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Breaching the epithelial barrier : Salmonella enterica serovar Typhimurium invasion and disruption of… Boyle, Colleen Erin 2007

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B R E A C H I N G T H E E P I T H E L I A L B A R R I E R : SALMONELLA ENTERICA S E R O V A R T Y P H I M U R I U M I N V A S I O N A N D D I S R U P T I O N OF T I G H T J U N C T I O N S by C O L L E E N E R I N B O Y L E B.Sc. (Specialized Honors), University of Guelph (2001) A THESIS S U B M I T T E D I N P A R T I A L F U L F I L L M E N T OF T H E R E Q U I R E M E N T S F O R T H E D E G R E E OF D O C T O R OF P H I L O S O P H Y in T H E F A C U L T Y OF G R A D U A T E S T U D I E S (Microbiology) T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A October 2007 © Colleen Er in Boyle, 2007 Abstract B y invading epithelial cells and disrupting cell-cell junctions, Salmonella enterica serovar Typhimurium (S. Typhimurium). employs both transcellular and paracellular mechanisms in order to overcome the barrier function of epithelial cell monolayers. The research presented in this thesis has focused on defining and elucidating both the host and bacterial factors required for S. Typhimurium to breach the epithelial barrier. Internalization of 5". Typhimurium absolutely requires the actin Cytoskeleton, yet only a few of the cytoskeletal components involved in this process have been identified. The recruitment of actin-associated proteins to the site of invasion was investigated in order to identify host proteins that may play a role in S. Typhimurium invasion. The role . of several key cytoskeletal proteins was assayed to determine whether they were required for S. Typhimurium invasion. The actin-severing protein, gelsolin, was found to inhibit the invasion process while ezrin did not play an important role during S. Typhimurium invasion. The contribution of the recruited Src homology (SH) 2 adaptors to invasion was ' further investigated. While not involved in bacterial internalization itself, the adaptors Nek and ShcA influenced adherence of S. Typhimurium to non-phagocytic cells. Using the murine model of S. Typhimurium-induced colitis we investigated whether S. Typhimurium disrupted epithelial tight junctions in vivo. S. Typhimurium infection of mice caused a significant increase in intestinal permeability and resulted in the redistribution of tight junction proteins ZO-1 and claudin-3 in the colon. Using Salmonella pathogenicity island-1 (SPI-l)-secreted effector mutants we found that SopB, SopE, SopE2, and S i p A a r e the specific effectors responsible for disruption of tight junction structure and function in vitro. Tight junction disruption by S. Typhimurium was i i prevented by inhibiting host protein geranylgeranylation but was not dependent on host protein synthesis or secretion of host-derived products. These data suggest that SPI-1-secreted effectors utilize their ability to stimulate Rho family GTPases to disrupt tight junction structure and function. Because the ability,of S. Typhimurium to penetrate the intestinal epithelium is key to its pathogenesis, these studies significantly enhance our understanding host-pathogen interaction and the molecular events leading to disease. i i i Table of Contents Abstract • ii Table of Contents : '. iv List of Tables : .' '. vii List of Figures -viii List of Abbreviations • • • •' ix Acknowledgements • xi Co-authorship Statement ; • xii CHAPTER 1: INTRODUCTION XII 1.1 S A L M O N E L L O S E S , 2 1.1.1 Salmonella enterica serovars'and salmonelloses : 2 1.1.2 Course of a S. Typhimurium infection 3 1.1.3 In vivo model systems to study salmonelloses 4 1.1.3.1 Calf model of enterocolitis 4 .1.1.3.2 Mouse typhoid model of systemic infection * 5 1.1.3.3 Mouse typhoid model of persistent systemic infection 5 1.1.3.4 Mouse streptomycin pre-treatment model of colitis 6 1.2 M O D U S OPERANDI: IN VITRO OBSERVATIONS O F S. T Y P H I M U R I U M INFECTION : 7 1.3 PATHOGENICITY I S L A N D - E N C O D E D V I R U L E N C E DETERMINANTS • ...8 1.4 INTESTINAL E P I T H E L I U M 13 1.4.1 Architecture of the intestinal epithelium : 14 1.4.2 Epithelial cell-cell contacts .....;...: 15 1.4.3 Actin polymerization 17 1.5 M E C H A N I S M S B Y W H I C H S. T Y P H I M U R I U M T A R G E T S , CROSSES, A N D DISRUPTS EPITHELIA ..19 1.5.1 Adherence. 19 1.5.2 Invasion 21 1.5.3 Tight junction disruption '. 24 1.6 R A T I O N A L E 25 1.7 S IGNIFICANCE : 26 CHAPTER 2: MATERIALS AND METHODS 27 2.1 C E L L LINES .- '. . - 2 8 2.1.1 Sources •. , ; 28 2.1.2 Growth conditions 28 2.1.3 Polarization of epithelial cells ...29 2.2 T R A N S F E C T I O N 29 2.3 SIRNA 29 2.4 B A C T E R I A L STRAINS 30 2.4.1 Growth conditions 30 2.4.2 Construction of bacterial mutants : ;............30 2.4.3 Construction of complementation plasmids 31 2.5 B A C T E R I A L INFECTIONS OF C E L L LINES : 32 2.6 G E N T A M I C I N PROTECTION A S S A Y ..33 2.7 I M M U N O F L U O R E S C E N T STAINING OF C E L L LINES 33 2.8 INSIDE/OUTSIDE IMMUNOSTAINING >. 34 2.9 C O L L E C T I O N O F HOST C E L L FRACTIONS 35 2.9.1 Soluble and insoluble fractions..' 35 2.9.2 Whole cell lysates ....'...36 2.10 SDS-PAGE AND WESTERN BLOTTING • 36 2.11 MEASUREMENT OF TER AND PARACELLULAR FLUX '. 37 2.12 IL-8 ELISA 38 2.13 IN vivo EXPERIMENTS 38 2.13.1 Mouse infections.... '. 38 2.13.2 Measuring intestinal permeability 38 2.13.3 Tissue preparation : i .39 2.13.4 Immunofluorescence ; .39 2.14 STATISTICAL ANALYSIS 40 C H A P T E R 3: C A T A L O G U E A N D F U N C T I O N A L A N A L Y S I S O F P R O T E I N S R E C R U I T E D T O S I T E S O F S. T Y P H I M U R I U M I N V A S I O N .'. . ................41 3.1 SUMMARY '. : 42 3.2 INTRODUCTION 42 3.3 RESULTS ...... 43 3.3.1 Recruitment of actin-associated proteins to sites of S. Typhimurium invasion 43 3.3.2 Ezrin is not required for invasion of S. Typhimurium into non-phagocytic cells ...47 3.3.3 Gelsolin inhibits invasion of S. Typhimurium into non-phagocytic cells i 48 3.3.4 Nek and ShcA, but not Crk, affect the extent of invasion of S. Typhimurium into non-phagocytic cells 50 3.3.5 Nek and ShcA affect adherence of S. Typhimurium to non-phagocytic cells 53 3.3.6 Nek and ShcA do not affect invasion efficiency of S. Typhimurium into non- phagocytic cells : ........54 3.4 DISCUSSION '. 56 C H A P T E R 4: S. T Y P H I M U R I U M D I S R U P T I O N O F T I G H T J U N C T I O N S T R U C T U R E A N D F U N C T I O N „ ........61 4.1 SUMMARY 62 4.2 INTRODUCTION '•. 63 4.3 RESULTS 64 4.3.1 S. Typhimurium disrupts tight junction structure in vivo ". 64 4.3.2 S. Typhimurium increases intestinal permeability in vivo .:.64 4.3.3 Inhibition of protein geranylgeranylation prevents barrier disruption by S. Typhimurium....66 4.3.4 Barrier disruption by S. Typhimurium does not require host protein synthesis or secretion of host-derived products : ....68 4.3.5 SopE, SopE2, SopB, and SipA disrupt the barrier function of polarized epithelial cell monolayers 69 4.3.6 Complementation analysis verifies that SopB, SopE, SopE2, and SipA disrupt the barrier function of polarized epithelial cell monolayers 71 4.3.7 AsopB/E/E2 and AsipA/sopE/E2 mutants do not alter the size-selective paracellular . permeability of Caco-2/TC7 cells : 73 4.3.8 SopE, SopE2, SopB, and SipA alter the localization of ZO-1 and occludin 74 4.3.9 SopE, SopE2, SopB, and SipA alter the expression levels of ZO : 1 and occludin 76 4.3.10 SopB, SopE, SopE2, and SipA disrupt epithelial cell polarity 78 4.4 DISCUSSION ; ; : 79 C H A P T E R 5: DISCUSSION. . . . . . . . . . . . . . . . . 87 5.1 EXPLOITATION OF CELL ADHESION MOLECULES BY ENTEROINVASIVE PATHOGENS ....89 5.1.1 "Zipper" invasion ;90 5.1.2 "Trigger" invasion 92 5.2 CYTOSKELETAL PROTEINS RECRUITED TO SITES OF INVASION: CYTOSKELETAL DYNAMICS WITHIN SALMONELLA-INDUCED RUFFLES .„ 95 v 5.3 CAN NCK AND SHCA TELL US ANYTHING ABOUT THE HOST CELL RECEPTOR FOR S. TYPHIMURIUM? 99 5.4 TIGHT JUNCTION DISRUPTION AND ITS CONTRIBUTION TO THE MANIFESTATION OF DIARRHEA 100 5.5 'FUTURE DIRECTIONS 103 Appendix 1 Publications Arising From Graduate Work 130 Appendix 2 Animal Ethical Approvals ; 132 v i Lis t of Tables Table 1.1 SPI-1 secreted effectors . 10 Table 1.2 SPI-2 translocated effectors . 12 Table 2.1 Bacterial strains used in this study...... 31 Table 2.2 Antibodies used in studies 35 Table 4.1 Conclusions regarding the bacterial effectors responsible for tight junction disruption based on mutant analysis '. 84 Table 5.1 Differences between Shigella and S. Typhimurium invasion. 95 Table 5.2 Host proteins recruited to sites of S. Typhimurium invasion. 97 vn Lis t of Figures Figure 1. 1 Organization of polarized epithelial cell monolayers 15 Figure 1. 2 Composition of tight junctions and adherens junctions. 17 Figure 1.3 SPI-1 effectors that mediate invasion of S. Typhimurium into non-phagocytic cells and the host cell pathways with which they interact 22 Figure 3.1 Recruitment of actin-associated proteins to sites of S. Typhimurium invasion 45 Figure 3.2 Ezr in does not play a functional role during S. Typhimurium invasion into non-phagocytic cells 48 Figure 3.3 Gelsolin inhibits invasion of S. Typhimurium 50 Figure 3.4 Nek and ShcA, but not Crk, affect the extent of invasion of S. Typhimurium into non-phagocytic cells 52 Figure 3.5 Nek and ShcA affect adherence of S. Typhimurium to non-phagocytic • cells 54 Figure 3.6 Nek and ShcA do not affect internalization efficiency of S. Typhimurium into non-phagocytic cells 55 Figure 4.1 S. Typhimurium causes redistribution of ZO-1 and claudin-3 in the colon of streptomycin-pretreated mice 65 Figure 4.2 Wild-type S. Typhimurium significantly increases intestinal permeability in streptomycin-pretreated mice 66 Figure 4.3 Inhibition of protein geranylgeranylation prevents tight junction disruption by S. Typhimurium 4 hours post infection 67 Figure 4.4 Barrier disruption by S. Typhimurium does not require host protein synthesis 69 Figure 4.5 S ipA, SopB, SopE, and SopE2 disrupt the barrier function of polarized epithelial cell monolayers -.. ....71 Figure 4.6 Complementation analysis verifies that S ipA, SopB, SopE, and SopE2 are the effectors that disrupt the epithelial barrier 72 Figure 4.7 AsopB/E/E2 and AsipA/sopE/E2 mutants do not alter the paracellular permeability of Caco-2/TC7 cells 74 Figure 4.8 S ipA, SopB, SopE, and SopE2 disrupt ZO-1 and occludin localization.. 75 Figure 4.9 SipA, SopB, SopE, and SopE2 modulates expression of ZO-1 and occludin 77 Figure 4.10 S ipA, SopB, SopE, and SopE2 are responsible for disrupting cell polarity .; 79 Figure 5.1 Model for the signaling pathways leading to Yersinia invasion 91 Figure 5.2 Signaling involved in internalin:E-cadherin-mediated uptake of L. monocytogenes 92 Figure 5.3 Signaling involved in the invasion of Shigella 94 v i i i List of Abbreviations A N O V A analysis of variance Arp2/3 actin-related protein 2/3 B S A bovine serum albumin C A M cell adhesion molecule C F T R cystic fibrosis transmembrane conductance regulator C F U colony forming units C H X cycloheximide D A P I 4',6-diamidino-2-phenylindole D M E M Dulbecco's Modified Eagle's Medium E C L enhanced chemiluminescence E C M extracellular matrix E D T A ethylenediamine tetraacetic acid E G T A ethylene glycol-bis(P-aminoethyl ether)-JV, N, N, N-tetraacetic acid E H E C enterohaemorrhagic Escherichia coU E L I S A enzyme-linked immunosorbant assay E P E C enteropathogenic Escherichia coli F-actin filamentous actin F A K focal adhesion kinase F C S fetal calf serum F I T C fluorescein isothiocyanate G-actin globular actin G E F G-nucleotide exchange, factor GGTase I geranylgeranyltransferase I G G T I geranylgeranyltransferase inhibitor Gsn gelsolin H E P E S 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid H I V human immunodeficiency virus H R P horseradish peroxidase IF immunofluorescence J A M junctional adhesion molecule L B Luria-Bertani L D 5 0 50% lethal dose L P P lipoma preferred partner L P S lipopolysaccharide M D C K Madin Darby canine kidney M E F mouse embryonic fibroblasts NG.S normal goat serum N - W A S P Neural Wiskott-Aldrich syndrome protein O C T optimal cutting temperature O R F open reading frame P A I pathogenicity island P B S phosphate buffered saline P F A paraformaldehyde ix PIPES piperazine-1,4-bis(2-ethanesulfonic acid) R T K receptor tyrosine kinase S C V Salmonella-containing vacuole SDS sodium dodecyl sulfate S D S - P A G E sodium dodecyl sulfate polyacrylamide gel electrophoresis S H Src homology S i f Salmonella-induced filament S G E F • Src homology 3-containing guanine nucleotide exchange factor SGI-1 Salmonella genomic island 1 s i R N A small interfering ribonucleic acid SPI Salmonella pathogenicity island T3SS type-3 secretion system T E R transepithelial resistance V A S P vasodilator-stimulated phosphoprotein WIP W A S P interacting protein ZO-1 zonula occludin 1 Acknowledgements I would first like to thank my supervisor, B . Brett Finlay ( B B F j , for his support and encouragement. Thank you to my supervisory committee members Yossef (Yossi) A v -Gay, Er in Gaynor, and Calvin (Cai) Roskelley for keeping my eye on the prize. Thank you to the Natural Science and Engineering Research Council , U B C Graduate Studies, and B B F for financial support. M u c h appreciation goes to past Finlay lab members. Thank you to Danika Goosney, who showed me the ropes in the early days. Thank you Sonya Kujat-Choy for letting me participate in her project. To my first "baymate" Carrie Rosenberger, thank you for listening to my experimental and personal woes. Phi l Hardwidge, you are one o f the most unique individuals I have ever met. I miss your giggles and lunch aliquots. To Ami t Bhavsar and Ol iv ia Champion: you're both hilarious and have made my last year here a blast. Special thanks go to the individuals who saw me through the bulk of my years here in the Finlay lab. I could not have done this without you: Brian Coombes (Coombes): Y o u set the example of how to be an excellent and prolific scientist. Y o u are a machine. To this day, many people in the lab can be heard saying, "What would Coombes do?". Jennifer Bishop (Jenna): M y baymate! Y o u were the lever, I was the fulcrum, and together we moved the world. Oh yes, we read it, photocopied it, and we're making t-shirts! Mark (Maak) Wickham: Y o u hold a soft place in my heart, my friend. You ' re such a lovable character!. Y o u were always there to tame my fiery temper, ease my broken heart, and f i l l my tummy with gourmet food. Y o u ' d better keep in touch. Nat Brown: You 've literally been there for me since day one. We met my first week of grad school and I instantly felt like we would be close friends. Whether it be over beer at Koerners, poutine at Zizanie's, or across the bench, you've been a wonderful friend, scientist, and mentor to me. Y o u have such a big heart. A n d finally, my deepest gratitude goes to my family. To my mom: Thank you for always supporting me despite thinking I was crazy for wanting a PhD! Thank you for instilling your work ethic while always encouraging me to. take care of myself along the way. To my dad: Thank you for forcing me to watch countless hours of P B S nature documentaries when I was young. Y o u fostered my love of animals and my curiosity for science. A n d although I regrettably inherited your susceptibility to stomach ulcers, I also inherited your stubbornness which served me very well when my research wasn't working. To Kevin : I have always looked up to you. You ' re a hard act to follow big brother! To Guntram (Gunti) Grassl: Schnecke, you are my love, you are my rock. xi Co-authorship Statement Chapter 3 I designed and performed all experiments described in this chapter. I analyzed all the data presented in this chapter and wrote the manuscript described in this chapter. Dr. John Brumell had the initial observation that Salmonella demonstrated reduced invasion into Nck l - /Nck2-ce l l s . Chapter 4 I designed and performed all experiments described in this chapter. I analyzed all the data presented in this chapter and wrote the manuscript described in this chapter. Dr. Guntram Grassl, Bryan Coburn, and Yul ing L i provided technical assistance for mouse infections and tissue harvesting. Chapter 5 . I prepared the manuscripts described in this chapter. x i i Chapter 1: Introduction 1 1.1 Salmonelloses 1.1.1 Salmonella enterica serovars and salmonelloses Salmonella enterica serovars are Gram negative enteropathogenic bacteria that cause diseases (salmonelloses) in humans ranging from mild gastroenteritis to life-threatening systemic infections. Salmonelloses are among the most common and widely distributed food-borne diseases in humans and represent a major public health and economical burden worldwide (World Health Organization, 2005). Typhoidal Salmonella serovars such as Typhi and Paratyphi cause systemic illness leading to an estimated 21 mil l ion cases and 200,000 deaths worldwide each year (Center for Disease Control, 2005). Non-typhoidal salmonelloses are caused by more than 2500 serovars of S. enterica, with Typhimurium and Enteritidis being the most frequently encountered. Intestinal disease caused by non-typhoidal Salmonella serovars is estimated to result in 1.3 bil l ion cases each year, leading to approximately 3 mil l ion deaths worldwide (Pang et al, 1995). Non-typhoidal Salmonella infections typically present as self-limiting gastroenteritis, although in immunocompromised individuals, systemic disease can occur. Human immunodeficiency virus (HIV) /AIDS patients are an especially high-risk population for contracting non-typhoidal Salmonella infections. The mortality rate for H I V patients infected with non-typhoidal Salmonella can be as high as 60% with bacterial recrudescence occurring in up to 45% of these individuals (Gordon et al, 2002; Kankwatira et al, 2004). A significant proportion of typhoid patients become chronic carriers of serovar Typhi, as do many people who have never had a clinical history of typhoid fever (Levine et al, 1982; Vogelsang & Boe, 1948). These individuals shed high numbers of bacteria in 2 their stools for long periods (up to their lifetime) without obvious signs of disease. Individuals infected with S. Typhimurium can shed bacteria in their feces for up to three months after resolution of gastroenteritis, while 1-3% of cases continue to shed the. bacteria for more than a year (Salyers & Whitt, 1994). Chronic carriage of Salmonella facilitates the spread of disease and predisposes sufferers to additional medical problems. 1.1.2 Course of a S. Typhimurium infection Although in humans S. Typhimurium infection is normally localized to the intestinal tract, systemic non-typhoidal Salmonella infections are an emerging problem. Both local and systemic infections w i l l be described although most observations on the systemic phase disease have been interpreted from S. Typhimurium infection of mice (see section 1.1.3.2). . S. Typhimurium gains entry into their host through oral ingestion of contaminated food or water. After passage through the stomach, S. Typhimurium primarily colonizes the ileum and colon, invading both M cells of the Peyer's patches and enterocytes. M cells are naturally phagocytic cells of the intestinal epithelium that sample antigens from the lumen of the gut and present them to the underlying antigen presenting cells (Jepson & Clark, 2001). Dendritic cells are also capable of intercalating enterocytes and phagocytosing luminal Salmonella (Rescigno et al, 2001). Crossing the intestinal epithelial barrier enables bacteria access to the lamina propria where phagocytic cells are thought to play an important role in determining the outcome of infection. I f macrophages and other immune cells are successful at ki l l ing Salmonella, infection results in self-limiting enteritis with disease manifesting as diarrhea, vomiting, and 3 abdominal pain. However, i f Salmonella survives and replicates within these cells, Salmonella can disseminate and cause severe systemic illness. Macrophages and dendritic cells have been proposed to be the cell types responsible for disseminating S. Typhimurium from the lamina propria to the lymph nodes (Vazquez-Torres et al, 1999). From the lymph nodes, bacteria can enter the bloodstream, eliciting a bacteremic state. S. Typhimurium then colonizes the liver and spleen where they survive and replicate within hepatic and splenic macrophages. From there, dissemination to the gallbladder is thought to result in reseeding of the intestine through the bile. In persistent infections, Salmonella reside primarily in the gallbladder, bone marrow, and mesenteric lymph nodes (Gaines et al, 1968; Monack et al, 2004; Sinnott & Teall, 1987; Wain et al, 2001). In severe systemic infections, ulceration and perforation of the intestine can occur, resulting in death due to massive internal injuries or septic shock. 1 . 1 . 3 In vivo model systems to study salmonelloses Animal models are of significant benefit to understand salmonelloses in humans and are thus key to developing new therapeutics. Animal models are available to study both the intestinal and systemic phases of salmonelloses. 1.1.3.1 Calf model of enterocolitis S. Typhimurium infection of calves results in enteric disease with clinical and pathological features that resemble human disease (Santos et al, 2001). In this model, a calf is infected either orally or bacteria are directly injected into ileal loops. Significant 4 intestinal secretory and inflammatory responses are induced, and upon oral infection, S. Typhimurium causes diarrheal disease. This model has provided valuable information regarding the bacterial factors required to cause enteric disease (Santos et al, 2001; Zhang et al, 2002; Zhang et al, 2003). However, methods for genetically manipulating the bovine host are extremely limited, and therefore, examining the host factors that contribute to disease is difficult. Unlike commercially-available mouse strains, cattle are outbred, making reproducibility between experiments a significant challenge. In addition, the cost and logistics involved in bovine experiments are significant disadvantages of this model. 1.1.3.2 Mouse typhoid model of systemic infection Oral infection of genetically susceptible mouse strains with S. Typhimurium leads-to systemic infection that resembles typhoid fever in humans. In mice, S. Typhimurium does not efficiently colonize the intestine, cause diarrhea, or cause significant intestinal inflammation. Accordingly, mouse infection with S. Typhimurium is not used to study enterocolitis. However; because S. Typhi is host-restricted to humans and higher primate hosts, the more amenable mouse typhoid model has been extensively used to study the immune responses to systemic infection and the bacterial factors necessary for systemic virulence. . 1.1.3.3 Mouse typhoid model of persistent systemic infection A recent model for persistent infection has been developed. In genetically resistant mouse strains, oral infection with 5*. Typhimurium results in chronic infection of the macrophages of mesenteric lymph nodes (Monack et al., 2004). This system provides a basis on which one can genetically dissect both pathogen and host factors required for persistent infection. 1.1.3.4 Mouse streptomycin pre-treatment model of colitis A s mentioned previously, mice do not normally exhibit intestinal inflammation in response to S. Typhimurium infection. However recently, a new model of Salmonella-induced colitis has been developed using streptomycin pre-treated mice (Barthel et al, 2003). Upon oral infection of streptomycin pre-treated mice, S. Typhimurium efficiently colonizes the large intestine and triggers severe inflammation in the cecum and colon. In humans, biopsies reveal acute enteritis characterized by mucosal edema and acute inflammation with polymorphonuclear leukocyte.(PMN) infiltrate (Santos et al, 2001). Similarly, in streptomycin pre-treated mice, S. Typhimurium infection causes mucosal edema and the inflammatory infiltrates are rich in P M N s (Barthel et al, 2003). One complicating factor of this model is that mice develop colitis and systemic infection in parallel: Secondly, unlike in humans where infection is localized primarily to the ileum and colon, inflammation in the mouse is only mild in the distal ileum and strongest in the cecum and colon. The reason for this difference is unknown. However, because both the host and pathogen are amenable to genetic manipulation, this model provides an excellent opportunity to investigate the specific host and bacterial mechanisms involved in intestinal inflammation induced by S. Typhimurium. 6 1.2 Modus operandi: in vitro observations of S. Typhimurium infection In vitro infection of cultured cells has proven to be a useful tool for studying Salmonella host-pathogen interaction. Although S. Typhimurium is rather promiscuous in its ability, to infect a wide variety of cell types including dendritic cells, macrophages, hepatocytes, neutrophils, epithelial cells and fibroblasts, the majority of studies have focused on macrophage arid epithelial cell infections. In the context of an infection, these represent two of the major cell types encountered by S. Typhimurium. Upon adherence to the surface of non-phagocytic cells, S. Typhimurium induces extensive cytoskeletal rearrangements that result in the formation of actin-driven membrane ruffles in the immediate vicinity of the invading organism and uptake via macropinocytosis (Finlay et al., 1991; Francis et al., 1992; Pace et al., 1993). Alternatively, upon contact with phagocytic cells, S. Typhimurium is internalized through a combination of bacterial-induced invasion and host cell-mediated phagocytosis. Subsequently, bacteria reside within membrane-bound vacuoles known as Salmonella-containing vacuoles (SCVs) (Garcia-del Portillo et al., 1992; Kihlstrom & Latkovic, 1978; Richter-Dahlfors et al., 1997). This entire process occurs within minutes and the host actin cytoskeleton returns to its normal distribution shortly thereafter (Finlay et al., 1991). In epithelial cell monolayers, a proportion of Salmonella crosses the monolayer via a transcellular route (Finlay et al, 1988). Intracellular S. Typhimurium rapidly uncouple themselves from normal endocytic maturation and avoid lysosomal degradation, creating a unique replicative niche inside the S C V [reviewed by (Abrahams & Hensel, 2006)]. Upon formation, the. S C V transiently interacts with early endosomal compartments as well as the endoplasmic 7 reticulum. Subsequently, the S C V has only limited interactions with late endosomes and lysosomes and matures into a modified late endosomal/lysosomal compartment rich in lysosomal glycoproteins (eg L a m p l , Lamp2). S. Typhimurium transiently induces an actin meshwork around the S C V which, through an unknown mechanism, is essential for intravacuolar replication (Meresse et al, 2001). Coincident with replication, extensive membrane tubules, known as Salmonella-induced filaments (Sifs), extend out from the S C V (Garcia-del Portillo et al, 1993; Knodler et al, 2003). Many of the bacterial factors involved in these processes, and required for virulence in various animal models, have been identified and wi l l be described below. 1.3 Pathogenicity island-encoded virulence determinants <S. Typhimurium virulence genes are scattered throughout the bacterial chromosome sometimes individually, but more often, in , large clusters called pathogenicity islands. Pathogenicity islands are distinct, often large regions of the bacterial chromosome that encode genes that contribute to virulence (Gal-Mor & Finlay, 2006). Pathogenicity islands are acquired by horizontal gene transfer and are therefore considered a means by which "quantum leaps" in bacterial evolution can occur. The fact that pathogenicity islands are acquired by horizontal gene transfer is reflected in their G + C content, which is distinct from the rest of bacterial chromosome, and their association with insertion sites such as t R N A genes. Certain pathogenicity islands also contain sequences associated with D N A mobility such as direct repeats, integrases, transposases, and bacteriophage genes. The majority of virulence genes of S. Typhimurium are 8 encoded within 7 to 8 Salmonella, pathogenicity islands (SPIs) (Hensel, 2004). Virulence factors are also harbored in smaller regions called "pathogenicity islets". Key to the pathogenic strategy of S. Typhimurium are its two SPI-encoded type 3 secretion systems (T3SSs). T3SSs are multi-protein complexes that inject specific bacterial proteins, called effectors, directly into the host cell cytoplasm. T3SSs have been described as molecular syringes due to their needle-like appearance by scanning electron microscopy. SPI-1 and SPI-2 each encode a T3SS that secretes bacterial effectors encoded either within, or outside of, their respective pathogenicity islands (Tables 1.1 and 1.2). SPI-1 was identified as a 40 kb D N A region inserted between genes that are consecutive in the E. coli K-12 chromosome (Mil ls et al, 1995). SPI-1 encodes a T3SS, as well as secretion system regulators, chaperones, and effectors. To date, the SPI-1 T3SS is known to secrete 12 effectors (Table 1.1). In cell culture, SPI-1-secreted effectors are responsible for nuclear responses induced upon infection, have anti-apoptotic effects on epithelial cells, and are essential for the cytoskeletal rearrangements necessary for bacterial invasion into non-phagocytic cells (Schlumberger & Hardt, 2006). In mice, bacterial mutants with non-functional SPI-1 T3SSs are highly attenuated for virulence when inoculated orally, but not systeniically, suggesting that SPI-1 is crucial in the intestinal, but not systemic, phase of disease (Galan & Curtiss, 1989; Jones & Falkow, 1994). In the mouse and calf model of colitis, SPI-1 plays a crucial role in inducing intestinal inflammation (Galan, 2001; Santos etal, 2001; Wallis & Galyov, 2000). 9 Table 1.1 • SPI-1 secreted effectors. Protein Gene Function in vitro Entercolitis models Reference Location Cow Mouse SipA SPI-1 Invasion; PMN recruitment + + (Hapfelmeier et al., 2004; Jepson et al.,2001; Lee et al., 2000; Raffatellu etal, 2005; Zhang etal, 2002; Zhouetal, 1999b) SipB SPI-1 Translocon component; macrophage apoptosis + ND (CoHazo & Galan, 1997; Hayward et al, 2000; Hersh etal, 1999; Santos etal, 2001; Zhang etal, 2002) SipC SPI-1 Translocon component; actin bundling and nucleation ND N D (Collazo& Galan, 1997; Hayward & Koronakis, 1999) SopA Chromosome Invasion; PMN recruitment + ND (Raffatellu etal, 2005; Wood et al, 2000; Zhang et-. al, 2002; Zhang etal, 2006) SopB/ SigD SPI-5 Invasion; perturbs endosome to lysosome trafficking; anti-apoptotic effect in epithelial cells; nuclear responses + (Dukes etal, 2006; Hapfelmeier et al, 2004; Knodler etal, 2005; Patel & Galan, 2006; Raffatellu et al, 2005; Zhang etal, 2002; Zhou etal, 2001) SopD Chromosome Invasion + ND (Raffatellu et al, 2005; Zhang etal, 2002) SopE phage Invasion; nuclear responses ND + (Hapfelmeier etal, 2004; Hardte^a/., 1998a; Patel & Galan, 2006; Wood etal, 1996) SopE2 phage remnant Invasion; nuclear responses + + (Bakshi etal, 2000; Hapfelmeier etal, 2004; Patel & Galan, 2006; Raffatellu etal, 2005; Zhangetal,2002) SptP SPI-1 Disrupts the actin cytoskeleton - ND (Fu & Galan, 1999; Zhang et al, 2002) SteA Chromosome ? - - (Geddes etal, 2005) AvrA SPI-1 Inhibits N F - K B activation - ND (Collier-Hyams etal, 2002; Zhangetal, 2002) SlrP Chromosome ? - ND (Zhang etal, 2002) SspHl Gifsy-3 Down-regulates NF- + (in the ND (Miao et al, 1999; Zhang et prophage KB-dependent gene expression absence of SspH2) al, 2002) ND: not determined +: this effector plays a role in this model system -: this effector does not play a role in this model system 10 SPI-2 is a 40 kb locus composed of two distinct regions: a 15 kb region with a G + C content of 43% and a 25 kb region with a G + C content of 54% (Hensel et al, 1999). These two regions were likely obtained in separate horizontal transfer events. The 15 kb region does not appear to contribute to systemic pathogenesis while the larger 25 kb region harbors genes important for virulence including genes for a T3SS apparatus as well as secretion system regulators, chaperones, and effectors (Hensel et al, 1999). Similar to the SPI-1 T3SS, the SPI-2 T3SS secretes effectors encoded both within and outside SPI-2 (Table 1.2). Expression of SPI-2 is up-regulated when bacteria are intracellular (Pfeifer et al, 1999). SPI-2 type 3 secretion is essential for intracellular replication within macrophages, which is thought to be necessary for systemic disease (Ciril lo et al, 1998; Hensel et al, 1998). In addition, modifications in endosomal trafficking and creation of the S C V are dependent on SPI-2-secreted effectors. Although many SPI-2 type 3-secreted effectors have been identified, the majority of their host cell targets and biochemical activities remain largely unknown. SPI-2 is essential for systemic infection in mice and has also recently been shown to play a role in colitis in the streptomycin-pretreated mouse model (Coombes et al, 2005a; Hapfelmeier et al, 2004; Kuhle & Hensel, 2004). Much less is known about the function of the other SPIs. SPI-3 is a 17 kb region that encodes a high-affinity M g 2 + uptake system that is assumed to be required for adaptation to the nutritional limitations of the S C V (Blanc-Potard & Groisman, 1997). SPI-4 is a 25 kb island that encodes a giant non-fimbral adhesin important for adherence of S. Typhimurium to the apical surface of polarized epithelial cells (Gerlach et al, 2007; Wong et al, 1998). SPI-5 encodes effectors secreted by both the SPI-1 and SPI-2 T3SSs 11 Table 1. 2 SPI-2 translocated effectors. Protein Gene location Function in vitro Role in typhoid model Role in long term systemic infection model Reference SifA chromosome Sif formation and SCV membrane integrity + (Beuzon etal., 2000; Stein et al., 1996) SifB chromosome Targeted to Sifs; Function unknown N D (Freeman et al., 2003) SseF SPI-2 Sif formation +/- (Hensel etal, 1998; Kuhle & Hensel, 2002) SseG SPI-2 Sif formation (Hensel et al, 1998; Kuhle & Hensel, 2002) SseJ phage deacylase; SCV membrane dynamics + + (Lawley et al, 2006; Ohlson etal, 2005; Ruiz-Albert et al, 2002) Ssel/ SrfH Gifsy-2 prophage Increases phagocyte motility + • (Lawley et al, 2006; Ruiz-Albert et al, 2002; Worley etal, 2006) SseKl chromosome Function unknown - (Kujat Choy etal, 2004) SseK2 chromosome Function unknown + (Kujat Choy etal, 2004; Lawley et al, 2006) SopD2 chromosome Sif formation + + (Jiang et al, 2004; Lawley etal, 2006) SpiC SPI-2 Effector translocation; Interferes with vesicular trafficking + (Freeman etal, 2002; Uchiy aet al, 1999; Yuet al, 2002) PipB SPI-5 Targeted to Sifs; Function unknown (Knodler et al, 2003;Knodler& Steele-Mortimer, 2005) PipB2 chromosome Sif formation (Knodler et al., ~ 2003; Knodler & Steele-Mortimer, 2005) SspHl Gifsy-3 prophage Down-regulates NF-KB-dependent gene expression N D (Miaoera/., 1999) SspH2 phage Function unknown N D (Miao etal, 1999) SlrP chromosome Function unknown + . (Tsolis etal, 1999) GogB Gifsy-1 ' prophage Function unknown ' N D (Coombes et al, 2005b) N D : not determined +: this effector plays a role in this model system -: this effector does not play a role in this model system 12 (Tables 1.1 and 1.2) (Hong & Mil le r , 1998;.Knodler et al, 2002; Wood et al, 1998)/ Salmonella chromosomal island (59 kb; also known as SPI-6 in S. Typhi) encodes fimbral operons (Folkesson et al, 2002) while SPI-9 (16 kb) encodes a type I secretion system and a single R T X (repeats in toxin)-like protein (Hensel, 2004). Analysis of multi-drug resistant strains of S. Typhimurium identified a 43 kb P A I , called Salmonella genomic island 1 (SGI-1), that contains genes conferring resistance to five antibiotics: tetracycline, ampicillin, chloramphenicol, streptomycin, and sulfonamides (Boyd et al, 2001). Significantly, one of the foremost obstacles tb administering effective treatment for S. Typhimurium infection is antibiotic resistance. 1.4 Intestinal Epithelium In addition to its functions in digestion, nutrient transport, water and electrolyte exchange, and hormone production, the intestinal epithelium functions as a barrier between the host and the external environment. The lumen of the human intestine hosts a vast microbial community known as the microbiota. With populations exceeding TOO trillion microorganisms, the gut microbiota achieves the highest cell densities recorded for any ecosystem (Backhed et al, 2005). The intestinal epithelial barrier is essentially devoted to protecting the body against invasion and systemic dissemination of both commensal and pathogenic microorganisms, as well as mediating nutrient uptake and fluid balance. 13 1.4.1 Architecture of the intestinal epithelium The intestinal tract is lined with a single layer of epithelial cells that are renewed every 3-4 days. Stem cells present in the crypts give rise to four types of epithelial cells: 1) absorptive enterocytes that make up more than 80% of all the small intestinal epithelial cells; 2) goblet cells which produce mucins and trefoil peptides needed for epithelial cells growth and repair; 3) enteroendocrine cells which export peptide hormones; and 4) Paneth cells which secrete antimicrobial peptides, digestive enzymes, and growth factors (Karam, 1999). Enterocytes, goblet cells and enteroendocrine cells migrate up from the crypts to the villus tip as they differentiate. Once at the villus tip, they undergo apoptosis and are sloughed off into the lumen. In contrast, Paneth cells differentiate as they move downward within the crypt and are removed at the base through phagocytosis. Intestinal enterocytes are polarized, meaning that they have distinct apical and basolateral poles characterized by different protein and lipid compositions. The apical surface forms a brush border composed of a regular, dense array of microvil l i . Mic rov i l l i are membranous actin-supported extensions of the apical membrane that allow the exchange of nutrients and fluids (Mooseker, 1985). Focal adhesions at the basal portion of the basolateral pole connect epithelial cells to the extracellular matrix (Lo, 2006). The lateral portion of the basolateral pole contains three specialized structures that are responsible for establishing cell-cell contacts: desmosomes, adherens junctions, and tight junctions (Figure 1.1). 14 [ l l l l l l l l l l l l l l l l l l l l l l l l CO fa-— o D D QD Basement membrane Microvili Tight junction Adherens junction Desmosome Focal contact Figure 1.1. Organization of polarized epithelial cell monolayers. This model illustrates the relative position of various cell-cell and cell-basement membrane contacts. Tight junctions delineate the apical and basolateral cell membranes. 1.4.2 Epithelial cell-cell contacts Desmosomes confer structural strength to epithelia through their association with the intermediate filament components of the cytoskeleton (Getsios et al, 2004). The transmembrane proteins desmoglein and desmocollin interact across the intercellular space while their cytoplasmic domains bind desmoplakin and desmoglobin, linking desmosomes to intermediate filaments. Adherens junctions are established through either Ca -dependent, homophilic interaction between cadherins or Ca 2 +-independent, homo- or heterophilic interaction between nectins (Miyoshi & Takai, 2005). Cadherins associate with the actin cytoskeleton through catenins while nectin-based junctions are linked to the actin cytoskeleton through afadin (Figure 1.2). Catenins and afadin in turn recruit other proteins involved in the development and regulation of intercellular junctions. Cadherin-based cell-cell adhesions establish and maintain the apical junction complex both 15 independently and cooperatively with nectin-based structures. Importantly, adherens junctions are required for the formation and integrity of tight junctions. Tight junctions are the most apical adhesion structure. On ultrathin section electron micrographs, tight junctions appear as a series o f fusion points (so-called "kissing points") between the plasma membranes of adjacent cells. A t the molecular level, tight junctions are highly regulated protein complexes that are intimately linked to the actin cytoskeleton (Miyoshi & Takai, 2005; Shin et al, 2006). Transmembrane cell adhesion molecules like occludin, claudins, and junctional adhesion molecules (JAMs) establish extracellular homophilic interactions between adjacent cells. Plaque proteins such as zonula occludens-1 (ZO-1), ZO-2 , and ZO-3 interact with cell adhesion molecules, acting as adaptors at the cytoplasmic surface of tight junctions, linking cell adhesion molecules to the actin cytoskeleton (Figure 1.2). Tight junctions restrict paracellular movement of harmful immunogenic materials while controlling the movement of water, solutes, and immune cells. Tight junctions thereby preserve the barrier or "gate" function of epithelial cell monolayers. Tight junctions also have what is known as a "fence" function in that they are required to maintain cell polarity. B y acting as diffusion barriers that physically separate apical and basolateral membrane components, tight junctions both establish and maintain cell polarity. 16 Figure 1. 2. Composition of tight junctions and adherens junctions. Composition of tight junctions and adherens junctions. Tight junctions are composed of transmembrane proteins (occludin, claudins, and J A M s ) which are linked to the actin cytoskeleton through Z O proteins. Adherens junctions are composed of nectin-afadin and cadherin-catenin complexes, a-catenin links cadherin-catenin to the actin cytoskeleton while afadin links nectin-based junctions to the actin cytoskeleton. 1.4.3 A c t i n polymerization Common to both adherens junctions and tight junctions is their linkage to the actin cytoskeleton. The actin cytoskeleton is a dynamic network that is essential for cellular processes such as movement, endocytosis, phagocytosis, and vesicular trafficking. A n d as w i l l be discussed, pathogens, including 5". Typhimurium, usurp the host actin cytoskeleton in order to breach the epithelial cell barrier. Ac t in exists in monomeric and polymeric pools in the cytoplasm. Act in trimers (nuclei) serve as the foundation upon which new actin filaments are built. Following 17 nucleation, actin monomers are added onto the fast-growing end of the actin filament (called the barbed end). Act in monomers are removed from the opposite, slow-growing end of the actin filament (called the pointed end). This process is referred to as actin "treadmilling". Production of free fast-growing ends of actin occurs in three main ways: 1) de novo synthesis of actin filaments; 2) uncapping of pre-existing filaments; and 3) severing of pre-existing filaments (Condeelis, 1993; Zigmond, 1998). De novo actin polymerization requires actin nucleation. A complex of seven proteins, known as the Arp2/3 complex (Machesky et al, 1994), is able to nucleate actin. Arp2/3 is regulated through proteins in the Wiskott-Aldrich syndrome protein ( W A S P ) family, including N - W A S P and W A V E (Takenawa & Suetsugu, 2007). Arp2/3-dependent actin polymerization is induced following activation of W A V E by the Rho G T P a s e R a c l or the Src homology (SH) 2/SH3 adaptor, Nek (Eden et al, 2002; M i k i et al, 1998). N - W A S P , on the other hand, is activated by Cdc42, R a c l , PIP 2 , or Nek (Prehoda et al, 2000; Rohatgi et al, 2000; Tomasevic et al, 2007). In addition, focal adhesion kinase ( F A K ) and Src family kinases regulate N - W A S P localization and activity through tyrosine phosphorylation (Cory et al, 2002; Suetsugu et al, 2002; Takenawa & Suetsugu, 2007; Torres & Rosen, 2003). Free, fast-growing, barbed ends can also be exposed by uncapping. CapG and capping protein are two proteins that cap the barbed end of existing filaments, thereby terminating polymerization (Isenberg et al, 1980; Y u et al, 1990). PIP2 causes dissociation of capping proteins, thereby making actin filaments available for extension. Act in severing can also lead to the extension of pre-existing actin filaments. For example, gelsolin is a potent actin-depolymerizing protein whose activity is induced in the 18 presence of Ca and inactivated by PIP2 (Siiacci et al, 2004). Gelsolin binds actin filaments, severs them, and caps the barbed end of the filament. Uncapping of gelsolin-capped F-actin by other factors such as the actin-binding protein tropomyosin (Nyakern-Meazza et al, 2002), can allow for extension of the newly severed filaments. In practice, cells likely use a combination of de novo synthesis, severing, and uncapping to elicit cytoskeletal changes in response to the plethora of external stimuli. 1.5 Mechanisms by which S. Typhimurium targets, crosses, and disrupts epithelia Epithelial cells that line the gastrointestinal tract function as the primary barrier preventing pathogens from escaping to systemic sites. Critical for S. Typhimurium virulence is its ability to attach to mucosal surfaces in the intestine and induce its own uptake into epithelial cells. In the context of disease, bacterial adherence and internalization are means by which pathogens colonize the host, spread to systemic sites, and evade both host defense mechanisms and antibiotic treatment. Accordingly, understanding the molecular mechanisms involved in these events is of the utmost importance. 1.5.1 Adherence Adherence is considered to be an important first step during the pathogenesis of 5*. Typhimurium. Firstly, attachment contributes to colonization of host tissues by preventing clearance of bacteria through the peristaltic activity of the bowel. Secondly, adherence to the host cell surface is a necessary prerequisite for type 3 secretion and hence, internalization of S. Typhimurium into non-phagocytic cells. 19 Multiple adhesins are involved in attachment of S. Typhimurium to epithelial cells in vitro and in vivo. Fimbriae (or pili) are a group of rigid filamentous appendages anchored to the outer membrane of bacteria (Gerlach & Hensel, 2007). A s many as 13 operons encoding putative fimbrial adhesins have been predicted in the S. Typhimurium genome; 11 of which have been confirmed to be expressed (Humphries et al, 2003). Mutations in single fimbrial operons results in only subtle decreases in mouse virulence (Baumler et al, 1996a; Baumler et al, 1996b), however, combined mutations in the fimbrial operons fim, Ipf, pef, and agf increase the 50% lethal dose (LD50) of S. Typhimurium 26-fold following oral infection (van der Velden et al, 1998). S. Typhimurium also has four known non-fimbrial adhesins. M i s L and ShdA are members of the autotransporter family, are expressed in the intestine during infection, and are reported to bind fibronectin (Dorsey et al, 2005; Kingsley et al, 2002). Bap A is a non-fimbral adhesin that, while not involved in attachment to host cells, is required for homophilic interactions during biofilm formation (Latasa et al, 2005). A giant non-fimbrial adhesin recently identified within SPI-4, called S i iE , is required for adherence to apical surface of polarized epithelial cell lines and is involved in enteropathogenesis of S. Typhimurium in the murine colitis model (Gerlach et al, 2007). The large number of adhesins possessed by S. Typhimurium may reflect the ability of this pathogen to adapt to different niches including various mammalian and non-mammalian hosts as well as non-host environments. For example, the bcf operon is required for colonization in cattle but not murine Peyer's patches (Tsolis et al, 1999). Adhesins also likely allow bacteria to target particular cell types once within a host. For example, mutations in Ipf and pef reduce the number of bacteria associated with murine 20 Peyer's patches and the villous intestine, respectively. In this way, the binding specificity of adhesins can confer tissue tropism within the host organism. In general, adherence involves the engagement of host cell receptors by bacterial adhesins. The cell surface receptor(s) for 5*. Typhimurium has/have been characterized as trypsin- and neuraminidase-sensitive (Finlay et al, 1989). Therefore, adherence of S. Typhimurium is likely a receptor-mediated phenomenon but surprisingly little is known about the host determinants that participate in this interaction. The closely-related pathogen, S. Typhi, but not S. Typhimurium, uses the cystic fibrosis transmembrane conductance regulator (CFTR) as a receptor for attachment to epithelial cells (Pier et al, 1998). To date, the host cell determinants required for S. Typhimurium adherence remain unknown. . 1.5.2 Invasion Upon adherence to the host cell surface, S. Typhimurium usurps the host cell cytoskeleton, initiating localized actin-driven lamellipodia-like extensions that facilitate internalization of bacteria into membrane-bound vacuoles (Schlumberger & Hardt, 2006). Modulation of the actin cytoskeleton during invasion of S. Typhimurium involves the SPI-1 type 3-secreted effectors S ipA, SipC, SopE, SopE2, and SopB. In cell culture, the SPI-1 type 3-secreted effector S ipA increases the efficiency of invasion by promoting actin polymerization (Jepson et al, 2001; Zhou & Galan, 2001). Extensive biochemical characterization has shown that S ipA binds actin (McGhie et al, 2001; Zhou et al, 1999a), stabilizes filamentous (F)-actin (Li l ic et al, 2003; Zhou et al, 1999a; Zhou et al, 1999b), reduces the critical globular (G)-actin concentration required 21 for polymerization (Galkin et al, 2002; Zhou et al, 1999a), increases the actin bundling activity of T-plastin (Zhou et al, 1999b), and inhibits depolymerization of F-actin (McGhie et al, 2004; Zhou et al, 1999a). A recent study has provided insight into the mechanism by which S ipA arrests actin depolymerization (McGhie et al., 2004). In cell-free extracts, S ipA inhibited ADF/cofilin-directed depolymerization, prevented gelsolin-directed severing of F-actin, and re-annealed gelsolin-severed F-actin fragments. S ipA can also potentiate the actin-bundling and -nucleating activity of SipC in vitro (McGhie et al, 2001). SipC is essential for the translocation of effectors and therefore determining the role of SipC in modulating actin polymerization during invasion is difficult. S o p E / E 2 S o p B fc* S G E F • R h o G Rac1 Figure 1. 3. SPI-1 effectors that mediate invasion of S. Typhimurium into non-phagocytic cells and the host cell pathways with which they interact. SopE and SopE2 act as G-nucleotide exchange factors for the Rho family GTPases RhoG and R a c l . SopB activates RhoG in and SGEF-dependent mechanism. Activation of N - W A S P and W A V E by R a c l stimulates Arp2/3 complex actin nucleation and polymerization: RhoG stimulates the Arp2/3 complex through an unknown mechanism. S ipA substantially stimulates SipC-directed actin nucleation and bundling. Furthermore, S ipA stabilizes actin filaments by preventing actin depolymerization and severing by ADF/co f i l i n and gelsolin, respectively. 22 SopE and the closely related protein SopE2, act as G-nucleotide exchange factors (GEFs) for a number of Rho GTPases including Cdc42, R a c l , and RhoG (Friebel et al, 2001; Hardt et al, 1998a; Patel & Galan, 2005; Stender et al, 2000; Zhou et al, 2001; Zhou & Galan, 2001). Previous studies using dominant-negative Rho GTPases indicated that Cdc42 was important for Salmonella-induced actin rearrangements and invasion (Chen et al, 1996; Hardt et al, 1998a). A n elegant new study has re-written previous concepts for the role of Rho GTPase activation by Salmonella effectors (Patel & Galan, 2006). Using R N A i , Cdc42 was found to be dispensable for Salmonella-induced actin remodeling and invasion. Instead, Cdc42 is required for signal transduction leading to nuclear responses upon infection. Conversely, R a c l is necessary for efficient actin remodeling and invasion, but not nuclear responses. The type 3-secreted effector SopB (also called SigD) is a potent inositol phosphatase. This new study also found that actin rearrangements by SopB are dependent upon the Rho GTPase RhoG (Patel & Galan, 2006), which has been implicated in both ruffling and macropinocytosis (Ellerbroek et al, 2004). SopB activates RhoG through stimulation of SH3-containing guanine nucleotide exchange factor (SGEF) , an exchange factor for RhoG (Ellerbroek et al, 2004; Patel & Galan, 2006). The mechanism by which SopB activates S G E F is unknown but it is presumably through phosphoinositide fluxes as a phosphatase mutant of SopB is incapable of S G E F activation (Patel & Galan, 2006). The Arp2/3 complex is absolutely required for invasion of 5*. Typhimurium into epithelial cells (Criss & Casanova, 2003; Unsworth et al, 2004). A s upstream activators of the Arp2/3 complex, N - W A S P and W A V E are also involved in S. Typhimurium invasion (Criss & Casanova, 2003; Shi et al, 2005). Interestingly however, when the 23 activity of both N - W A S P and W A V E is blocked, invasion is not completely abolished (Unsworth et al, 2004). Accordingly, there are likely other, unidentified N - W A S P - and WAVE-independent mechanisms for Arp2/3 complex activation. More recently, the focal adhesion proteins pl30cas and focal adhesion kinase ( F A K ) have also been implicated in invasion of S. Typhimurium (Shi & Casanova, 2006). 1.5.3 Tight junction disruption In the context of disease, epithelial cells represent the first line of defense, yet many pathogens are capable of disrupting cell-cell adhesion. Pathogens can disrupt junction structure and function by a) directly altering or degrading adhesion proteins; b) modulating the actin cytoskeleton; c) activation of cellular signal transduction; or d) triggering cytokine production or P M N transmigration (reviewed by Sears, 2000). Many diarrheal pathogens target cell-cell junctions. For example, a type 3-secreted effector of enteropathogenic Citrobacter rodentium disrupts the barrier by causing relocalization of claudins 1, 3, and 5 (Guttman et al, 2006) while Vibrio cholera produces a metalloprotease that directly degrades the extracellular domain of occludin (Wu et al, 2000). Infection of polarized epithelial cell monolayers by S. Typhimurium disrupts localization of occludin and ZO-1 and disrupts both the gate and fence function of the barrier (Finlay & Falkow, 1990; Jepson et al, 1995; Jepson et al, 1996; Jepson et al, 2000; Tafazoli et al, 2003). Disruption of tight junction structure and function is dependent upon the SPI-1 T3SS (Jepson et al, 1996) yet, the specific effectors responsible are not known. 24 1.6 Rationale B y invading epithelial cells and disrupting cell-cell junctions, S. Typhimurium employs two different mechanisms in order to overcome the barrier function of epithelial cell monolayers. Internalization of S. Typhimurium absolutely requires the actin cytoskeleton yet only a few of the cytoskeletal components involved in this process have been identified. This leads to several questions: To what extent is the actin cytoskeleton engaged during S. Typhimurium invasion?- What components of the actin cytoskeleton play a functional role during the invasion process? S. Typhimurium disrupts tight junctions in an SPI-1 T3SS-dependent manner. This observation led us to ask: Which SPI-1 type 3-secreted effectors are responsible for disrupting tight junction structure and function?; Through what mechanism do these effectors function?; Furthermore, does S. Typhimurium compromise intestinal barrier permeability in vivo? Thus, my doctoral research has focused oh defining and elucidating the mechanisms by which S. Typhimurium breaches the epithelial barrier. Three approaches were explored to meet this objective: 1) identification of cytoskeletal proteins recruited to sites of S. Typhimurium invasion; 2) identification of cytoskeletal proteins required for efficient S. Typhimurium invasion; and 3) investigation into the mechanism of intercellular junction disruption by S. Typhimurium and identification of the specific SPI-1 secreted effectors involved. In Chapter 3, the hypothesis tested is that S. Typhimurium recruits proteins necessary for the invasion process. Recruitment of 14 structural and signaling proteins to the site on invasion was examined. Given the extent that S. Typhimurium engaged the cytoskeleton, a functional role for five of the recruited proteins in invasion was explored. 25 In Chapter 4, the hypothesis tested is that S. Typhimurium disrupts the intestinal barrier function in an in vivo infection model. Using an in vitro cell culture model, the mechanism of S. Typhimurium-induced tight junction disruption is further dissected and, using various SPI-1-secreted effector mutants, the specific bacterial factors required for disruption of tight junction structure and function were determined. 1.7 Significance The ability of S. Typhimurium to penetrate the intestinal epithelium is key to its pathogenesis. Accordingly, understanding the molecular mechanisms involved in epithelial cell invasion and tight junction disruption is of the utmost importance. Moreover, tight junction disruption likely represents an important aspect of S. Typhimurium enteritis and may be central to the clinical manifestation of diarrhea. Collectively, these studies are relevant to understanding host-pathogen interaction, the molecular events leading to disease, and w i l l thereby aid our efforts to design effective therapeutics for salmonelloses. 26 Chapter 2: Materials and Methods 2.1 Cell lines 2.1.1 Sources The Madin Darby canine kidney ( M D C K ) epithelial cell line and human uterine epithelial line H e L a (CCL-2) were obtained from the American Type Culture Collection cell biology stock centre (Rockville, M D ) . The human enterocyte-like cell line Caco-2/TC7 (Chantret et al, 1994) was established from the parental Caco-2 cell line (Pinto et al, 1983). Christopher McCul loch (University of Toronto, Toronto, ON) kindly provided . fibroblasts obtained from either wild-type (Gsn + / + ) or gelsolin null (Gsn" / _) 12-day mouse fetuses as described previously (Witke et al, 1995). Mouse embryonic fibroblast ( M E F ) cell lines generated from Nckl+/+;Nck2+/+ (Nckl+/Nck2+), Nckl-/- ;Nck2+/+ ( N c k l -/Nck2+), Nckl+/+;Nck2-/- (Nckl+/Nck2-) and Nck l - / - ;Nck2- / - (Nckl- /Nck2-) knockout embryos were kindly provided by Tony Pawson (Samuel Lunenfeld Research.Institute, Toronto, ON) and have been described elsewhere (Bladt et al, 2003; Gruenheid et al, 2001). N c k l - / N c k 2 - cells complemented with wild-type human N c k l or a dominant-negative version of N c k l with inactivating mutations in all three SH3 domains (Kail) have been previously described (Rivera et al, 2006; Tanaka et al, 1995). Monique Arpin (Institut Curie, Paris) kindly provided stable transfectants of L L C - P K 1 porcine kidney epithelial cells expressing either full-length ezrin (E l7 ) or the N-terminal domain of ezrin (amino acids 1 to 309), serving as a dominant-negative (N12) (Skoudy et al, 1999). 2.1.2 Growth conditions Cel l lines were incubated at 37°C in a humidified atmosphere with 5% CO2 and were cultured in Dulbecco's minimal Eagle medium ( D M E M ; HyClone, Logan, U T ) 28 supplemented with 10% fetal calf serum (FCS). Caco-2 T C 7 cells were grown in D M E M - F C S supplemented with 1% M E M non-essential amino acids. Stable transfectants were grown in the presence of 0.7 mg/ml G418 (Stratagene, L a Jolla, C A ) . 2.1.3 Polarization of epithelial cells To obtain polarized monolayers, cells were grown on permeable Transwell filters (polyester; 6.5 or 24 mm diameter; 3 pm pore size; Corning Inc., N Y ) . Caco-2/TC7 cells were seeded onto Transwells at an initial density of 7.6 x 10 cells/cm and were grown for 18-22 days, until transepithelial resistance (TER) stabilized between 250-300 Ohms/cm 2 . M D C K cells were initially seeded at 1.0 x 10 5 cells/cm 2 and were grown for 4 days. Culture medium was replaced every 2-3 days. 2.2 Transfection The Nek 1-GFP construct was kindly provided by Tony Pawson. The profil in-GFP construct was kindly provided by Bob Heinzen (University of Wyoming, Laramie, W Y ) , the W I P - G F P was kindly provided by Michael Way (London Research Institute, London). Plasmid D N A was prepared using Qiagen (Mississauga, ON) columns and was transfected into cells using Fugene 6 (Roche, Basel, Switzerland) in accordance with the manufacturer's instructions. Cells were transfected 18 hours prior to infection. 2.3 siRNA Small interfering R N A (s iRNA) SMARTpool™ (Dharmacon R N A Technologies, Lafayette, CO) sequences targeting Crkl/II , ShcA, or non-targeting scrambled control • 29 s i R N A were diluted and stored according to the manufacturer's instructions. The Crkl/II and ShcA targeting sequences are published (Kisielow et al, 2002; Nagashima et al, 2002). H e L a cells were transfected 24 h after seeding with 40 pmol of s iRNA/we l l using Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions. Cells were trypsinized and seeded 48 h post-transfection and bacterial infections were carried out 72 h post-transfection since knockdown of ShcA and Crk protein levels were maximal at this time-point (see Figure 3.4b). 2.4 Bacterial strains A l l bacterial strains and plasmids used in this study are described in Table 2.1. 2.4.1 Growth conditions Bacteria were grown in Luria-Bertani (LB) medium that was supplemented with streptomycin (100 p-g/ml), kanamycin (25 u,g/ml), chloramphenicol (25 u.g/ml) tetracycline (12.5 u.g/ml), and ampicillin (100 ug/ml), where appropriate. A l l antibiotics were purchased from Sigma. 2.4.2 Construction of bacterial mutants Unmarked non-polar internal deletions of sopB and sipA were introduced by allelic exchange as previously described by Steele-Mortimer et al. (Steele-Mortimer et al, 2000) and Zhang et al. (Zhang et al, 2002), respectively. A l l chromosomal deletions were confirmed by P C R and sequencing. 30 Table 2.1 Bacterial strains used in this study. Plasmid or Strain Description Source/Reference S. Typhimurium SL1344 Wild-type (Hoiseth & Stacker, 1981) SB103 SL1344, invAwk&n, defective in a structural gene of (Galan & Curtiss, 1991) the SPI-1 type 3 apparatus (Brumell et ai, 2001) AssaR SL1344, in frame deletion of a structural gene of the SPI-2 type 3 apparatus AsopB SL1344, in frame deletion of sopB (Steele-Mortimer et al., 2000) AsipA SL1344, in frame deletion of sipA This study AsopE/E2 SL1344, sopEy.kan and an in frame deletion of J. Galan sopE2 AsopB/E/E2 SL1344, AsopE/E2 mutant with an in frame deletion This study of sopB AsipA/sopE/E2 SL1344, AsppE/E2 with an in frame deletion of sipA This study SL1344, AsopB with an in frame deletion of sipA AsipA/sopB This study Plasmids pMOB Ampr, high copy number cloning vector J. Galan; (Strathmann et al, 1991) pACYC184 Tef, Cm r; low copy number cloning vector New England BioLabs J. Galan, (Hardt et al., pSB1122 Tetr; sopE cloned into pACYC 184 1998b) J. Galan, (Kaniga et al., pSB0415 Ampr; sipA cloned into pMOB . 1995) This study psopE2 Tef; pA CYC 184 containing the entire sopE2 ORF plus 1038 bp upstream DNA sequence (promoter region) psipA Cm r; pACYC184 containing the entire sipA ORF Jepson etal., 2001 plus the -40 bp upstream and downstream 2.4.3 Construction of complementation plasmids The sip A plasmid (psipA) utilized p A C Y C 1 8 4 (New England BioLabs, Ipswich, M A ) and was constructed as previously described (Jepson et al, 2001). To construct the psopE2 plasmid, sopE2 and its promoter region were PCR-amplified from SL1344 chromosomal D N A using Elongase (Invitrogen, Burlington, ON) and the oligonucleotide primers (Nucleic A c i d Protein Service Unit, U B C , Vancouver, B C ) S O P E 2 F W D (5'-31 G A C C C A T G G C T T T C G A C C A G T A T T G T C G A G T - 3 ' ) and S 0 P E 2 R E V (5'-G A C G A A T T C C C A T C A G G A G G C A T T C T G A A G - 3 ' ) to generate a 1761 bp fragment. This fragment was cloned into pCR2.1 (Invitrogen) and its sequence confirmed by D N A sequencing. This plasmid was subsequently digested with EcoRI and N c o l (New England BioLabs) and the insert was cloned into the corresponding restriction enzyme sites in p A C Y C 184. 2.5 Bacterial infections of cell lines Non-polarized cells were seeded into 24-well plates with or without 12-mm-diameter coverslips at a density of 1.0 x 10 5 cells/ml 24 hours prior to infection. Polarized Caco-2/TC-7 cells were treated with GGTI-298 (Calbiochem, L a Jolla, C A ) 40 hours prior to infection or cycloheximide (Sigma) 15 minutes prior to infection. Late-log bacterial cultures were prepared by growing bacteria for 16 h shaking at 37 °C, sub-culturing (1:33) in L B broth without antibiotic, and incubating for an additional 3 h. Bacteria, were then centrifuged at 8000 rpm, re-suspended (equal volume) in phosphate buffered saline (PBS), and diluted 1:100 in growth medium. Cells were infected for 10 minutes at 37 °C with a multiplicity of infection of ~50. 32 2.6 Gentamicin protection assay After a 10 minute infection, cells were washed three times with P B S and incubated with D M E M - F C S for 20 minutes at 37 °C. Thirty minutes post-infection, media was replaced with D M E M - F C S containing 50 ug/mL gentamicin. Two hours post-infection, cells were washed three times with P B S and lysed in-PBS containing 1% Triton X-100 and 0.1% SDS. Bacterial dilutions were made in P B S and plated onto L B agar for enumeration of bacterial colony forming units (CFU) . 2.7 Immunofluorescent staining of cell lines Cells were washed three times with P B S and fixed with 2.5% paraformaldehyde (PFA) for 20 minutes at room temperature. After fixation, the cells were washed three times with P B S and permeabilized with 0.1% Triton X-100 in P B S . Following permeabilization, the cells were blocked with 10% normal goat serum (NGS) in P B S and probed with the primary antibody in the blocking buffer. For gelsolin staining, cells were blocked in 3% B S A in P B S and probed in 3% BSA/0.1%Tri ton X-100. For GP135 staining, cells were fixed with ice cold methanol for 20 minutes at - 2 0 ° C , re-hydrated i n . ( P B S for 10 minutes,.blocked in 1% B S A / 1 0 % N G S for 30 minutes, and probed for GP135 in P B S . Following incubation with primary antibodies, the cells were washed three times with P B S and probed with Alexa and Cy5 dye-conjugated antibodies and Alexa-conjugated phalloidin to detect actin (Molecular Probes). A l l primary and secondary antibodies used in immunofluorescence experiments are listed in Table 2.2. Samples were mounted on glass slides in M o w i o l (Sigma) and samples were viewed on a Zeiss Axiovert SI00 T V microscope attached to a BioRad Radiance Plus confocal scanhead. 33 Confocal sections were projected using Image J v. 1.3 6b and were imported into Adobe Photoshop and Illustrator CS2 (San Jose, C A ) . 2.8 Inside/outside immunostaining Quantification of bacterial adherence and invasion using the inside/outside differential staining assay has been described previously (Pentecost et al, 2006). Cells were infected for 10 minutes, washed three times with P B S , fixed with P F A , and blocked in 10% N G S in P B S . Cells were probed with polyclonal anti-Salmonella L P S , washed with P B S , and incubated with Alexa 568-conjuagted donkey anti-rabbit to label the bacteria outside the cell. Cells were washed with P B S and permeabilized with 0.1% Triton X-100 in P B S . Cells were probed with rabbit polyclonal anti-Salmonella L P S , washed with 0.1% Triton X-100 in P B S , and incubated with a 350-conjugated donkey anti-rabbit antibody. Coverslips were mounted as described above and viewed on a Zeiss Axiophot epifluorescence microscope. Twenty fields of view were selected at random and analyzed for each experiment. "Invasion" was defined as the number of intracellular bacteria per cell while "adherence" was defined as the number of intracellular and cell-associated bacteria per cell. "Invasion efficiency" was calculated by: (Invasion/Adherence)x 100. 34 Table 2. 2 Antibodies used in studies. Antibody Species Dilution in Dilution Source (anti-) Western blot for IF a-actinin mouse - 1:200 Sigma, St. Louis, MO Crkl/II mouse 1:2500 1:200 BD Biosciences claudin-3 rabbit - 1:100 Zymed, San Francisco, CA • ezrin mouse - 1:100 ICN Biomedical, Aurora, OH gelsolin mouse - 1:200 Sigma GP135 mouse - 1:10 G. Ojakian, SUNY Downstate Medical Centre, Brooklyn, NY LPP rabbit - 1:100 R. Golsteyn, Institut Curie, Paris occludin mouse 1:500 1:200 Zymed Salmonella LPS mouse - 1:1000 BioDesign, Saco, ME Salmonella LPS rabbit - 1:200 BD Biosciences ShcA rabbit 1:1000 1:200 Upstate Biotechnology talin mouse - 1:100 Sigma tropomyosin mouse - 1:100 Sigma VASP mouse - 1:200 Transduction Labs vinculin mouse - 1:100 Sigma ZO-1 rabbit 1:250 1:200 Zymed zyxin mouse - 1:100 Synaptic Systems, Gottingen calnexin rabbit 1:2000 - Stressgen Bioreagents, E-cadherin mouse 1:200 BD Biosciences Mouse-HRP goat 1:5000 Jackson ImmunoResearch, West Grove, PA Rabbit-HRP goat 1:5000 Sigma Rabbit AlexaFluor® goat 1:200 Molecular Probes Cy5 Mouse AlexaFluor® goat 1:200 Molecular Probes Cy5 Rabbit AlexaFluor® goat 1:200 Molecular Probes 350 Mouse AlexaFluor® goat 1:200 Molecular Probes 350 Rabbit AlexaFluor® goat 1:200 Molecular Probes 488 Mouse AlexaFluor® goat 1:200 Molecular Probes 488 Rabbit AlexaFluor® goat 1:200 Molecular Probes 568 Mouse AlexaFluor® goat 1:200 Molecular Proves 568 2.9 Collection of host cell fractions 2.9.1 Soluble and insoluble fractions Cells were washed three times with P B S . Extractions were performed by overlaying cells with ice cold C S K buffer [10 m M piperazine-l,4-bis(2-ethanesulfonic 35 acid) (PIPES), p H 6.8; 50 m M N a C l ; 300 m M sucrose; 3 m M M g C l ; 0.5% Triton-X-100] containing a Complete protease inhibitor tablet (Roche). Cells were extracted for 20 minutes on ice, collected, and spun at 13 200 rpm at 4 °C for 15 minutes. The supernatant (soluble fraction) was collected. The pellet (insoluble fraction) was disrupted by adding SDS-1P buffer [1% SDS; 10 m M Tris-Hcl , p H 7.5; 2 m M ethylenediamine tetraacetic acid (EDTA)] containing a Complete protease inhibitor tablet. The insoluble fraction was boiled for 5 minutes and sonicated until the pellet was resuspended. 2.9.2 Whole cell lysates Cells were washed three times with P B S and lysed in N P buffer (20 m M Tris-H C l , p H 7.5, 150 m M N a C l , 1% Nonidet P-40, 10 m M Na4P207, 50 m M NaF) supplemented with a Complete protease inhibitor tablet (Roche). 2.10 S D S - P A G E and Western blotting The protein concentration of each sample was quantified using a bicinchoninic acid assay (Sigma). Equal amount of total protein (15 u.g) was loaded in each lane of the gel. Samples were electrophoresed through a 6% (for ZO-1), 10% (for occludin), or 12% (ShcA and Crk) SDS polyacrylamide gel and transferred onto nitrocellulose membrane (Millipore, Bedford, M A ) . The membranes were incubated for one hour at room temperature in blocking buffer [Tris-buffered saline, 0.1% Tween 20 (TBST) , 5% skim milk] and then incubated overnight at 4 °C with primary antibodies diluted in blocking buffer. After washing in T B S T , the membranes were incubated with the appropriate HRP-conjugated secondary antibodies diluted in blocking buffer for 1 hour at room 36 temperature. After washing in T B S T , the bands were detected using an enhanced chemiluminescence ( E C L ) kit (Amersham) according to the manufacturer's instructions. ZO-1 and occludin signals were quantified using ImageJ v. 1.36b software. A l l Western blots are representative of at least two experiments carried out. 2.11 Measurement of TER and paracellular flux Transepithelial resistance (TER) was measured using an epithelial voltohmmeter (World Precision Instruments Inc., Sarasota, Fla.). To measure paracellular flux, 4 and 40 kDa fluorescein isothiocyanate (FITC)-dextran (Sigma, St. Louis, M O ) was dissolved in P buffer [10 m M 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES) , p H 7.4; 1 m M sodium pyruvate; 10 m M glucose; 3 m M CaC12; 145 m M NaCl ) or P / E G T A buffer [10 m M H E P E S , p H 7.4; 1 m M sodium pyruvate; 10 m M glucose; 145 m M N a C l ; 2 m M ethylene glycol-bis((3-aminoethyl ether)-7V, TY, N, TV-tetraacetic acid ( E G T A ) ] . To measure paracellular flux, cells were allowed to equilibrate in P buffer one hour prior to infection. Immediately prior to infection, FITC-dextran was added to the apical compartment to give a final concentration of 10 mg/mL. Uninfected cells served as a negative control while uninfected cells incubated in P / E G T A buffer served as the positive control for tight junction disruption. After a 4 hour infection, the basolateral medium was collected and the concentration of FITC-dextran was measured with a fluorometer (excitation 492 nm, emission 520 nm). 37 2.12 IL-8 ELISA The basolateral culture media was collected and IL-8 concentration was determined by E L I S A ( B D Biosciences) as recommended by the manufacturer. 2.13 In vivo experiments 2.13.1 Mouse infections A l l animal experiments were conducted in a manner consistent with the ethical requirements of the Animal Care Committee at the University of British Columbia and the Canadian Council on the Use of Laboratory Animals. Eight to 10 week old male C57B16 mice were purchased from Jackson Laboratories (Bar Harbor, M E ) . Each mouse was given 20 mg of streptomycin by oral gavage 24 hours prior to infection. Mice were infected with 3x10 bacteria in 100 pi L B by oral gavage. Control mice were given 100 pi L B . Mice were euthanized with CO2 at designated time points. Blood was collected by cardiac puncture and tissues were harvested aseptically for bacterial enumeration and staining. 2.13.2 Measuring intestinal permeability To measure intestinal permeability, mice were given 8.8 mg of FITC-dextran (FD40; Sigma) by oral gavage 4 hours prior to sacrifice. Collected blood was immediately combined with citrate dextrose (20 m M citric acid, 110 m M sodium citrate, 5 m M dextrose) and centrifuged at 1000 xg for 15 min at 4°C. The concentration of FITC-dextran in the plasma was determined with a fluorescence spectrophotometer ^excitation, e^mission. 5 j5 ^ p e r ] d n E l m e r > p a l o A k o ? C A ) 38 2.13.3 Tissue preparation Tissues were placed into 3% P F A in P B S for 3 hours. Three 10 min P B S washes followed the fixation to wash out any remaining fixative. Fixed tissues were frozen (using liquid nitrogen) onto an aluminum stub with optimal cutting temperature (OCT) compound (Sakura Finetek U S A , C A , U S A ) . Five um frozen colon sections were cut by Wax-It Histology Services (Vancouver, British Columbia), attached to glass slides, immediately plunged into cold (-20°C) acetone for 5 min, air dried, and then processed for immunofluorescence. 2.13.4 Immunofluorescence Tissue cryo-sections were blocked with 5% N G S in T P B S - B S A (PBS containing 0.05% Tween-20 and 0.1% B S A ) for 20 min at room temperature. Primary antibodies were diluted in T P B S - B S A with 1% N G S , and incubated overnight at 4°C. Tissue sections were washed extensively with the T P B S - B S A (wash buffer) then incubated for 90 min at 37°C with secondary antibodies conjugated to Alexa 568 and 488. The slides were washed, incubated with 2 fxg/ml 4',6-diamidino-2-phenylindole (DAPI) for 1 minute, and then washed again and mounted in ProlongGold (Invitrogen). Samples were visualized by confocal. microscopy .using a Bio-Rad Radiance 2000 Multiphoton microscope. 39 2.14 Statistical analysis Data were analyzed using PRISM 4.0. One-way A N O V A s , Student unpaired t tests, and Mann-Whitney U non-parametric tests- were performed using a 95% confidence interval. Bonferroni's and Dunnett's tests were applied post-hoc. 40 Chapter 3: Catalogue and functional analysis of proteins recruited to sites of S. Typhimurium invasion1 1 A version of this chapter has been published. Boyle E . C , Brown, N . F. , Brumell, J. H . , and Finlay, B . B . (2007) Src homology domain 2 adaptors affect adherence of Salmonella enterica serovar Typhimurium to non-phagocytic cells. Microbiology. 153: 3517-3526. 41 3.1 Summary The ability of S. Typhimurium to penetrate the intestinal epithelium is key to its pathogenesis. Invasion of S. Typhimurium absolutely requires the actin cytoskeleton yet only a few of the cytoskeletal components involved in the process have been identified. In order to identify host proteins that may play a role in S. Typhimurium invasion, the recruitment of actin-binding proteins; focal adhesion proteins; proteins associated with actin polymerization and depolymerization; and Src homology 2 (SH2) adaptors was investigated. Alpha-actinin, Crk, ezrin, gelsolin, lipoma preferred partner (LPP), Nek, profilin, ShcA, talin, tropomyosin, vasodilator-stimulated phosphoprotein ( V A S P ) , WASP-interacting protein (WIP), and zyxin were recruited to sites of S. Typhimurium invasion while vinculin was not. The actin severing protein, gelsolin, was found to inhibit the invasion process while ezrin did not play an important role during S. Typhimurium invasion. The contribution of the recruited SH2 adaptors to invasion was further investigated and it was found that, while not involved in bacterial internalization itself, the adaptors Nek and ShcA influenced adherence qf S. Typhimurium to non-phagocytic cells. 3.2 Introduction Critical for S. Typhimurium virulence is its ability to attach to mucosal surfaces in the intestine and induce its own uptake into epithelial cells. Upon adherence to the host cell surface, S. Typhimurium usurps the host cell cytoskeleton, initiating localized actin-driven lamellipodia-like extensions that facilitate internalization of bacteria into membrane-bound vacuoles. Internalization of S. Typhimurium absolutely requires the 42 actin cytoskeleton yet only a few of the host proteins involved in this process have been identified. In this section of my doctoral thesis, I test the hypothesis that S. Typhimurium • recruits cytoskeletal and signaling proteins necessary for the invasion process. In order to identify host proteins that may play a role in S. Typhimurium invasion, the recruitment of actin-associated proteins was investigated. Whether recruited proteins played an important functional role in the invasion process was also assessed for several of the recruited proteins. 3.3 Results 3.3.1 Recruitment of actin-associated proteins to sites of S. Typhimurium invasion In order to identify new actin-associated proteins that may play a role in S. Typhimurium invasion, recruitment of actin-binding proteins, focal adhesion proteins, SH2 adaptors, and proteins associated with actin polymerization and depolymerization, was investigated. Recruitment of these proteins was visualized using immunofluorescent staining of epithelial cells infected with S. Typhimurium for 10 minutes. O f the proteins screened, the following were recruited to S. Typhimurium-induced membrane ruffles: ct-actinin, Crk, ezrin, gelsolin, lipoma preferred partner (LPP), Nek, profilin, ShcA, talin, tropomyosin, vasodilator-stimulated phosphoprotein ( V A S P ) , WASP-interacting protein (WIP), and zyxin (Figure 3.1). A s shown previously, vinculin did not accumulate at sites of invasion (Finlay et al, 1991) (Figure 3.1). These data demonstrate the extent to which S. Typhimurium engages the host cell actin cytoskeleton during invasion. Proteins recruited to sites of invasion have the potential to.play an important role in bacterial internalization. 43 Figure^ 3.1. Recruitment of actin-associated proteins to sites of S. Typhimurium invasion. Profi l in-GFP, N c k - G F P , and W I P - G F P constructs were transfected into H e L a cells 18 hours prior to infection. H e L a cells were infected with wild-type S. Typhimurium for 10 minutes and stained with phalloidin to label actin (red), an anti-LPS antibody (blue) to label bacteria, and antibodies towards endogenous actin-associated proteins (green), a-actinin, Crk, ezrin, gelsolin, L P P , Nek, profilin, ShcA, talin, tropomyosin, V A S P , WIP, and zyxin were recruited to sites of invasion while vinculin was not. Images are en face Z-projections of confocal slices and are representative of at least 3 different experiments. 44 3.3.2 E z r i n is not required for invasion of S. T y p h i m u r i u m into non-phagocytic cells The membrane-cytoskeleton linker, ezrin, is highly enriched in the cellular protrusions induced by 5*. Typhimurium and plays an important role during epithelial cell invasion by the highly-related pathogen, Shigella flexneri (Skoudy et al, 1999). Accordingly, a functional role for ezrin during S. Typhimurium invasion was investigated. To study the potential role of ezrin in bacterial internalization, gentamicin protection assays were performed with stable L L C - P K 1 transfectants expressing either full-length (E l7 ) or dominant-negative (N12) ezrin. S. Typhimurium invaded to the same extent into E17 and N12 cells (Figure 3.2, N .S) . Therefore, although recruited by 5". Typhimurium, ezrin is not essential for invasion into host cells. 47 N.S. 3 L i . o E 9.0x10s-8.0x10s-7.0x10s-6.0x10s-5.0x10s-4.0x10s-3.0x10s-2.0x10s-1.0x10s-0 ezrin DN-ezrin Figure 3.2. . Ezr in does not play a functional role during S. Typhimurium invasion into non-phagocytic cells. Cells were infected with S. Typhimurium for 10 minutes and the number of intracellular bacteria was quantified using a gentamicin protection assay. Values represent colony-forming units (cfu) and are expressed as means +/- the standard error of the mean of 3 different experiments, each performed in duplicate. N . S., not significant; unpaired Student's t test, 95% confidence interval. 3.3.3 Gelsolin inhibits invasion of S. Typhimurium into non-phagocytic cells A recent study has demonstrated that in cell-free extracts, the bacterial effector S ipA prevents severing by gelsolin and re-anneals gelsolin-severed F-actin fragments (McGhie et al., 2004). We have demonstrated that gelsolin was strongly recruited to sites of S. Typhimurium invasion (Figure 3.1), therefore, a potential role for gelsolin during an S. Typhimurium infection was investigated using a gentamicin protection assay with fibroblasts generated from wild-type (Gsn + / + ) and gelsolin knockout (Gsn"A) mice (Witke et al., 1995). Invasion of wild-type S. Typhimurium into Gsn" / _ cells was significantly higher than invasion into G s n + / + cells (Figure 3.3; P< 0.001). The extent of invasion can be influenced by changes in bacterial adherence to the cell surface. Accordingly, adherence of wild-type S. Typhimurium to G s n + / + and Gsn _ /" cells was assessed by infecting cells as above, yet in this case, gentamicin was excluded. Therefore, upon host 48 cell lysis, both intracellular and host cell-associated bacteria were quantified. Adherence of wild-type S. Typhimurium to Gsn _ /" cells was 107±9.42% relative to adherence to Gsn+/+ (N.S. by Student's t test). Therefore, gelsolin inhibits the internalization of S. Typhimurium into non-phagocytic cells as bacterial uptake was significantly greater in the absence of gelsolin while adherence remained unaffected. Interestingly, in a •cell-free system, the type 3-secreted effector S ipA has been shown to protect F-actin from severing by gelsolin (McGhie et al, 2004). Accordingly, to investigate the biological consequence of this potential SipA-gelsolin interplay, we infected Gsn"7" and G s n + / + cells with the AsipA mutant strain. Gelsolin did not significantly inhibit invasion of AsipA bacteria (Figure 3.3; N.S.) . In order to confirm this observation was attributable to specific deletion of the gene encoding S ipA, we expressed S ipA from a plasmid in an AsipA mutant. Fibroblasts were infected as above with the AsipA mutant strain harboring either psipA or the empty vector (pACYC184) . Gelsolin significantly inhibited invasion of bacteria expressing S ipA (Figure 3.3; P O . 0 0 1 ) yet did not significantly inhibit S ipA null bacteria (Figure 3.3, N .S) . Therefore, it appeared as though the inhibitory effect of gelsolin was lost in the absence of S ipA. To test whether this effect was specific to SipA, Gsn"7" and G s n + / + cells were infected with a AsopE/sopE2 mutant strain of S. Typhimurium. Like the AsipA strain, gelsolin did not significantly inhibit invasion of the AsopE/sopE2 strain (Figure 3.3, N . S). Because SipA, SopE, and SopE2 are SPI-1-secreted effectors involved in 5*. Typhimurium invasion, we propose that the inhibitory role of gelsolin is simply not as apparent in these already invasion-deficient strains. 49 ** wt AsipA AsipA+ AsipA+ psipA vector AsopE/ sopE2 Figure 3.3. Gelsolin inhibits invasion of S. Typhimurium. G s n + / + and Gsn"'~ cells were infected with S. Typhimurium strains and invasion was quantified using a gentamicin protection assay. Values represent invasion of each strain as a percentage normalized to invasion into G s n + / + cells. Values are expressed as means ± the standard error of the mean. With the exception of the L\sopE/sopE2 mutant, all data represents at least 3 independent experiments, each performed in triplicate wells. The AsopE/sopE2 mutant was tested once, in triplicate wells. The number of intracellular bacteria ± the standard error of the mean are as follows: wildtype, 2.4x10 5 ± 3 .0x l0 4 (Gsn + / + ) and l . l x l O 6 ± ' 2 .3x l0 5 (Gsn"7"); AsipA, 1.3xl0 5 ± 1.4xlG 4 (Gsn + / + ) and 1.7xl0 5 ± 6 .1x l0 3 (Gsn"A); AsipA+psipA, 9 .8x l0 4 ± 6 .4x l0 3 (Gsn + / + ) and 3 .7xl0 5 ± 6 .7x l0 4 (Gsn" / _); AsipA+vector, 5JxW± 5 .5xl0 3 (Gsn + / + ) and 8 .4xl0 4 ± 9 .2x l0 3 (Gsn"7"); AsopE/E2, 2 .7x l0 4 ± 2 .5x l0 3 (Gsn + / + ) and 3 .0x l0 4 ± 2 .9x l0 3 (Gsn"A). N . S., not significant, **P < 0.001 using a Bonferroni post-hoc test. 3.3.4 Nek and ShcA, but not Crk, affect the extent of invasion of S. Typhimurium into non-phagocytic cells SH2 adaptors are often exploited by viral and bacterial pathogens in order to manipulate the host actin cytoskeleton (Gruenheid & Finlay, 2003). Accordingly, a functional role for the recruited SH2 adaptors Nek, ShcA, and Crk during S. Typhimurium invasion was investigated. Mammals possess two Nek family members, N c k l / a and Nck2/|3 (also called Grb4) (Braverman & Quill iam, 1999). Gentamicin 50 protection assays were performed using cell lines deficient in N c k l and/or Nck2 to determine the requirement for different Nek family members in S. Typhimurium invasion. Compared to wild-type cells, the number of intracellular S. Typhimurium was significantly reduced in cells lacking both N c k l and Nck2 (Figure 3.4a; P < 0.001). However, the absence of either N c k l or Nck2 did not significantly reduce the number of intracellular S. Typhimurium (Figure 3.4a), demonstrating that N c k l and Nck2 can functionally compensate for the absence of the other. These data reveal that N c k l and Nck2 play an important role in the invasion process of S. Typhimurium. A functional role for the recruited SH2 adaptors Crk and ShcA was also explored. Alternate splicing of the human crk gene generates two Crk proteins: CrkI, consisting of an SH2 domain and an SH3 domain, and C r k l l , which has an additional carboxy-terminal SH3 domain (Buday, 1999). ShcA exists in 3 isoforms, namely p66, p52, and p46, which differ only in their N-terminal regions (Ravichandran,. 2001). Using commercially-available s i R N A directed towards sequences of ShcA and Crk that were common to all isoforms, expression of all isoforms of ShcA and Crk were successfully knocked down (Figure 3.4b). Importantly, expression of ShcA and Crk were not affected when cells were transfected with scrambled (control) s i R N A (Figure 3.4b). We infected s i R N A -treated cells with S. Typhimurium 72 hours post-transfection as knockdown of ShcA and Crk was maximal at that time point (Figure 3.4b). Invasion of 5*. Typhimurium was quantified using the gentamicin protection assay. While knockdown of Crk did not affect the extent of invasion, knockdown of ShcA significantly increased the level of S. -Typhimurium invasion (P < 0.001; Figure 3.4c). These results suggest that ShcA inhibits the invasion process of S. Typhimurium. 51 A. 175 150 j 125 £ 100 B. H>" # # rfr j*f rfF ^ • • • control siRNA She siRNA 0 24 48 72 0 24 48 72 hours * - . -p66Shc ™ : - -p46Shc wmm*Fm*"+ calnexin control siRNA Crk siRNA 0 24 48 72 0 24 48 72 hours -Crkll N.S. 300 £ 200 i 100 Figure. 3.4. Nek and ShcA, but not Crk, affect the extent of invasion of S. Typhimurium into non-phagocytic cells, (a) Nckl+/Nck2+, N c k l - / N c k 2 , Nckl+/Nck2- , and Nck l - /Nck2+ M E F s were infected with wild-type S. Typhimurium for 10 minutes and invasion was quantified using a gentamicin protection assay. Invasion is reported as a percentage normalized to invasion into Nckl+/Nck2+ cells and values are expressed as means +/-the standard error of the mean of 3 different experiments, each performed in duplicate. **, P < 0.001 using a Bonferroni post-hoc test, (b) H e L a cells were transfected with either scrambled (control), anti-ShcA, or anti-Crkl/II s i R N A and assessed at various time points post-transfection for expression of ShcA or Crkl/II by Western immunoblotting. A corresponding calnexin immunoblot is shown as a loading control, (c) H e L a cells were transfected with either scrambled (control), anti-ShcA, or anti-Crkl/II s i R N A and infected with wild-type S. Typhimurium 72 hours post-transfection. Invasion was quantified using a gentamicin protection assay. Invasion is reported as a percentage normalized to invasion into control s i R N A -transfected cells and values are expressed as means +/- the standard error of the mean of 3 different experiments, each performed in duplicate. N . S., not significant; **, P < 0.001 using a Bonferroni post-hoc test. 52 3.3.5 Nek and ShcA affect adherence of S. Typhimurium to non-phagocytic cells The extent of invasion can be influenced by changes in bacterial adherence to the cell surface and/or by changes in the ability of bacteria to trigger actin-dependent internalization. In order to distinguish between these two possibilities, cells were infected with wild-type S. Typhimurium and intracellular and extracellular (cell-associated) bacteria were quantified by immunoflourescence. Compared to wild-type cells, S. Typhimurium adhered significantly less to N c k l - / N c k 2 - cells (Figure 3.5a; P < 0.001). In order to confirm that the inability of S. Typhimurium to adhere to N c k l - / N c k 2 - cells was attributable to specific deletion of the Nek genes, we performed complementation analysis. Adherence of 5". Typhimurium to N c k l - / N c k 2 - cells was restored to near wi ld-type levels when N c k l was expressed in the N c k l - / N c k 2 - cells (Figure 3.5a). However, the defect was not restored when a dominant-negative form of N c k l was expressed (Kai l , Figure 3.5a). Therefore, Nek does indeed contribute to the adherence of S. Typhimurium to non-phagocytic cells. Conversely, in cells where ShcA expression had been knocked down, bacterial adherence significantly increased (Figure 3.5b, P < 0.0001), demonstrating that ShcA inhibits this early interaction of S. Typhimurium with host cells. 53 A. B. Figure 3.5. Nek and ShcA affect adherence of S. Typhimurium to non-phagocytic cells, (a) M E F s [Nckl+/Nck2+, N c k l - / N c k 2 - , N c k l - / N c k 2 - expressing N c k l , and N c k l -/Nck2- expressing dominant-negative N c k l (Kail)] were infected for 10 minutes with wild-type S. Typhimurium and adherence was quantified using inside/outside immunostaining. Adherence is reported as a percentage normalized to adherence into wild-type Nckl+/Nck2+ cells and values are expressed as means +/- the standard error of the mean of 3 different experiments, each performed in duplicate. **, P < 0.001 using a Bonferroni post-hoc test, (b) H e L a cells were transfected with either scrambled (control) or anti-ShcA s i R N A and infected with wild-type S. Typhimurium 72 hours post-transfection. Adherence was quantified using inside/outside immunostaining and is reported as a percentage normalized to adherence into control siRNA-transfected cells. Values are expressed as means +/- the standard error of the mean of 3 different experiments, each performed in duplicate. ***, P < 0.0001 using a Student's unpaired t test. 3.3.6 Nek and ShcA do not affect invasion efficiency of S. Typhimurium into non-phagocytic cells Because Nek and ShcA could affect both bacterial adherence and internalization, we investigated whether these adaptors also influenced internalization efficiency -defined here as the proportion of adherent bacteria that are internalized. The internalization efficiency of S. Typhimurium into N c k l - / N c k 2 - cells did not significantly differ from wild-type cells (Figure 3.6a). Similarly, internalization efficiency into ShcA 54 r knockdown cells was not significantly different from control siRNA-treated cells (Figure 3.6b). Therefore, Nek and ShcA do not play significant roles in internalization in that, once bound to the cell surface, S. Typhimurium can be internalized to a similar extent with or without these SH2 adaptors. Therefore, the differences in invasion observed in N c k l - / N c k 2 - and She knockdown cells (Figure 3.4a and c) are likely solely attributable to differences in the ability of S. Typhimurium to adhere to host cells: A. B. Figure 3.6. Nek and ShcA do not affect internalization efficiency of S. Typhimurium into non-phagocytic cells, (a) Nckl+/Nck2+ and N c k l - / N c k 2 - M E F s were infected with wild-type S. Typhimurium for 10 minutes and internalization efficiency was quantified using inside/outside immunostaining. Internalization efficiency is reported as a percentage normalized to internalization efficiency into wild-type Nckl+/Nck2+ cells and values are expressed as means +/- the standard error of the mean of 3 different experiments, each performed in duplicate. N . S., not significant using a Student's unpaired t test, (b) H e L a cells were transfected with either scrambled (control) or anti-ShcA s i R N A and infected with wild-type S. Typhimurium 72 hours post-transfection. Internalization efficiency was quantified using inside/outside immunostaining and is reported as a percentage normalized to internalization efficiency into control s i R N A -transfected cells. Values are expressed as means +/- the standard error of the mean of 3 different experiments, each performed in duplicate. N . S., not significant using a Student's unpaired t test. 55 3.4 Discussion Bacterial invasion can be seen as a two-step process first requiring adherence to the host cell surface followed by internalization into the host cell. In the context of disease, bacterial adherence and internalization are means by which pathogens colonize the host, spread to systemic sites, and evade both host defense mechanisms and antibiotic treatment. Accordingly, understanding the molecular mechanisms involved in these events is of the utmost importance. Invasion of S. Typhimurium is absolutely dependent on the actin cytoskeleton yet few of the cytoskeletal components involved have been identified. In order to identify new host determinants involved in S. Typhimurium invasion, the recruitment of various actin-associated proteins to the site of invasion was investigated. To test whether recruited proteins were necessary for efficient invasion, this study focused on the contribution of the actin-membrane linker, ezrin, the actin-severing protein, gelsolin, and SH2 adaptor proteins Nek, Crk, and ShcA. Ezr in links actin filaments to membranes and is important for invasion of the closely-related bacterial pathogen, Shigella flexneri (Bretscher et al., 2002; Skoudy et al., 1999). Despite being recruited to sites of S. Typhimurium invasion (Figure 3.1), we have been unable to establish a role for ezrin during S. Typhimurium invasion (Figure 3.2). This observation illustrates the fact that some of the proteins recruited to sites of S. Typhimurium invasion may not perform a functional role at this site but are rather recruited because their binding partners are actively recruited to ruffles. In addition, one might suspect a certain amount of functional redundancy with these proteins. For example, the ezrin paralogues, radixin and moesin, could compensate for ezrin during S. Typhimurium invasion (Bretscher et al, 2002). 56 Gelsolin is an actin-binding protein that severs F-actin and caps the barbed end (Silacci et al., 2004). Since gelsolin was strongly recruited to Salmonella-induced ruffles (Figure 3.1), we hypothesized that it may play an important role in the invasion process. The role of gelsolin during S. Typhimurium invasion was intriguing for two additional reasons. Firstly, recent evidence suggests that actin turnover is important for the formation of lamellipodia (Loisel et al., 1999). Gelsolin plays an important role in membrane ruffling as gelsolin null cells display reduced ruffling in response to serum and epidermal growth factor (Azuma et al., 1998). In the case of Salmonella-induced ruffles, severing of F-actin by gelsolin and subsequent uncapping by factors such as tropomyosin- which is also recruited to ruffles (Figure 3.1), would expose filaments' barbed ends, enabling rapid actin polymerization (Nyakern-Meazza et al., 2002). On the contrary, gelsolin could counteract actin polymerization at the site of invasion by breaking down actin filaments. Secondly, we were also interested in gelsolin since, in a cell-free system, the bacterial effector, S ipA, prevents gelsolin-directed severing of F-actin, and re-anneals gelsolin-severed F-actin fragments (McGhie et al, 2004). S ipA ' s antagonism of gelsolin was demonstrated in a cell-free system, however, the role of gelsolin during S. Typhimurium invasion into host cells had not been explored. Our results clearly demonstrate that gelsolin is recruited to Salmonella-induced ruffles where it inhibits the invasion process (Figure 3.1 and 3.3). We speculate that one way in which gelsolin inhibits invasion is by promoting depolymerization of actin at the site of entry thereby decreasing the efficiency of Salmonella-driven actin-dependent ruffle formation. S. Typhimurium strains lacking S ipA display a reduced capacity to invade non-phagocytic cells (Jepson et al, 2001; Zhou et al, 1999a). I f the ability of S ipA to inhibit 57 F-actin severing by gelsolin contributed to Salmonella's ability to invade host cells, one might have expected that the AsipA strain could invade Gsn"A cells significantly better than G s n + / + cells. However, the inhibitory role o f gelsolin was only apparent upon infection by wild-type S. Typhimurium and not upon infection by invasion-deficient strains like AsipA and AsopE/E2 strains. Accordingly, a role for S ipA in countering the inhibitory effect of gelsolin could not be definitively demonstrated. SH2 and SH3 domain-containing adaptor proteins facilitate proteimprotein interaction (Buday, 1999; Pawson, 2007). SH2 domains bind specific phosphorylated tyrosine residues while SH3 domains bind proline-rich sites. SH2-containing proteins act as adaptors that modulate the actin cytoskeleton and signal transduction, often linking receptor tyrosine kinases (RTKs) to downstream effectors (Buday, 1999; Pawson, 2007). Interestingly, during infection of host cells, SH2 adaptors are often exploited by viral and bacterial pathogens. For example, interaction between the SH2 adaptor Nek and the vaccinia virus protein A 3 6 R facilitates actin-based motility of the virus (Frischknecht et al, 1999). Enteropathogenic E. coli inserts its own receptor, translocated intimin receptor (Tir), into the host cell plasma membrane and the binding of Nek to Tir is required for actin pedestal formation underneath the bacterium (Gruenheid et al, 2001). In addition, the adaptor Crk is involved in invasion of Shigella and Yersinia into epithelial cells (Bougneres et al, 2004; Bruce-Staskal et al, 2002; Burton et al, 2003). Whether S. Typhimurium targets SH2 adaptors during invasion had not been explored. This study examined the involvement of the recruited SH2 adaptors Nek, Crk, and ShcA during & Typhimurium invasion. 58 The SH2-SH3 adaptor Nek often links R T K s with the actin cytoskeleton and is best known for its roles in growth factor signaling, cell motility, and integrin signaling (Buday et al, 2002). We were initially interested in investigating the role of Nek as it is a potent upstream regulator of N - W A S P and W A V E (Eden et al, 2002; Rohatgi et al, 2001; Tomasevic et al, 2007), both of which are important for S. Typhimurium invasion (Shi et al, 2005; Unsworth et al, 2004). Nek was recruited to sites of S. Typhimurium invasion and was required for efficient invasion by affecting bacterial adherence to the host cell surface. In these studies, N c k l and Nck2 were found to be functionally redundant, which is consistent with other studies (Bladt et al, 2003; Braverman & Quill iam, 1999). The SH2-SH3 adaptor Crk is involved in cell migration, signaling pathways from R T K s , and invasion of the bacterial pathogens Yersinia and Shigella into epithelial cells (Bougneres et al, 2004; Bruce-Staskal et al, 2002; Buday, 1999; Burton et al, 2003). Although enriched in Salmonella-induced ruffles (Figure 3.1), Crk was not required in the invasion process (Figure 3.4). The SH2 adaptor ShcA is best known for its roles in signaling events controlling cell proliferation, survival, and apoptosis and has been implicated in signaling via many different types of receptors including growth factor receptors, antigen receptors, G-protein coupled receptors, hormone receptors, cytokine receptors, and integrins (Ravichandran, 2001). ShcA has been linked to cytoskeletal organization of cells by regulating integrin-mediated random cell migration (Gu et al., 1999). Interestingly, the recruitment of ShcA to sites of S. Typhimurium invasion appeared to inhibit adherence of S. Typhimurium to epithelial cells. The Arp2/3 complex, N - W A S P , W A V E , F A K , and pl30cas have all been shown to be involved in invasion of S. Typhimurium into non-phagocytic cells (Criss & 59 Casanova, 2003; Shi et al, 2005; Shi & Casanova, 2006; Unsworth et al, 2004). With the exception of the W A V E study, these studies have not addressed whether the observed decreases in invasion were attributable to decreases in S. Typhimurium attachment. We dissected the two factors that could contribute to differences in the number of intracellular bacteria: adherence and internalization. A n d although the role of Nek and ShcA in bacterial adherence was unexpected, future studies should address the role of additional cytoskeletal proteins in S. Typhimurium adherence. Because Nek and ShcA affect S. Typhimurium adherence, they are clearly affecting the host cell surface. The mechanisms though which adaptors may modulate plasma membrane proteins are discussed in Chapter 5. Significantly, this is the first study to identify host molecules involved in S. Typhimurium adherence and although this involvement is presumably indirect, these findings may provide clues as to the identity of the cell-surface receptor for S. Typhimurium. Clearly more work must to be done in order to identify the mechanism by which Nek and ShcA modulate the host cell surface. In addition, this study has identified many new actin-associated proteins recruited to sites of S. Typhimurium invasion that may play important roles during the invasion process. Understanding the molecular mechanisms underlying bacterial adherence and internalization is key to understanding how 5*. Typhimurium causes disease. 60 Chapter 4 : S. Typhimurium disruption of tight junction structure and 2 function 2 A version of this chapter has been published. Boyle, E . C., ' Brown, N . F., and Finlay, B . B . (2006) Salmonella enterica serovar Typhimurium effectors SopB, SopE, SopE2, and S ipA disrupt tight junction structure and function. Cel l Microbiol . 8: 1946-1957. 61 4.1 Summary Infection of epithelial monolayers by S. Typhimurium disrupts tight junctions that normally seal.the monolayer and maintain cell polarity. It is thought that disruption of tight junctions in the intestine could contribute to the manifestation of diarrhea. Accordingly, we examined whether tight junction structure and function were perturbed in a murine model of S. Typhimurium-induced colitis. S. Typhimurium infection caused a significant increase in intestinal permeability to 40 k D a FITC-dextran and resulted in the redistribution of tight junction proteins ZO-1 and claudin-3 in the colon. This is the first report of barrier disruption by S. Typhimurium in an in vivo model and prompted us to further investigate the molecular mechanisms involved in tight junction disruption in vitro. In cell culture, tight junction disruption is dependent on the SPI-1 T3SS but the specific effectors involved had not been identified. In this study we demonstrate that SopB, SopE, SopE2, and S ipA are the SPI-1-secreted effectors responsible for disruption of tight junction structure and function. Tight junction disruption by S. Typhimurium was prevented by inhibiting host protein geranylgeranylation but was not dependent on host protein synthesis o r . secretion of host-derived products. Unlike wild-type S. Typhimurium, AsopB, AsopE/E2, AsipA, or AsipA/sopB mutants, AsopB/E/E2 and AsipA/sopE/E2 mutants were unable to increase the permeability of polarized epithelial monolayers, did not disrupt the distribution or levels of ZO-1 and occludin, and did not alter cell polarity. These data suggest that SPI-1-secreted effectors utilize their ability to stimulate Rho family GTPases to disrupt tight junction structure and function. 62 4.2 Introduction S. Typhimurium is capable of disrupting tight junctions that normally serve to seal the epithelial barrier and maintain cell polarity. Tight junction disruption by S. Typhimurium is observed in vitro, however, whether cell junctions are compromised in vivo has not been assessed. Importantly, disruption of tight junctions in the intestine is thought to contribute the manifestation of diarrhea. Recently, a new model of S. Typhimurium-induced colitis has been developed using streptomycin pre-treated mice (Barthel et al, 2003). Upon oral infection of streptomycin pre-treated mice, S. Typhimurium efficiently colonizes the large intestine and triggers severe inflammation in the cecum and colon. Using this model, we investigated whether the structure and function of tight junctions were perturbed in vivo upon infection by S. Typhimurium. The molecular mechanism through which S. Typhimurium disrupts tight junctions is not known. In vitro, disruption of the epithelial barrier by S. Typhimurium is SPI-1-dependent (Jepson et al, 1996; Tafazoli et al, 2003) yet the specific SPI-1-secreted effectors responsible have not been identified. Rho family GTPases are key regulators of tight junctions and their activation leads to disruption of tight junction structure and function (Braga, 2002). Interestingly, S. Typhimurium possesses several SPI-1 T3SS-delivered effectors that activate Rho family GTPases. Using polarized epithelial monolayers, we have investigated the specific SPI-1-secreted effectors responsible for S. Typhimurium's ability to compromise the epithelial barrier. 63 4.3 Results 4.3.1 S. Typhimurium disrupts tight junction structure in vivo ZO-1 and claudin-3 are cytoplasmic and transmembrane tight junctions proteins, respectively. While ZO-1 is known to localize specifically to tight junctions, claudin-3 is known to also localize along the entire length of the lateral membrane (Rahner et al, 2001). A s expected, in the colon of streptomycin-pretreated mice, claudin-3 localized to tight junctions and to the lateral membrane while ZO-1 appeared exclusively at tight junctions (Figure 4.1). A 6 hour infection was chosen in order to minimize alterations in the epithelium architecture that results from 5*. Typhimurium-induced inflammation. After a 6 hour infection with wild-type S. Typhimurium, both ZO-1 and claudin-3 were no longer apparent at tight junctions (Figure 4.1). These observations suggest that S. Typhimurium disrupts tight junction structure in vivo. 4.3.2 S. Typhimurium increases intestinal permeability in vivo To determine whether structural changes in tight junctions were accompanied by functional changes in the barrier integrity of the epithelium, intestinal permeability was assessed by measuring the appearance of orally-delivered FITC-dextran (40 kDa) in the blood. Compared to uninfected mice, intestinal permeability was significantly elevated after a 6 hour infection with Wild-type S. Typhimurium (Figure 4.2). These results indicate that S. Typhimurium functionally disrupts the intestinal barrier function early during infection. 64 LPS DNA ZO-1 Merge •o c. 'c 3 CD a *-* 5 LPS DNA Claudin-3 Merge A * J v-, *J,'. * ' . •.. s M... • * % i | 1 ••• r? .... J ••' • ' ..• T i .' % . J , Figure 4.1. S1. Typhimurium causes redistribution o f ZO-1 and claudin-3 in the colon of streptomycin-pretreated mice. Mice were gavaged with 20 mg streptomycin 24 hours prior to gavage with either 3x10 S. Typhimurium or L B (uninfected). Mice were sacrificed 6 hours post-infection and tissue preparations were stained for Salmonella L P S (red), nuclei (blue), and ZO-1 or claudin-3 (green). The claudin-3 antibody appears to cross-react with luminal contents in infected mice. Images are en face Z-projection of confocal slices. 65 ** 11000-1 10000-9000-8000-2 _ 7000 J tt I CD C 6000-"? g> 5000-£ — 4000-0 3000-I 2000-I 1000-| 0 uninfected wildtype Figure 4.2. Wild-type S. Typhimurium significantly increases intestinal permeability in streptomycin-pretreated mice. Mice were gavaged with 20 mg streptomycin 24 hours prior to gavage with either 3 x l 0 8 S. Typhimurium or L B (uninfected). Four hours prior to sacrifice, mice were gavaged with 8.8 mg of 40 kDa FITC-dextran. Six hours post-infection, mice were sacrificed, blood was collected by cardiac puncture, and F I T C -dextran concentration in the plasma was measured. **, P < 0.01 using a Mann-Whitney U non-parametric test. n = 5 mice per group. 4.3.3 Inhibition of protein geranylgeranylation prevents barrier disruption by S. Typhimurium 5. Typhimurium possesses several effectors that activate Rho family GTPases. Because Rho family GTPases regulate cellular junctions, we determined whether they were involved in S. Typhimurium-induced disruption of tight junctions using the well-characterized human intestinal epithelial cell line, Caco-2/TC7. Prior to infection by invasive S. Typhimurium, polarized monolayers were treated with GGTI-298, an inhibitor of geranylgeranyltransferase I (GGTase-I) which is required for Rho GTPase activation (Casey & Seabra, 1996). Transepithelial resistance (TER) , a measure of the integrity of the epithelial barrier, was monitored over the course of a 4 hour infection. Both 15 and 50 u M GGTI-298 alone had no effect on the barrier (Figure 4.3). A t 4 hours 66 post-infection, wild-type S. Typhimurium decreased T E R to 57.84 ±2 .36% of the T E R at time zero (Figure 4.3). Pretreatment of cells with either concentration of GGTI-298 significantly reduced the disruption of the epithelial barrier by S. Typhimurium (P < 0.001; Figure 4.3). Accordingly, these results demonstrate that protein geranylgeranylation, and likely Rho GTPase activation, is involved in barrier disruption by 5*. Typhimurium. *** Figure 4.3. Inhibition of protein geranylgeranylation prevents tight junction disruption by S. Typhimurium 4 hours post infection. Caco-2/TC7 cells were grown on Transwells for 18-21 days, until T E R stabilized between 250-300 Ohms/cm 2 . Polarized Caco-2/TC7 monolayers were pretreated with media alone or the indicated concentration of GTTI-298 (GGTI), a geranylgeranyltransferase 1 inhibitor, 40 hours prior to apical infection with S. Typhimurium. Values are reported as a percentage of the initial T E R value and are expressed as means +/- the standard error of the mean of 3 different experiments, each performed in duplicate. ***, P < 0.001 using a Bonferroni post-hoc test. .67 4 . 3 . 4 Barrier disruption by S. Typhimurium does not require host protein synthesis or secretion of host-derived products Activation of Rho family GTPases can trigger reorganization of the actin cytoskeleton as well as initiate gene transcription (Benitah et al., 2004; Sorokina & Chernoff, 2005). To determine which function of Rho family GTPases was required for ( barrier disruption by S. Typhimurium, cells were pretreated with 5 u M cycloheximide, an inhibitor of eukaryotic protein synthesis. To confirm this concentration of cycloheximide blocked protein synthesis during infection, IL-8 production was assayed in the presence and absence of the inhibitor. A s expected, infection resulted in a considerable increase in IL-8 production as compared to uninfected cells (Figure 4.4A) (Hobbie et al., 1997; Mynott et al, 2002). Pretreatment of Caco-2/TC7 cells with 5 u M cycloheximide significantly inhibited IL-8 production upon infection (P < 0.05) indicating that this concentration of cycloheximide effectively blocked protein synthesis during a 4 hour infection (Figure 4.4A). Cycloheximide alone had no effect on T E R and pretreatment of cells prior to infection did not prevent disruption of the barrier by 5*. Typhimurium over the course of a 4 hour infection (Figure 4.4B). Therefore, host protein synthesis is not required for barrier disruption by S. Typhimurium. To test whether soluble epithelial-derived products produced upon infection were acting in an autocrine or paracrine fashion on the barrier, basolateral supernatants from cells infected for 4 hours were filter sterilized and placed on the basolateral side of polarized Caco-2/TC7 monolayers. Little to no change in T E R was detected when monitored for 10 hours ( T E R remained at 95.77% ± 0.83 of the initial value). Therefore, S. Typhimurium-induced barrier disruption is not dependent on host protein synthesis or secretion of host-derived products. These 68 data provide evidence that barrier disruption by S. Typhimurium is a result of the effect of Rho GTPases on the actin cytoskeleton. A. B. Figure 4.4. Barrier disruption by S. Typhimurium does not require host protein synthesis. Caco-2/TC7 cells were grown on Transwells for 18-21 days. Polarized Caco-2/TC7 monolayers were pretreated with media alone or 5 u M cycloheximide ( C H X ) 20 minutes prior to apical infection with wild-type S. Typhimurium. (A) E L I S A s performed on basolateral supernatants confirm that 5 u M cycloheximide effectively blocks IL-8 production during a 4 hour infection by S. Typhimurium. Values are expressed as means +/- the standard error of the mean of 3 different experiments, each performed in duplicate. *, P < 0.05 using a Bonferroni post-hoc test. (B) T E R 4 hours post infection reported as a "percentage of the initial T E R . Values are expressed as means +/- the standard error of the mean of 3 different experiments, each performed in duplicate. N . S., not significant using a Bonferroni host-hoc test. 4.3.5 SopE, SopE2, SopB, and SipA disrupt the barrier function of polarized epithelial cell monolayers Disruption of the epithelial barrier by S, Typhimurium is SPI-1-dependent (Jepson et al, 1996; Tafazoli et al, 2003) yet the specific SPI-1-secreted effectors responsible have not been identified. We focused on three SPI-1-secreted effectors that activate Rho family GTPases (ie. SopB, SopE, and SopE2) and another that directly interacts with actin (ie. SipA). Polarized Caco-2/TC7 monolayers were infected with various bacterial 69 mutants and the barrier function was assessed by measuring T E R . To confirm the role of SPI-1 in barrier disruption, cells were infected with an AinvA mutant which lacks a functional SPI-1 T3SS (Galan & Curtiss, 199.1). Over the course of 4 hours, T E R remained unchanged in uninfected and Awvv4-infected cells (Figure 4.5), confirming that the drop in T E R is indeed SPI-1-dependent. In order to evaluate the role of the SPI-2 T3SS, cells were infected with an AssaR mutant which is defective for secretion of SPI-2 effectors (Brumell et al, 2001). After 4 hours, the T E R of cells infected with wild-type bacteria or the AssaR mutant decreased to 41.02 ± 2.479% and 45.37 ± 5.338% of the T E R at time zero, respectively (Figure 4.5). Thus, SPI-2-secreted effectors are not involved in barrier disruption. Next, we investigated the role of specific SPI-1 effectors. The AsopB and AsopE/sopE2 mutants were able to decrease the T E R to a similar extent as wild-type S. Typhimurium, while T E R was similar to the uninfected control cells upon infection with the AsopB/sopE/E2 mutant (Figure 4.5). Therefore, SopB, SopE, and SopE2 are collectively required for barrier disruption by S. Typhimurium. Infection by a AsipA mutant had the same effect on the barrier as wild-type bacteria, as did a AsipA/sopB mutant (Figure 4.5). However, a role for S ipA in harrier disruption was revealed as the AsipA/sopE/E2 mutant strain was unable to decrease T E R (Figure 4.5). Collectively, these data indicate that SopB, SopE, SopE2, and S ipA are the effectors responsible for decreasing the T E R of polarized epithelial cells, and while S ipA and SopB are involved, they are insufficient for barrier disruption. 70 UJ 120-1 110-100-90-80-70-60-50-40-30-20-10 0 ** ** ** 1 I Figure 4.5. S ipA, SopB, SopE, and SopE2 disrupt the barrier function of polarized epithelial cell monolayers. Caco-2/TC7 cells were grown on Transwells for 18-21 days. Polarized Caco-2/TC7 monolayers were apically infected with wild-type S. Typhimurium and mutant derivatives. T E R 4 hours post infection reported as a percentage of the initial T E R value and expressed as means +/- the standard error of the mean of 3 different experiments, each performed in duplicate. **, P < 0.01 compared to wild-type-infected cells using a Dunnett's post-hoc test. 4.3.6 Complementation analysis verifies that SopB, SopE, SopE2, and SipA disrupt the barrier function of polarized epithelial cell monolayers In order to confirm that the inability of AsipA/sopE/E2 and AsopB/sopE/E2 mutants to disrupt the barrier was attributable to specific deletion of the genes encoding SipA, SopB, SopE, and SopE2, we performed a complementation analysis. Polarized Caco-2/TC7 cells were infected, as above, with the complemented strains and T E R was measured over the course of a 4 hour infection. Transformation of the AsopB/sopE/E2 mutant with the low-copy-number vector expressing either SopB, SopE, or SopE2 significantly restored the strain's ability to decrease the T E R when compared to the 71 *** — I T 1 1 1 I I I I I I I A? & <$> & # A * v<P\<>^ & (#> Figure 4.6. Complementation analysis verifies that SipA, SopB, SopE, and SopE2 are the effectors that disrupt the epithelial barrier. Caco-2/TC7 cells were grown on Transwells for 18-21 days. Polarized Caco-2/TC7 monolayers were apically infected with wild-type S. Typhimurium and mutant derivatives. T E R 4 hours post infection reported as a percentage of the initial T E R value and expressed as means +/- the standard error of the mean of 3 different experiments, each performed in duplicate. ***, P < 0.001; **, P < 0.01; *, P < 0.05 using a Bonferroni post-hoc test. empty vector control (P < 0.05 for SopB; P < 0.001 for SopE and SopE2; Figure 4.6). Similarly, transformation of the AsipA/sopE/E2 mutant with plasmids expressing SopE or SopE2 significantly restored this strain's ability to disrupt T E R when compared to the empty vector control (P < 0.05 for SopE; P < 0.01 for SopE2; Figure 4.6). O f note, while AsopE or AsopEl mutants were not tested, ectopic expression of either SopE or SopE2 restored the mutant strains' ability to decrease T E R . This confirms that both SopE and SopE2 can disrupt tight junctions. Expression of S ipA from a high-copy-number plasmid 72 within the AsipA/sopE/E2 strain also significantly complemented this strain's ability to decrease the T E R when compared to the empty vector control (P < 0.001; Figure 4.6). These results verify that the SPI-1-secreted effectors S ipA, SopB, SopE, and SopE2 are indeed responsible for the decrease in T E R upon infection by S. Typhimurium. 4.3.7 AsopB/E/E2 and AsipA/sopE/E2 mutants do not alter the size-selective paracellular permeability of Caco-2/TC7 cells T E R is an instantaneous measure of the tightness of tight junctions however, in some instances, T E R can remain undisturbed while the paracellular permeability of the monolayer to small molecules can increase over time (Balda et al., 1996). So to definitively conclude that the AsopB/E/E2 and AsipA/sopE/E2 mutants were incapable of disrupting the barrier function of Caco-2/TC7 cells, we measured the diffusion df fluorescently-labeled dextrans (FITC-dextran) with average molecular masses of 4 and 40 kDa. A s a positive control, diffusion of FITC-dextran was measured in cells that had been treated with E G T A . After 4 hours, E G T A treatment resulted in an approximate 400-fold increase in the monolayer's permeability to both 4 and 40 kDa FITC-dextran. A 4.5-fold and 3.3-fold increase in the permeability to 4 and 40 kDa FITC-dextran, respectively, was observed in cells infected with wild-type S. Typhimurium when compared to uninfected cells (Figure 4.7). Permeability to 4 and 40 k D a FITC-dextran was significantly reduced upon infection with the AsopB/E/E2 (P < 0.05) and AsipA/sopE/E2 mutants (P < 0.01; Figure 4.7). Together with the T E R data, this data confirms the role of SopB, SopE, SopE2 and S ipA in the disruption of epithelial barrier function. 73 A. B. 4kDa FITC-dextran 4 0 k D a FITC-dextran Figure 4.7. AsopB/E/E2 and AsipA/sopE/E2 mutants do not alter the paracellular permeability of Caco-2/TC7 cells. Caco-2/TC7 cells were grown on Transwells for 18-21 days. Polarized Caco-2/TC7 monolayers were apically infected with wild-type S. Typhimurium and mutant derivatives. Paracellular flux of (A) 4 kDa FITC-dextran and, (B) 40 k D a FITC-dextran was measured 4 hours post-infection. Values are normalized to uninfected cells and are expressed as means +/- the standard error of the mean of 3 different experiments, each performed in duplicate. **, P < 0.01; *, P < 0.05 using a Bonferroni post-hoc test. 4.3.8 SopE, SopE2, SopB, and SipA alter the localization of ZO-1 and occludin Disruption of the localization of tight junction proteins ZO-1 (cytoplasmic) and occludin (transmembrane) is SPI-1-dependent (Tafazoli et al, 2003). Because SopB, SopE, SopE2, and S ipA were involved in the disruption of epithelial barrier, we examined whether these effectors were also responsible for changes in the localization or expression levels of tight junction proteins. Polarized Caco-2/TC7 monolayers were infected at the apical pole with various strains of S. Typhimurium for 4 hours, and the localization of ZO-1 and occludin was examined by confocal microscopy. In uninfected and Az«v^4-infected cells, ZO-1 and occludin localized to the lateral cell membrane and formed a characteristic, contiguous web-like pattern (Figure 4.8). In contrast, ZO-1 and occludin were highly disrupted in cells infected with wild-type S. Typhimurium, 74 appearing in a discontinuous bead-like pattern, with increased levels appearing in the cytoplasm (Figure 4.8). A n AssaR mutant strain disrupted ZO-1 and occludin to the same extent as wild-type, further supporting the above data that tight junction disruption occurs independently of SPI-2. Uninfected Wildtype Asso/? MnvA AsopE/E2 Figure 4.8. S ipA, SopB, SopE, and SopE2 disrupt ZO-1 and occludin localization. Caco-2/TC7 cells were grown on Transwells for 18-21 days. Polarized Caco-2/TC7 monolayers were apically infected with wild-type S. Typhimurium and mutant derivatives for 4 hours whereby cells were immunostained for occludin and Z O - 1 . E n face Z-projection o f confocal slices. Scale bar = 5 um. The AsopB and AsopE/sopE2 mutant strains were able to displace ZO-1 and occludin, however, when all three effectors were absent, S. Typhimurium was no longer 75 able to disrupt tight junction structure (Figure 4.8) indicating that SopB, SopE, and SopE2 are collectively required for disruption of tight junction structure. The AsipA and AsipA/sopB strains disrupted ZO-1 and occludin however, a role for S ipA in tight junction disruption was identified as the AsipA/sopE/E2 mutant was unable to disrupt localization of these two proteins (Figure 4.8). These data demonstrate that together SopB, SopE, SopE2, and S ipA are collectively responsible for the disruption ZO-1 and occludin localization, and that both SopB and S ipA are insufficient to disrupt tight junction structure. 4.3.9 SopE, SopE2, SopB, and SipA alter the expression levels of ZO-1 and occludin Detergent solubility of tight junction proteins can be used as an indicator of their membrane and cytoskeletal association (Nusrat et al, 2000; Stuart & Nigam, 1995). Accordingly, we assessed the Triton-X solubility properties of ZO-1 and occludin during an infection by S. Typhimurium. Western blot analysis of Triton-X-soluble and -insoluble fractions from Caco-2/TC7 revealed that ZO-1 was detected exclusively in the Triton-X-insoluble protein fraction (Figure 4.9). Infection by wild-type S. Typhimurium decreased ZO-1 in the Triton-X-insoluble fraction ~66% of basal levels after 4 hours (Figure 4.9). Interestingly, over the course of an infection, ZO-1 remained undetectable in the Triton-X-soluble protein fraction, indicating perhaps that ZO-1 was not redistributed to cellular compartments accessible by the Triton-X lysis buffer or that soluble ZO-1 was rapidly degraded, making increased soluble levels difficult to observe. In the same protein extractions, occludin was localized primarily in the Triton-X-insoluble fraction 76 ZO-1 Occludin 0 2 4 Q 2 4 hours S I S I S I S I S I S I W i u m wi m • L w » i — wildtype M • * MnvA M l m m ^sipA *-« I ASOpS M » W M - * AsopE/E2 ^ | | \sopB/E2/E2 b| B 1 I Mi Mi *"> ^sipA/sopB m m ±sipA/sopE/E2 U m mi Figure 4.9. S ipA, SopB, SopE, and SopE2 modulates expression o f ZO-1 and occludin. Western blot analysis of Triton-X-soluble (S) and -insoluble (I) fractions at the indicated time points post-infection. Equal amounts of protein (15 u,g) were loaded in each lane. Results shown are representative of at least two independent experiments. with a minimal amount appearing in the -soluble fraction (Figure 4.9). Upon infection with wild-type S. Typhimurium, occludin levels in the Triton-X-insoluble protein fraction decreased to - 4 6 % of basal levels after 4 hours (Figure 4.9). Although expression of occludin decreased over time, the ratio of Triton-X-soluble and -insoluble was maintained. Over the course of 4 hours, infection by AsipA, AsopB, AsipA/sopB, AsopE/E2 mutants decreased ZO-1 and occludin expression to a similar extent as wild-type bacteria (Figure 4.9). Conversely, infection with either a AsopB/E/E2 or AsipA/sopE/E2 mutant did not appreciably perturb the expression level of ZO-1 or occludin (Figure 4.9). In accordance with our other measurements of tight junction 77 structure and function, SopB, SopE, SopE2, and S ipA are the bacterial effectors modulating disruption of tight junctions. 4.3.10 SopB, SopE, SopE2, and SipA disrupt epithelial cell polarity Next we determined whether infection by 5". Typhimurium altered the polar distribution of proteins. For these experiments, we used the canine epithelial cell line M D C K as it is arguably the best-characterized polarized cell line and because many reagents for cell polarity studies are canine-specific. Polarized M D C K cells were infected with S. Typhimurium and monitored for redistribution of apical and basolateral markers. In uninfected cells, GP135 and E-cadherin were restricted to apical and basolateral surfaces, respectively (Figure 4.10). However, 4 hours after infection with wild-type bacteria, E-cadherin and GP135 staining was apparent on both the apical and basolateral membranes as well as in the cytoplasm (Figure 4.10). Therefore, S. Typhimurium infection results in the intermixing of apical and basolateral membrane components causing loss of cell polarity. Next, we investigated which bacterial effectors were responsible for the disruption of cell polarity. The AsipA, AsopB, AsopE/E2, AsipA/sopB mutants all altered the polar distribution of GP135 and E-cadherin (Figure 4.10). In contrast, and in agreement with our other measures of tight junction function, infection of polarized monolayers with AinvA, AsopB/E/E2, AsipA/sopE/E2 mutants did not affect the fence function of the monolayer (Figure 4.10). Accordingly, S. Typhimurium disrupts cell polarity via SopB, SopE, SopE2, and SipA. 78 0> JC XI ra V LU * m <• * * * ' > # - , • * * , 1 | co CL O Uninfected Wildtype \s /pA AsopB AsopE/E2 AsipA/sopB \sopB/E/E2 AsipA/sopE/E2 Uninfected Wildtype AinvA AsipA AsopB AsopE/E2 AsipA/sopB AsopB/E/E2 \sipA/sopE/E2 Figure 4.10. S ipA, SopB, SopE, and SopE2 are responsible for disrupting cell polarity. M D C K cells were grown on Transwells for 4 days. Polarized monolayers were apically infected with wild-type S. Typhimurium or mutant strains for 4 hours. Cells were immunostained for the basolateral marker, E-cadherin, or the apical marker, GP135. X Z -slice using confocal microscopy. Arrows indicate areas where cell polarity has been lost. Scale bar = 5 urn. 4.4 Discussion The intestinal epithelium functions as a physical barrier. Tight junctions seal epithelial cell layers, performing a "gate" function that restricts paracellular passage of 79 ions, water, solutes, and immune cells. Tight junctions also perform a "fence" function in that they regulate cell polarity by acting as diffusion barriers that physically separate apical and basolateral membrane components. Infection by S. Typhimurium causes a progressive increase in the permeability of polarized epithelial monolayers and causes alterations in the localization of the tight junction-associated proteins ZO-1 and occludin (Finlay & Falkow, 1990; Jepson et al, 1995; Tafazoli et al, 2003). Tight junction disruption likely represents an important aspect of Salmonella enteritis and may be central to the clinical manifestation of diarrhea (Fasano, 2002; Hecht, 2001; Sandle, 2005), yet whether S. Typhimurium disrupts tight junctions in vivo had not previously been assessed. In this study, we demonstrate that S. Typhimurium disrupts intestinal tight junction structure and function in vivo. These findings prompted us to return to an in vitro system in order to dissect the specific bacterial effectors responsible for disrupting tight junctions. Our results identify a novel role for SopB, SopE, SopE2, and S ipA in the pathogenesis of S. Typhimurium. Modulation of the actin cytoskeleton through activation or inhibition of R a c l , Cdc42, or Rho can perturb the gate and fence function of tight junctions (Braga, 2002). Co-opting host cell Rho GTPases in order to disrupt cell-cell junctions is a common strategy used among several diarrheal pathogens (Sousa et al, 2005). For example, toxins of the diarrheagenic pathogen Clostridium difficile monoglucosylate Rho family GTPases thereby inducing disruption of cellular junctions through disorganization of the actin cytoskeleton (Ciesla & Bobak, 1998). Escherichia coli increases the permeability of epithelial monolayers via its cytotoxic necrotizing factor 1 which causes the constitutive activation of Rho GTPases (Flatau et al, 1997; Schmidt et al, 1997). During the 80 preparation of this work, the role of protein geranylgeranylation in tight junction disruption by S. Typhimurium was identified using M D C K cells (Tafazoli et al, 2003), however, we thought it important to corroborate these findings a cell line relevant to human disease. Accordingly, for these studies we used the well-characterized human intestinal epithelial cell line, Caco-2/TC7. These monolayers have defined apical and basolateral surfaces, well-developed brush borders, and functional tight junctions. Dominant-negative or constitutively-active constructs of Cdc42, R a c l , or Rho cannot be used to address, the role of Rho GTPases in tight junction disruption by S. Typhimurium as their expression alone w i l l perturb the barrier function of the epithelium (Braga, 2002). Our studies utilized a pharmacological inhibitor of GGTase-1-dependent protein geranylgeranylation that, at the concentrations utilized, blocks Rho GTPase activity without altering tight junctions. Inhibition of protein geranylgeranylation effectively prevented tight junction disruption by S. Typhimurium. Inhibition was significant yet incomplete and therefore we recognize there may be another Rho GTPase-independent mechanism of T J disruption. However, the results presented here strongly suggest that in human intestinal cells, Rho family GTPases are involved in tight junction disruption by S. Typhimurium. Rho family GTPases mediate diverse cellular activities that include not only reorganization of the actin cytoskeleton but also modulation of gene transcription, primarily through their effects on mitogen-activated protein kinase and N F - K B signaling cascades (Benitah et al, 2004; Sorokina & Chernoff, 2005). Therefore, it was possible that S. Typhimurium-induced barrier disruption was caused by Rho GTPase-dependent epithelial-derived products. Using a eukaryotic-specific inhibitor of protein synthesis, we 81 establish that de novo protein synthesis is not required for loss of barrier function by S. Typhimurium. Soluble products such as IFNy, T N F a , I L - l p \ IL-4, IL-8, and IL-13 have been reported to disrupt tight junctions (Clayburgh et al, 2004). In polarized endothelial cells infected with dengue virus, secreted IL-8 acts in an autocrine manner and contributes to tight junction disruption in these cell monolayers (Talavera et al, 2004). In this study, we demonstrate that secreted soluble factors from S. Typhimurium-infected epithelial cells do not play a role in intercellular disruption. B y discounting the role of epithelial-derived products, our findings support the idea that tight junction disruption by S. Typhimurium is a result of the effect of Rho GTPases on the actin cytoskeleton. The effects of S. Typhimurium on tight junctions are dependent upon SPI-1 (Jepson et al, 1996; Tafazoli et al, 2003) yet the contribution of specific SPI-1-secreted effectors to these events had not previously been addressed. Accordingly, using various SPI-1-secreted effector mutants, we investigated the mechanism of S. Typhimurium-induced intercellular disruption. Because of the involvement of Rho family GTPases in tight junction disruption by S. Typhimurium, the Rho GTPases activators SopB, SopE, and SopE2 were considered likely candidate effectors to be involved. In the absence of SopB or SopE/E2, T E R decreased to the same extent as wild-type-infected cells. However, compared to wild-type-infected cells, a AsopB/E/E2 mutant did not disrupt T E R . Due to partial functional overlap, studying the role of single effectors has proven to be difficult since disruption of a single effector often results in only a minor change in a cellular phenotype (Ehrbar et al, 2002). The synergistic activation of Rho family GTPases by SopB, SopE, and SopE2 is known to contribute to invasion into host cells (Ehrbar et al, 2002; Zhou et al, 2001). Consequently, due to their complementary effects 82 on Rho family GTPases, tight junction disruption by S. Typhimurium was prevented only in the absence of all three effectors. Together, we have shown that tight junction disruption by S. Typhimurium can be prevented by inhibiting Rho GTPases or S. Typhimurium's ability to activate Rho GTPases via SopB, SopE, and SopE2. S ipA is another SPI-1-secreted effector that modulates the cytoskeleton by directly binding .to actin, stabilizing F-actin filaments, and increasing the bundling activity of the host protein, T-plastin (Zhou et al, 1999a). In the absence of SipA, or S ipA and SopB, T E R decreases to the same extent as wild-type-infected cells. However, when the effect of a L\sopE/E2 mutant (TER drops) is compared to that of a AsipA/sopE/E2 mutant (TER remains undisturbed), an important role for S ipA in tight junction disruption is revealed. Interestingly, the fact that the AsipA/sopE/E2 mutant was unable to disrupt T E R demonstrates that SopB alone is insufficient to disrupt tight junctions. Conversely, the lack of tight junction disruption by the AsopB/E/E2 mutant reveals that S ipA is also insufficient to alter tight junctions (Figure 4.1). Previous transfection studies have demonstrated that SopB and S ipA expression is sufficient to modulate the actin cytoskeleton (Higashide et al, 2002; Zhou et al, 2001). Whether S ipA and SopB cooperate or whether either functions with SopE or SopE2 in order to disrupt tight junctions is unknown, however, a recent study has provided evidence that cooperation exists between S ipA and SopB during invasion into polarized and non-polarized epithelial cells (Raffatellu et al, 2005). Tight junctions do not appear to be simple seals but rather allow selective passage of certain components through the paracellular pathway (Miyoshi & Takai, 2005). Therefore, the existence of selective channels within tight junctions had been proposed. 83 Table 4.1. Conclusions regarding the bacterial effectors responsible for tight junction disruption based on mutant analysis. Strain Effectors present Tight junction disruption Conclusions regarding tight junction disruption ASopB SipA/SopE/E2 Yes ASipA SopB/E/E2 Yes ASopE/E2 SipA/SopB Yes ASipA/SopB SopE/E2 Yes ASopB/E/E2 SipA No SopB, SopE, and SopE2 are involved. SipA is not sufficient. ASopB/E/E2 + pSopE SipA/SopE Yes SopE is sufficient. ASopB/E/E2+ . pSopE2 SipA/SopE2 Yes SopE2 is sufficient. ASipA/SopE/E2 SopB No SipA is also involved. SopB is not sufficient. ASipA/SopE/E2+ pSopE SopB/SopE Yes SopE is sufficient. ASipA/SopE/E2+ pSopE2 SopB/SopE2 Yes SopE2 is sufficient. Under some experimental conditions, there can be a functional dissociation of paracellular flux from T E R in that T E R can remain undisturbed while paracellular diffusion of small molecules increases over time (Balda et al, 1996). These observations suggest that T E R and paracellular flux may not measure the same characteristics of epithelial permeability. T E R is an instantaneous measurement of ionic conductivity that reflects epithelial integrity as well as tight junction ion selectivity, while paracellular flux reflects permeability over longer periods of time and allows the determination of the size selectivity of the paracellular diffusion barrier. Therefore, in order to definitively prove that the AsopB/E/E2 and AsipA/sopE/E2 mutants were not able to affect the epithelial permeability, we measured paracellular flux of FITC-dextran during a 4 hour infection. Compared to wild-type S. Typhimurium, paracellular flux was significantly reduced with both mutants thereby confirming that SipA, SopB, SopE, and SopE2 are the, effectors responsible for increasing the permeability of epithelial monolayers. 84 The formation of tight junctions is associated with an increase in the resistance of tight junction complexes to solubility in detergent-salt extractions. Conversely, disassembly of tight junction complexes correlates with internalization of tight junction proteins into the cytoplasm, making them more extractable with detergent-salt solutions (Nusrat et al, 2000; Stuart & Nigam, 1995). Epithelial barrier function can be affected by changes in the distribution or expression levels of tight junction proteins (Miyoshi & Takai, 2005). Accordingly, upon infection, we investigated ZO-1 and occludin localization using immunofluorescence and investigated the detergent solubility of ZO-1 and occludin to assess the association of each protein with either the membrane or the cytoskeleton. Upon infection by wild-type S. Typhimurium, we did not observe a redistribution of ZO-1 or occludin from the insoluble to the soluble cell fractions despite observing significant changes in localization by immunofluorescence. There was, however, a reduction in the steady state levels of these proteins. These results suggest that the observed redistribution of ZO-1 and occludin by immunoflourescent staining is the result of a redistribution of these proteins within the insoluble, tight junction-associated network. These studies also identified SipA, SopB, SopE, and SopE2 as the SPI-1 -secreted effectors responsible for alterations in the cellular distribution and the reduction in steady state levels of both ZO-1 and occludin. While Salmonella-induced tight junction alterations have profound effects on the epithelial barrier, we also wanted to address the impact of S. Typhimurium on epithelial fence function. Tight junctions play key roles in establishing and maintaining epithelial polarity. Despite this, under certain circumstances, fence function can remain intact when gate function has been compromised (Braga, 2002; Takakuwa et al, 2000). We 85 demonstrate that infection by wild-type S. Typhimurium leads to disruption of protein polarity and, consistent with our results on the disruption of epithelial gate function, SipA, SopB, SopE, and SopE2 are involved in perturbing epithelial fence function. Other diarrheal pathogens have similar effects on cell polarity. For example, GP135 shows lateral mobility into the basolateral membrane following rotaviral infection (Nava et al., 2004). Enteropathogenic E. coli disrupts epithelial cell polarity enabling the basolateral protein (31-integrin.to migrate to the apical surface where it is proposed to act as a receptor for bacterial adhesins (Muza-Moons et al, 2003). Future studies should address the physiological consequences of S. Typhimurium-induced loss of cell polarity. In summary, we have demonstrated that S. Typhimurium disrupts tight junction structure and function in vivo and have identified SopB, SopE, SopE2, and S ipA as the specific SPI-1-secreted effectors responsible for tight junction disruption in vitro. Our data support that these effectors are signaling through Rho family GTPases in order to exert their effect on epithelial barrier function. Our results identify a novel role for SopB, SopE, SopE2, and S ipA in the pathogenesis of S. Typhimurium and these findings have important implications for Salmonella enteritis as disruption of tight junctions is likely to contribute to the production of diarrhea. 86 Chapter 5: Discussion3 3 This chapter contains material what has been published. Boyle, E . C. and Finlay, B . B . (2003) Bacterial pathogenesis: exploiting cellular adherence. Curr Opin Cel l B i o l . 15: 633-639. Boyle E . C , Brown, N . F. , Brumell, J. H . , and Finlay, B . B . (2007) Src homology domain 2 adaptors affect adherence of Salmonella enterica serovar Typhimurium to non-phagocytic cells. Microbiology. 153: 3517-3526. 87 The underlying theme of this research has been to understand the mechanisms by which Salmonella is able to breach the epithelial barrier. Whether it be through adherence, invasion, or disrupting epithelial cell monolayers, the goal was to understand, at the molecular level, both the host and bacterial components that contribute to Salmonella overcoming this first line of host defense. A t the time this research was initiated in 2001, very few cytoskeletal components were known to be recruited to S. Typhimurium-induced ruffles. Other than actin, no other cytoskeletal proteins were known to be required for invasion. The first part of the work presented here focused on the identification and functional analysis of cytoskeletal components recruited to sites of S. Typhimurium invasion. Together with the novel observation that SH2 adaptors alter the host cell surface and thereby modulate S. Typhimurium adherence, this research has expanded our knowledge of the molecular mechanisms involved in Salmonella pathogenesis and has added to. our knowledge of cytoskeletal dynamics and signaling. While it was known since the mid to late 1990s that 5*. Typhimurium disrupted tight junctions in cell culture, many questions still remained, one of which was whether tight junction disruption occurred in vivo. The second part of this research described functional disruption of tight junctions in vivo using a model of S. Typhimurium-induced colitis, while in vitro work identified the host cells pathways and specific bacterial effectors responsible for tight junction disruption. These results are significant to our understanding of Salmonella pathogenesis since the alteration of intestinal permeability is most likely a key contributor to diarrheal disease. 88 5.1 Exploitation of cell adhesion molecules by enteroinvasive pathogens Engagement of host cell receptors by bacterial adhesins can be a means of targeting a pathogen to a particular niche, co-opting underlying signaling pathways, establishing persistent infections, and inducing invasion. Invasion affords bacteria protection from immune detection and facilitates access to deeper tissues. Many bacterial pathogens have evolved the capacity to adhere to host cell adhesion molecules ( C A M s ) (Boyle & Finlay, 2003). C A M s are cell-surface receptors that mediate cell-cell and cell-extracellular matrix interactions. Bacteria are able to bind to C A M s by possessing adhesins that mimick or act in place of host cell receptors or their ligands. Ubiquitously-expressed and intimately-associated with downstream signaling pathways, C A M s make ideal anchors for pathogen adherence and effective mediums for communication with host cells. Adherence of bacteria to C A M s can result in relatively modest cytoskeletal rearrangements and membrane extensions. This type of invasion involves sequential engagement of C A M s by bacterial adhesins and results in membrane "zippering" around the invading pathogen. This mode of invasion is exemplified by Yersinia and Listeria. In contrast, Shigella and Salmonella typify the "trigger" mechanism of invasion which is characterized by extensive cytoskeletal rearrangements and membrane extensions upon contact with the host cell surface. Formation of a macropinocytic pocket at the entry focus results in bacteria being taken up within spacious vacuoles. Using the aforementioned pathogens as examples, I w i l l now discuss and compare their mechanisms of invasion to what we currently know about Salmonella invasion. 89 5.1.1 "Zipper" invasion Perhaps the best characterized bacterial adhesin is invasin, an outer membrane protein of the Gram negative diarrhea-causing pathogens Yersinia enterolitica and Y. pseudotuberculosis (Isberg & Barnes, 2001). During,the initial stages of infection, Yersinia translocates rapidly from the intestinal lumen to the underlying lymph nodes via invasion into M cells. Invasin mediates high-affinity binding to a subset of (3i integrins presented at the apical surface of M cells (Isberg & Leong, 1990; Schulte et al., 2000). Yersinia invasin efficiently mimics and competes with host molecules by binding |3i integrins with 100 times higher affinity than its natural substrate, fibronectin (Tran V a n Nhieu & Isberg, 1993). High-affinity binding by invasin and invasin self-association mediate integrin recruitment and multimerization (Dersch & Isberg, 1999; Tran V a n Nhieu & Isberg, 1993), facilitating circumferential binding of integrins about the bacterial surface culminating in a zipper-like mode of entry. Multimerization of (3i integrins also seems to be important for recruitment of cytoskeletal and signaling proteins required for transmission of downstream signals. Both the actin cytoskeleton and microtubules are involved in uptake, as well as the tyrosine kinases Pyk2 and Src, the Rho GTPase Rac, the Arp2/3 complex, F A K , Cas, and the adaptor Crk (Alrutz et al., 2001; Bruce-Staskal et al., 2002; Isberg & Barnes, 2001; McGee et al., 2003; Weidow et al., 2000) (Figure 5.1). 90 p p A r p 2/3 S r c C A S F A K \ • S r c / • W A V E ? F i g u r e 5 . 1 . Model for the signaling pathways leading to Yersinia invasion. Yersinia infection increases autophosphorylation of the protein tyrosine kinase Pyk2. Phosphorylated Pyk2 likely activates Src family kinases which phosphorylate Cas. The adaptor protein Crk is recruited to phosphorylated Cas which results in a downstream signal that activates R a c l . In a parallel pathway, engagement of Pi integrins (blue) by invasin leads to F A K autophosphorylation. Both F A K and Src are required for Yersinia invasion. Src is likely recruited via F A K , and through an indirect mechanism, F A K - S r c interaction can lead to R a c l activation. Both pathways require R a c l . R a c l likely activates W A V E which can then bind the Arp2/3 complex, leading to actin polymerization and bacterial engulfment. L. monocytogenes is a facultative intracellular pathogen and can induce its own uptake into cells that are normally non-phagocytic using a zipper-like mechanism similar to Yersinia (Ireton, 2007). Internalin (also called InlA) is a surface-exposed bacterial adhesin that mediates entry into epithelial cells by binding the epithelial cell receptor, E -cadherin. The binding site of internalin on E-cadherin was mapped to the extracellular N -terminal domain of E-cadherin which is responsible for the adhesive specificity o f homotypic interactions between neighboring cells. Figure 5.2 illustrates the host components involved in internalin-mediated uptake of Listeria. Unt i l recently, it was not known how Listeria could access E-cadherin in vivo due to the fact it is sequestered 91 within the basolateral membrane of epithelial cells. A recent study found that in vivo, Listeria specifically adheres and invades at sites of cell extrusion where E-cadherin is transiently exposed to the lumen (Pentecost et al., 2006). Figure 5.2. Signaling involved in internalin:E-cadherin-mediated uptake of L. monocytogenes. The bacterial adhesin, internalin, binds the epithelial C A M , E-cadherin (blue), a - and (3-catenin act as adaptors between the cytoplasmic tail o f E-cadherin and the actin cytoskeleton. The transmembrane protein vezatin (grey) bridges myosin V i l a (black) to the cadherin-catenins-actin complex. Vezatin, myosin V i l a , and the interaction between A R H G A P 1 0 (yellow) and a-catenin are necessary for Listeria invasion (Ireton, 2007). 5.1.2 "Tr igger" invasion Although both Shigella and Salmonella induce lamellipodia-like structures during entry, these pathogens have evolved different strategies of invasion (Pizarro-Cerda & Cossart, 2006). Unlike Salmonella, Shigella invades enterocytes exclusively at the basolateral pole after translocating across the epithelium via M cells. Invasion and adherence of Shigella depends on its T3SS which is encoded in a pathogenicity island 92 located in its virulence plasmid. The translocon proteins IpaB and IpaC are homologous to Salmonella's SipB and SipC, arid have been shown,to play a role in adherence to host cells. IpaB and IpaC bind a$\ integrins while IpaB also binds the hyaluronic receptor CD44 with high affinity (Lafont et al, 2002; Watarai et al, 1996). Both ct5|3i integrin and CD44 are highly expressed on the basolateral surface of enterocytes which may account for Shigella's preference for invading at this pole. L ike Salmonella invasion, Rho family GTPases play important roles in Shigella invasion, however, Shigella does not possess homologues of SopE or SopE2 that could directly act as G E F s (Mounier et al., 1999). The C-terminus of IpaC is important for activation of Cdc42 and R a c l through an unknown mechanism (Tran V a n Nhieu et al, 1999). L ike SipC, IpaC is capable of nucleating the formation of actin filaments beneath invading bacteria (Kueltzo et al, 2003). Another type 3-secreted effector, V i r A , destabilizes microtubules which also contributes to activation of R a c l (Yoshida et' al, 2002). The membrane-actin linker ezrin and the SopB homologue IpaD promote membrane extension during ruffling (Niebuhr et al, 2002; Skoudy et al, 1999). A b l tyrosine kinase, Crk, cortactin, and W A V E 2 are all involved in Arp2/3-dependent invasion of Shigella (Bougneres et al, 2004; Burton et al, 2003; Yoshida et al, 2002) (Figure 5.3). Although 5". Typhimurium is closely-related to Shigella, invades in a phenotypically similar manner, and has several homologous effector proteins, there are numerous differences in the molecular mechanisms through which these two pathogens invade into host cells (Table 5.1). 93 Figure 5.3. Signaling involved in the invasion of Shigella. Translocon components IpaB (B) and IpaC (C) bind CD44 and a5(3i integrins. IpaC and V i r A trigger the activation of Cdc42 and R a c l , which in turn activates N - W A S P and W A V E , resulting in Arp2/3-dependent actin polymerization. A b l tyrosine kinases phosphorylate Crk, which presumably activates a G E F for Cdc42 and R a c l . Crk recruits cortactin which is phosphorylated by Src and is implicated in stimulating the Arp2/3 complex. 94 Table 5.1 Differences between Shigella and S. Typhimurium invasion. Shigella S. Typhimurium References Invades enterocytes exclusively basolaterally Invades enterocytes both apically and basolaterally (Criss et ai, 2001; Finlay et al., 1988; Pizarro-Cerda & Cossart, 2006) Translocon components are important for adherence Translocon components may be important for adherence (Lafont et al, 2002; Watarai et al, 1996) Ezrin is necessary for efficient invasion Ezrin does not play an important role in invasion (Skoudy et al, 1999); this study Crk is required for efficient invasion > Crk is not required for efficient invasion (Bougneres etal, 2004); this study Cortactin is required for efficient invasion Cortactin is not required for efficient invasion (Bougneres etal, 2004; Unsworth et al, 2004) Vinculin is recruited to sites of invasion Vinculin is not recruited to sites of invasion (Tran Van Nhieu et al, 1997); this study Cdc42 plays a major role in invasion Cdc42 is not required for invasion (Mounier et al, 1999; Patel & Galan, 2006) Microtubules important for invasion Microtubules not important for invasion (Garcia-delPortilloe?a/., 1994; Yoshida etal, 2002) 5.2 Cytoskeletal proteins recruited to sites of invasion: cytoskeletal dynamics within Salmonella-induced ruffles Morphologically, Salmonella-induced ruffles are very similar to lamellipodia. Many of the proteins assessed for recruitment to sites of S. Typhimurium invasion in Chapter 3 were chosen for study because they have important functional roles in lamellipodia formation and cell motility and therefore may be important for Salmonella-induced ruffles and bacterial invasion. Characterization of Salmonella-induced ruffle components revealed a surprising variety of actin-associated proteins recruited upon infection including actin-binding proteins, SH2 adaptors, focal adhesion proteins, and proteins involved in actin polymerization and depolymerization (Table 5.2). We investigated the role of gelsolin during invasion since it is an important cytoskeletal protein involved in the membrane ruffling response to various stimuli (Azuma et al., 1998) and was shown, in a cell-free system, to be inhibited by the S. Typhimurium effector S ipA (McGhie et al., 2004). A s described in Chapter 3, gelsolin was recruited to 95 S. Typhimurium-induced ruffles whereby it inhibited invasion (Figure 3.1 and 3.3). We propose that gelsolin counteracts actin polymerization at the site of invasion by breaking down actin filaments. WIP is a multifunctional protein that participates in the formation of lamellipodia by regulating the activity of N - W A S P (Anton & Jones, 2006). This study found WIP to be localized at sites of S. Typhimurium invasion (Figure 3.1) and, because of the importance of N - W A S P during S. Typhimurium invasion (Unsworth et al., 2004), it is tempting to speculate that WIP ' s regulation of N - W A S P may also play a key role during S. Typhimurium invasion. Profilin localizes to lamellipodia where it binds and sequesters globular actin and regulates actin polymerization (Witke, 2004). We have identified profilin as a novel component of Salmonella-induced ruffles where it may also contribute to the efficiency of actin polymerization at the site of invasion. Interestingly, many of the proteins recruited to sites of invasion are also important components of focal adhesions ( L i et al., 2005). Focal adhesions are the sites of cellular attachment to the extracellular matrix ( E C M ) . Attachment is mediated through a family of integral membrane proteins called integrins that link the E C M to the actin cytoskeleton. Recruitment of actin to integrins is achieved via a complex of cytoskeleton proteins. The most well-defined linkage between (3-integrins and actin occurs through ct-actinin and talin. Both of these proteins were localized to sites of S. Typhimurium invasion, confirming findings by Finlay et al. (Finlay et al, 1991). V A S P , another key component of focal adhesions, enhances N-WASP/Arp2/3-dependent actin polymerization and is essential for invasion of Listeria monocytogenes via the Met 96 Table 5. 2 ' Host proteins recruited to sites of S. Typhimurium invasion. Protein AB* • FA T Actin polymer-ization* SH2 adaptor Recruited to sites of invasion Functional role in invasion Reference a-actinin + + , + N D ( ' this study, (Finlay etal, 1991) Arp2/3 + + + required (Criss & Casanova, 2003; Unsworth et al, 2004) cortactin + + + no (Unsworth et al, 2004) Crkl/II + + no this study ezrin + + N D this study gelsolin + inhibits this study F A K + + enhances; may affect adherence (Shi & Casanova, 2006) LPP + + N D this study Nek + + + + enhances; affects adherence this study N-WASP + + enhances (Criss & Casanova, 2003; Unsworth et al, 2004) pl30cas + + enhances; may affect adherence (Shi & Casanova, 2006) paxillin + + inhibits (Shi & Casanova, 2006) profilin + + + N D . this study ShcA + + + inhibits; affects adherence this study talin + + + N D this study, (Finlay etal, 1991) tropomyosin + + N D this study, (Finlay etal, 1991) V A S P + + + + N D this study vinculin + • + - .ND this study, (Finlay etal, 1991) W A V E + + enhances (Shi et al, 2005; Unsworth et al, 2004) WIP + + + N D this study zyxin + N D this study A B : actin-binding protein * FA: focal adhesion protein + Proteins involved in actin polymerization N D : not tested 97 receptor (Bierne et al, 2005; Sechi & Wehland, 2004). Zyxin , and its homologue L P P , also normally localize to both focal adhesions and lamellipodia where they are proposed to contribute to cell motility by targeting V A S P to sites of actin polymerization (Hoffman et al, 2003). Because all three proteins were enriched in Salmonella-induced ruffles, we hypothesize that zyxin and L P P target V A S P to sites of S. Typhimurium invasion whereby V A S P enhances the activity of N - W A S P and Arp2/3, both of which are important for S. Typhimurium invasion. The focal adhesion proteins F A K , pl30cas, and paxill in have recently been implicated in the invasion process of S. Typhimurium (Shi & Casanova, 2006). Using gentamicin protection assays^ Shi and Casanova (2006) found that S. Typhimurium invasion into FAK" 7 " and Cas"7" knockout fibroblasts was significantly reduced, while in paxillin"7" knockout cells, invasion was significantly increased. These results were, confirmed using inside/outside immunostainings. Photographs of some of these immunostainings were presented and reveal that, unlike the authors' conclusion that only internalization was affected, adherence to FAK" 7 " and Cas"7" knockout cells appears to be drastically reduced (Shi & Casanova, 2006). In the case of paxillin"7" knockout cells, no immunostainings were shown and therefore it is not known whether paxil l in plays a role in inhibiting adherence of 5*. Typhimurium to non-phagocytic cells. In Chapter 3, we demonstrate that Nek and ShcA alter overall invasion levels by affecting adherence of S. Typhimurium to non-phagocytic cells (Figure 3.4). Together with the likely role of F A K and Cas in S. Typhimurium adherence, these results may reveal clues as to the identity of the host cell receptor for S. Typhimurium. 98 5.3 Can Nek and ShcA tell us anything about the host cell receptor for 5. Typhimurium? Because Nek and ShcA affect S. Typhimurium adherence, they are clearly affecting the host cell surface. One way Nek and ShcA could affect the host cell surface is through modulating protein expression. N c k l has been shown to enhance protein translation through its interaction with eukaryotic initiation factor 2 (eIF2) (Kebache et al, 2002; Kebache et al, 2004; Latreille & Larose, 2006). Accordingly, perhaps Nek is involved in the expression of the 5*. Typhimurium receptor. Alternatively, Nek and ShcA could affect the composition of the cell surface is by influencing the trafficking of the 5*. Typhimurium receptor(s) to and/or from the cell surface. The cell surface pool of any given receptor can be influenced by internalization of the receptor into endocytic vesicles (endocytosis) and the redistribution of internalized receptors back to the plasma membrane (endocytic recycling) (Soldati & Schliwa, 2006). Is there evidence that Nek and She are involved in receptor trafficking? The actin cytoskeleton actively participates in the recycling of certain G-protein coupled receptors, the transferrin receptor, glucose transporter 4, and C F T R (Ganeshan et al, 2007; Jiang et al, 2002; Stanasila et al, 2006; Y a n et al, 2005). Accordingly, Nek and She could affect receptor trafficking through their interactions with the actin cytoskeleton. For example, N - W A S P has recently been shown to be critical for surface localization of C F T R by affecting both endocytosis and endocytic recycling of C F T R (Ganeshan et al., 2007). Because Nek is a potent upstream regulator of N - W A S P (Tomasevic et al, 2007) it may also be involved in these processes. The actin cytoskeleton is also known to actively participate in the transport and fusion of secretory vesicles to the plasma membrane (Malacombe et al, 2006). Nek 99 localizes to certain endo- and exocytic vesicles (Benesch et al, 2002) and recent evidence suggests that Nek is required for actin-based translocation of exocytic vesicles to the cell surface (Lettau et al, 2006). The ability of cytoplasmic proteins to directly or indirectly regulate the affinity of receptors for their ligands is referred to as "inside-out signaling". Once thought to be exclusive to integrin activation (Ginsberg et al., 2005), inside-out signaling has also been characterized for Fc (Bracke et al, 2000) and hormone (Caunt et al, 2004) receptor activation. Could Nek modulate adherence of S. Typhimurium through its influence on the activation state of a cell surface receptor? Nek has been implicated in inside-out signaling through its involvement in integrin activation (Becker et al., 2000). Integrins are absent from the apical surface of polarized enterocytes and are therefore poor candidates to be the initial S. Typhimurium receptors on enterocytes. In contrast, integrins are present on the apical surface of M cells which have been shown to be important portals of entry for S. Typhimurium (Jepson & Clark, 2001). 5.4 Tight junction disruption and its contribution to the manifestation of diarrhea In this work, we. demonstrate that S. Typhimurium perturbs the intestinal barrier in an in vivo model of colitis. In this model, S. Typhimurium induces significant inflammation in the colon and cecum of streptomycin pretreated mice (Barthel et al, .2003). In vivo, inflammation induced by bacterial infection can play a large role in the loss of mucosal integrity, and vice versa- maintaining mucosal integrity is important for preventing inflammation that occurs as a result of exposure of luminal contents to the 100 underlying immune system. For instance, at sites of bacterial infection, P M N s are recruited and transepithelial migration of P M N s causes physical damage to the intestinal epithelial barrier (Nash et al, 1987; Parkos et al, 1992). Pro-inflammatory cytokines produced upon infection can also perturb tight junction structure and function. Evidence from both cell culture and animal models suggests that exposure of the epithelium to pro-inflammatory cytokines such as IFNy, T N F a , IL-1|3, IL-4, IL^6, IL-8, and IL-13 compromises intestinal barrier function (Clayburgh et al, 2004). These effects are mediated at the molecular level through modulation of the actin cytoskeleton and via altered expression and/or redistribution of tight junction proteins. Moreover, after tight junctions are disrupted, exposure of the luminal contents to the underlying immune system serves to amplify the inflammatory response, and in turn, barrier destruction. Based on the work described in Chapter 4, future studies should address the role of S. Typhimurium-induced inflammation in tight junction disruption in vivo. Not only does tight junction disruption contribute to inflammation upon bacterial infection, it also likely enhances diarrhea through fluid loss. In the gut, water is normally passively absorbed following the absorption of solutes. Disruption of tight junctions alters electrolyte processes such as the sodium gradient, whereby the luminal and serosal sodium concentrations equilibrate. Consequently, when luminal sodium concentrations increase, water accumulates in the intestinal lumen, enhancing diarrhea. Diarrheagenic pathogens have evolved various strategies to target intercellular junctions during infection. A s mentioned in Chapter 1, pathogens can disrupt junction structure and function by a) directly altering or degrading adhesion proteins; b) modulating the actin cytoskeleton; c) activating cellular signal transduction; or d) 101 triggering cytokine production or P M N transmigration (reviewed by Sears, 2000). Cholera is a severe watery diarrhea caused by Vibrio cholerae. Through secretion of a metalloprotease called hemagglutinin/protease, V. cholera directly degrades the extracellular domain of occludin, thereby perturbing the barrier function. Clostridium spp. target the actin cytoskeleton in order to weaken the intestinal barrier. For example, the etiological agent of botulism, C. botulinum, directly targets the actin cytoskeleton via its C2 toxin that ADP-riboslyates G-actin. C. difficile infections cause pseudomembrous colitis, a potentially severe post-antibiotic diarrhea. This pathogen produces two toxins (A and B) that inactivate Rho family GTPases Rac, Cdc42, and Rho through monoglucoslyation thereby causing disorganization of the actin cytoskeleton and disruption of the barrier. The diarrheagenic pathogens enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli ( E H E C ) also affect tight junctions by activating host cell signaling pathways. Infection results in phosphorylation of myosin light chain kinase, dephosphoryation of occludin, activation of ezrin, and redistribution of ZO-1 and occludin away from junctions. Through an unknown mechanism, the type 3-secreted effectors EspF and Map are responsible for these effects. Disruption of tight junctions by E P E C and E H E C had been observed in cell culture, however, two recent studies have shown that disruption of tight junction also occurs in animal models (Guttman et al., 2006; Shifflett et al., 2005). In Chapter 5, the molecular mechanisms of S. Typhimurium tight junction disruption in vitro were dissected in the absence of most inflammatory mediators. We identified the specific SPI-1-secreted effectors responsible for disruption of tight junction structure and function. These data suggested that these effectors utilize 102 their ability to stimulate Rho family GTPases to disrupt tight junction structure and function. 5.5 Future directions M y research has led us to ask new questions about S. Typhimurium pathogenesis. There are several avenues for future research that can be pursued. Listed below are several questions that could be addressed in the future. 1. What are the roles of other key actin regulators during 5. Typhimurium invasion? Work presented in Chapter 3 demonstrates that many actin-associated proteins are recruited to sites of S. Typhimurium invasion. A functional role for additional recruited proteins during S. Typhimurium invasion should be explored. For instance, WIP is an actin-binding protein that participates in filopodia and lamellipodia formation. It interacts with N - W A S P , regulating N - W A S P function in Arp2/3-mediated actin polymerization. V A S P is also an actin-binding protein involved in filopodia and lamellipodia formation. V A S P has been implicated in Listeria motility and localizes to the tip of E P E C and E H E C pedestals. Reagents, including dominant-negative constructs and knockout cell lines have recently become available to study the role of both of these proteins in S. Typhimurium invasion. 103 2. Which host cell surface proteins serve as receptors for S. Typhimurium adherence? The data presented in Chapter 3 provides evidence that a host cell receptor for S. Typhimurium requires the adaptor Nek for expression, trafficking to the cell surface, or regulating its affinity via inside-out signaling. To date, the host cell receptor(s) required for adherence of S. Typhimurium to non-phagocytic cells is/are not known. From previous studies we know that the apical epithelial cell receptor is a glycoprotein since trypsin and neuramidase treatment of the apical surface abolished adherence of Salmonella (Finlay et al, 1989). The receptor for S. Typhimurium is likely expressed on both the apical and basolateral surfaces of epithelial cells since S. Typhimurium readily adheres and invades at both poles. Alternatively, S. Typhimurium could use different cell surface receptors depending on the pole it infects. This raises the possibility that perhaps the reason why the receptor has been so hard to identify is because S. Typhimurium is capable of exploiting multiple receptors on a single cell type. In addition, S. Typhimurium may exploit multiple receptors on different cell types as it is able to infect virtually all cell types it encounters. Alternatively, i f S. Typhimurium uses a single host receptor, it must be ubiquitously expressed in all cell types and conserved across host species. Future studies should focus on host cell surface components capable of binding to S. Typhimurium's translocon components or adhesins. 3. How do Nek and ShcA affect the host cells surface? In Chapter 3 we found that Nek and ShcA are capable of modulating the host cell surface in such a way as to affect S. Typhimurium adherence. Using mass spectrometry, 104 one could compare the protein composition of plasma membranes isolated from Nckl+/Nck2+ and N c k l - / N c k 2 - cells. I f Nek is involved in receptor expression or trafficking, the S. Typhimurium receptor w i l l be absent from the N c k l - / N c k 2 - plasma membrane samples when compared to the.Nckl+/Nck2+ samples. Similarly, one could compare the plasma membranes isolated from She knockdown and control cells. In this case, the receptor would be enriched in the She knockdown plasma membrane samples when compared to the control. If, however, Nek and ShcA are involved in inside-out signaling, there would be no difference in receptor density in the plasma membrane. 4. How does Salmonella transcytose through epithelial cells? What are the "escape determinants"? Once internalized, S. Typhimurium is capable of transcytosing across the monolayer. This is likely a very important means by which S. Typhimurium breaches the epithelial barrier in vivo but there has been little to no research on the molecular mechanisms behind such an event. How is the S C V trafficked to the basolateral pole of epithelial cells? H o w does the S C V fuse with the basolateral membrane? Does S. Typhimurium use a similar mechanism to not only cross the monolayer but to infect adjacent cells (ie. cell-to-cell spread)? In vivo, as bacterial numbers increase in tissues, rarely does one observe more than one or two bacteria per cell (Brown et al, 2006; Sheppard et al, 2003). Recently, elegant studies have provided evidence that growth of 5. Typhimurium in tissues is the result of the spread of the microorganisms from cell-to-cell rather than simply replication within the same cell (Brown et al, 2006). Therefore, 105 identification of bacterial "escape determinants" w i l l be key to understanding disease progression. 5. How do SipA and SopB work together? In Chapter 4, we observed cooperation between effectors S ipA and SopB during tight junction disruption. In the future, it would be interesting to determine specifically which domain(s) of S ipA and SopB are required for this cooperation. Also important to determine would be: Do S ipA and SopB interact directly? Do S ipA and SopB bind different host proteins when expressed together rather than in. isolation? Do S ipA and SopB localize to different places within the host cells when expressed together rather than in isolation? 6. What are the physiological consequences of loss of cell polarity? In Chapter 4, we identified the specific bacterial effectors responsible for the loss of cell polarity during S. Typhimurium infection, however, the physiological consequences of loss of cell polarity. during infection are unknown. For example, disruption of tight junctions by E P E C disrupts cell polarity. E P E C exploits the loss of cell polarity by using normally basolaterally-sequestered (3i integrins as receptors for adherence (Muza-Moons et al, 2003). Loss of cell polarity induced by C a 2 + chelation leads to intermixing of apical and basolateral membrane components. When C a 2 + is restored, tight junctions seal once again, leaving cell polarity temporarily disrupted with the barrier intact. In this way, the physiological consequence of loss of cell polarity during S. Typhimurium infection could be assessed in vitro. 106 7 . What is the role of inflammation in in vivo tight junction disruption? In Chapter 4, we demonstrate that wild-type S. Typhimurium disrupts localization of tight junction proteins and increases intestinal permeability. To tease out the role of inflammation in tight junction disruption in vivo, streptomycin pre-treated mice could be infected with the AsopB/E/E2 mutant. In vitro, this mutant was incapable of disrupting tight junctions (Figure 4.5), however, in streptomycin-pretreated mice, this mutant would be predicted to produce significant inflammation (Hapfelmeier et al., 2004). Therefore, i f this mutant is incapable of perturbing intestinal barrier function, one could conclude that inflammation does not play a significant role in barrier disruption by S. Typhimurium. Overall, this thesis has significantly enhanced our knowledge of how S. Typhimurium interacts with epithelial cells. 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(2007) Src homology 2 adaptors affect adherence of Salmonella enterica serovar Typhimurium and non-phagocytic cells. Microbiology. 153: 3517-3526. Wickham, M . E . * , Brown, N . F.*, Boyle, E. C., Coombes, B . K . , and Finlay, B . B . (2007) Virulence is positively selected by transmission success between mammalian hosts. Curr B i o l . 17:783-788. Bishop, J. L . * , Boyle, E. C.*, and Finlay, B . B . (2007) Deception point: peptidoglycan modification as a means of immune evasion. Proc Natl Acad Sci U S A . 104: 691-692. Boyle, E. C.*, Bishop, J. L . * , Grassl, G . A . * , and Finlay, B . B . (2007) Salmonella: From pathogenesis to therapeutics. J Bacteriol. 189: 1489-1495. Boyle, E. C , Brown, N . F. , and Finlay, B . B . (2006) Salmonella enterica serovar Typhimurium effectors SopB, SopE, SopE2, and SipA disrupt tight junction structure and function. Cel l Microbiol . 8: 1946-1957. Boyle, E. C. and Finlay, B . B . (2005) Leaky guts and lipid rafts. Trends Microbiol . 13: 560-563. Kujat-Choy, S. L , Boyle E. C , Gal-Mor, O., Goode D . L . , Valdez Y . , Vallance B . A . , and Finlay, B . B . (2004) S s e K l and SseK2 are novel translocated proteins of Salmonella enterica serovar Typhimurium. Infect Immun. 2: 5115-5125. Boyle, E. C. and Finlay, B . B . (2003) Bacterial pathogenesis: exploiting cellular adherence. Curr Opin Cel l B io l . 15: 633-639! * These authors contributed equally to this work. Abstracts Boyle, E. C. and Finlay, B . B . (2006) Salmonella enterica serovar Typhimurium effectors SopB, SopE, SopE2, and S ipA disrupt tight junctions structure and function. Presented at the 2 n d A S M Conference on Salmonella: From Pathogenesis to Therapeutics. Boyle, E. C. and Finlay, B . B . (2005) Salmonella Typhimurium effectors SopB, SopE, SopE2, and S ipA are responsible for disruption of tight junction structure and function. Presented at Cold Spring Harbor Laboratory Microbial Pathogenesis & Host Response Meeting, Cold Spring Harbor, U S A . 130 Boyle, E . C , Brumell, J. H . , Pawson, T., and Finlay, B . B . (2003) Cytoskeletal recruitment and the role of Nek during invasion of Salmonella Typhimurium into epithelial cells. Presented at the E M B O / F E B S Workshop on Frontiers in Cytoskeleton Research, Gosau, Austria. 131 Appendix 2 Animal Ethical Approvals https://rise.ubc.ca/rise/Doc/0/35FHAEI43M6KT2DF01603A0D43/.. THE UNIVERSITY OF BRITISH COLUMBIA ANIMAL CARE CERTIFICATE Application Number: A05-1082 Investigator or Course Director: Brett B. Finlay Department: Michael Smith Laboratories Animals: Mice C57/BL6 408 Mice Transgenic mice - various 147 MiceBALB/c 160 Mice C3H/He 114 Mice nramp -/- 40 Mice CD1 286 Mice 129/Sv40 Mice SHIP -/- 43 Approval Date: Start Date: October 1,2005 Funding Sources: Canadian Institutes of Health Research (CMR) September 17,2007 Funding Agency: Funding Title: Novel therapeutics that boost innate immunity to treat infectious disease The Foundation for the National Institutes of Health Funding Agency: Funding Title: Novel therapeutics that boost innate immunity to treat infectious diseases The Foundation for the National Institutes of Health Funding Agency: Funding Title: Novel therapeutics that boost innate immunity to treat infectious diseases 1 of 2 09/10/2007 1:28 PM 132 

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