipid A was not required for growth and persistence of B. bronchiseptica in the murine respiratory tract when introduced in a high dose inoculum. RB50ΔarnT Is Unable to Colonize Mice When Inoculated in Low Dose. Although RB50ΔarnT grew and persisted similarly to the wild type strain following high dose inoculation challenge, we hypothesized that high dose infections may not accurately reproduce the interactions taking place when B. bronchiseptica initially seeds the respiratory mucosa. For example, CAMPs may not be sufficiently potent to control the initial influx of infectious organisms, or the epithelial cells that produce CAMPs may be rapidly damaged by bacterial toxins. A functional defect resulting from decreased CAMP resistance could therefore be more effectively determined using low dose inoculations. To approximate a mean infectious dose, 5, 50 or 200 CFU of either RB50 or RB50ΔarnT were inoculated in a 5 µl droplet onto the external nares of C57BL/6 mice. Seven days post- inoculation, the presence of B. bronchiseptica in the nasal cavity was determined (Figure 2.5A). Five CFU of wild type

57 45 RB50 was sufficient to infect 5 of 10 mice whereas 50 CFU resulted in stable colonization of 11/12 mice. Additionally, 12/12 mice became infected following inoculation with 200 CFU of RB50. By comparison, 5 CFU RB50ΔarnT infected only 1/16 mice, 50 CFU infected 4/12 mice and 200 CFU infected 12/ 12 mice (Figure 2.5B). Using the Reed Muench calculation (27), we determined that the ID50 of wild type RB50 was approximately 6.2 CFU whereas the ID50 of RB50ΔarnT was 30 CFU. RB50ΔarnT Fails to Transmit between Mice. Although the presence of ArnT did not affect the infectious burden following high dose inoculation, subtle functional deficiency resulting from mutation of arnt would be more likely resolved by analysis of transmission efficiency. We have previously demonstrated that RB50 transmits efficiently between C3H/HeJ mice (28). To evaluate whether RB50ΔarnT can be transmitted between animals, mice were inoculated with 500 CFU of RB50 or RB50ΔarnT. Inoculated individuals (index mice) were placed in a cage with 2-3 naïve mice (secondary mice). After co-housing for 21 days, mice were dissected and bacterial numbers in the nasal cavity were determined (Figure 2.6). Five out of 6 secondary mice exposed to RB50-inoculated index mice became colonized, whereas none of the secondary mice exposed to RB50ΔarnT-infected index mice became colonized (Figure 2.6). RB50ΔarnT Is not Defective at Being Shed by the Host. Although the inability of RB50ΔarnT to be transmitted corresponded with an increased ID50, this did not exclude the possibility that RB50ΔarnT might also be shed at a reduced rate. We have demonstrated that the shedding intensity of index mice correlated with the probability of transmission to other hosts (28). To determine whether

58 46 RB50ΔarnT was shed at a lower rate as compared to wild type RB50, C57BL/6 mice were inoculated with 500 CFU of either RB50 or RB50ΔarnT. Shedding was monitored by swabbing the external nares at multiple points over the course of 14 days. Neutrophil recruitment to the nasal cavity, previously shown to correlate with shedding intensity (10) was also determined at 7 and 14 days after inoculation. Shedding intensity of RB50ΔarnT was not different from that of RB50 at any time over a 14 day course (Figure 2.7B). Furthermore, neutrophil numbers in the nasal cavity of mice inoculated with RB50ΔarnT were similar to those obtained from RB50 inoculated mice at day 7 and day 14 post-inoculation (Figure 2.7A). These results demonstrate that ArnT is not required to induce neutrophil recruitment or enhance bacterial shedding from the murine nasal cavity. Discussion Phenotypic assessment of an arnt-deficient derivative of B. bronchiseptica strain RB50 showed that this gene was required for the modification of lipid A with GlcN (15). Deletion of arnt was associated with decreased resistance to killing mediated by cationic antimicrobial peptides (CAMPs). ArnT did not affect the growth or persistence within the host but was required for transmission. The failure of mice to transmit RB50ΔarnT corresponded with a five-fold increase in the mean infectious dose. Together these results suggested that ArnT-mediated addition of glucosamine to lipid A confers resistance to certain CAMPs and increases the frequency with which pathogens that seed the nasal mucosa are able to successfully colonize and initiate infections.

59 47 The protein encoded by arnt shares homology with Salmonella ArnT, a periplasmic enzyme that catalyzes the transfer of aminoarabinose from an undecaprenyl donor to the phosphate groups of lipid A and results in resistance to CAMPs (34). This protein is 100% identical at the amino acid level to the B. pertussis ArnT homologue, encoded by the gene designated BP0398. While the B. pertussis ArnT homologue mediates substitution of lipid A by GlcN (18), this activity was not associated with decreased resistance to CAMP-mediated attack but rather with increased TLR4 agonist potency (11). Deletion of the B. bronchiseptica ortholog did not alter the TLR4 stimulatory activity of the bacteria, suggesting that ArnT activity is not required for maximal stimulation of TLR4 by B. bronchiseptica. The differences between the observed effects of ArnT in B. bronchiseptica and B. pertussis may relate to their relative TLR4 stimulatory activity, which differ by an order of magnitude. The other structural differences between the LPS (actually LOS for B. pertussis) of these two organisms, and the basis for these differences, remain to be elucidated. B. bronchiseptica is strongly resistant to the bactericidal activity of porcine β- defensin 1 (6). In the absence of arnt, B. bronchiseptica is more susceptible to the bactericidal effects of polymyxin B and pbd1. These results are analogous to findings reported for deletion mutants of arnt in S. typhimurium. As with 4-amino L-arabinose, substitution of the phosphate group by GlcN could decrease the anionic character of the outer membrane. The charge neutralizing effect of GlcN may therefore represent an adaptation that destabilizes CAMP binding within the membrane and thus mitigates its activity. It is unclear why RB50ΔarnT is more susceptible to killing by pbd1 but is unaffected in its sensitivity to mbd3. Although CAMPs are hypothesized to have a

60 48 common mechanism of action based on conserved structural motifs, the multitude of genes encoding CAMPs in eukaryotic genomes, and the divergence of sequences from one host species to another suggests that individual CAMPs may have some specificity regarding the microbes they target and/or their mechanisms of action (8). Alternatively, it is possible that mbd3 is not highly active on its own but acts in cooperation with other antimicrobial mechanisms in vivo. Since ArnT activity is increased in the Bvg + phase, which is thought to contribute to growth during infection, one would expect this gene to contribute to fitness within the host. When comparing RB50 and RB50ΔarnT in mouse infections, differences between the two strains were observed upon titration of the infectious dose toward the lower limits. When mice were inoculated with RB50ΔarnT in a volume and dose sufficient to seed the entire respiratory tract with high bacterial numbers, no difference between the wild type parental strain and the arnt mutant was detected. When comparing the fitness of the wild type and the mutant introduced in very low numbers (50 CFU or less), the mean infectious dose required to stably infect 50% of mice (ID50) with RB50ΔarnT was approximately five-fold greater than the ID50 of the wild type strain. These data suggest that the likelihood of B. bronchiseptica successfully colonizing the respiratory tract of a new host is greatly enhanced by ArnT. The lipid A modification mediated by ArnT is likely an adaptation to host antimicrobial defenses, which while effective against small numbers of pathogens, may be overwhelmed by increased doses of bacteria. When pathogens seed mucosal surfaces and begin the process of invasion and colonization, CAMP molecules, in particular β-defensins, are among the first elements of host resistance encountered. Resistance to CAMP killing may play a role in the survival of

61 49 bacteria during the initial colonization process. However, due to the number of genes encoding CAMPs, knockout systems may not be a practical way of demonstrating that ArnT activity enables B. bronchiseptica to colonize mice by conferring resistance to CAMP mediated attack. Our results indicate that ArnT mediated GlcN modification of lipid A and contributes significantly to the infectiousness and transmissibility of B. bronchiseptica. We also demonstrated that directly quantifying transmission and infectivity using low dose infection models can be a more sensitive approach for probing interactions that are important for the initial colonization of new hosts, aspects not clearly observed when higher dose inocula are delivered. Whether this inocula-based difference occurs because important host defense measures are overwhelmed or because of some other mechanism remains to be determined. However, an approach focusing on lower, more natural, doses and on transmission between animals, revealed phenotypes dependent on ArnT that are likely to have important implications to the spread of infection, and therefore the burden of this infectious disease at the population scale.

62 50 Author Contributions Olivier Rolin 1,2*, Sarah J. Muse 1,3*, Chetan Safi 1, Shokrollah Elahi 4, Volker Gerdts 5, Lauren E. Hittle 6, Robert K. Ernst 6, Eric T. Harvill 1, and Andrew Preston 7 1 Department of Veterinary and Biomedical Sciences, The Pennsylvania State University, University Park, PA USA. 2 Graduate Program in Immunology and Infectious Disease. 3 Graduate Program in Biochemistry, Microbiology and Molecular Biology. 4 Seattle Biomedical Research Institute, Seattle, WA USA. 5 Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, SK Canada. 6 Department of Microbial Pathogenesis, University of Maryland School of Dentistry, Baltimore, MD, USA. 7 Department of Biology and Biochemistry, University of Bath, Bath, U.K. * Contributed equally to this manuscript Conceived and designed experiments: OR, SJM, CS, SE, VG, LEH, RE, ETH, AP Performed experiments: OR, SJM, CS, SE, LEH, AP Analyzed the data: OR, SJM, CS, SE, VG, LEH, RE, AP Wrote the paper: OR, SJM, ETH, AP

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66 54 Figure 2.1. Ions corresponding to glucosamine additions in RB50 are not seen in RB50ΔarnT.. Mass spectra of (A) B. bronchiseptica RB50, ions corresponding to glucosamine additions mass-to-charge ratio (m/z) of 1651, 1667, and 1731 are indicated (*) (B) RB50ΔarnT mutant strain no glucosamine peaks are present. Lipid A structures corresponding to m/z 1570 (C), 1808 (D) and 1651 (E).

67 55 Figure 2.2. ArnT was not required for induction of TNF TNFα in murine macrophages, complement resistance, or adherence to the lung epithelial cells. (A) J774 macrophages were incubated for 2 hours in the presence of RB50 (white bars) or RB50ΔarnT (black bars) at an MOI of 0.001, 0.01, 0.1 or 1.0. Grey bars represent cultures treated with media only. (B) 10 3 CFU of either B. bronchiseptica strain RB50 (white squares), RB50ΔarnT (black diamonds) or RB50Δwbm (black circles) were incubated for 1 hour in PBS only or PBS with 5, 10 or 15 percent naive mouse serum. Symbols represent the mean log10 CFU ± standard error of four individual samples recovered after incubation. The limit of detection is marked by a dashed line. (C) Increasing MOI of RB50 (white squares) or RB50ΔarnT (black diamonds) were inoculated onto cultured L2 rat lung epithelial cells. The quantity of CFU of RB50 (white squares) or RB50ΔarnT (black diamonds) adhering to epithelial cells following incubation was determined by culture. Symbols represent the mean CFU ± standard error of four individual samples.

68 56 Figure 2.3. ArnT was required for resistance to killing by cationic antimicrobial peptides CFU of bacteria were incubated for 2 hours in PBS (white bars) or PBS with increasing concentrations (striped bars, black bars) of (A) polymyxin B (B) pbd1 or (C) synthetic mbd3. B. bronchiseptica strains RB50 or RB50ΔarnT were incubated with mbd3, or E coli K12, whose susceptibility to mbd3 was previously shown, was used as a control to determine whether mbd3 was active (B). Bars represent the mean log10 CFU ± standard error of 4 individual samples in each group. * indicates a p value of <0.05, ** indicates a p value of <0.01. The limit of detection is marked by a dashed line.

69 57 Figure 2.4. ArnT did not contribute to growth or resistance in the respiratory tract in a high dose model of respiratory infection. Groups of 4 mice were inoculated with 5x10 4 CFU of RB50 (white squares) or RB50ΔarnT (black diamonds) in 50 µl of PBS. Symbols represent the mean log10 CFU ± standard error recovered from either the (A) nasal cavity, (B) trachea, or (C) lungs at 3, 7, 14 and 28 days following inoculation. The limit of detection is marked by a dashed line.

70 58 Figure 2.5. ArnT decreased the mean infectious dose of B. bronchiseptica. (A) C57BL/6 Mice were inoculated with 5 CFU in 5 µl of either RB50 (left) or RB50ΔarnT (right). Dots represent the CFU recovered from the nasal cavity of individual mice dissected seven days after inoculation. The whiskers span the 1 st and 4 th quartile. The limit of detection is marked by a dashed line. The proportion of infected mice in each group is listed above the box and whisker plot. (B) Percent of C57BL/6 mice infected after inoculation with either 5 CFU (10 mice/group), 50 CFU (12 mice/group) or 200 CFU (16 mice/group) of RB50 (white squares) or RB50ΔarnT (black diamonds).

71 59 Figure 2.6. ArnT was required for transmission of B. bronchiseptica between mice. Two index C3H/HeJ mice per group were inoculated with 500 CFU of either RB50 or RB50ΔarnT in 5μl PBS. Index mice were housed with 2-3 secondary mice for 3 weeks following inoculation. The quantity of CFU of RB50 (left) or RB50ΔarnT (right) recovered from the nasal cavity of individual secondary mice is represented by dots. Box spans the interquartile range, and the median value is represented by a bar. The whiskers span the 1 st and 4 th quartile. The proportion of infected mice in each group is listed above the box and whisker plot. The limit of detection is marked by a dashed line.

72 60 Figure 2.7. ArnT did not affect neutrophil recruitment or shedding from the host. C57BL/6 mice were infected with 500 CFU in 5 µl PBS of either RB50 or RB50ΔarnT. (A) Mice were sacrificed at either 7 or 14 days after inoculation and the mean ± standard error neutrophil counts from nasal cavities of mice infected with RB50 (white bars) or RB50ΔarnT (black bars) were obtained by flow cytometric detection of CD45 + / CD11b + / Ly6G + cells. (B) Shedding was detected by culture of bacteria from a 10 second swab of the external nares. Symbols represent the mean log10 CFU ± standard error shed from four mice/group infected with RB50 (white squares) or RB50ΔarnT (black diamonds). The limit of detection is marked by a dashed line.

73 Chapter 3 B. bronchiseptica and the Caspase-1 Inflammasome.

74 62 Abstract Successful bacterial pathogens have evolved numerous strategies to evade and manipulate host immune responses. These can include the ability to overcome early innate immune responses in order to prevent detection and favorably shift the type of immune responses elicited by the infection. Caspase-1 is a key inflammasome component that activates a pro-inflammatory signaling cascade and has been shown to be important for the control of invading pathogens. Using the animal pathogen B. bronchiseptica as a model, we investigated how bacterial infection can result in decreased caspase-1 levels; we also uncovered a novel requirement for the recently characterized Type VI Secretion System (T6SS). Although no differences in colonization or cytokine expression were detected between wild-type and Casp1 -/- mice during infection, exposing cultured macrophages to a functional B. bronchiseptica T6SS resulted in a reduction of Nlrp3 protein, an important NLR bacterial sensing protein, indicating the involvement of multiple components of the inflammasome. The presence of the T6SS did not affect Casp1or Nlrp3 mrna levels, indicating that the decrease did not take place at the level of transcription. Tissue culture inhibitor assays revealed that the B. bronchiseptica T6SS was required for the induction of autophagy and that this process caused the decrease in caspase-1. Understanding this mechanism illustrates the ability of B. bronchiseptica to manipulate host microbe sensing pathways to reduce inflammasome function and may allow for the future development of novel treatments for bacterial disease.

75 63 Introduction The presence of pathogenic bacteria in the body initiates a series of molecular signaling events that lead to activation of resident immune cells, the expression of proinflammatory cytokines, chemokine cascades, cell recruitment to the site of infection, and the generation of an adaptive immune response. Bacterial infections initiate an acute inflammatory response in the respiratory tract. Damage caused by the pathogen results in the expression of pro-inflammatory cytokines such as members of the IL-1 family, TNFα, and IL-6 (43) by resident phagocytic cells as well as epithelial tissue (49), resulting in chemokine signaling via CXCL10 (60), CCL20 (34), KC, LIX, or MIP-2 (58) and leukocyte extravasation through the microvasculature. The cytokines produced will also determine the type of immune response (ex. type 1 or 17 versus type 2) (44), T cell responses at the site of infection (2), and the generation of an adaptive immune response (26, 28) (Figure 3.1A). Understanding the mechanisms and interactions underlying the innate immune response will create a more complete picture of the dynamics of the overall immune system response to infection. The IL-1 signaling pathway (Figure 3.1B) consists of the generation and processing of precursor cytokines IL-1α and IL-1β, which in turn signal through the IL- 1R. This results in activation of NF-κB signaling as well as JNK, ERK1/2, p38 pathways, which induce the expression of IL-1 target inflammatory genes as well as Il1a

76 64 and Il1b (55). IL-1β precursor protein has been shown to be cleaved and prepared for secretion by protein complexes called inflammasomes, which assemble in response to pro-inflammatory stimuli (19). In this work, we used the respiratory pathogen B. bronchiseptica to study IL-1 and inflammasome signaling. B. bronchiseptica is closely related to the subspecies B. pertussis and B. parapertussis, the causative agents of whooping cough in patients (37). Therefore, researching the pathogenesis of B. bronchiseptica infection can reveal bacterial strategies for evading and manipulating the host immune response and lead to novel treatments for both human and veterinary respiratory diseases. Murine survival assays have shown that the IL-1R is necessary during B. bronchiseptica infection (Figure 3.2 A,B; L. Bendor and A. Karanikas, unpublished). IL- 1R -/- mice were shown to succumb to lethal bordetellosis, with entire cohorts of mice inoculated with B. bronchiseptica strain RB50 at doses of 5 x 10 5 and 1 x 10 5 CFU dying by days 3 and 4 post-inoculation respectively. The LD50 of RB50 in IL-1R -/- mice appears to be between 5 x 10 4 and 1 x 10 5 CFU, whereas death of wild-type mice was not observed with the infectious dose used in this experiment during the 12 day time period. Measurement of early colonization in the respiratory tract of inoculated mice revealed that RB50 colonized to significantly higher levels in the absence of IL-1R in the lungs and nasal cavity (Figure 3.3A,C), but no changes in colonization were observed in the trachea (Figure 3.3B). These results suggest that IL-1R is required for the control of bacterial numbers, which, if uncurbed, could be the cause of death in the mice. Interestingly, no mortality was observed in either wild-type or IL-1R deficient mice when they were inoculated with bacteria lacking a functional Type VI secretion

77 65 system (T6SS) (Figure 3.2 C,D). These data indicate that the T6SS is necessary for the lethal phenotype and could play an important role in interacting with the IL-1 signaling pathways. The genome of B. bronchiseptica laboratory strain RB50 contains a contiguous 26 gene locus that is predicted to encode T6SS associated genes (57). Type VI secretion systems are present in Gram negative bacteria and are structurally similar to T4 bacteriophage tail spikes (Figure 3.4) (23). The secretion apparatus consists of a membrane spanning protein complex, including core components Hcp, a multimerized protein that forms the tube-like structure for effector secretion; VgrG, which forms the tip of the complex that enables it to interact with the membranes of other cells; and ClpV, the AAA+ ATPase acting as the primary power source for the system (47). In the absence of clpv, the T4 phage-like sheath is unable to properly assemble and contract (3), rendering the secretion apparatus stuck and nonfunctional. The T6SS has been linked to virulence in eukaryotic systems. In Aeromonas hydrophila (51) and Legionella pneumophila (41), the T6SS is associated with increased macrophage cytotoxicity; and in Vibrio cholerae (35) and H. hepaticus (9) infections in the gut, the T6SS has been shown to lead to an increase in inflammation. In B. bronchiseptica, infections with the mouse model have shown that a functional T6SS is necessary for colonization of the respiratory tract as well as levels of lung pathology (57). These data indicate that the B. bronchiseptica T6SS could influence inflammatory processes during infection. In addition, the presence of the clpv component is required for production of key proinflammatory cytokines in cultured

78 66 macrophages, indicating that this system could influence the host innate immune response. Due to the superior natural host infection model and dramatic phenotypes observed, the knowledge acquired from studying how the T6SS in B. bronchiseptica interacts with the innate immune system can be used to better understand how human pathogens (Y. pestis, S. typhimurium, E. tarda, F. tularensis, P. aeruginosa, V. cholerae (40) ) that also have Type VI secretion systems, manipulate the immune system. One potential mechanism by which the T6SS could affect murine survival is by inducing increased levels of IL-1 cytokine, leading to localized inflammation and the generation of immune responses that could control the numbers and location of B. bronchiseptica. Therefore, in the absence of IL-1 signaling, the bacteria would be unchecked by this response and could spread systemically, leading to septic shock and death. However, no significant T6SS dependent changes in total IL-1β levels were detected in either the early cytokine response (Figure 3.5A) in the lungs or over the course of infection (Figure 3.5B). A possible explanation could be that the T6SS affects changes in active IL-1β levels, facilitated by caspase-1 inflammasome function (56). Work by Dunne et al. (15) has shown that adenylate cyclase toxin induces Nlrp3-caspase- 1 inflammasome mediated activation of IL-1β during B. pertussis infection, resulting in a robust, protective Th17 immune response. These data highlight the potential importance of inflammasome signaling during host-immune interactions with bordetellae species. One of the key mediators of inflammation is the cysteine protease caspase-1 (Figure 3.6A). Caspase-1 activation results in cleavage and activation of IL-1β and IL-18 (14) pro-inflammatory cytokines, as well as alterations in NF-kB signaling (31) and

79 67 changes in cell death pathways (1). These localized signaling events can affect many important immune processes which include promoting T cell differentiation (10) and upregulating cytokine production in immune cells (42). The caspase-1 inflammasome consists of a Nod-like receptor (prominent members include Nlrp1, Nlrp3, and Nlrc4 (21)) or Aim2 (24) cytosolic sensor protein (Figure 3.6B), which interacts though a Pyrin-domain with the scaffolding protein Asc (50), and caspase-1, which is recruited by Asc through interaction with the CARD domain and cleaved for activation (16). Activation of various members of the NLR family and assembly of the inflammasome complexes are stimulated by different ligands, such as lethal toxin produced by Bacillus anthracis and the muramyl dipeptide peptidoglycan component for Nlrp1 (17, 33); a wide variety of microbial toxins and ionophores or endogenous danger molecules for Nlrp3 (62); bacterial flagellin and T3SS structural components for Nlrc4 (38); and cytosolic DNA for Aim2 (18). One of the major inflammasome functions involves detection of microbial components (Figure 3.7A). This process enables the detection of pathogens, leading to an appropriate immune signaling response to facilitate their clearance. The Nlrp3 (nucleotide-binding domain and leucine-rich repeat protein 3) inflammasome is one of the best characterized complexes; is involved in detecting physiological stresses such as arthritis or gout as well as microbial threats; and is induced in a two-step process in which an initial signal through the TLR or other proinflammatory stimulatory pathways results in increased expression of the genes encoding Nlrp3 and Il1b. The second signal (one of the aforementioned bacterial or endogenous

80 68 ligands) results in the activation of the Nlrp3 complex formation (29). In Nlrp3 -/- macrophages, caspase-1 activation is decreased and susceptibility to killing is increased during V. parahaemolyticus and Yersinia infection (22, 61), indicating that Nlrp3 is necessary for control of complex assembly in response to microbes. However, pathogenic bacteria have developed ways to inhibit inflammasome function and favorably alter the mechanics of the subsequent host immune response to counteract detection and the subsequent immune response (Figure 3.7B). A M. tuberculosis zinc metalloprotease, Zmp1, is required to prevent caspase-1 activation (36). The presence of F. tularensis genes mvin and ripa decreases AIM2 inflammasome activation (54). In addition, Type III secretion system (T3SS) effectors play prominent roles in inflammasome inhibition. P. aeruginosa PA103 T3SS effector ExoU acts to inhibit the activation of caspase-1 (52) Yersinia T3SS effector protein, YopK, impedes recognition of bacterial components by the Nlrp3 and Nlrc4 inflammasome (4), and YopM binds directly to caspase-1 to prevent the formation of the complex (32). Another manner through which inflammasome activity can be limited is via activation of autophagy (46). Selective macroautophagy is a specialized degradative process which results in the targeted destruction of large cellular protein complexes, organelles, and even foreign bodies (30). Key steps in the process include autophagosome formation through the class III PI3K and Beclin-1 ;sequestration of the ubiquitinylated protein of interest; autophagosome-lysosomal fusion; and ultimately degradation and transport of components (Figure 3.8A) (53). Selective autophagy has also been shown to be involved in xenophagy to remove invading microorganisms (27).

81 69 Autophagy interfaces with the phagocytosis pathway by ubiquitinylating bacteria taken up or detected in the cell and targeting them for destruction during the autophagosomal fusion. Numerous intracellular bacteria have evolved effectors to avoid this destructive process, such as M. tuberculosis, that secretes ESAT-6 to block autophagosomal fusion, or cholera toxin from Vibrio cholera that presents the assembly of autophagy machinery (25). However, other bacteria utilize autophagy in a different survival strategy by using it to destroy inflammasome complexes and thus eliminate the pro-inflammatory downstream signals. Manipulation of autophagy by bacterial pathogens has been shown to influence bacterial interactions with the inflammasome and is a likely candidate to play a role in this system. Pseudomonas aeruginosa prevents caspase-1 from cleaving Trif, resulting in less inflammasome activation, lower autophagy, and higher bacterial numbers (26). Conversely, Vibrio parahaemolyticus secretes effector protein VopQ to induce autophagy and prevent Nlrc4 inflammasome activation (Figure 3.8B) (22). Our results indicate that B. bronchiseptica strain RB50 caused a clpv dependent decrease in caspase-1 protein in cultured macrophages as well as a decrease in Nlrp3 protein, an important inflammasome component. This clpv dependent process occurred due to selective autophagy. These novel findings implicate the Type VI secretion system in enabling B. bronchiseptica to manipulate inflammatory pathways and alter hostpathogen dynamics.

82 70 Materials and Methods Ethics Statement This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee at The Pennsylvania State University at University Park, PA (#31297 Bordetella-host Interaction). All animals were euthanized using carbon dioxide inhalation to minimize animal suffering. Bacterial Strains. Bordetella bronchiseptica strains RB50 (12) and RB50ΔclpV (57) were grown on Bordet- Gengou (BG) agar (HiMedia) supplemented with 15% defibrinated sheep blood (Hema Resources & Supply, Inc.) and 20 μg/ml streptomycin (Sigma-Aldrich) or grown to mid-log phase in Stainer-Scholte broth (48) supplemented with 500μg/mL heptakis (Sigma-Aldrich) shaking overnight at 37 C. Animal experiments. Wild type C57BL/6 mice were obtained from Jackson Laboratories, Bar Harbor, ME and and Casp1 -/- mice were obtained as a gift from the Flavell Lab at Yale University. Mice were bred and maintained at a specific pathogen-free facility at The Pennsylvania State University, University Park, PA, and all experiments were carried out in accordance with all institutional guidelines. All animal experiments were done as previously described (57). Mice were sedated with 5% isoflurane (IsoFlo, Abbott Laboratories) in oxygen and inoculated by pipetteing 50 µl PBS containing the indicated number of CFU onto the external nares of the mouse. For quantification of bacterial numbers, mice were euthanized with CO2 inhalation, and the respiratory organs were harvested. Tissues were homogenized in PBS, serially diluted and plated onto BG (Hi-Media) agar plates with 20 µg/ml streptomycin. Colonies were enumerated after 2

83 71 days of growth at 37 C. Survival curves were generated as previously described (6). Mice were observed over a 28 day period; any mouse exhibiting lethal bordetellosis, indicated by ruffled fur, labored breathing, and diminished responsiveness, was euthanized immediately to prevent unnecessary suffering. Bone Marrow Derived Macrophages. Bone marrow-derived macrophages were generated from bone marrow isolated from the femurs of C57BL/6 mice as previously described (60). Briefly, to extract the marrow, the bones were crushed with a sterile pestle through a 70 μm nylon cell strainer (BD Biosciences), and the cell suspension was pelleted and resuspended in Dulbecco s Modified Eagle s Medium/ F-12, 1:1 Mixture, with g/l Glucose, 15 mm HEPES, and L-Glutamine (BioWhittaker) containing 10% FBS (Gemini) and 20% L-929 cell supernatant media before seeding the plates. The L-929 supernate contains protein factors such as GM-CSF for macrophage differentiation. Cells were seeded into 100 x 15 mm non-tissue culture treated Petri dishes (VWR) and grown in 10mL of 20% L-929 supernate differentiation media in 5% CO2 incubator (Binder) at 37 C. After 6 days, the cells were checked for morphology and adherence. The cells were then removed from the plates using Cellstripper (Cellgro), resuspended in media and seeded into 6-well (Greiner Bio- One) or 24-well tissue culture-treated plates (Costar) overnight at densities of 1 x 10 6 and 2 x 10 5 cells per well, respectively. Cell culture infections. Bone marrow-derived macrophages were primed with 100ng/mL E. coli 011:B4 LPS (Sigma-Aldrich) for three hours. After three hours, all wells were aspirated and replenished with fresh media, or media containing RB50 or RB50ΔclpV at MOIs of 10. In assays to inhibit autophagy, cells were pre-treated for 30 minutes with media containing 10mM 3-methyladenine (3-MA) (Sigma-Aldrich). To ensure contact between the bacteria and the cultured macrophages, the plates were centrifuged at 500 x g for 5 minutes. After 2 or 4 hours, the medium was

84 72 removed, wells were washed with 1 x PBS, and cell lysates were harvested. All assays were performed in triplicate. RNA Extraction. Cell lysates were harvested in 1mL Trizol (Life Technologies) for RNA extraction. Trizol extraction was performed according to previously established methods (8). The extracted nucleic acid was treated with RNase free DNAse I (Ambion) for 20 minutes at 37 C to remove DNA contamination, and then the concentration of RNA was measured by Nanodrop (Thermo Scientific). RT-qPCR. 1μg of RNA from each sample was used to synthesize cdna using the Superscript III First Strand Synthesis System (Invitrogen) according to manufacturer s instructions. The resulting cdna was used as a template for qpcr. SYBR GreenER (Invitrogen) Master Mix was used with primers for Casp1 (5 -GCC CAC TGC TGA TAG GGT GA-3 (forward) and 5 -CCC GGG AAG AGG TAG AAA CG-3 (reverse)), Nlrp3 (5 -ATC AAC AGG CGA GAC CTC TG- 3 (forward) and 5 -GTC CTC CTG GCA TAC CAT AGA-3 (reverse) ), or Gapdh (5 -TGA CCT CAA CTA CAT GGT CTA CA-3 (forward) and 5 -CTT CCC ATT CTC GGC CTT G-3 (reverse)). ROX was used as a passive reference dye. 2 μl cdna and 23 μl Master Mix were added to each well of a 96-well optical reaction plate (Denville), and each plate was run using an Applied Biosystems StepOne Real-Time PCR System. All reactions were performed with 3 biological replicates in technical duplicate with no-template and no-reverse transcriptase controls. The data were analyzed using the 2 -ΔΔCt method (45), and expression of all genes was normalized to the Gapdh reference gene. The data are mean increases relative to samples harvested after 0 hours treatment.

85 73 Western Blots Cell culture lysates were harvested in Laemmli sample buffer (BioRad) with 2- Mercaptoethanol (Sigma-Aldrich), and protein concentrations were quantified using the Pierce 660 nm Protein assay in conjunction with the Nanodrop (Thermo Scientific). For detection of IL- 1β, cell culture supernatants were concentrated by a methanol/chloroform precipitation method described previously (15). Samples were run on a 12% polyacrylamide gel under reducing conditions and transferred to a polyvinylidene difluoride (PVDF) membrane (BioRad). Membranes were probed with primary antibodies against caspase-1 p10 (Santa Cruz, sc-514, 1:1000), Nlrp3 (Abcam, ab4207, 1:500), anti-il-1β (Abcam, ab9722) or β-actin (Sigma Aldrich, A2006, 1:12,000) and secondary goat anti-rabbit-hrp (Southern Biotech, , 1:10,000). The membrane signals were visualized using Immobilon Chemiluminescence detection reagent (Millipore) and the ChemiDoc XRS+ System (BioRad). Statistical Analyses. Significant differences between groups were determined using the 1-way ANOVA using MINITAB and Student s t-test with p values <0.05 considered significant. Results Caspase-1 is not required for B. bronchiseptica respiratory tract colonization. To determine if the previously observed IL-1R dependent increases in B. bronchiseptica colonization and lethality occurred due to the effects of caspase-1 signaling, colonization by wild-type RB50 (which has an intact T6SS) and RB50ΔclpV bacteria in C57BL/6 was measured (Figure 3.9 A, B, C), as well as RB50 in C57BL/6 and Casp1 -/- mice inoculated with CFU (Figure 3.9 D, E, F). The levels of

86 74 colonization of RB50 were significantly higher in comparison those of RB50ΔclpV, illustrating that clpv is required for colonization of the respiratory tract. However, no statistically significant differences in bacterial numbers were observed between the wildtype and Casp1 -/- mice, and none of the Casp1 -/- mice succumbed to bordetellosis during the 28 day time course. Therefore, it is unlikely that the T6SS dependent ability of bacteria to grow to large numbers in the absence of IL-1R is dependent on the caspase-1 mediated ability to activate IL-1β. It is also possible, because the IL-1R -/- mice were generated on the F2.129 background and were not C57BL/6 mice, that the previous phenotype was not observed due to genetic background specific effects (due to strain specific enhancers or alleles in inflammatory genes). However, it is possible that the T6SS could act on multiple points of the pathway. These results could also indicate that the T6SS is able to downregulate caspase-1 in the wild-type mice, thus enabling it to colonize at equivalent levels regardless of the presence of caspase-1. Caspase-1 could act on specialized immune cells such as macrophages and the result of the signaling involves a more specific, localized response. Therefore, the effect of the T6SS on caspase-1 in cultured macrophages was assayed (Figure 3.10). B. bronchiseptica reduces caspase-1 protein in a clpv-dependent manner. To determine the effect of B. bronchiseptica strain RB50 on the levels of caspase- 1 protein in macrophages, primed bone marrow-derived macrophages (BMDM) cells were inoculated at an MOI of 10 with RB50, RB50ΔclpV (lacks a functional T6SS), or medium as a control. Western blot analysis showed that there were lower levels of procaspase-1 and active caspase-1 protein in the macrophages treated with RB50 in comparison to medium alone (Figure 3.11 A,B ). However, the macrophages treated with

87 75 RB50ΔclpV did not show any changes in either form of caspase-1 in comparison to those treated with only medium, indicating that clpv was required for lower levels of caspase- 1. One of the canonical functions of caspase-1 is its ability to proteolytically cleave the precursor form of IL-1β, an important proinflammatory cytokine, into the active form, enabling the secretion of the mature IL-1β from the cell (59). To determine the effect of the B. bronchiseptica T6SS component clpv on IL-1β, BMDM cells were stimulated with LPS and treated with media, RB50, or RB50ΔclpV at an MOI of 10 for 2 or 4 hours. As a positive control, additional cells were treated with 5mM ATP to provide the second signal necessary for IL-1β secretion, and unprimed cells were included as a negative control (Figre 3.12A). Higher levels of total IL-1β were present in samples treated with RB50 in comparison to those treated with RB50ΔclpV, but the ratio of active IL-1β to total did not change in a clpv dependent manner (Figure 3.12B). Based on these findings, clpv is necessary for increased levels of total IL-1β protein. However, these data indicate that the clpv dependent decrease in caspase-1 does not result in a downstream decrease in active IL-1β. This is likely processed through one of the noncanonical activation pathways. clpv dependent reduction in caspase-1 occurred at the protein level rather than reduction in Casp1 mrna levels. One possible mechanism by which B. bronchiseptica affected caspase-1 protein levels was to alter the mrna levels of caspase-1. RT-qPCR analysis performed on BMDMs treated with RB50 or RB50ΔclpV (Figure 3.13). Analysis was performed using the 2 -ΔΔCt method (45), and expression of all genes was normalized to the Gapdh

88 76 reference gene. The data are mean increases relative to samples harvested after 0 hours treatment. The infected macrophages showed no significant clpv dependent changes in Casp1 mrna levels, signifying that the lower levels of the caspase-1 protein occurred in translation or are due to degradation. Reduction in caspase-1 protein correlates with lower levels of Nlrp3 protein. Because caspase-1 functions as a key member of canonical inflammasomes, it is likely that if caspase-1 protein levels were decreased in a clpv dependent manner, other protein components of the complex would be affected as well. Nlrp3 is a NOD-like receptor that serves as a sensor for the complex and has been shown to be stimulated by numerous pathogens (5, 11, 39). Western blot analysis to detect caspase-1, Nlrp3, and beta actin was performed on whole cell lysate collected from LPS primed BMDMs treated with media, RB50, and RB50ΔclpV (Figure 3.14 A,B). Densitometry analysis indicated that there were lower levels of Nlrp3 in cells treated with RB50 in comparison to those treated with RB50ΔclpV. Therefore, the decrease in caspase-1 protein levels may have been due to the decrease in components of assembled Nlrp3 inflammasomes. To determine if the presence of the T6SS affected the mrna levels of inflammasome components, RT-qPCR analysis was performed on samples extracted from BMDMs treated with RB50 or RB50ΔclpV. Fold induction of Nlrp3 (Figure 3.14C) relative to untreated macrophages showed no statistically significant clpv dependent changes in mrna levels. These data indicated that the decrease in Nlrp3 occurred at the level of the protein.

89 77 clpv is required for induction of autophagy and degradation of caspase-1 protein. A major protein degradation pathway that interacts with immune signaling components is selective autophagy (30). Recent findings have shown that inflammasomes can be selectively targeted for destruction in autophagosomes. To determine whether autophagy played a role in the clpv dependent decrease of caspase-1, BMDMs were incubated with medium or 10mM 3-methyadenine (3MA) and then treated with RB50 or RB50ΔclpV. Treatment with 3MA inhibited the process of autophagosomal formation, and prevented autophagy from occurring. Western blot analysis performed to detect caspase-1 and actin (Figure 3.15A,B) showed that the clpv dependent decrease in caspase-1 when cells were treated with RB50 did not occur when the 3MA inhibitor was added. The presence of 3MA also does not affect IL-1β activation levels. Taken as a whole, these results indicate that the presence of the B. bronchiseptica T6SS component clpv is required for the activation of the selective autophagy pathway and decreased the levels of caspase-1. Discussion Caspase-1, a prominent pro-inflammatory signaling protein, has been shown to influence the overall immune response through signaling at the site of infection and leading to larger shifts in the global immune response type. In culture, clpv, a gene encoding a core component of the B. bronchiseptica T6SS, was shown to be necessary for the induction of autophagy and decrease in caspase-1. These results suggest a novel requirement for T6SS in bacterial interaction with the inflammasome as well as

90 78 autophagosomal degradation. Changes in Nlrp3 indicated that the T6SS mediated effect could act on the Nlrp3 inflammasome complex as a whole and result in the possible ubiquitinylation and degradation of the entire complex via autophagy. The clpv dependent decrease in caspase-1 could be due to direct interaction between T6SS effectors acting on host cell or disruption of bacterial systems by dysregulating the T6SS and other B. bronchiseptica virulence factors. For example, in Burkholderia mallei, the T6SS shares regulatory pathways with the T3SS, which has previously been shown to influence inflammasome function (7). The T3SS might also be required for decreases in caspase-1 protein levels, and future work will be conducted to further explore these possibilities. In addition, this alteration in immune signaling could affect the intracellular phase for B. bronchiseptica (20). Bacterial pathogens have been shown to manipulate autophagy to survive intracellularly in addition to promoting inflammasome degradation (28). The T6SS induction of autophagy could come at the expense of the ability of B. bronchiseptica to occupy intracellular niche. However, the potential alteration of the inflammatory immune response could be more beneficial for the entire bacterial population. Identification of a T6SS effector that could induce the decrease of the inflammasome complex could be used as a potential recombinant protein treatment, which could reduce inflammation by decreasing Nlrp3 inflammasome signaling in chronic inflammatory conditions such as arthritis and gout. Chemical inhibitors utilizing a similar strategy of decreasing inflammasome activation are currently undergoing clinical trials (13); however, care must be taken to prevent subjecting the patient to a risk

91 79 of septic shock if the resident bacteria can no longer be properly controlled, such as was observed in the IL-1R -/- mouse model. Overall, bacterial manipulation of the autophagy pathway correlated to a decrease the levels of a major inflammasome component. This decrease could allow bacteria to change the response of resident macrophages and thus affect larger, more global immune responses in the respiratory tract. These data have uncovered a novel bacterial immune interaction involving a functional T6SS and its requirement for an autophagy dependent decrease in caspase-1, a key inflammatory signaling protein. These results can contribute to our understanding of immune-pathogen dynamics and may aid in the development of novel treatments for infectious disease.

92 80 Author Contributions Sarah J. Muse 1,2, David E. Place 1,3, Laura L. Goodfield 1,3, Sara E. Hester 1,2, Alexia T. Karanikas 1,3, Liron Bendor 1,4, Laura S. Weyrich 1,2, Eric T. Harvill 1 1 Department of Veterinary and Biomedical Sciences, The Pennsylvania State University, University Park, Pennsylvania, USA 2 Graduate Program in Biochemistry, Microbiology and Molecular Biology 3 Graduate Program in Immunology and Infectious Diseases 4 Graduate Program in Cell and Developmental Biology 5 Graduate Program in Genetics Conceived and designed experiments: SJM, DEP, LLG, SEH, ATK, LB, ETH Performed experiments: SJM, DEP, LLG, ATK, LB Analyzed the data: SJM, DEP, LLG, ATK, LB Wrote the paper: SJM, ETH

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97 85 Figure 3.1. IL-1 and the pro-inflammatory immune response. A) Pro-inflammatory cytokine and cell type specific response to infection. B) IL-1α and IL-1β synthesis and signal transduction pathway. Reprinted with permission from Nature Publishing Group, (A) Nat Rev Immunol Nov;8(11):829-35, (B) Nat Rev Rheumatol Jan;10(1):44-56.

98 86 Figure 3.2. clpv is required for mortality in B. bronchiseptica infected IL-1R -/- mice. Groups of wild-type F2.129 or IL-1R -/- mice were inoculated with 5 x 10 5 CFU RB50 (A,B) or RB50ΔclpV (C,D), and monitored for survival up to 12 days post-inoculation. *Courtesy of A. Karanikas and L. Bendor

99 87 Figure 3.3. IL-1R is required for control of B. bronchiseptica respiratory colonization. Groups of wild-type (diamond) and IL-1R -/- mice (square) were inoculated with 5 x 10 5 CFU RB50, and bacteria were enumerated in the lungs A), trachea B), and nasal cavity C) after 0, 12, 24, 36, and 48 hours. Symbols represent the mean CFU ± standard error of four individual samples. * indicates p<0.05. *Courtesy of A. Karanikas

100 88 Figure 3.4. The Type VI Secretion System (T6SS). A model for type VI secrection system assembly and function. A putative model that integrates the current data is proposed. An inner membrane channel formed by the IcmF like and IcmH like proteins interacts at the cytoplasmic side of the IM with a complex composed of the probable cytosolic type VI secrection (T6S) subunits and the ClpV AAA + ATPase. Recruitment of the ClpV multimer is induced by the regulation of forkhead associated (FHA) phosphorylation through the activities of PpkA and PppA, and by the presence of the Hcp protein. A multimer of the putative lipoprotein in association with periplasmic subunits is shown at the outer membrane. Putative routes for substrate translocation are depicted through the cell envelope and the host cell membrane (blue arrow) including a one step mechanism through a unique channel, and a two step mechanism, in which both steps are catalysed by T6S subunits with transient accumulation in the periplasm (P). This hypothetical model shows a trimeric VgrG inserted into the OM through the amino terminal domain and puncturing the host cell through the needle like structure formed by the central domains, releasing the activity domain into the host cytosol (for eukaryotic like activities) or in the medium (for binding or adhesion activities). J, L, M and H (TssJ, TssL, TssM and TssH respectively) represent the T6S core components, following the nomenclature of Shalom et al, Reprinted with permission from Wiley Online Library, EMBO Rep Aug;9(8):

101 89 Figure 3.5. Deletion of clpv does not affect induction of IL-1β in murine lungs during infection. Groups of C57BL/6 mice were inoculated with 5 x 105 CFU RB50 (black bars) or RB50ΔclpV (white bars). The early induction of IL-1β protein levels in the lungs was measured 4, 24, or 48 hours post-inoculation A), and the IL-1β levels were also measured over the course of infection 0,3,7,14, 28, and 49 days post-inoculation B). Bar represent the mean CFU ± standard error of four individual samples

102 90 Figure 3.6. Caspase-1 Inflammasome Complex. A) Mechanism of Inflammasome complex formation. The auto-repression of NLRP3 is removed in the presence of pathogen associated molecular patterns (PAMPs) from microorganisms or damage-associated molecular patterns (DAMPs) from endogenous danger signals. This results in exposure of the NACHT domain. In turn, NLRP3 oligomerizes and recruits apoptosis-associated speck-like protein containing a CARD (ASC; also known as PYCARD) and pro-caspase 1, triggering the activation of caspase 1 and the maturation and secretion of pro-inflammatory cytokines such as interleukin-1β (IL-1β) and IL-18. B) The three biochemically characterized inflammasomes. The NLRP1 inflammasome consists of NLRP1, ASC, and caspases-1 and -5. Little is known about the agonists that activate NLRP1. Anthrax lethal toxin, MDP, and decreased cytosolic ATP have been reported to stimulate this inflammasome. NAIP and NLRC4 form a caspase-1 inflammasome in response to bacterial flagellin and T3SS rod proteins. NLRP3, on the other hand, is activated by a wide range of agonists including a number of MAMPs and DAMPs. Reprinted with permission from Nature Publishing Group, Nat Rev Immunol Mar;10(3):210-5 and via the Creative Commons Attribution (CC BY) license, Front Immunol Oct 16;4:333.

103 Figure 3.7. Pathogen Interaction with the Inflammasome. 91

104 92 A) Pathogen components that activate inflammasomes. B) Microbial and endogenous inhibitors of inflammasome activation. Reprinted with permission from Nature Publishing Group, Nat Immunol Mar 19;13(4): and Nat Immunol Mar 19;13(4):333-42

105 93

106 94 Figure 3.8. Autophagy and the Inflammasome. A) Model overview of autophagy pathway. B) Degradation of inflammasome through autophagy pathway. Reprinted with permission from Nature Publishing Group, Nat Rev Microbiol Feb;12(2): and via the Creative Commons Attribution (CC BY) license, Front Immunol Mar 12;3:44. Figure 3.9. Effect of clpv and Caspase-1 during B. bronchiseptica Colonization in the Mouse Model. Colonization of RB50 verses RB50ΔclpV in C57BL/6 mice at an inoculation dose of CFU in 50 µl in the nasal cavity A), trachea B), and lung C). Colonization of RB50 in C57BL/6 and Casp1 -/- mice at an inoculation dose of CFU in 50 µl in the nasal cavity D), trachea E), and lungs F). Symbols represent the mean CFU ± standard error of four individual samples *= p<0.05

107 95

108 96 Figure Model of T6SS affecting the inflammasome in macrophages. Model of formation of the T6SS Apparatus.

109 97 Figure clpv was required for reduction of capase-1 protein in cultured macrophages. A) Western blot analysis to detect caspase-1 and actin (reference) was performed on whole cell lysates from bone marrow derived macrophages that were treated for 2 hours with medium, RB50, or RB50ΔclpV (MOI 10). B) Quantification was performed to normalize signal to reference levels (caspase-1: actin).

110 98 Figure clpv is required for IL-1β production. LPS primed bone marrow-derived macrophages were treated with media, RB50, RB50ΔclpV (MOI 10) for 2 hours with or without the presence of the 3MA inhibitor. Macrophages treated with 5mM ATP and cells that were not primed with LPS were included as controls. Western blot analysis was used to measure IL-1β in methanol-precipitated supernatant and whole cell lysate.

111 99 Figure clpv did not affect Casp1 mrna levels. RT-qPCR analysis was performed on samples extracted from bone marrow derived macrophages. Cultured macrophages were treated with RB50 or RB50ΔclpV (MOI 10) for 0.5, 1, or 2 hours, and fold induction of Casp1 was measured relative to untreated macrophages using the 2 -ΔΔCt method with Gapdh as a reference gene. Relative expression is shown as mean + standard error, * indicates p<0.05.

112 100 Figure clpv is necessary for in decreased levels of Nlrp3 protein. (A) Western blot analysis to detect caspase-1 and NLRP3 was performed on whole cell lysate collected from LPS primed bone marrow derived macrophages treated with media, RB50, or RB50ΔclpV. ). B) Quantification was performed to normalize signal to reference levels (Nlrp3: actin). RT-qPCR analysis was performed on samples extracted from cultured macrophages treated with RB50 or RB50ΔclpV (MOI 10) for 0.5, 1, or 2 hours, and fold induction of Nlrp3 C) in bone marrow- derived macrophages was measured relative to untreated macrophages using the 2 -ΔΔCt method with Gapdh as a reference gene. Relative expression is shown as mean + standard error, *p<0.05.

113 101 Figure clpv is required for induction of autophagy and degradation of caspase-1. Bone marrow derived macrophages were incubated with media or 10mM 3MA and then treated with RB50 or RB50ΔclpV (MOI 10) for 2 hours. Western blot analysis was performed to detect caspase-1 A) and actin B).

114 102 Chapter 4 Hcp is necessary for normal B. bronchiseptica growth and morphology.

115 103 Abstract The Type VI secretion system (T6SS) is a T4 phage-like apparatus that is present in numerous species of Gram negative bacteria and has been shown to affect bacterial interspecies competition as well as virulence against eukaryotic host cells. Previously, we described a T6SS locus in B. bronchiseptica, and have shown that clpv, which encodes an ATPase that provides power for the system, is required for manipulation of macrophages and colonization in a mouse model. To determine the role of a gene encoding another conserved core component of the T6SS apparatus, hcp, an in-frame deletion was generated in B.bronchiseptica strain RB50. Deletion of hcp increased lag phase when grown in Stainer-Scholte media but not when the strain was grown in LB broth or medium supplemented with yeast extract. In addition, RB50Δhcp was more susceptible than wild type to polymyxin B, suggesting membrane alterations. Finally, hcp was required for normal morphology in B. bronchiseptica; the absence of hcp resulted in enlarged, filamentous bacteria, indicating dysfunctional cell replication. These results showed the importance of the tube component of the T6SS for maintenance of homeostasis within the bacterium.

116 104 Introduction The Type VI secretion system (T6SS) is found in a wide range of Gram negative bacteria (25% of proteobacteria) (25) and has been shown to affect bacterial interspecies competition (1) as well as virulence against eukaryotic host cells (4). Type VI secretion systems are encoded by a locus of conserved genes and share a large degree of homology with a T4 phage tail (5, 16). The proteins form a membrane spanning complex, complete with an ATPase /chaperone and secretion tube, and topped by a potential cell puncturing multimeric protein (Figure 4.1A) (16). The conserved core components include ClpV (24), VgrG (20), VipA/B (7), and Hcp (38). Hcp (haemolysin coregulated protein) dimerizes to form a hexameric ring structure (15, 31) that has been shown in vitro to polymerize and form the tube portion of the T6SS (3, 8). Hcp shares homology with the T4 phage gp19 tail protein (25) and forms a tube structure analogous to the contractile phage sheath (6). VgrG interacts with the polymerized Hcp tube, and the VipA/B polymerized sheath assembles in the cytoplasm, requiring ClpV ATPase activity (2). The VipA/B tail sheath has been shown to act as a scaffold for head-to-tail Hcp subunit assembly and is thought to propel the assembled Hcp nanotube and VgrG spike out of the cell and through the target cell membrane (9, 19). Hcp has been shown to be required for secretion of multiple classes of effectors, and its secretion is considered the hallmark of a functioning T6SS (41). Hcp can act as a chaperone for effectors, such as the Tse2 toxin in P. aeruginosa, as well as serving to stabilizing effectors that interact with VgrG through the PAAR motif, such as Tse5 and

117 105 Tse6 (37, 41). Hcp is thought to enable the transport and release of these effectors and is not thought to interact with them in their final, active conformation. Hcp is also required for virulence against amoeba (44), yeast (18), host cells (45), and in some systems, plays a role in quorum sensing (21) and biofilm formation when the T6SS is necessary (22). In addition, Hcp has been shown to be necessary for bacterial growth (1) as well as under stressed environmental conditions (17, 42). We investigated the role of hcp in B. bronchiseptica strain RB50. B. bronchiseptica is closely related to the subspecies B. pertussis and B. parapertussis, the causative agents of whooping cough (28). Therefore, understanding the pathogenesis of B. bronchiseptica infection can reveal bacterial strategies for evading and manipulating the host immune response and can lead to novel treatments for both human and veterinary respiratory diseases. Previous research in the lab has been performed to characterize the T6SS locus in RB50 (Figure 4.1 B). The locus consists of 26 genes, including all the core components as well as 13 novel, Bordetella specific genes (40). SWISS-Model analysis indicates that the B. bronchiseptica Hcp shares strong structural similarity (RMSD=0.06) with the P. aeruginosa PAO1 Hcp crystal structure (Figure 4.2A) (36). Recombinant Hcp (Figure 4.2B, L. Bendor, unpublished) was imaged using negative stain TEM and shown to have the characteristic hexameric shape associated with the protein complex. Preliminary experiments with the protein showed that it was immunogenic, indicating that its presence could shape the immune response against the T6SS (36). Previous work has shown that clpv is required for in vitro macrophage cytotoxicity as well as induction of interleukin (IL)-1β, IL-6, IL-17, and IL-10 production

118 106 in J774 macrophages (40). clpv is necessary for alterations in cytokine production, lung pathology, delayed lower respiratory tract clearance, and long term nasal colonization in the mouse model (40). Therefore, we are interested in determining the role played by hcp, another key T6SS component and have examined how disruption of this gene can influence membrane stability and bacterial growth and morphology. Materials and Methods Bacterial Strains and Growth B. bronchiseptica strain RB50 (12), RB50ΔclpV (40), and RB50Δhcp were maintained on Bordet Gengou (BG) agar plates (Difco) containing 10% sheep blood (Hema Resources) with 20 µg/ml streptomycin (Sigma). E. coli K-12 were maintained on Luria Bertani (LB) plates and cultured in LB broth. B. bronchiseptica strains were cultured in Stainer-Scholte (39) or LB liquid media until mid-log phase, shaking overnight at 37ºC. Growth curves were performed as previously described (32). Construction of RB50Δhcp Strain The RB50Δhcp strain was constructed using an allelic exchange strategy as previously described (40). The first 3 codons of hcp (BB0802) and the 755 base pairs (bp) upstream were amplified via PCR using primers flanked with BamHI on the 5 end and EcoRI on the 3 end (Table 1). The last 6 codons of hcp and the 584 bp downstream were amplified via PCR using primers flanked with EcoRI on the 5 end and BamHI on the 3 end (Table 1). These fragments were PCR purified (Qiagen), BamHI digested

119 107 (New England Biolabs), gel purified (Qiagen), and ligated overnight at 4 C (New England Biolabs), and amplified with the 5 F and 3 R primers as described above. The 1,364 bp knock-out construct was then ligated into the TOPO-TA vector (Invitrogen), transformed into Mach1 DH5α cells (Invitrogen), and verified by sequencing. The 1,364 bp construct was digested from TOPO-TA, gel-purified, and ligated overnight into the BamHI-digested pss4245 allelic exchange vector (courtesy of S. Stibitz). Triparental mating was conducted with DH5α containing pss4545δhcp, DH5α containing pss1827, and B. bronchiseptica strain RB50 grown on BG plates with 50 mm MgSO4 (Bvg conditions). The mating was performed for 4 hrs on a BG-10 mm MgCl2-50 mm MgSO4 plate at 37 C. B. bronchiseptica containing pss4245δhcp was positively selected by growth on BG-streptomycin-kanamycin-50 mm MgSO4 plates and incubated for 2 days at 37 C, which was performed twice. The resulting colonies were streaked onto BG plates and incubated for 2 days at 37 C, which resulted in colonies lacking pss4245 and containing either the wild type hcp or knockout construct. Colonies were then screened for the presence of either the wild type or knockout construct using PCR. The absence of pss4245 was confirmed by growth on BG-streptomycin plates and lack of growth on BG-kanamycin plates. Polymyxin B Susceptibility Assay Cultures of RB50, RB50ΔclpV and RB50Δhcp were diluted to 10 4 CFU/mL into a final 100 µl volume of PBS or PBS containing 0.09, 0.19, 0.39, 0.78, 1.56, 3.12, 6.25, 12.5, or 25 µg/ml polymyxin B. Suspensions were incubated for 2 hours at 37 C, following which, the number of organisms remaining in each sample was determined by quantitative culture on BG agar plates.

120 108 Brightfield and Transmission Electron Microscopy RB50 and RB50Δhcp cells grown to mid-log phase in either LB or Stainer- Scholte broth were visualized using an Olympus BX61 microscope. One ml of RB50 and RB50Δhcp cells grown to mid-log phase in either LB or Stainer-Scholte broth were centrifuged at 10,000 x g for 1 minute. The cells were fixed overnight at 4ºC in 2% glutaraldehyde in 0.05 M sodium cacodylate, ph 7.2. The pellets were washed 3 times in 0.1 M sodium cacodylate and incubated for 1 hour in 2% osmium tetroxide in 0.1 M sodium cacodylate, ph 7.2. Then the pellets were washed once with 0.1 M sodium cacodylate, 2 times with dh20 water, and stained for 1 hour in 2% aqueous uranyl acetate. The samples were dehydrated in acetone and then infiltrated with Epon araldite resin for incubation overnight at 60ºC. Ultrathin sections of the embedded sample were cut onto grids, and samples were visualized using the JEM 1200 EXII electron microscope (JEOL). Statistical Analysis Data analysis was performed using a two-way Student s T-test, with significant values of p>0.05. Results Deletion of hcp from B.bronchiseptica strain RB50 To investigate the role of hcp in B. bronchiseptica, we constructed an in-frame deletion of hcp (BB0802) in B. bronchiseptica strain RB50 using the pss4245 allelic exchange vector (10) to generate RB50Δhcp (Figure 4.3A). PCR was performed to

121 109 confirm the deletion; amplification of the wild type hcp gene resulted in a 1,945 bp product, and amplification of the region after deletion of hcp resulted in a 1,364 bp product (Figure 4.3B). These results indicate that the RB50Δhcp strain was successfully generated and could be used for further experimentation. hcp contributes to bacterial growth in a Bvg independent, media specific manner When assessing the growth of RB50Δhcp in Stainer-Scholte broth (39), a defined medium used to culture bordetellae and optimize production of virulence factors, we observed that the mutant lacking hcp had an increased time in lag phase in comparison to wild type RB50 (Figure 4.4A). Because bacteria use the lag phase of growth to adapt to new environmental conditions and synthesize proteins and metabolites necessary for cell replication, it is possible that the presence of hcp is necessary for the function of these pathways. Therefore, RB50 and RB50Δhcp were cultured in the nutrient rich LB broth, and growth was assessed over time (11). When RB50Δhcp was cultured in LB broth, the lag phase was decreased (6 hours versus 12 hours), indicating that the hcp dependent alteration in growth dynamics was medium specific (Figure 4.4B). The BvgAS two-component system in Bordetella serves as a master regulator of gene sets, including virulence factors such as adenylate cyclase toxin in the Bvg + phase as well as motility genes such as flagellin in the Bvg - phase, and bipa in the Bvg i phase (12, 32). Because Stainer-Scholte media was developed to maximize virulence factor production, the bordetellae grow in the Bvg + phase when cultured in Stainer-Scholte broth. However, different sets of genes expressed in the various Bvg phases, including the Bvg - phase, which enables the bordetellae to grow under nutrient limiting conditions (13, 33). To determine whether the Bvg phase of the bacteria contributed to the effect of

122 110 hcp on length of lag phase, RB50 and RB50Δhcp were cultured in Stainer-Scholte broth supplemented with 40mM MgSO4 to shift the phase to Bvg- (Figure 4.4C. However, the change in Bvg phase did not decrease the lag phase of RB50Δhcp, indicating that the effect of hcp on bacterial growth kinetics was not due to Bvg-dependent production of virulence factors. RB50Δhcp showed decreased lag phase when grown in a 1:1 mixture Stainer-Scholte and LB broth (6 hours versus the original 12 hours) (Figure 4.4D), illustrating that the mutant adapted more rapidly to growth in LB broth. The addition of yeast extract to Stainer-Scholte also for a shorter lag phase for RB50Δhcp to 4 hours (Figure 4.4E). hcp is required for resistance to polymyxin B Changes in growth kinetics could be caused by disruption of the bacterial membrane. In the absence of hcp, the other T6SS structural components could form a dysfunctional complex, changing architecture, composition, or properties of the outer and inner membrane. Alternatively, without hcp acting as a chaperone, T6SS effectors could be released into the cytoplasm, causing the misfolding of proteins or oxidative stress responses which have been shown to alter membrane properties (34). To test whether hcp is required for membrane stability, RB50 and RB50Δhcp were treated with multiple concentrations of polymyxin B (Figure 4.5). Polymyxin B is an amphipathic molecule that works through indirect interaction with the lipid A and subsequently acts to perturb the bacterial membrane (14). Susceptibility to polymyxin B has been shown to be associated with changes in outer membrane protein expression (26, 27). RB50Δhcp was significantly more susceptible to polymyxin B than RB50 (decreased below the threshold of detection after exposure to 0.39µg/mL). These data show that hcp was necessary for

123 111 resistance to killing with this cationic protein and suggested that hcp plays a role in membrane stability, which could explain effects on growth. hcp is required for normal morphology of B. bronchiseptica To examine the growth defect more closely, RB50Δhcp was grown in LB and Stainer-Scholte media, and the culture turbidity and number of colony forming units (cfu) were measured over time (Figure 4.6 A,B). Although RB50Δhcp grown in Stainer- Scholte media has an extended lag phase in comparison to RB50Δhcp grown in LB broth, the bacteria grown in Stainer-Scholte media have equivalent numbers of cfu/ml to those grown in LB after 12 and 24 hours and significantly higher numbers of viable bacteria at the 6 and 18 hour timepoints. RB50Δhcp grown in Stainer-Scholte has an increased ratio of cfu/ml to optical density (Figure 4.6 C) in comparison to RB50Δhcp grown in LB broth. These data suggest that growing RB50Δhcp in LB broth could enable the bacteria to increase in biomass without the generation of more viable cells. These data could indicate that in the absence of hcp, when grown under nutrient rich conditions, B. bronchiseptica could be replicating normally and then dying, or the bacteria could be enlarged due to continued cell growth without division. Gram stain pictures and quantification of area indicated that RB50Δhcp were filamentous in shape and were 4 times larger in area than the wild type when grown on BG agar (Figure 4.7 A,B). Therefore, in the absence of hcp, filamentation occurs in nutrient rich conditions. These data added further evidence to the possibility of growth without cell division by illustrating that the presence of hcp affected cell morphology. Therefore, we investigated whether the RB50Δhcp bacteria grown in LB broth became elongated and filamentous (increased nutrients partially enabling the bacteria to

124 112 increase in size but still unable to divide), while RB50Δhcp grown in Stainer-Scholte medium did not (no increase in cell size or division). Transmission electron microscopy analysis was performed and showed that RB50Δhcp bacteria did grow as filaments in comparison to wild type when cultured in LB broth (Figure 4.8 A,B). The RB50Δhcp cells cultured in Stainer-Scholte broth comprised a more heterogeneous population of both fibrous bacteria and bacteria which were closer to wild type in size but with a more rounded shape (Figure 4.8 C,D). These findings could indicate that when grown in Stainer-Scholte, the bacteria lacking hcp do not form filaments as readily, or they become filamentous but can undergo some cell division as they reach the end of lag phase. Overall, these results suggest that hcp is required for normal B. bronchiseptica cell replication. Discussion This work has uncovered a role for Hcp, a core component of the T6SS, in bacterial function and viability of B. bronchiseptica. These data underscore the importance of understanding the roles of individual secretion system components in relation to the maintenance of global homeostasis of multiple bacterial systems rather than simply in the context of the system function and secretion of effectors. In other systems, bacterial filamentation has been shown to occur in response to the presence of bacterial stress (23). One possible explanation for the observed phenotype is that the disruption of a secretion system that spans the outer and inner membrane could create membrane stress. Also, because of the Hcp chaperone activity,

125 113 T6SS effector proteins could be released into the cytoplasm, resulting in damage or even triggering the misfolded protein response. This possibility could be tested by pulling down Hcp in the cytoplasm to determine which proteins interact, or putative effectors in the cytoplasm could be detected using antibodies generated against recombinant proteins encoded by the T6SS locus. Future directions also include determining how the absence of hcp creates problems with cell replication. To directly show that hcp is required for normal septal formation, visualization of the cell division process could be performed via microscopy using a membrane dye, such as FM 46-4 (43) or immunofluorescence with antibodies against FtsZ, a tubulin like protein that enables Z ring assembly (30). The absence of hcp could result in induction of SOS response, which has been shown to halt cell division in the presence of DNA damage. The initiation of this pathway could occur through potential damaging agents generated by cytoplasmic effectors, direct damage to the nucleoid (tssd) encodes a putative effector with nuclease activity), or via crosstalk with the aforementioned stress pathways. Overall, these findings suggested that Hcp, a single secretion system component, can alter other critical functions besides virulence and illustrates the drastic alterations which can be facilitated by subtle disruptions of protein apparati. These data open up new avenues of research in defining how components of the T6SS can affect integral processes of bacterial physiology.

126 114 Author Contributions Sarah J. Muse 1,2, Yury V. Ivanov 1, Kai Hu 1,3, Liron Bendor 1,3, Eric T. Harvill 1 1 Department of Veterinary and Biomedical Sciences, The Pennsylvania State University, University Park, PA Graduate Program in Biochemistry, Microbiology and Molecular Biology 3 Graduate Program in Genetics Conceived and designed experiments: SJM, YVI, ETH Performed the experiments: SJM, YVI, KH, LB Analyzed the data: SJM, YVI, KH, ETH Wrote the paper: SJM, ETH

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130 118 Figure 4.1. The Type VI Secretion System (T6SS). A) Model of formation of the T6SS Apparatus. B) B. bronchiseptica strain RB50 T6SS locus.

131 119 Figure 4.2. Hcp protein. A) Crystal Structure of Hcp1 protein from P. aeruginosa. B) Negative Stain TEM imaging of recombinant B. bronchiseptica Hcp protein. C) SWISS Model Alignment of Hcp1 from P. aeruginosa PAO1 (PDB entry 1Y12) and model of B. bronchiseptica BB802. Reprinted with permission from American Association for the Advancement of Science, Science Jun 9;312(5779):

132 120 Figure 4.3. Construction and confirmation of RB50Δhcp. A) Schematic for generating RB50Δhcp using the pss4245 allelic exchange vector. B) PCR confirmation of knock out generation. Size markers are designated on the left.

133 121 Figure 4.4. hcp contributed to bacterial growth. OD600 values were measured for RB50 and RB50Δhcp grown in Stainer-Scholte broth (A) and LB broth (B) as well as C) Stainer Scholte broth supplemented with 40mM MgS04. OD600 values were measured for RB50 and RB50Δhcp grown in D) a 1:1 mixture of Stainer Scholte broth and LB broth and E) Stainer Scholte supplemented with yeast extract. * indicates p<0.05

134 122 Figure 4.5. hcp is required for polymyxin B resistance. RB50 and RB50Δhcp were incubated for 2h at 37 C in PBS with multiple concentrations of polymyxin B (0-25 µg/ml, twofold dilutions) and plated on BG agar for bacterial enumeration.

135 123 Figure 4.6. RB50Δhcp optical density did not correspond to colony forming units. Growth curve of RB50Δhcp in LB broth and Stainer Scholte measuring A) OD600and B) CFU/mL. Ratio of CFU/mL vs. OD600 is represented by C). * indicates p<0.05

136 124

137 125 Figure 4.7. hcp is required for normal morphology in B. bronchiseptica. A) Gram-stain of RB50 (left) and RB50Δhcp (right) grown on BG agar. B) Quantification of bacterial volume of RB50 and RB50Δhcp using ImageJ for length measurement and approximating cylindrical shape. Transmission electron microscopy images of RB50 and RB50Δhcp grown to mid-log phase in Stainer Scholte (C,D) and LB broth (E,F).

138 Figure 4.8. Model of hcp in B. bronchiseptica growth. 126

139 127 Table 4.1. Primer Sequences. Primer Name Sequence Hcp KO 5 F BamHI AGA GGA TCC GTT CGT TCA AGC TGT ACG Hcp KO 5 R - EcoRI Hcp KO 3 F - EcoRI Hcp KO 3 R Bam HI CAC AAG GAC TGG ACC GAT GAA TTC TAG AAA GAA TTC CTG GTC GCG TGG GAC ATC TTC TCG ACG GCA GCT ATG GGA TCC TAA

140 Chapter 5 Summary and Significance.

141 129 Synopsis The development of new treatments to fight infection depends on understanding the mechanisms by which bacteria use virulence factors to survive and propagate between host systems. The goal of this work was to investigate how B. bronchiseptica uses its virulence factors to survive and propagate. Multiple strategies pursued by the bacterium were investigated, such as novel virulence and transmission properties for arnt, a mechanism by which B. bronchiseptica can alter inflammasome cell signaling in host immune cells, and how hcp, a Type VI secretion system component, can have important functions for the bacterium in the absence of host interaction. These experiments shed light on strategies used by B. bronchiseptica to survive, manipulate the host in a beneficial manner, and facilitate transmission to another host. Enzymatic Modification of the Lipid A by an ArnT Protects B. bronchiseptica Against Cationic Peptides and Is Required for Transmission By investigating a strategy used by pathogens to adapt to their host environment, we uncovered a novel virulence property for arnt, which encodes a protein that modifies the lipid A by adding glucosamine. As previously observed in other systems, arnt is required for resistance to cationic antimicrobial peptides; however, novel findings from our studies show that arnt is also required for transmission between hosts, indicating how virulence factors can influence multiple aspects of a pathogen s life cycle.

142 130 Summary and Implications To investigate the role played by the glucosamine (GlcN) lipid A modification, we generated the RB50ΔarnT deletion mutant and verified that the arnt was required for the GlcN addition using mass spectrometry (Figure 2.1A-E). Interestingly, the presence of arnt did not affect TLR4 signaling (measured by TNF-α levels) in J774 cells (Figure 2.2A), which differed from the phenotype observed from the B. pertussis arnt deficient mutant which was used to infect human THP-1 cells (12). However, this distinction could be due to the fact that infection with B. bronchiseptica stimulates TLR4 more highly than infection with B. pertussis (12, 13), as well as the fact that there are other structural differences (i.e. B. pertussis has a lipooligosaccharide) (2). The lipid A modification provided resistance to antimicrobial peptides (Figure 2.3), which is a trait that may have evolved to counter the barrage of cationic peptides encountered as the bacteria initially try to colonize the mucosa (6, 8). However, the ability of arnt to affect colonization in the respiratory tract depends on the dose of bacteria administered. Using low dose model of infection illustrates that glucosamine modification could enable a smaller number of bacteria to establish an infection in the nasal cavity (Figure 2.5), whereas a difference is not detected using the more standard high dose model (Figure 2.4). This fact draws attention to subtle bacterial properties that may ensure survival during a natural infection but would not be detected using the high dose infection model. One of the most interesting findings indicates that arnt is required for transmission between hosts (Figure 2.6) without affecting cell recruitment (Figure 2.7A)

143 131 or bacterial shedding (Figure 2.7B). Pursuing how bacteria can influence transmission can lead to the development of better treatments that prevent the spread of disease rather than limiting pathology or symptoms. Examination of how virulence factor modifications could affect transmission could also be important for vaccine development (17), especially given the recent B. pertussis resurgence (14) and the consideration of using detoxified endotoxin (4) as a potential additional component for improving the current acellular vaccine. Future Directions The results of these experiments are the first to indicate that modifications of the bacterial lipid A could influence transmission. A major question in the study of infectious disease, which is difficult to model, concerns how pathogens spread between hosts. Previous work in the field has shown a correlation between neutrophil recruitment to the site of infection and levels of transmission and shedding (9, 20). However, the influence of lipid A modifications, if not altering TLR4 signaling, indicates that other factors could influence B. bronchiseptica transmission, such as the promoting the ability to reliably colonize a potential host rather than increasing the numbers of bacteria released from the index animal through immune system manipulation. One possible future direction includes determining whether the lipid A modification can affect transmission using defensins or other antimicrobial peptide deficient mice as well as assaying for changes in local cytokine profiles linked to transmission rather than broader cell recruitment.

144 132 Bordetella bronchiseptica and the Inflammasome Another aspect of bacterial utilization of virulence factors is how they can alter the immune inflammatory response to their benefit. IL-1 signaling has been shown to be required during bordetellae infection (7, 16). Previous research using the mouse model has shown that IL-1R is required for control of B. bronchiseptica colonization (Figure 3.3 A,B,C) as well as survival of the host during infection (Figure 3.2 A,B), preventing rapid expansion of bacterial numbers that presumably escape the respiratory tract and kill the host through septic shock. However, when the mice are inoculated with a B. bronchiseptica strain lacking a functional T6SS, the subsequent infection in the absence of IL-1R is no longer lethal (Figure 3.2 C,D). No T6SS specific differences in IL-1β levels were detected early in infection or throughout the time course (Figure 3.4 A,B). However, activation of IL-1β could still be involved in the T6SS specific response. To investigate the involvement of IL-l signaling pathway upstream of the IL-1R, we explored the relationship between the T6SS and the caspase-1 inflammasome (22) in mouse model as well as in cultured macrophages. Summary and Implications The findings of these experiments indicate that a complex relationship exists between caspase-1 and the B. bronchiseptica T6SS (Figure 3.10). In cultured macrophages, a decrease in caspase-1 protein was observed in the presence of a functional T6SS (Figure 3.11) which did not result in a corresponding decrease in activated IL-1β (Figure 3.12). However, caspase-1 has multiple other targets (10, 11),

145 133 and these data could indicate that the decrease in caspase-1 could affect the host immune response in other ways besides the hypothesized mechanism. The presence of the T6SS does not result in a decrease in Casp1 or Nlrp3 mrna levels (Figure 3.13, 3.14C) but does affect levels of Nlrp3 protein (Figure 3.14A,B), an important inflammasome component (5, 19, 22). These data raise the possibility that the entire inflammasome complex is being decreased. After treatment with the 3MA autophagy inhibitor(18), the decrease in caspase-1 is abrogated (Figure 3.15), suggesting that the autophagy pathway is required for the clpv-mediated decrease in caspase-1 levels. Results from the murine infectionshint that the clpv dependent decrease in caspase-1 observed in culture could also affect infection in the animal model..during B. bronchiseptica infection, clpv is required for colonization of the respiratory tract. However, in mice deficient in caspase-1, no reduction in colonization is observed. One explanation of this phenomenon is that wild-type B. bronchiseptica, which have a functional T6SS, are able to decrease caspase-1 levels in wild type mice, a future possibility to be explored. Future Directions These findings open up many new avenues in pursuing how a newly characterized secretion system can interact with an important immune signaling pathway. One of the main points of interest would be determining how the T6SS influences autophagy and thus levels of inflammasome proteins. If the observed effect occurs due to a direct interaction between the T6SS and the host immune pathways, creating a series of deletion

146 134 mutants lacking genes encoding putative T6SS effector proteins could determine whether a specific secreted protein is responsible for manipulating host responses. Identifying the T6SS effectors responsible for the decrease in inflammasome levels could also be accomplished by generating and screening a transposon library. It is also possible that disrupting the T6SS locus affects other bacterial virulence systems, so determining if other known virulence factors can mediate a similar effect is another strategy of interest. For example, mutants deficient in Type III secretion system function or Bvg phase locked mutants could be tested to determine if they affect inflammasome levels. Another important research aspect would be to determine the mechanism underlying the caspase-1 degradation, including measuring ubiquitinylation state and using pulldown assays to determine if bacterial effector proteins are interacting directly with the inflammasome. Pursuing these avenues of research would enable the scientific community to gain knowledge about important early immune responses as well as understanding novel bacterial secretion system mechanisms. Hcp is necessary for normal B. bronchiseptica growth and morphology The final aspect of this study involves determining how regulation of a virulence factor also affects bacterial systems and growth. In the simplest terms, a virulence factor is a component that enables the pathogen to inflict damage on the host (3). This perspective highlights the importance of bacterial function and viability in determining whether or not designated virulence systems are required for host-pathogen interactions.

147 In this study, we investigate a major component of the T6SS, Hcp, and how it affects the growth of B. bronchiseptica. 135 Summary and Implications The in-frame deletion strain of B. bronchiseptica that lacks hcp (Figure 4.3) has an increased lag phase in comparison to wild-type when grown in Stainer-Scholte broth which is compensated for by growth in the rich LB broth (Figure 4.4). However, RB50Δhcp has different optical density to CFU ratios in Stainer-Scholte and LB media (Figure 4.5) due to filamentous morphology (Figure 4.7). Altered resistance to Polymyxin B (Figure 4.5) could also indicate a membrane perturbation, possibly due to stress caused by a misassembled secretion system apparatus or due to a buildup of unchaperoned effector molecules in the cytoplasm. These results illustrate how disruption of one system can have large scale effects, such as filamentation, and can result in negative consequences for the pathogen. Future Directions Future directions include determining the mechanism underlying how the absence of hcp creates problems with bacterial growth and morphology. Directly testing whether hcp is required for normal septal formation could be performed using a membrane dye, such as FM 46-4, to directly visualize the division process (21) or immunofluorescence to visualize FtsZ polymerization during Z ring assembly (15).

148 136 Another important question to investigate would be whether the absence of hcp could result in induction of SOS response, which has been shown to halt cell division in the presence of DNA damage (1) or potentially cell envelope stress. Microarray analysis and comparisons of expression profiles would indicate an increase in genes having promoters with LexA boxes in RB50Δhcp versus wild type or possibly induction of the RpoS regulon in the case of an SOS response and an increase in expression of genes under the control of alternate sigma factors in the case of membrane or oxidative stress. The response pathway initiation could occur through potential damaging agents generated by cytoplasmic effectors, direct damage to the nucleoid (tssd encodes a putative effector with nuclease activity), or via crosstalk with the aforementioned stress pathways. This possibility could be tested by creating double mutant strains of bacteria lacking both hcp and the putative effector proteins and observing morphology and growth parameters. Conclusion In conclusion, this research has investigated multiple aspects of bacterial virulence and how the B. bronchiseptica pathogen can interact with the immune response. Pathogens use multiple strategies to ensure their survival, such as the ArnT mediated glucosamine lipid A modification, which alters a pathogen associated molecular pattern to improve resistance to an innate immune defense as well as enable them to transmit to a new host. The T6SS enables the bacterium to manipulate host processes to eliminate a key pro-inflammatory response. Absence of Hcp, a T6SS component generally considered in the context of secretion system specific functions, results in severe changes

149 137 in bacterial growth and morphology. These different aspects of pathogenesis illustrate the diversity and complexity of the ways in which bacterial pathogens interact with their host and the host immune response to survive. As always, further work is necessary to better understand bacterial virulence strategies and to develop disease prophylactics and treatments in the ever evolving fight against infection.

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152 Appendix A Parameters of B. bronchiseptica Infection in Relation to Caspase-1 Figure A.1. Measuring T6SS dependent caspase-1 activity. Bone marrow derived macrophages were incubated with media, RB50, or RB50ΔclpV (MOI 10) for 2 hours and treated with medium alone or the BafA1 autophagy inhibitor. Activity of caspase-1 was determined using fluormetric assay based on the detection of AFC (7-amino-4-trifluoromethyl coumarin). Bars represent the mean fluorescent units + standard error.

153 141 Figure A.2. The Type III Secretion System is required for decreased levels of caspase-1 protein Immunoblot of Caspase-1 using RAW whole cell lysates from cells inoculated with RB50, RB50ΔclpV (inactive T6SS), and RB50ΔbscN (WD3) (inactive T3SS) at an MOI of 10 for 2 hours using β-actin loading control (S. Hester, unpublished).