MOLECULAR MECHANISMS OF LISTERIA MONOCYTOGENES INVASION OF THE INTESTINAL EPITHELIUM

A DISSERTATION SUBMITTED TO THE DEPARTMENT OF MICROBIOLOGY & IMMUNOLOGY AND THE COMMITTEE ON GRADUATE STUDIES OF STANFORD UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

Mickey Joseph Pentecost September 2009

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Copyright by Mickey Joseph Pentecost 2009 All Rights Reserved

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I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. ________________________________ (Manuel R. Amieva) Principal Advisor

I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. ________________________________ (Stanley Falkow)

I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. ________________________________ (Julie A. Theriot)

I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. ________________________________ (W. James Nelson)

Approved for the Stanford University Committee on Graduate Studies. ________________________________

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ABSTRACT

Listeria monocytogenes causes invasive disease by crossing the intestinal epithelial barrier. This process depends on the interaction between the bacterial surface protein Internalin A (InlA) and the host protein E-cadherin. A second L. monocytogenes invasin Internalin B (InlB) promotes invasion of numerous non-phagocytic cell types, but has not been shown to promote oral infection. The receptor for InlB is c-Met, a receptor tyrosine kinase and the endogenous receptor for Hepatocyte Growth Factor (HGF). E-cadherin and c-Met are localized to the basolateral side of polarized epithelial cells and are not thought to be accessible to the apical (lumenal) side across functional tight junctions. We used polarized MDCK cells as a model epithelium to determine how L. monocytogenes gain access to basolateral receptors. We found that L. monocytogenes do not actively disrupt the tight junctions, but find E-cadherin at a morphologically distinct subset of intercellular junctions. We identified these sites as naturally occurring regions where single senescent cells are extruded from the epithelium. The surrounding cells reorganize to form a multicellular junction (MCJ) that maintains epithelial continuity. We found that E-cadherin is transiently exposed to the lumenal surface at MCJs during and after cell extrusion. We hypothesized that L. monocytogenes utilize analogous extrusion sites for epithelial invasion in vivo. By infecting rabbit ileal loops, we found that the MCJs at the cell extrusion zone of villus tips are the specific target for InlA-mediated L. monocytogenes adhesion and invasion. L. monocytogenes expressing a modified InlA capable of binding murine E-cadherin (InlAm) specifically invade and replicate within villous tips of orally infected of mice. We hypothesized that InlB functions synergistically with InlA to promote intestinal invasion. Utilizing L. monocytogenes expressing InlAm, we found that InlB promotes oral infection of mice and colonization of mouse villous tips.

v We investigated the mechanism by which InlB mediates Listeria invasion at MCJs using polarized MDCK monolayers. Following InlA-mediated attachment at MCJs, cMet activation by InlB increases the rate of bacterial uptake. The efficiency of invasion is also controlled by intrinsic epithelial properties since MCJs undergo rapid remodeling and are naturally more endocytic than other junctional sites; MCJs endocytose fluorescent dextran, a fluid phase marker, from the apical surface into unique cytoplasmic puncta containing both tight- and adherens junction proteins. Apical HGF or InlB increase the number and size of dextran puncta at MCJs, but do not increase endocytosis at other junctions, suggesting that c-Met is apically exposed at MCJs and that L. monocytogenes can modulate cellular endocytosis during invasion of this specific site. Thus, L. monocytogenes exploit the dynamic nature of junctional remodeling and epithelial renewal to target exposed receptors and hijack host cell processes for epithelial invasion and intestinal barrier breach.

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ACKNOWLEDGMENTS

Throughout this thesis I use the pronoun we rather than I when discussing results because this work has been possible only though the assistance and intellectual contributions of many people. First, I thank my Advisor Manuel Amieva. His patience and personal generosity have made my graduate career enjoyable and rewarding. I will rely on him throughout my life for both personal and professional guidance. Manuel has also created a very interactive, collaborative and friendly lab environment. I thank my friends and colleagues Michael Howitt, Josephine Lee, Shumin Tan, Lee Shaughnessy, Brooke Lane, Fabio Bagnoli, Roger Vogelman and Elizabeth Joyce for experimental and intellectual help and guidance, as well as for the coffee breaks, costume parties, soccer games, travels, and baked goods. I wish to thank the rest of my Thesis Committee. Stanley Falkow, James Nelson and Julie Theriot are dedicated educators and wonderful people who have always been constructive, available and generous with their time and expertise. In addition to my committee, Denise Monack, Glen Otto, Susanne Rafelski, Alex Nielsen, Ana Bakardjiev and Jonathan Hardy have been mentors and collaborators. I acknowledge the people who made my academic and intellectual growth possible. My parents Lyn and Dave always provided love and encouragement, but more importantly, they let me pursue all of my interests. They also gave me my younger brother and Best Man Will, a chef/artists/musician/handball champ who I admire. I inherited an interest in science from my grandfathers, Joe Pentecost and Bill Tiefenbacher, and inherited a love of literature and a sense of civic responsibility from my grandmothers, Maxene Pentecost and Sally Tiefenbacher. My aunts and uncles, especially Wendy, Ken, Moira, Tom and June have always given love and support in every possible way, including housing, warm meals and emotional support during my schooling.

vii I would like to thank a scientific mentor, role model, and dear friend, Issar Smith. Smitty and his former graduate student Ben Gold introduced me to biological research and began my training microbiology. They also showed me that scientists dont have to sacrifice a full life and dedication to family and friends to be professionally successful and admired. Finally, I thank my best friend and gorgeous wife, Tina Huang. Tina is the most talented, warm and generous person in the world. I thank her for love and support through all the successes, challenges, and changes of homes we have had. I also thank her for keeping art and excitement in my life.

viii DEDICATION

The author wishes to dedicate this dissertation to Grandma Sally.

ix TABLE OF CONTENTS

List of Tables ............................................................................................................... xiii List of Figures.............................................................................................................. xiv List of Videos ............................................................................................................. xvii Chapter 1 : Introduction and Literature Review ............................................................... 1 Research Rationale .................................................................................................... 1 History ...................................................................................................................... 2 Listeria monocytogenes, an Enteroinvasive Bacterial Pathogen............................ 2 Identification ....................................................................................................... 3 Listeriosis: a Disease of Humans and Animals..................................................... 5 Pathogenesis and Epidemiology........................................................................... 6 Listeria in Basic Biological Research .................................................................. 8 Intracellular Parasitism............................................................................................ 10 Stages and Mechanisms of Listerias Intracellular Life-Cycle............................ 10 Genetic Regulation of Intracellular Infection ..................................................... 12 Adherence to the Cell Surface............................................................................ 16 Internalin A, Internalin B and the Internalin Family........................................... 16 Internalin A Co-opts the Epithelial Junctions ..................................................... 20 Internalin B Co-opts Growth Factor Signaling ................................................... 23 Synergy Between Internalin A and Internalin B ................................................. 26 Species Specificities of Internalin A and Internalin B ........................................ 27 Animal Models Permissive for Internalin A and Internalin B............................. 29 Tissue Specificities of Internalin A and Internalin B .......................................... 30 Challenging the Internalin Tissue Specificity Dogma......................................... 30 Anatomical and Subcellular Site of Epithelial Invasion: The Polarity Paradox......... 31 The Epithelial Barrier, Cell Renewal and Gastrointestinal Pathogens................. 31 E-cadherin and c-Met are Basolateral Receptors ................................................ 32 The Peyers Patch Paradigm .............................................................................. 33 Challenging the Peyers Patch Paradigm............................................................ 34

x Chapter 2 : Internalin A Targets Listeria monocytogenes to Epithelial Junctions at Sites of Cell Extrusion................................................................................................... 36 Introduction............................................................................................................. 36 Materials and Methods ............................................................................................ 38 Results .................................................................................................................... 43 L. monocytogenes Invade The Epithelium at Distinct Multicellular Junction Sites............................................................................................................. 43 Apical Attachment to Multicellular Junctions is Dependent on Internalin A....... 48 Quantitative Analysis of L. monocytogenes Adhesion Sites ............................... 50 Multicellular Junctions Form and Persist at Sites of Cell Extrusion.................... 52 L. monocytogenes Attachment Sites are Sites of Cell Extrusion ......................... 55 L. monocytogenes Adhere to Transiently Exposed E-cadherin at Sites of Cell Extrusion.............................................................................................. 59 Discussion............................................................................................................... 65 Chapter 3 : Internalin A targets L. monocytogenes to the Villus Tip Extrusion Zone...... 69 Introduction............................................................................................................. 69 Materials and Methods ............................................................................................ 72 Results .................................................................................................................... 80 L. monocytogenes Invade Multicellular Junctions at the Villus Tip Extrusion Zone ............................................................................................ 80 Analysis of L monocytogenes Infection of the Villous Tip Extrusion Zone by Transmission Electron Microscopy ......................................................... 87 Rational Design of Internalin A variants Predicted to Bind Murine Ecadherin....................................................................................................... 90 Mutations in Internalin Differentially Affect the Tropism of Listeria for Epithelial Cells of Different Species ............................................................ 90 InlA S192N Y369S (InlAm) Permits Oral Infection of Mice............................... 95 InlAm Specifies Invasion of the Villus Tips, But Not of Peyers Patches ............ 98 Discussion............................................................................................................. 101 Chapter 4 : InlB Targets c-Met and Modulates Endocytosis at Multicellular Junctions ..................................................................................................................... 103

xi Introduction........................................................................................................... 103 Materials and Methods .......................................................................................... 105 Results .................................................................................................................. 111 Construction of GFP-expressing Listeria Strains.............................................. 111 inlB GFP L. monocytogenes Express InlA ..................................................... 113 inlB L. monocytogenes Recruit E-cadherin and -catenin to Sites of Bacterial Attachment ................................................................................. 116 Internalin B promotes Apical Invasion of Multicellular Junctions .................... 118 InlB Targets c-Met Locally During Apical Invasion of Polarized MDCK Monolayers................................................................................................ 120 InlB Does Not Influence Listeria Intracellular Replication and Cell-to-cell Spread Within a Polarized Epithelium........................................................ 122 Junctional Remodeling at Multicellular Junctions Correlates with Increased Apical Endocytosis .................................................................................... 124 InlB and HGF Modulate Dextran Endocytosis at Multicellular Junctions......... 128 Apical Endocytosis AND L. monocytogenes Invasion at Multicellular Junctions Require Common Endocytic Machinery ..................................... 128 Discussion............................................................................................................. 132 Chapter 5 : InlB Promotes L. monocytogenes Oral Infection and Colonization of the Villus Tip Extrusion Zone ..................................................................................... 136 Introduction........................................................................................................... 136 Materials and Methods .......................................................................................... 139 Results .................................................................................................................. 145 Development and Verification of L. monocytogenes Strains expressing InlAm, InlB and GFP .................................................................................. 145 InlB Promotes Oral Infection of Mice.............................................................. 148 InlB Promotes Colonization of the Villus Tip Extrusion Zone ......................... 150 Discussion............................................................................................................. 154 Chapter 6 : Questions for Future Research................................................................... 157 Do Other Enteric Bacteria Utilize Cell Extrusion Zones?....................................... 157

xii Are Multicellular Junctions Inherently Permissive for Bacterial Uptake In Vivo? ............................................................................................................... 159 What are the Cellular Molecular Mechanisms of Listeria Invasion or Junctional endocytosis at Multicellular Junctions? ........................................... 159 How are Basolateral Receptors Exposed to the Lumenal Surface at Multicellular Junctions?................................................................................... 161 How Do Listeria and Host Cells Interact at the Villous Tips? ................................ 162 Are the Villus Tips a Site of Colonization or Asymptomatic Carriage?.................. 162 Bibliography ............................................................................................................... 165

xiii LIST OF TABLES

Number......................................................................................................................Page Table 3.1: Oligonucleotides Used in Chapter 3.............................................................. 78 Table 3.2: L. monocytogenes Strains Used in Chapter 3................................................. 79 Table 4.1: Oligonucleotides Used in Chapter 4............................................................ 110 Table 4.2: L. monocytogenes Strains Used in Chapter 4............................................... 110 Table 5.1: Oligonucleotides Used in Chapter 5............................................................ 143 Table 5.2: L. monocytogenes Strains Used in Chapter 5............................................... 144

xiv LIST OF FIGURES

Number......................................................................................................................Page Figure 1.1: Schematic Representation of the Pathophysiology of Listeria Infection......... 2 Figure 1.2: Molecular and Genetic Requirements for Listerias Intracellular LifeCycle (Following Page) ............................................................................... 14 Figure 1.3: Domain Organization of Internalins and Sequence Alignments of Internalin LRR Domains (Following Page) ................................................. 18 Figure 1.4: Structural and Molecular Aspects of InlA/E-cadherinmediated Listeria Invasion (Following Page) .............................................................. 21 Figure 1.5: Structural Aspects of InlB/c-Met-mediated Listeria Invasion (Following Page) ......................................................................................... 24 Figure 1.6: Listeria versus The Apical Junctional Complex of Polarized Epithelia ........ 35 Figure 2.1: Preservation of Barrier Function During L. monocytogenes Infection of MDCK Cells Polarized on Transwell Filters (Following Page)..................... 44 Figure 2.2: Invasion and Replication of L. monocytogenes at Multicellular Junction Sites (Following Page)................................................................................. 46 Figure 2.3: Internalin A-dependent Apical Adhesion and Invasion of Polarized Epithelia ...................................................................................................... 49 Figure 2.4: Tropism of L. monocytogenes for Multicellular Junctions............................ 51 Figure 2.5: Multicellular Junctions Created by Cell Extrusion ....................................... 53 Figure 2.6: L. monocytogenes Adhesion to Sites of Cell Extrusion ................................ 56 Figure 2.7: E-cadherin Associated with L. monocytogenes at Multicellular Junctions...................................................................................................... 57 Figure 2.8: L. monocytogenes Attachment to Accessible E-cadherin at Multicellular Junctions of Cell Extrusion Sites............................................. 60 Figure 2.9: Increased E-cadherin Exposure and L. monocytogenes Adhesion in Calcium Depleted MDCK Monolayers ........................................................ 63 Figure 2.10: L. monocytogenes Adherence to Single Cell Polarity Defects .................... 64

xv Figure 3.1: L. monocytogenes Invasion of the Intestinal Epithelium at the Villustip Extrusion Zone (Following Page)............................................................ 81 Figure 3.2: Lack of Association of L. monocytogenes Invasion with the Intestinal Crypts or the Peyers Patches; inlA-mutant is Noninvasive (Following Page) ........................................................................................................... 83 Figure 3.3: L. monocytogenes Associate with Intercellular Junctions Prior to Villus Tip Invasion................................................................................................. 88 Figure 3.4: L. monocytogenes Infect Cells Adjacent to Apoptotic Cells at the Villous Tip................................................................................................... 89 Figure 3.5: Internalin Variants with Altered Tropism for Canine and Murine Cells ....... 93 Figure 3.6: InlA R168S E170G Q190G Promotes Invasion of the Murine Intestine....... 94 Figure 3.7: InlA S192N Y369S Reconstitutes Oral Infection in Mice (Following Page) ........................................................................................................... 96 Figure 3.8: Functional InlA Targets Listeria to the Villus Tip Epithelium (Following Page) ......................................................................................... 99 Figure 4.1: Construction of GFP Expression Constructs and Verification of InlA and InlB Expression in GFP Listeria Strains .............................................. 112 Figure 4.2: Activation of c-Met and MDCK Cell Scattering by Purified InlB .............. 115 Figure 4.3: Recruitment of E-cadherin and -catenin by Listeria to Sites of Attachment ................................................................................................ 117 Figure 4.4: InlB Mediated Apical Invasion of Polarized MDCK Monolayers. ............. 119 Figure 4.5: Invasion of Listeria Through Local c-Met Activation ................................ 121 Figure 4.6: Lack of a Role for InlB in Intracellular Replication and Cell-to-Cell Spread ....................................................................................................... 123 Figure 4.7: Unique Para-endocytosis of Junctional Components at Sites of Cell Extrusion (Following Page) ...................................................................... 125 Figure 4.8: Enhancement of Dynamin-dependent Endocytosis at Multicellular Junctions by HGF and InlB (Following Page) ............................................ 130 Figure 5.1: Construction of Expression Constructs and Verification of InlAm and InlB Expression in Listeria ........................................................................ 147

xvi Figure 5.2: Analysis of the role of InlB in Listeria Oral Infections by Bioluminescence Imaging.......................................................................... 149 Figure 5.3: Single Infection of Mice with inlAB inlAmB gfp or inlAB InlAm gfp........ 151 Figure 5.4: InlB-mediated Invasion of the Intestinal Villus Tips.................................. 152 Figure 5.5: Coinfection with inlAB inlAmB and inlAB inlAm gfp............................... 153

xvii LIST OF VIDEOS

Number......................................................................................................................Page Video 2.1: Cell Extrusion.............................................................................................. 54 Video 2.2: L. monocytogenes Invasion at a Multicellular Junction................................. 58 Video 3.1: Villus Tip Infected with Wild Type Listeria monocytogenes ........................ 85 Video 3.2: Villus Tip Infected with inlA L. monocytogenes......................................... 86 Video 4.1: Junctional Endocytosis at Cell Extrusion.................................................... 127

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CHAPTER 1 : INTRODUCTION AND LITERATURE REVIEW

RESEARCH RATIONALE In the United States, food-borne pathogens have been estimated to be responsible for 76 million illnesses, 323,914 hospitalizations, and 5,194 deaths each year (Mead et al., 1999). Invasive microorganisms that infect the intestinal epithelium and breach the intestinal barrier cause more than 75% of these deaths. Listeria monocytogenes has emerged as a significant cause of mortality due to food-borne illness in the United States since it was responsible for 27.6% of deaths from enteric infection (Mead et al., 1999). Other important pathogens that invade the epithelium include rotavirus, Salmonella, Shigella, Yersinia, Enteroinvasive E. coli, and Campylobacter. How these organisms breach the gastrointestinal epithelial barrier is not fully understood. Specific interactions between microbial adhesins and cell surface receptors are known to be critical for invasion (Boyle and Finlay, 2003). Paradoxically, many of the known adhesin-receptor interactions involve cellular receptors that are not typically present on the apical (lumenal) side of the gastrointestinal epithelium because intact intercellular junctions prevent the diffusion of these molecules from the basolateral to the apical side of the epithelial cells. For example rotavirus, Shigella and Yersinia are known to use integrin receptors for attachment and entry through the basolateral (interstitial) surfaces of epithelial cells (Ciarlet et al., 2002; Graham et al., 2003; Guerrero et al., 2000; Hewish et al., 2000; Isberg and Leong, 1990; Mounier et al., 1992; Watarai et al., 1996). Similarly, Listeria monocytogenes use the basolateral junction protein E-cadherin and the basolateral signaling protein c-Met for epithelial cell invasion (Mengaud et al., 1996; Shen et al., 2000). How and where L. monocytogenes find receptors for attachment and entry are important questions in the pathogenesis and clinical outcomes of microbial gastroenteritis and enteric fever.

2 HISTORY , AN ENTEROINVASIVE BACTERIAL PATHOGEN The Gram-positive, facultative intracellular bacterium is a cause of human and animal food-borne infection (Vazquez-Boland et al., 2001). The initial steps in the pathogenesis of Listeriosis involve colonization and growth in the intestinal tissue, followed by spread to other organs via the lymphatics or blood stream (Figure 1.1) (Vazquez-Boland et al., 2001). Invasive Listeriosis is one of the most deadly bacterial infections with a mortality of ~30%, and the ability of to survive within hosts is attributed to the organisms sophisticated intracellular infection cycle: hijacks endocytic machinery to invade cells, escapes from the vacuole to replicate within the host cell cytosol, and recruits components of the host cell cytoskeleton to translocate to neighboring cells, all the while avoiding the humoral immune system (Figure 1.2A) (Pamer, 2004; Portnoy et al., 2002; Vazquez-Boland et al., 2001).

Figure 1.1: Schematic Representation of the Pathophysiology of Figure from (Vazquez-Boland et al., 2001).

Infection

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IDENTIFICATION Listeria monocytogenes was identified and described in two independent reports in 1926 and 1927. In the first, Murray, Webb and Swann isolated the bacterium during epidemic outbreak of lethal disease in their rabbit colony in Cambridge, England (Murray et al., 1926). The disease, affecting young animals within the first few months of age and pregnant animals, was characterized by development of a distended belly, rapid weight loss and lethargy interrupted by convulsive struggles. Necropsies revealed edema of subcutaneous tissues, accumulation of fluids in pleural, pericardial and peritoneal cavities, enlarged and edematous mesenteric lymph nodes, foci of necrosis in the liver, and enlarged spleens. These are now well-known pathologies caused by invasive Listeria. Although the gastrointestinal tract would not be recognized as a site of L. monocytogenes host invasion until the 1960s and 1970s, Murray et al. made at least two important observations that could have put the field in the right direction. First, the authors stated that the that outbreaks had always occurred at times when the fresh food upon which the breeding establishment largely depended either became scarce or rank, and noted that adequate food would terminate the epidemic. Second, they identified the location of L. monocytogenes invasion within the gastrointestinal tract since they could trace the most affected mesenteric lymph nodes to terminal ileum via the connecting mesenteries. However, they did not understand the significance of these observations since recreating infection in rabbits and guinea pigs through the oral route was inefficient. The strength of the gastrointestinal tract as a barrier to Listeria invasion hampered the recognition of this site as the natural route of infection, and continues to hamper research into the gastrointestinal phase of Listeriosis. The feature of the disease that was most compelling to Murray et al. was the ability of the bacterium to elicit a huge increase in the number of circulating monocytes in the blood. For this reason, they named the organism Bacterium monocytogenes. Unfortunately, this characteristic also resulted in the erroneous belief, held through the 1960s, that Listeria was the (or a) cause of infectious mononucleosis despite evidence

4 of viral etiology since the late 1930s and despite the fact that monocytosis was never a marked feature of human Listeriosis (Gray and Killinger, 1966; Murray, 1955; Schultz, 1945). Without knowledge of Bacterium monocytogenes, in 1927 Pirie identified the bacterium from the livers of a South African gerbil (known as the African Jumping Mouse) suffering from a plaque-like disease with necrotizing hepatic infection (Gray and Killinger, 1966; Pirie, 1927). Pirie named the bacterium Listerella hepatolytica in honor of Joseph Lister (1827 1912), the British surgeon and one time president of the Royal Society who promoted sterile surgery. In 1940, Pirie suggested the use of the current name Listeria monocytogenes, since Bacterium monocytogenes and Listerella hepatolytica were identified as the same, and since the genus Listerella had already been assigned to a group of slime molds in 1906 (Pirie, 1940). In retrospect, L. monocytogenes was probably identified a number of times prior to 1926. In the 1890s, Gram-positive rods were isolated from tissue sections from patients who died of Listeriosis-like disease in Germany and In 1911 an organism named Bacterium hepatitis was isolated from necrotic foci in the liver of a rabbit in Sweden (Gray and Killinger, 1966; Murray, 1955). In 1917 a diptheroid with L. monocytogenes characteristics was isolated from 5 children with meningitis in Australia and in 1921 a bacterium later confirmed as L. monocytogenes was isolated from the cerebrospinal fluid of an Italian soldier (Schultz, 1945). Even after 1926, the differences in symptoms and diseased hosts, and the difficulty of strain characterization led identification of a number of species only later confirmed as L. monocytogenes. These include hemolytic Corenybacterium, Listerella hepatolytica, Listerella monocytogenes hominis, Corenybacterium parvulum, Listerella ovis, Corenybacterium infantisepticum, Listeria infantiseptica, Listerella bovina, L. gallinarium, L. cuniculi, L. suis, and L. gerbilli (Gray and Killinger, 1966; Schultz, 1945).

5 LISTERIOSIS: A DISEASE OF HUMANS AND ANIMALS In the three decades following Murray et al. and Pirie, only sporadic cases of human Listeriosis were reported (Murray, 1955). Rather, Listeriosis was a curious disease of animals. Listeria was found associated with numerous species including rabbit, hare, guinea pig, gerbil, lemming, mouse, rat, hamster, vole, sheep, goat, cattle, pig, horse, dog, ferret, raccoon, fox, chicken, canary duck, goose and eagle (Gray and Killinger, 1966; Murray, 1955; Schultz, 1945). It should be noted that a number of these associations are based on fecal shedding as in mice and rats rather than disease. Despite the initial identification of L. monocytogenes in rabbits, domestic livestock were recognized as the major victims of Listeriosis, though with some differences in symptoms and pathology. In contrast to rabbits and some other monogastric animals, ruminants do not develop monocytosis. Rather, young sheep and cattle can develop septicemia with or without encephalitis (Gray and Killinger, 1966). Listeriosis of ruminants was often called circling disease since encephalitic animals were observed walking in circles (Vazquez-Boland et al., 2001). Between the first confirmed identification of neonatal human Listeriosis in 1933 and the early 1950s, encephalitis, meningitis and meningoencephalitis in non-pregnant humans and animals was given the majority of medical and experimental attention (Gray and Killinger, 1966). However, pregnancy-associated Listeria infection soon became a great concern when in the early 1950s, hundreds of tragic reports of neonatal deaths came from hospitals of bombed cities in East Germany being reconstructed after the war. Gray and Killinger wrote that, life, or even existence, was difficult. Food was poor, meager, and rationed, and essentials, such as milk for pregnant women, were found only in the black markets. Among the many who died were the yet unborn. Some of these stillborn infants showed characteristic, distinctive focal necrosis throughout their tiny bodies (Gray and Killinger, 1966). This generalized infection with extensive focal necrosis of the liver, infection of the lungs, central nervous system and skin was named granulomatosis infantiseptica. It is now known that some infants can also be born apparently well and develop disease within days or weeks post partum. Although the majority of cases of Listeriosis have been associated

6 with pregnancy, attention is returning to adult disease, which is on the increase due to immune suppression by HIV and immune suppressant therapies associated with organ transplants (Farber and Peterkin, 1991).

PATHOGENESIS AND EPIDEMIOLOGY Prior to the use of antibiotics, mortality of invasive Listeriosis was at least 70% (Gray and Killinger, 1966). By the late 1960s, that had dropped to roughly 50% and now stands at roughly 30% (Farber and Peterkin, 1991; Gray and Killinger, 1966; Mead et al., 1999; Vazquez-Boland et al., 2001). We note that the statistics are skewed by the high mortality rates in the very young and very old; The case fatality rates is thought to be as high as 50% for infants and as high as 20% for people over 60 years of age (Bortolussi, 2008). Despite the decrease in percent mortality, Listeriosis remains one of the most deadly food-borne illnesses. Furthermore, over the decades there has been an increase in both incidence of infection and in total death, even when accounting for improved detection and diagnosis (Farber and Peterkin, 1991). By the 1960s only ~ 500 human deaths due to L. monocytogenes were identified throughout the world (Gray and Killinger, 1966). More recent estimates suggest approximately 500 deaths per year in the U.S. (Mead et al., 1999). Factors contributing to the increase in Listeriosis include industrialized farming and industrialized food production which has increased prevalence of L. monocytogenes in the environment and in food. Because of industrialization of cattle production, cows now represent 80-90% of all animal Listeriosis, and livestock infections can be traced to contaminated feed, notably poorly fermented silage (Fenlon, 1985, 1986). The epidemiological connection between silage feed and infection had been made by the early 1960s, although confirmation of the food-borne route of infection would wait nearly 20 years with the advent of human epidemics (Farber and Peterkin, 1991; Gray and Killinger, 1966; Vazquez-Boland et al., 2001). Infected animals perpetuate the expansion of L. monocytogenes in the environment and at least 10% of asymptomatic animals are known to shed L. monocytogenes in their feces (Esteban et al., 2009; Husu, 1990; Unnerstad et al., 2000) Thus, livestock amplify and shed L.

7 monocytogenes in the farm environment, leading to new or sustained infections and potential contamination of animal and human foodstuffs (Farber and Peterkin, 1991; Gray and Killinger, 1966; Nightingale et al., 2005; Nightingale et al., 2004). Industrialized food processing of ready to eat food products has led to increased exposure because L. monocytogenes grows at refrigeration temperatures and is highly resistant to food preservation techniques such as smoking, curing or added chemical preservatives. A study surveying luncheon meats, deli salads, fresh soft "Hispanicstyle" cheeses, bagged salads, blue-veined and soft mold-ripened cheeses, smoked seafood, and seafood salads detected Listeria in nearly all food types as high as 106 CFU / g (Gombas et al., 2003). L. monocytogenes contamination has resulted in numerous disease epidemics and costly food recalls (Gottlieb et al., 2006). Protection of food from L. monocytogenes is of such great financial and public health importance that the FDA recently approved bacteriophage (listeriocidal virus) as a food additive to ready to eat meat and poultry products (Lang, 2006). Frequent and severe outbreaks of Listeriosis from ready to eat foods beginning in the 1980s provided the first unequivocal epidemiological evidence that Listeria is a foodborne pathogen. Previously proposed routes of invasion included inhalation, ocular inoculation, cutaneous infection (either by tick bite or by handling contaminated animal material), through sexual transmission, and from mothers to newborns through the vaginal canal during child-birth (Gray and Killinger, 1966). Because many thought that Listeria had to be transmitted directly from animal to human, Gray and Killinger made the off-color joke: it is fortunate that L. monocytogenes has not been isolated from a stork, or surely this poor bird would be blamed not only for his big bill but also for transmitting the bacterium to newborn infants (Gray and Killinger, 1966). The link to food was made in 1981 after an epidemic in Canada involving 41 people (34 perinatal and 7 adult) was linked to consumption of prepackaged ready-to-eat coleslaw. A sample of coleslaw from a patients refrigerator was contaminated with the epidemic strain, and the cabbage was traced to a farm that fertilized with manure from a flock of sheep that had two members die of Listeriosis (Schlech et al., 1983).

8 More recently, an outbreak of Listeriosis in Canada in the summer of 2008 resulted in 57 confirmed cases and 22 deaths. The outbreak was traced to contaminated meat from the processing plant of Maple Leaf food products and caused a massive nationwide recall of 220 products from the company (Austen, 2008; Canada, 2009). In addition to epidemiological correlations, molecular genetics and phylogenetics show that L. monocytogenes evolved to invade the intestinal epithelium (see below). The emergence of the AIDS epidemic was also a major factor influencing the increase in incidence of Listeriosis in the 1980s. As an intracellular pathogen, L. monocytogenes avoids a humoral immune response, and antibodies are not protective against L. monocytogenes (Cerny et al., 1988; Miki and Mackaness, 1964). Rather clearance of L. monocytogenes requires components of cell-mediated immunity including neutrophils and activated macrophages, and protective immunity requires CD8 T-cells (Pamer, 2004). A majority of adults with Listeriosis have underlying conditions that suppress T-cell or other cellular immune responses (Farber and Peterkin, 1991; Vazquez-Boland et al., 2001). These include leukemias, lymphomas, chemotherapy, immunosuppressant therapy, cirrhosis of the liver, alcoholism, kidney disease, diabetes, lupus, advanced age, and HIV infection. HIV as a predisposing factor accounts for as much as 20% of adult Listeriosis (Farber and Peterkin, 1991; Vazquez-Boland et al., 2001). That it is not higher is probably due to frequent treatment of AIDS patients with antimicrobials for numerous infections (Farber and Peterkin, 1991). These data also suggests that L. monocytogenes should be considered an opportunistic pathogen, targeting the very young, the very old and the immune compromised. The corollary is that invasive disease may be a distraction from the real or evolved natural biology of Listeria infection, which probably includes subclinical carrier states in as yet unrecognized natural hosts.

LISTERIA IN BASIC BIOLOGICAL RESEARCH Immunologists were interested in L. monocytogenes long before the emergence of the organism as a public health risk. Initially, L. monocytogenes-induced monocytosis in rabbits was used to investigate the origin and development of monocytes (Gray and

9 Killinger, 1966). Since the 1960s, L. monocytogenes has been used as a model intracellular parasite and was instrumental in understanding innate and protective cell mediated immunity, including the roles of T-cells and activated macrophages in intracellular parasite clearance (Mackaness, 1962, 1969; Miki and Mackaness, 1964; North, 1970, 1978; Pamer, 2004; Shaughnessy et al., 2007). In the past 20 years, with the increasing power of biochemical and genetic approaches, L. monocytogenes has contributed to molecular dissection of intracellular (e.g. TLRs, NODs, NFB, Caspase-1, Myd88) and intercellular (e.g. CCL2, TNF, IFN-, IFN-) immune signaling (Pamer, 2004). In the 1990s and 2000s, L. monocytogenes emerged as a tool for studying cell biology (Hamon et al., 2006). (There are now numerous books and journals devoted to this approach; e.g. Cellular Microbiology and Cell Host and Microbe.) Stanley Falkow has stated that bacteria are the worlds best cell biologists and Julie Theriot writes on her lab website, we spy on them [L. monocytogenes] as they've spied on cells, trying to learn what they know. For example, the ability of L. monocytogenes ActA to polymerize actin forming a propulsive actin comet tail has shed great light on mechanisms of eukaryotic cell motility and cytoskeletal force generation, the biomechanics and biochemistry of actin polymerization, and the physical properties of the eukaryotic cytosol (Auerbuch et al., 2003; Cameron et al., 2000; Chakraborty et al., 1995; Dabiri et al., 1990; Domann et al., 1992; Kocks et al., 1992; Lacayo and Theriot, 2004; Niebuhr et al., 1997; Rafelski and Theriot, 2002, 2004; Robbins et al., 1999; Shenoy et al., 2007; Skoble et al., 2000; Tilney and Portnoy, 1989). The ability of L. monocytogenes InlA to bind the junctional protein E-cadherin has shed light on the components and function of the intercellular junctions. For example, ARHGAP10 (Rho GTPase-activating protein 10) was found to be necessary for InlA mediated bacterial invasion and then shown to be a novel regulator of the epithelial junctions (Sousa et al., 2005a). A second Invasin, InlB targets c-Met, a receptor kinase, to induce bacterial uptake by Clathrin-mediated endocytosis and has been used to study c-Met trafficking (Li et al., 2005; Veiga and Cossart, 2005). We believe that the study

10 of InlA/InlB mediated Listeria invasion will provide a fuller understanding of how intercellular junctions are regulated by endocytosis.

INTRACELLULAR PARASITISM Although some normally extracellular bacteria are capable of survival and replication within the cytosol of cells, e.g. when given access by microinjection, it should not be assumed that the cytosol is necessarily hospitable (Goetz et al., 2001). Although little is known about the specific chemical makeup of the cytosol, intracellular bacteria have taught us that the cytosol is a reducing environment limiting in free iron and aromatic amino acids (Ray et al., 2009). Nor is the cytosol necessarily a protective environment. Intracellular bacteria must find a new niche before significant intracellular immune detection or host cell killing that would expose the bacteria to humoral and inflammatory host responses. Thus, intracellular bacteria like Shigella flexneri, Burkholderia pseudomallei, Listeria monocytogenes, Francisella tularensis and Rickettsia species have evolved mechanisms to invade cells, escape the primary vacuole, acquire nutrients, modulate intracellular immune detection, and in some cases spread directly to neighboring cells, avoiding exposure to the extracellular milieu (Ray et al., 2009).

STAGES AND MECHANISMS OF LISTERIAS INTRACELLULAR LIFE-CYCLE L. monocytogenes is capable of infecting phagocytic and nonphagocytic cells. Surface proteins like Internalin A (InlA) and Internalin B (InlB) bind host cell receptors and induce internalization of bacteria by nonphagocytic cells. Internalization of Listeria occurs through a so-called zipper-like mechanism where host cell plasma membrane is closely opposed to the bacterium during internalization (Figure 1.2A) (Karunasagar et al., 1994). Following initial internalization, cytosolic bacteria escape from the vacuole/endosome by secreting enzymes that disrupt the vacuolar membrane. Listeria uses the enzymes listeriolysin O (LLO) and two type C phospholipases (Figure 1.2A) (Portnoy et al., 1988; Portnoy et al., 1994; Portnoy et al., 1992; Smith et al., 1995a; Smith and Portnoy, 1993). LLO binds cholesterol in the vacuolar membrane and forms

11 pores, which serve to prevent vacuolar maturation into a lysosome and to destabilize the membrane for bacterial escape (Beauregard et al., 1997; Bielecki et al., 1990; Henry et al., 2006; Portnoy et al., 1992; Shaughnessy et al., 2006). hly, encoding LLO, is transcribed only after cell invasion (see below) and LLO is activated by acidification of the vacuole and by the host derived enzyme gamma-interferoninducible lysosomal thiol reductase (Glomski et al., 2002; Singh et al., 2008). Following vacuolar disruption LLO is rapidly degraded in the cytosol (Glomski et al., 2003; Glomski et al., 2002; Schnupf et al., 2006; Schnupf et al., 2007). This temporal and spatial compartmentalization of LLO expression and activity prevents disruption of cell plasma membranes that would cause cytotoxicity and expose Listeria to the extracellular environment (Glomski et al., 2003; Schnupf and Portnoy, 2007). The secreted phosphatidylinositol-specific phospholipase C (PI-PLC, encoded by plcA) functions with LLO to disrupt the single membrane primary vacuole (Figure 1.2A). Some cytosolic bacteria like Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Rickettsia spp., Mycobacterium marinum and viruses like Vaccinia virus have evolved mechanisms to hijack the host cell cytoskeleton for intracellular and intercellular motility. Either by expressing proteins that directly bind actin, or by expressing proteins that bind actin nucleators, these microbes polymerize and elongate actin filaments to generate propulsive actin comet tails (Cudmore et al., 1995; Ray et al., 2009; Stamm et al., 2003). Upon entry into the cytosol L. monocytogenes expresses the surface protein ActA, which directly binds actin (Figure 1.2A) (Kocks et al., 1992; Theriot et al., 1992; Tilney and Portnoy, 1989). Listeria actin comet tail formation requires ActA concentrated on a single pole of the bacterium (Kocks and Cossart, 1993; Kocks et al., 1993; Smith et al., 1995b). This occurs only after intracellular L. monocytogenes have undergone a few rounds of replication since polarization of ActA is linked to new ActA synthesis and to cell wall growth (Moors et al., 1999; Rafelski and Theriot, 2005, 2006). Thus, ActA expression and actin tail formation is also a good indication of bacterial viability in the cytosol. When actin comet tails propel the bacterium into host cell plasma membrane, a neighboring cell may internalize the resulting protrusion, or listeriopod (Figure

12 1.2A). Presented but unpublished research from Keith Iretons lab suggests that the virulence factor InlC may promote cell-to-cell spread by interacting with the apical junctions and making them more slack and permissive for protrusion formation (Engelbrecht et al., 1996; Rajabian, 2008). Successful uptake of the protrusion also requires cooperation of the recipient cell and may depend on the state of cell-cell adhesion and/or the organization of the submembranous cytoskeleton (Robbins et al., 1999). Once the protrusion is fully internalized in the recipient cell, the resultant double membrane vacuole is disrupted by LLO and phosphatidylcholine-specific phospholipase C (PC-PLC, encoded by plcB) (Camilli et al., 1993; Marquis and Hager, 2000; Smith et al., 1995a). A Listeria metalloprotease (Mpl) regulates this process by proteolytically activating PC-PLC upon acidification of the secondary vacuole (Marquis et al., 1997). Free bacteria can now repeat the intracellular infectious cycle, which is critical for Listeria pathogenesis. Loss of any stage of the intracellular infectious cycle severely attenuates Listeria pathogenicity (Portnoy et al., 2002).

GENETIC REGULATION OF INTRACELLULAR INFECTION Many of the genes required for the intracellular infection cycle of L. monocytogenes are organized in a genetic island, LIPI-1 (Figure 1.2B). Following cell invasion, this core set of genes is upregulated by the master regulator of virulence genes, Positive Regulatory Factor A (PrfA). (Chakraborty et al., 1992). PrfA also regulates some virulence genes outside LIPI-1, such as the inlAB locus, inlC and hpt (Figure 1.2B) (Chico-Calero et al., 2002; Engelbrecht et al., 1996). InlC is required for full virulence and may promote cell-to-cell spread (Engelbrecht et al., 1996; Rajabian, 2008). hpt encodes a glucose-6-phosphate translocase that allows pathogenic Listeria species to use hexose phosphates from the host cell cytosol as a carbon energy source (ChicoCalero et al., 2002). To regulate genes, PrfA dimers bind a 14-bp (7-bp invariant) consensus sequence or a PrfA-box directly upstream of promoters (Figure 1.2B). PrfAs activity is regulated on numerous levels including autoregulation of its own transcription and allosteric regulation of DNA binding activity by either a cofactor or a repressor (Scortti et al.,

13 2007). In addition, translation of prfA is temperature-dependent since a transition to 37 dissolves a secondary structure in prfA RNA that otherwise prevents ribosome binding (Scortti et al., 2007). The degree of PrfAs rgulation of a given promoter is in part determined by the degree of homology of the PrfA-box to the canonical PrfA-box sequence. For example the PrfA-box upstream of inlAB has mutations that result in only weak regulation and InlA is nearly undetectable during intracellular growth (Engelbrecht et al., 1996; Kazmierczak et al., 2003; Lingnau et al., 1995; McGann et al., 2007a; McGann et al., 2008; Scortti et al., 2007). In contrast, actA has a canonical PrfA-box and is upregulated by as much as 300-fold following cell invasion, making ActA the most abundant surface or secreted protein during intracellular growth (Figure 1.2B) (Brundage et al., 1993; Moors et al., 1999; Scortti et al., 2007; Shetron-Rama et al., 2002). Although InlA and InlB are only weakly regulated by PrfA, they are strongly regulated by the general stress response sigma factor, B (Figure 1.2B) (Kazmierczak et al., 2003; Kim et al., 2004; Kim et al., 2005). Sigma factors are dissociable protein subunits of prokaryotic RNA polymerase (RNAP) that provide promoter recognition specificity to the RNAP holoenzyme and contribute to DNA strand separation during transcription initiation. Most transcription in exponentially growing Listeria is mediated by an RNAP holoenzyme carrying the housekeeping sigma factor A, which is similar in function to E. coli 70. In contrast, B is activated is response to a variety of stresses including heat, high osmolarity, high ethanol concentrations, high and low pH, and oxidizing agents leading to transcription of the B regulon (van Schaik and Abee, 2005). B increases InlA expression in response to acid and osmotic stress simulating the intestinal environment, and B is also required for InlA/InlB expression and cell invasion in the absence of a specific stress (Kim et al., 2005; McGann et al., 2008; McGann et al., 2007b; Sue et al., 2004). prfA is partially regulated by B. A-RNAP transcribes prfA from both prfA promoters, while B-RNAP shares one prfA promoter (Figure 1.2B). In addition, PrfA regulates prfA transcription from a PrfA-box upstream of plcA (Figure 1.2B). Another gene regulated by both PrfA and B is bsh encoding a bile salt hydrolase that contributes to

14 L. monocytogenes survival within the intestinal lumen and fecal shedding in a guinea pig model of oral infection (Dussurget et al., 2002; Kazmierczak et al., 2003). Thus, it appears that the core virulence genes are regulated as two sets. First, the genes required for survival in the gastrointestinal tract and needed in preparation for cell invasion (inlA, inlB, bsh) are regulated by B and partially regulated by PrfA. The second set includes the genes required for intracellular parasitism (hly, mpl, plcA, plcB, actA, hpt, inlC), which are strongly regulated by PrfA, but not influence by a stress response. Given this model, it is tempting to speculate that a third, independent set regulated by B, but not PrfA, might be important for L. monocytogenes in an environmental reservoir or during noninvasive persistence in the gastrointestinal tract. This set includes the genes encoding the surface internalins InlC2, InlD, and InlE, which have not been found to be important for invasive disease (Dramsi et al., 1997; Kazmierczak et al., 2003).

Figure 1.2: Molecular and Genetic Requirements for Listerias Intracellular LifeCycle (Following Page) (A). Inside, a cartoon depicting key Listeria proteins and stages in the intracellular life-cycle of L. monocytogenes, which include entry, escape from a vacuole, actin nucleation, actin-based motility, and cell-to-cell spread. Outside, electron micrographs from which the cartoon was derived (Tilney and Portnoy, 1989). Figure from (Portnoy et al., 2002). (B) Physical and transcriptional organization of Listeria pathogenicity island-1 (LIPI-1), genes the inlAB operon, and the inlC and hpt monocistrons. PrfAboxes are indicated by black squares, known promoters indicated by P and transcripts are indicated by dotted lines. Adapted from a figure in (Scortti et al., 2007) and data in (Garner et al., 2006; Kim et al., 2005; McGann et al., 2008; McGann et al., 2007b; Ollinger et al., 2009; Ollinger et al., 2008).

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16 INVASION OF NONPHAGOCYTIC CELLS ADHERENCE TO THE CELL SURFACE From the bacterial perspective, phagocytic cells are not necessarily a preferential cell type to infect since immune cells, especially activated macrophages, can kill Listeria (Shaughnessy and Swanson, 2007). ActA- mediated cell-to-cell spread allows Listeria to translocate between cell types and Listeria infects nonphagocytic cells such as hepatocytes, endothelial cells, fibroblasts and neurons. At least in tissue culture, Listeria can directly invade many non-phagocytic cell types through the interaction of surface adhesins with host cell surface receptors. Putative Listeria adhesins include Internalin A (InlA), Internalin B (InlB), InlJ, ActA, Listeria adhesion protein (LAP), P60 (Iap), Ami, FbpA, and Vip (Cabanes et al., 2005; Dramsi et al., 2004; Jaradat et al., 2003; Milohanic et al., 2001; Pilgrim et al., 2003; Sabet et al., 2005; Suarez et al., 2001; Wampler et al., 2004). Only Internalin A (InlA), Internalin B (InlB) efficiently promote invasion, while the other proteins appear to function as adhesins primarily in the absence of InlA and InlB or if overexpressed. In addition, some have known functions or spatiotemporal patterns of expression that suggests that they evolved for purposes other than invasion (e.g. p60, Ami, ActA, FbpA) (Domann et al., 1992; Dramsi et al., 2004; Kocks et al., 1992; Milohanic et al., 2001; Pilgrim et al., 2003). InlA is necessary and sufficient for invasion of epithelial cells (Gaillard et al., 1991; Lecuit et al., 1997; Mengaud et al., 1996). While InlB promotes invasion of multiple cell types including epithelial cells, endothelial cells, fibroblasts and hepatocytes (Banerjee et al., 2004; Copp et al., 2003; Dramsi et al., 1995; Greiffenberg et al., 1998; Ireton et al., 1999; Li et al., 2005; Lingnau et al., 1995; Marino et al., 2002; Marino et al., 1999; Niemann et al., 2007; Parida et al., 1998; Shen et al., 2000). INTERNALIN A, INTERNALIN B AND THE INTERNALIN FAMILY InlA and InlB are expressed from adjacent genes transcribed both independently and biciscronically from the inlAB locus (Figure 1.2B) (Gaillard et al., 1991). They were identified in a genetic screen of L. monocytogenes transposon-insertion mutants unable

17 to invade the enterocyte-like colon carcinoma cell line Caco-2 (Gaillard et al., 1991). In the study, InlA was found to be necessary for attachment and invasion, and InlA was sufficient to reconstitute invasion when expressed in the non-invasive species L. innocua. Southern Blot analysis with an inlA-based probe suggested that inlA and inlB were members of a larger highly homologous family (Gaillard et al., 1991). The family now includes at least eight additional members: inlC, inlC2, inlE, inlF, inlG, inlH, inlI, and inlJ. In addition, there are also at least 15 Internalin-like genes identified through genomic analyses (Figure 1.3A) (Bierne and Cossart, 2007; Bierne et al., 2007; Cabanes et al., 2002; Domann et al., 1997; Dramsi et al., 1997; Engelbrecht et al., 1996; Lingnau et al., 1996; Raffelsbauer et al., 1998; Sabet et al., 2008). Only InlA and InlB are well understood. The defining characteristic of Internalins is a leucine rich repeat (LRR) domain of 3 to 28 repeats of 22 amino acids each (Figure 1.3A, 1.3B). Each repeat contains a short strand and a spatially larger 310-helix and each LRR wraps in a right-handed direction to stack upon one another. The entire LRR domain takes a solenoid sickle shape with parallel stacked -strands forming the concave face and stacked 310-helices forming the convex face (Figure 1.3B, Figure 1.4A, 1.4B, Figure 1.5B) (Bierne et al., 2007; Marino et al., 1999, 2000; Schubert and Heinz, 2003). In addition, each repeat is rotated ~5 degrees with respect to its predecessor giving the sickle-shaped solenoid a superhelical twist (Figure 1.4A, 1.4B, Figure 1.5B (Bierne et al., 2007; Marino et al., 1999, 2000; Schubert et al., 2001; Schubert and Heinz, 2003; Schubert et al., 2002). An N-terminal cap and an Ig-Like IR domain always flank the LRR domain and it is thought that these domains stabilize the LRR domain by shielding the hydrophobic core from an aqueous environment (Schubert and Heinz, 2003). Internalin and Internalin-like proteins all have an N-terminal signal sequence suggesting that these proteins are processed to the bacterial surface by the general secretory pathway (Figure 1.3A) (Bierne et al., 2007; Rafelski and Theriot, 2006). All but InlC, a secreted Internalin, are attached to the bacterial surface, generally through a Cterminal peptidoglycan-anchoring sequence (e.g. LPXTG) or C-terminal domains that

18 associate noncovalently with the bacterial cell wall (e.g. GW domains that bind lipoteichoic acid) (Figure 1.3A) (Engelbrecht et al., 1996).

Figure 1.3: Domain Organization of Internalins and Sequence Alignments of Internalin LRR Domains (Following Page)

(A) The three families of internalins by reference to their association with the bacterial surface are as follows: I, LPXTG-internalins; II, GW- or WxL-internalins; III, secreted internalins. InlH results from a recombination event between InlC2 and InlD. Figure from (Bierne et al., 2007). (B) Sequence alignments of L. monocytogenes Internalin LRR regions for InlB, InlA, InlC, InlC2, InlD, InlE, InlF, InlG, and InlH. Asterisks show conserved Internalin LRR residues, and bars show the position and extent of bstrands 310-helices. Hydrophobic, negatively charged, and positively charged residues predicted to be surface exposed are highlighted in yellow, red, and cyan, respectively. Figure from (Marino et al., 2000).

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INTERNALIN A CO-OPTS THE EPITHELIAL JUNCTIONS Using affinity chromatography E-cadherin was identified as the cellular receptor for InlA (Mengaud et al., 1996). E-cadherin is a transmembrane cell surface glycoprotein and the dominant adhesion molecule of epithelial adherens junctions (Figure 1.4A) (D'Souza-Schorey, 2005; Hartsock and Nelson, 2008). Like other classical cadherens (N, P, and R-cadherin), E-cadherin contains five Ig-like extracellular domains (ECs), and the most N-terminal E-cadherin EC1 makes a trans-pairing interaction with Ecadherin on adjacent cells (Hartsock and Nelson, 2008). Internalin A co-opts Ecadherin by binding EC1 within the concave face the LRR domain (Figure 1.4A, 1.4B) (Schubert et al., 2002). The E-cadherin-E-cadherin interaction is Ca2+ dependent as is the interaction between InlA and E-cadherin (Schubert et al., 2002). Each Ecadherin-E-cadherin interaction is relatively weak, Kd = 720 M (Haussinger et al., 2004). The InlA-E-cadherin interaction is ~100X stronger with a Kd = 8+/-4 M (Wollert et al., 2007a; Wollert et al., 2007b). The total strength of cell-cell or Listeriacell adherence is due to the high density of the individual protein-protein interactions, like a molecular Velcro. Although InlA binds the extracellular domain of E-cadherin, it is the function of the intracellular domain that is required for bacterial uptake. It appears that many, if not all of the intracellular components required for maintaining the integrity, tension or endocytic recycling of the intercellular junctions are also involved in generating the forces that reorganize cell membrane and internalize the bacterium. Experiments with cytochalasin first demonstrated that Listeria internalization requires a functional actin cytoskeleton (Wells et al., 1998). The cytoplasmic domain of E-cadherin dynamically interacts with the actin cytoskeleton through interactions with - and -catenin and both of these proteins are recruited to the site of bacterial attachment (the endocytic cup) and are required for internalization (Drees et al., 2005; Hartsock and Nelson, 2008; Lecuit et al., 2000; Yamada et al., 2005). p120, which binds the juxtamembrane region of E-cadherin and regulates E-cadherin stability at the junctions, is also recruited to the endocytic cup (Hartsock and Nelson, 2008; Lecuit et al., 2000). A study of InlA-dependent invasion identified ARHGAP10 as a novel regulator of -

21 and -catenin at cell-cell junctions, possibly through regulation of RhoA and CDC42 (Sousa et al., 2005a). Myosin VIIA and its ligand Vezatin, which generate tension required to hold cells together, were found to be involved in Listeria internalization (Sousa et al., 2004). Hakai, a ubiquitin ligase involved in Clathrin-dependent Ecadherin internalization is recruited to the site of Listeria invasion and is required for InlA mediated internalization (Bonazzi et al., 2008; Fujita et al., 2002). Finally, Ecadherin can be internalized through Clathrin-dependent and Caveolin-dependent pathways and InlA mediated invasion also utilizes both for efficient invasion (Bonazzi et al., 2008). All combined, these results suggest that regulation of E-cadherin stability at the membrane and Listeria binding and internalization via E-cadherin are mechanistically related.

Figure 1.4: Structural and Molecular Aspects of InlA/E-cadherinmediated Listeria Invasion (Following Page) (A) Bacterial surface attached InlA binds the N-terminal EC1 domain of the transmembrane junctional protein E-cadherin. Figure from (Schubert et al., 2002). (B) Ribbon and Space fill model of Wt InlA- human E-cadherin. Figure from (Schubert and Heinz, 2003). (C) Detailed View of the Interactions between InlA and hEC1. All residue side chains involved in direct interactions or as ligands to bridging ions/water are indicated in ball-and-stick representation. InlA strands and adjacent coils are shown in violet. Figure from (Schubert et al., 2002). (D) View of the hydrophobic pocket in InlA, which accommodates P16 of hEC1. Hydrogen bonds are indicated by green dotted lines. In mice, E-cadherin P16 is replaced by glutamate (yellow model). Figure from (Schubert et al., 2002). (E) Superposition of InlA-hEC1 and InlAm-mEC1 complexes. Figure from (Wollert et al., 2007b). (F) The carboxylate of E16 mEC1 (yellow) occupies the same hydrophobic pocket of InlAm as P16 hEC1 (violet) in InlA. Figure from (Wollert et al., 2007b). (G) Partial alignment of E-cadherin sequences from various species. Note critical residue 16. (H) Cartoon diagram of molecular components involved in formation of the adherens junction (AJ). Figure from (Ireton, 2007). (I) Cartoon diagram of molecular components involved in InlA-mediated Listeria entry. Proteins or domains known to contribute to both uptake of Listeria and AJ formation appear in yellow. Molecules that promote bacterial entry, but are not yet known to participate in AJ formation are in orange. Proteins that regulate AJ assembly, but have not yet been directly demonstrated to participate in internalization of Listeria, are in green. Figure from (Ireton, 2007).

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INTERNALIN B CO-OPTS GROWTH FACTOR SIGNALING InlB promotes L. monocytogenes invasion of cells by binding the extracellular domain of c-Met, a receptor tyrosine kinase (Banerjee et al., 2004; Copp et al., 2003; Dramsi et al., 1995; Greiffenberg et al., 1998; Ireton et al., 1999; Li et al., 2005; Lingnau et al., 1995; Marino et al., 2002; Marino et al., 1999; Niemann et al., 2007; Parida et al., 1998; Shen et al., 2000). Although InlB is unrelated by sequence or structure to the endogenous c-Met ligand, Hepatocyte Growth Factor (HGF), it acts as an exogenous c-Met agonist. Binding of InlB to c-Met results in phosphorylation and ubiquitination of c-Met, leading to recruitment of Clathrin, protein adaptors such as Gab1, Shc or Cbl, and activation of type I phosphatydylinositol 3-kinase (PI3K) (Ireton, 2007; Ireton et al., 1996; Ireton et al., 1999; Li et al., 2005; Shen et al., 2000; Veiga and Cossart, 2005; Veiga et al., 2007). PI3K can regulate Rac, Cofilin and the Arp2/3 complex, which control cytoskeletal dynamics and are required for bacterial uptake (Bierne et al., 2001; Ireton et al., 1999). c-Met is a disulfide-linked two-chain heterodimer that is initially translated as a 1390 amino acid precursor. The c-Met precursor polypeptide is cleaved between residues 307 and 308 to yield a small, extracellular chain and a large, multidomain transmembrane chain. The chain and amino acids 308514 of the chain form the N-terminal semaphorin (Sema) domain, which is the binding site for HGF. The extracellular portion of the chain also contains a small cysteine-rich domain and four Ig-like domains (Ig1Ig4). A transmembrane helix links the extracellular portion of cMet to the cytoplasmic juxtamembrane and tyrosine kinase domains (Niemann et al., 2007). Biochemical studies of InlB / c-Met binding demonstrated that InlB it does not compete with HGF for c-Met binding, yet both agonists result in seemingly identical kinase signaling and endocytic trafficking of c-Met (Li et al., 2005; Shen et al., 2000). A recent crystal structure of InlB bound to c-Met shows that InlB acts as a molecular clamp that forces the flexible Met receptor into a signaling-competent conformation (Figure 1.5A) (Niemann et al., 2007). Like InlA, the concave surface of the LRR domain is the binding interface in InlB (Figure 1.5B). InlB makes two important

24 interactions with c-Met via two interfaces: The InlB LRR region and Met Ig1 are the primary interface (Figure 1.5B, 1.5C, 1.5D), while a secondary less extensive contact involves the InlB IR region and the Sema domain of c-Met (Figure 1.5B). The primary binding interface approximately encompasses residues 599-660 in c-Met. The residues in c-Met thought to be critical for binding are indicated in Figure 1.5C, 1.5D (red and pink) and shown in alignments with arrows in Figure 1.5E. Other receptors for InlB have been identified, but play only supporting roles in promoting invasion. Negatively charged cell surface heparin sulfate proteoglycans (HGPGs) can bind the InlB GW domains and promote invasion. The GW domains noncovalently anchor InlB to lipoteichoic acid in the bacterial cell wall (Jonquieres et al., 1999; Marino et al., 2002). It is thought that HSPGs on the host cell surface might locally displace InlB from adherent bacteria, thereby presenting InlB to c-Met (Banerjee et al., 2004; Ireton, 2007). Using affinity chromatography gC1qR, the receptor for complement component C1q, was also found as a receptor for the InlB GW domains (Braun et al., 2000). gC1qR may also present InlB to c-Met. However, there is no conclusive role for gC1qR in Listeria invasion (Ireton, 2007). Figure 1.5: Structural Aspects of InlB/c-Met-mediated Listeria Invasion (Following Page)

A) HGF and InlB non-competitively bind and induce conformational changes in c-Met promoting kinase signaling. Figure from (Veiga and Cossart, 2007). (B) The GW domains of cell-dissociated InlB induce clustering via interaction with heparin sulfate proteoglycans (HSPGs) on the host cell. Adapted from a Figure in (Niemann et al., 2007). (C) Close-up showing Y170 and Y214 of InlB interacting with K599 and K600 of c-Met. Y170 makes hydrogen bonds (dotted orange lines) to K599 and the R602 side chain. Intra- and intermolecular salt bridges (dotted purple lines), hold the side chains of K599 and K600 in place. Figure from (Niemann et al., 2007). (D) Side chains of residues from strands C, F, and G of the c-Met Ig1 domain form a hydrophobic pocket into which W124 from the concave face of the InlB LRR binds. Figure from (Niemann et al., 2007). (E) Clustal X alignment of c-Met binding interface from C and D, with critical residues indicated (arrows). Boxed residues represent differences that may account for InlB insensitivity of Guinea pig and Rabbit c-Met. (F) Phosphorylation of c-Met cytoplasmic tail recruits adaptors and signaling molecules. Figure from (Ireton, 2007). (G) InlB-mediated entry. Proteins or domains shown to be involved in InlB-mediated bacterial uptake are in orange. PI 3-kinase and

25 its lipid product PIP3 might affect F-actin through (1) actin polymerization, (2) recruiting WAVE2 (3) inducing membrane association of a guanine nucleotide exchange factor (GEF) for Rac1. Figure from (Ireton, 2007).

26

SYNERGY BETWEEN INTERNALIN A AND INTERNALIN B Because InlA is covalently attached to the bacterial cell wall, it acts as an adhesin (promotes adhesion) and an invasin (promotes bacterial uptake). In contrast, InlB is only loosely associated with the bacterial surface and appears to function as an invasin, but not an adhesin (at least of epithelial cells) (Pentecost et al., 2006). InlB acts synergistically with InlA during invasion of cultured epithelial cells (Bergmann et al., 2002; Dramsi et al., 1995; Lingnau et al., 1995). However, the mechanism of synergy between the two proteins is poorly understood because the invasion pathways are often investigated independent of one another. This is generally accomplished by genetic deletion of one protein from L. monocytogenes, expression of one protein in the closely related L. innocua, which lacks internalins, or by use of beads coated with only one protein at a time. In addition, most of what we know about InlB signaling is the result of an experimental trick where InlB is made adhesive for the bacterium by artificially linking the protein to the bacterial cell wall through genetic addition of a cell wall anchor sequence (Bierne et al., 2001; Bierne et al., 2005; Braun et al., 1999; Dramsi and Cossart, 2003; Jonquieres et al., 2001; Khelef et al., 2006; Seveau et al., 2004; Seveau et al., 2007; Veiga and Cossart, 2005; Veiga et al., 2007). Yet, whether InlB signals through c-Met local to the attached bacterium, and whether InlB is dissociated, remains attached, or diffuses across an epithelium is critical to understanding how and why InlB promotes invasion. We hypothesize that InlB regulates uptake by imitating the regulatory role of RTKs on endocytosis of the epithelial junctions, and more specifically the role of c-Met in regulating E-cadherin endocytosis. For example, growth factor activation of receptor tyrosine kinases has been shown to induce macropinocytosis of E-cadherin (Bryant et al., 2007). c-Met and E-cadherin are co-endocytosed in HGF treated MDCK cells (Kamei et al., 1999). InlB mimics HGF for c-Met activation and internalization (Li et al., 2005). InlB can promote Clathrin-mediated internalization of Listeria while HGF similarly promotes Clathrin-mediated internalization of E-cadherin (Izumi et al., 2004; Veiga and Cossart, 2005; Veiga et al., 2007). c-Met signaling regulates p120, Hakai, and Clathrin, which in turn have been shown to regulate E-cadherin endocytosis or

27 InlA-mediated Listeria invasion (Bonazzi et al., 2008; Cozzolino et al., 2003; Fujita et al., 2002; Lecuit et al., 2000; Veiga and Cossart, 2005). Thus InlA and InlB should be studied in the same context to determine how Listeria invasion is mechanistically related to growth factor regulation of E-cadherin endocytosis.

SPECIES SPECIFICITIES OF INTERNALIN A AND INTERNALIN B Internalin A Does Not Bind Murine E-cadherin Although L. monocytogenes has been cultured in association with mice and rats, these species have never been found to acquire natural disease (Gray and Killinger, 1966; Lecuit et al., 1997; Murray, 1955). Furthermore, it has long been recognized that mice and rats are not easily or consistently infected via the oral route. Oral infections have generally required extremely high doses, which often failed to produce lethal infection (Gaillard et al., 1996; Huleatt et al., 2001; MacDonald and Carter, 1980; Marco et al., 1992; Pron et al., 1998; Roll and Czuprynski, 1990; Zachar and Savage, 1979). Although internalins are critical for host cell invasion in tissue culture, a role for internalins in intestinal invasion could not be established until relatively recently (Lecuit et al., 2001). For example, infections of mice found no role for InlA or InlB and initial rates of translocation of the rat intestine by L. monocytogenes is low, independent of inlAB, hly or actA, and is similar to translocation by L. innocua, which lacks internalins (Gaillard et al., 1991; Gaillard et al., 1996; Pron et al., 1998). It was found that mouse epithelial cells were resistant to L. monocytogenes invasion because InlA does not bind murine E-cadherin (Lecuit et al., 1999). The species specificity of InlA was shown to depend primarily on the difference of a single amino acid in E-cadherin, the 16th, which is a proline in permissive species (human, rabbit, guinea pig) but a glutamic acid in mice and rats (Figure 1.4 G). A P16E mutation in human E-cadherin is sufficient to prevent InlA binding (Figure 2.10C, 2.10D) (Lecuit et al., 1999). Crystal structures of InlA bound with human E-cadherin then revealed the nature of the specificity: P16 of EC1 adopts a strained cis-conformation to fit in a hydrophobic pocket between -strands 6 and 7 in InlA (Figure 1.4C, D) (Schubert et al., 2002). The bulky and charged nature of glutamic acid at residue 16 in mouse and

28 rat E-cadherin prevents a close association of InlA (Figure 1.4C, D) (Wollert et al., 2007a; Wollert et al., 2007b). A transgenic mouse was developed where human Ecadherin is expressed from the promoter of the intestinal fatty acid binding protein (iFABP) gene, which is turned on in non-proliferative small intestinal enterocytes. This model demonstrated that InlA could promote L. monocytogenes invasion of the intestinal epithelium by interacting with permissive E-cadherin (Lecuit et al., 2001). We note that canine E-cadherin is also expected to bind InlA. Canine E-cadherin is identical to human E-cadherin in the first 30 amino acids, which also contains the critical proline at position 16 required for InlA interaction. Furthermore, the Ecadherin residues in closest contact with InlA (V3, I4, P5, P6, K14, P16, F17, P18, K19, Q23, K25, N27, V48, W59, E64, M92) are all conserved in the human and canine sequences (Figure 1.4G) (Schubert et al., 2002). Furthermore L. monocytogenes efficiently infects Madin Darby Canine Kidney cells, and dogs, unlike mice and rats, are susceptible to Listeriosis (Gray and Killinger, 1966; Robbins et al., 1999). Internalin B Does Not Activate Guinea Pig or Rabbit c-Met InlB has also been found to be species specific. Following intravenous inoculation of guinea pigs and rabbits, an inlB mutant exhibits no attenuation in the liver (Khelef et al., 2006). In contrast, InlB appears to promote colonization of the livers of mice given a high infectious dose intravenously (Dramsi et al., 1995; Dramsi et al., 2004; Gaillard et al., 1996; Khelef et al., 2006). It was shown that transfection of guinea pig and rabbit cells with human c-Met restores the ability of InlB to stimulate c-Met and promote Listeria invasion (Khelef et al., 2006). Thus InlB activates c-Met on human, canine mouse and rat cells but not guinea pig or rabbit (Khelef et al., 2006; Shen et al., 2000). Cow and sheep c-Met is also permissive for InlB according to unpublished data in (Disson et al., 2008). The structural basis for InlB species specificity is not known. However, there are some intriguing differences between the c-Met sequence from permissive and nonpermissive species within the InlB binding region (Figure 1.5E). For example, I639 in human c-Met is mutated to tyrosine in rabbit and leucine in guinea pig and T646 is mutated to arginine in rabbit and is absent in guinea pig. We

29 hypothesize that humanizing these residues will restore binding to InlB by rabbit and guinea pig c-Met.

ANIMAL MODELS PERMISSIVE FOR INTERNALIN A AND INTERNALIN B The humanized mouse expressing hE-cadherin in the intestinal epithelium limits the study of InlA-mediated invasion to the intestine (Lecuit et al., 2001). Furthermore, if InlB function requires InlA-mediated adhesion, this mouse model could also be insufficient to establish a role for InlB at extra-intestinal sites. For example, a role for InlB in crossing the fetoplacental barrier was not found in mice even though mice are permissive for InlB (Le Monnier et al., 2007). However, a second humanized transgenic mouse was recently developed by knocking-in murine E-cadherin with E16P mutation (Disson et al., 2008). Both InlA and InlB were implicated in fetoplacental infection in this model where InlA is adhesive. Although this model appears to be sufficient to study InlA and InlB in the same context, we are concerned by the fact that mE-cadherinE16P binds InlA with a lower affinity (96 M) than hEcadherin (8 M) (Wollert et al., 2007b). It was recently found that gerbils are permissive for InlA and InlB functions (Disson et al., 2008). However, in contrast to mice, this model lacks the power of forward genetics and also lacks well characterized reagents. Rather than making permissive mice, we were interested in generating L. monocytogenes strains with InlA mutations that would allow binding to murine Ecadherin. Our efforts are detailed in Chapter 3. In addition, a recent independent effort in Germany recently succeeded in generating a mouse-adapted InlA. In designing mutations that would increase the binding of InlA to human E-cadherin, InlA S192N Y369S (InlAm) was found to have an equivalent binding affinity for mouse E-cadherin (Kd = 10+/-2 M) as wild type InlA has for human E-cadherin (8+/-4 M) (Figure 1.4E) (Wollert et al., 2007b). The S192N mutation displaces a water molecule and introduces a direct hydrogen bond between N192 in InlA and F17 in human EC1. As an unexpected consequence, S192N allows E16 of mEC1 to adopt a relaxed trans conformation and the carboxy group of E16 of mEC1 occupies the same hydrophobic

30 pocket of InlA as P16 of hEC1 in InlA/hEC1 (Figure 1.4E, 1.4F) (Wollert et al., 2007a; Wollert et al., 2007b). The second mutation improves overall binding at the major interface of the InlA-E-cadherin interaction. Y369S replaces the bulky tyrosine sidechain with a serine, which makes a water-mediated hydrogen bond to N27 in EC1 (Wollert et al., 2007a; Wollert et al., 2007b). The binding of InlAm and murine Ecadherin promotes oral infection of mice through the intestinal epithelium (Wollert et al., 2007b).

TISSUE SPECIFICITIES OF INTERNALIN A AND INTERNALIN B Loss of Internalin A abrogates intestinal invasion of guinea pigs, but has no effect on pathogenesis when mutant bacteria are administered intravenously (Lecuit et al., 2001). Whether InlB also promotes efficient invasion of the intestine in permissive hosts has implications for the success of Listeria invasion and colonization at this site. However, InlB has not been found to be important for intestinal invasion, but rather for colonization of mouse livers after intravenous infection (Dramsi et al., 2004; Khelef et al., 2006). Furthermore InlB did not appear to affect invasion of the intestine of transgenic mice expressing human E-cadherin in the intestine (Khelef et al., 2006). Thus, the current dogma is that InlA and InlB have evolved to target different tissues at different stages of infection: InlA is required for intestinal infection and is subsequently dispensable for invasive disease in non-pregnant animals, while InlB primarily mediates invasion of other tissues, notably the liver (Ireton, 2007; Schubert and Heinz, 2003).

CHALLENGING THE INTERNALIN TISSUE SPECIFICITY DOGMA There is reason to hypothesize a role for InlB in intestinal invasion. First, c-Met is present on many tissues, including epithelia suggesting that InlB may also promote infection of the gastrointestinal tract or other barriers (Di Renzo et al., 1991; Disson et al., 2008; Fukamachi et al., 1994; Ishikawa et al., 2001; Kato et al., 1997a, b; Neo et al., 2005; Nusrat et al., 1994; Sunitha et al., 1999; Wormstone et al., 2000). Indeed, as mentioned above, InlB appears to play a role in crossing of fetoplacental barrier after

31 intravenous inoculation, but only with the coexpression of a functional InlA (Disson et al., 2008). Second, InlA and InlB are coregulated and are upregulated in the intestinal tract and under conditions simulating the gastrointestinal tract (Kim et al., 2005; McGann et al., 2007b; Sue et al., 2004; Toledo-Arana et al., 2009). Third, InlB promotes infection of cultured epithelial cells, including primary intestinal epithelial cells from permissive animal models, like gerbils (Disson et al., 2008).

ANATOMICAL AND SUBCELLULAR SITE OF EPITHELIAL INVASION: THE POLARITY PARADOX THE EPITHELIAL BARRIER, CELL RENEWAL AND GASTROINTESTINAL PATHOGENS The gastrointestinal epithelium fulfills two seemingly incompatible tasks. On the one hand, it maintains a tight epithelial barrier that controls fluid and solute transport, separates the external (lumenal) environment from the internal (interstitial) environment, and prevents invasion of potentially harmful microbes. The tight and adherens junctions that comprise the apical junctional complex (AJC) are the intercellular glue that maintains this barrier (Anderson et al., 2004; Balda and Matter, 1998; Laukoetter et al., 2006). On the other hand, the epithelium continuously disassembles the barrier in a conveyor belt of rapid cell renewal and cell death. 1010 cells are shed per day and a new epithelial monolayer is generated every 3-6 days as a continuous flow of cell division, differentiation, migration and cell loss along the crypt-villus axis (Bullen et al., 2006). Epithelial renewal continuously threatens the integrity of the epithelial barrier since dead cells must be removed and detached through junction disassembly followed by epithelial junction reassembly. The epithelium also needs to maintain its functions and integrity in the face of continuous exposure to potentially invasive microbes, and their metabolic products and toxins. Most bacterial-epithelial relationships in the intestine are benign, and in some cases symbiotic (the gastrointestinal lumen is thought to be home to more bacterial cells than the total number of cells in the body). However a number of invasive bacterial and viral pathogens have evolved mechanisms to breach the

32 intestinal barrier. Interestingly, many of these invasive microbes cross the epithelial barrier by utilizing cellular receptors that are found only on the basolateral membrane of the epithelial cell, and thus should not be available on the lumenal surface of an intact epithelium since the AJC also allows cells to separate the plasma membrane into distinct apical versus basolateral domains. For example, rotaviruses and Yersiniae bind integrins, a class of adhesion and signaling molecules found solely on the basolateral sides of enterocytes (Guerrero et al., 2000; Isberg and Leong, 1990).

E-CADHERIN AND C-MET ARE BASOLATERAL RECEPTORS L. monocytogenes receptors E-cadherin and c-Met are also basolateral proteins that co-localize at the adherens junction, below the epithelial tight junction (Boller et al., 1985; Boyle and Finlay, 2003; Crepaldi et al., 1994; Nusrat et al., 1994; Sousa et al., 2005b). Even prior to identification of these receptors, it was known that L. monocytogenes invades polarized epithelial cells most efficiently from the basolateral side (Gaillard and Finlay, 1996; Temm-Grove et al., 1994). For example, when cultured epithelial cells are plated sparsely and grown as small islands of cells, the edge cells do not have a continuous tight junction to prevent mixing of apical and basolateral proteins and L. monocytogenes preferentially infects these cells (Gaillard and Finlay, 1996; Temm-Grove et al., 1994). Some cultured epithelial cell lines will become more differentiated and polarized over time in culture and L. monocytogenes invasion of confluent monolayers of Caco-2 cells decreases with epithelial monolayer maturity. The epithelial cell-to-cell junctions are Ca2+-dependent and disrupting the intercellular junctions with calcium chelators, there by exposing lateral cell membranes, increases L. monocytogenes invasion of polarized epithelial monolayers (Gaillard and Finlay, 1996). These data present a paradox that L. monocytogenes has evolved to use receptors that are not accessible during infection of the gastrointestinal epithelium from the lumen.

33 THE PEYERS PATCH PARADIGM The prevailing hypothesis to explain the discrepancy between receptor localization and the site of infection has been that enteric pathogens may not directly enter enterocytes from the lumenal side. Rather, they first invade the intestine by taking advantage of the function of M-cells (Clark and Jepson, 2003). M-cells are modified epithelial cells overlying the intestinal lymphoid follicles known as Peyers patches (PP). They are capable of phagocytosis and also express basolateral receptors on their lumenal surface. M-cells normally function in immune surveillance by engulfing lumenal antigens and presenting them to dendritic cells and macrophages found underneath the Peyers patch epithelium. Some pathogens, like Salmonella typhi, which causes enteric fever in humans, and Salmonella typhimurium which causes a similar disease in mice, have been shown to enter through M-cells before spreading systemically through the lymphatics and blood stream (Jensen et al., 1998; Jones et al., 1994). Yersinia pseudotuberculosis expresses the protein Invasin to attach to and stimulate entry through integrin receptors (Marra and Isberg, 1997). Although integrins are found only on the basolateral surface of enterocytes, they have been detected on the apical surface of M-cells (Clark et al., 1998). In humans, Yersinia pseudotuberculosis causes a self-limited enteritis and occasionally mesenteric adenitis. In mice this pathogen is able to invade and spread systemically and cause an enteric fever-like syndrome. Similarly Shigella, which also use integrins for basolateral enterocyte invasion has been proposed to first cross the intestinal epithelial barrier through M-cells (Mounier et al., 1992; Zychlinsky et al., 1994). The same model has been proposed for Listeria monocytogenes (Gaillard and Finlay, 1996; Jensen et al., 1998; Pron et al., 1998). For example, Jean-louis Gaillard and B. Brett Finlay postulated: because invasion of these highly differentiated cells [enterocytes] is thought to be exclusively basolateral, L. monocytogenes must utilize another site of entry. This could be the M cell, as reported for other bacterial pathogens. This hypothesis is in agreement with previous results showing that listeriae given to rodents penetrate mostly into the Peyers patches (Gaillard and Finlay, 1996).

34 CHALLENGING THE PEYERS PATCH PARADIGM