ENTEROHEMHORRHAGIC ESCHERICHIA COLI O157:H7 INITIAL ADHERENCE FACTORS AND INTERACTIONS WITH THE POLYMERIC IMMUNOGLOBULIN RECEPTOR DURING ADHERENCE TO INTESTINAL EPITHELIAL CELLS

Abstract

Escherichia coli O157:H7 is the most notorious and well-studied serotype of the enterohemorrhagic E. coli (EHEC) class of E. coli intestinal pathogens. E. coli O157:H7 and other EHEC strains are responsible for multiple outbreaks across the world each year, with those afflicted suffering mainly from diarrhea, vomiting, and hemorrhagic colitis; a substantial fraction of those infected require hospitalization. Serious complications like hemolytic uremic syndrome (HUS) contribute to the significant morbidity and mortality caused by EHEC infection. Virulence varies greatly across outbreak strains and much remains to be studied regarding EHEC pathogenesis. E. coli O157:H7 remains a relevant foodborne pathogen due in part to its complexity and variety of virulence factors. Adherence mechanisms are a critical component of pathogenesis, persistence in natural reservoirs, and environmental contamination. E. coli O157:H7 has a highly effective adherence operon (the LEE-Locus of Enterocyte Effacement) and its encoded intimate adherence mechanism is well characterized. However, factors involved in initial attachment are not well understood and adhesins provide potential targets for intervention and treatment strategies.

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In this study, we describe several factors involved with the initial adherence of E. coli O157:H7 in vitro. Primarily, we describe a bacterial protein not previously reported to be involved in adherence, Slp (starvation lipo-protein); and its interactions with the human host protein polymeric immunoglobulin receptor (pIgR). Following the observation of significant colocalization phenotypes by immunofluorescence microscopy, a co-immunoprecipitation (Co-IP) assay was done with a human recombinant Fc-tagged pIgR protein and E. coli O157:H7 proteins, which led to the identification of a bacterial protein (Slp) attaching to the pIgR. Slp is a small lipoprotein found in E. coli and Shigella species. Disruption of Slp expression through deletion of its encoding gene slp in E. coli O157:H7 produced a significant adherence deficiency to Caco-2 cells, especially at early time points associated with initial adherence. Plasmid complementation of the slp gene fully restored the wild-type phenotype Furthermore, immunofluorescence microscopy revealed evidence that this interaction is specific for extra-intestinal pathogenic strains of E. coli and specific for adherence to human colonic cells in vitro. Additionally, deletion of slp gene resulted in the absence of the corresponding protein band in further Co-IP assays, with the slp complemented mutant strain restored the wild-type binding behavior. These data support the proposition that Slp significantly and directly contributes to initial adherence, with the pIgR protein as its proposed receptor.

Table of Contents

Abstract

List of Figures

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List of Tables

List of Abbreviations

Acknowledgements

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Chapter 1: Literature Review

1.1. Pathogenic Escherichia coli

1.2. Clinical Presentation and Outbreaks

1.3. Molecular Pathogenesis

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1.4. Genetics

1.5. Expression of Virulence and Adherence Factors

1.6 Acid Resistance and Gene Regulation

1.7. The Polymeric Immunoglobulin Receptor (pIgR)

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1.8. Bovine Hosts and EHEC

Chapter 2: Materials and Methods

Table 2.1. Bacterial Strains and Plasmids

2.2. Cell Culture and Media

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2.3. Infection

2.4. Microscopy

2.5. Adherence

2.6. RNA Preparation

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2.7. Quantitative PCR (qPCR)

2.8. RNA Sequencing

2.9. Gene Deletions and Complementations

Table 2.2. Primer Sequences

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2.10. Co-immunoprecipitation (Co-IP) and Protein Gels

2.11. Protein Identification

Chapter 3: pIgR and Slp

3.1. Introduction

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3.2. Results

3.3. Discussion

Chapter 4: RNA Sequencing and Gene Deletions

4.1. Introduction

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4.2. Results

4.3. Discussion

Chapter 5: Summary and Future Directions

5.1. Summary and Future Work

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Appendix

Appendix A. Additional Materials and Methods

Appendix B. Colocalization and Statistics

Appendix C. List of 686 genes upregulated in E. coli  O157:H7 during adherence to Caco-2 cells.

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References

List of Figures

Figure 3.1. Initial Adherence Timeline in E. coli O157:H7

Figure 3.2. Colocalization and Covariance of pIgR with E. coli K12 and E. coli O157:H7 Over Time

Figure 3.3. Colocalization and Covariance of pIgR with Pathogenic E. coli Strains

Figure 3.4. Relative Gene Expression Profiles and Growth Rates of ……..???

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Figure 3.5. Co-immunoprecipitation of E. coli O157:H7 Proteins With Human Recombinant Fc-Tagged pIgR Protein

Figure. 3.6. Co-Immunoprecipitation of Fc-Tagged pIgR Protein with E. coli O157:H7 Δslp and E. coli O157:H7 Δslp(pKD3::slp) Strains

Figure 3.7. Colocalization and Covariance of pIgR with E. coli O157:H7 Δslp and E. coli O157:H7 Δslp(pKD3::slp) Strains

Figure 3.8. Adherence of E. coli Strains (what strains??) to Caco-2 Cells

Figure 3.9. Bovine Intestinal Cells and the pIgR (should be more descriptive)

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Figure 4.1. Heat Map of E. coli O157:H7 Gene Expression During Initial Adherence

Figure 4.2. Quantitative Adherence and Growth Curves of E. coli O157:H7 Strains

List of Tables

Table 1.1. Select Signals Affecting Virulence Gene Expression in E. coli O157:H7 Within the Host

Table 1.2. AFI???? Genes

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Table 2.1. Bacterial Strains and Plasmids used in the Study

Table 2.2. Primer Sequences Used in the Study

Table 3.1. Protein Identification Using LC MS/MS???

Table 4.1. Genes Upregulated in E. coli O157:H7 Adhered to Caco-2 Cells

List of Abbreviations

AFI: Acid fitness island

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AR: Acid resistance

Co-IP: Co-immunoprecipitation

EHEC: Enterohemorrhagic Escherichia coli

IEC: Intestinal epithelial cell

LC MS/MS: Liquid chromatography tandem mass spectroscopy

LEE: Locus of enterocyte effacement

MOI: Multiplicity of infection

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pIgR: polymeric immunoglobulin receptor

pIgR-Fc: Fc-tagged human recombinant polymeric immunoglobulin receptor protein

RAJ: recto-anal junction

RSE: Recto-anal junction squamous epithelial cells

T3SS: Type III secretion system

Acknowledgements

Chapter 1: Literature Review

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1.1. Pathogenic Escherichia coli

1.1.1. Identification of Escherichia coli and its Pathogenic Varieties (Pathovars)

In 1885, German physician and bacteriologist Theodor Escherich first described a bacterium isolated from the digestive tracts of ill children, which he called Bacillus communis coli. In 1919, after his death, it was renamed Escherichia coli to acknowledge Escherich’s contributions to bacteriology. Because of its easy propagation and versatility in the laboratory, it has become one of the most widely studied and well-characterized bacterial species and is considered a model organism in microbiology. Most E. coli strains are harmless or beneficial commensal gastrointestinal (GI) organisms in the microflora of humans and animals, but there are pathogenic varieties. Pathogenic E. coli can be classified as either extraintestinal (ExPEC) or diarrheagenic pathotypes. ExPEC cause diseases outside of the GI tract,  such as urinary tract infections and neonatal meningitis, while diarrheagenic E. coli cause a spectrum of GI illnesses (Stenutz, Weintraub, & Widmalm, 2006). Currently, there are five commonly referenced classifications of diarrheagenic E. coli: enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC), enterotoxigenic E. coli (ETEC), and enterohemorrhagic E. coli (EHEC) (Clements, Young, Constantinou, & Frankel, 2012) [Pangenome Structure of Escherichia coli 2008]?? Several other unstandardized classifications have come into use, such as diffusely adherent E. coli (DAEC) and adherent invasive (AIEC) (Croxen et al., 2013). The EHEC classification is used to describe a subset of diarrheagenic E. coli pathovars that produce hemorrhagic colitis during infection. In literature, it is common to see enterohemorrhagic E. coli (EHEC), Verotoxin-Producing E. coli (VTEC), and Shiga Toxin-Producing E. coli (STEC) used interchangeably; however, these terms may sometimes be used to denote subtle differences between pathovars. For the purposes of <clarity in this writing> three genes are used to define each type: Shiga toxin (Stx) genes stx1 and stx2, and adhesin gene eae. STEC and VTEC (used here as synonyms) refer to any E. coli strain that produces one or both types of Stx. Within this classification of STEC, there are pathovars that also contain the gene eae, and any E. coli strain that is positive for stx1 and/or stx2 and for eae are referred to as EHEC. EHEC strains cause disease in humans and some animals, with prototypical symptoms range from diarrhea to hemorrhagic colitis, but can include many other and more serious complications (Etcheverría, Padola, & Stec, 2013). EHEC serotype O157:H7 is the most well-known and best-studied EHEC strain and is the main subject of this study.

1.1.2. EHEC Biology and Characteristics

E. coli is a species within the family Enterobacteriaceae, a common family in the intestinal microflora. Like all E. coli, EHEC serotype O157:H7 is Gram-negative, facultatively anaerobic, motile, and stress tolerant (Croxen et al., 2013). It is also metabolically versatile, which allows it to survive and/or grow in harsh habitats with a variety of challenges (nutrient and oxygen-poor conditions, low temperature, low pH, exposure to antimicrobial agents, etc.). Conversely, this bacterium is well-adapted to thrive in a variety of host environments while competing with commensal organisms and immune responses. E. coli O157:H7 is also able to produce anti-microbial compounds, such as colicins that inhibit the growth of competing bacterial species (including other E. coli) creating additional survival and growth advantages.

Among other characteristics as a Gram-negative species, the E. coli O157:H7 cell membrane is composed of peptidoglycan and lipopolysaccharide (LPS or endotoxin). The combined membrane structure is composed of three different layers: the cytoplasmic or inner membrane (IM), the peptidoglycan layer, and the outer membrane (OM) (Chatterjee & Chaudhuri, 2012). The IM layer is composed of a phospholipid bilayer adjacent to the cytosol, with assorted trans-membrane proteins. The peptidoglycan layer is located in the periplasmic space, between the IM and OM (Schwechheimer & Kuehn, 2015). The OM is also a bilayer, with the periplasm-adjacent layer being composed of phospholipids like those found in the IM, but also containing three components of LPS: lipid A, the core polysaccharide region, and the O antigen polysaccharide chains (Chatterjee & Chaudhuri, 2012). Lipid A, which serves as an anchor for the core polysaccharides and O antigens, is highly conserved among E. coli strains, and is the toxic component of endotoxin (Frirdich & Whitfield, 2005). The core region of the OM is responsible for maintaining a barrier between the cell’s interior and its environment, with some specific subtypes being associated with higher virulence. The O antigens share a basic structure, but contain enough variability to be used to classify E. coli strains using serology. O antigens are chains of approximately 10 to 25 repeating sugar residues, with a range of two to seven residues in a chain (Stenutz et al., 2006). The type (or absence) of the H antigen (flagellar proteins) is combined to define E. coli serotypes using the Kauffman method. Anti-sera that contain antibodies specific for each O and H antigen will agglutinate in a positive reaction, and the specificity of antibody-based identification makes serotyping a useful method for identifying unknown isolates and tracking outbreaks, but are also crucial in the study of virulence (Croxen et al., 2013) (Clements et al., 2012)

1.1.3. E. coli O157:H7 and Non-O157 EHEC Serotypes

E. coli O157:H7 was declared a nationally reportable food contaminant in the United States by the United States Department of Agriculture (USDA) in 1993, after a large outbreak linked to undercooked beef in fast-food restaurants. E. coli O157:H7 remains one of the most prominent causes of E. coli outbreaks, but six other EHEC serogroups have been significant causes of illness as well and are now tracked along with E. coli O157:H7: O26, O111, O103, O121, O45, and O145 (Hegde et al., 2012). Study of EHEC strains shows distinct clusters of more closely-related serotypes within the classification. It is highly likely that <at least most>?? EHEC strains originated from progenitor clones and evolved independently, and in the case of E. coli O157:H7 from an EPEC progenitor clone containing the eae (and eae-related) genes. The EHEC strains are presumed to be a case of convergent evolution, with multiple progenitors acquiring similar sets of virulence genes, such as stx genes, the plasmid pO157 (or other virulence-associated plasmids), and other virulence traits (Law, 2000). Examination of EHEC genomes using multi-locus sequence typing (MLST) concluded that O157 strains are actually more closely related to EPEC serotype O55:H7 than to other non-O157 EHEC strains (Law, 2000) (Croxen et al., 2013). The variability of sequence, virulence genes, and other traits between EHEC serotypes leads to interesting questions in the study of EHEC pathogenesis.

1.2. Clinical Presentation and Outbreaks

1.2.1. Clinical Presentation and Symptoms

After ingestion, symptoms of EHEC infection can take 1-3 days to manifest, and can be incited by the ingestion of as few as 100 bacterial cells (Melton-celsa, Mohawk, Teel, & Brien, n.d.) (Gyles, 2007). Characterized mainly by symptoms of watery diarrhea and hemorrhagic colitis, infection can also present with abdominal cramping, headache, nausea, vomiting, and fever (Hunt, 2010). Many cases require hospitalization and the infection can progress to much more serious complications. EHEC infection can strike anyone, but the most serious sequelae are primarily in children under the age of five, the elderly, and immunocompromised individuals (Mohawk & O’Brien, 2011) (Smith, Fratamico, & Gunther, 2014). According to data collected prior to 2013, 4% of overall confirmed E. coli O157:H7 cases in the U.S. progressed to hemolytic uremic syndrome (HUS), while 14% of children under the age of ten developed HUS (Croxen et al., 2013). The national mortality rate in the United States for all patients is 0.5%, while children under the age of ten had an increased mortality rate of 1-4% (Croxen et al., 2013). HUS is a serious complication resulting in kidney damage and is characterized by acute renal failure, thrombocytopenia, and microangiopathic hemolytic anemia (Farfan & Torres, 2012)(Nguyen & Sperandio, 2012). In addition to Stx, LPS  is found in the bloodstream and causes an acute inflammatory response, causing upregulation of proinflammatory cytokines and chemokines (Melton-celsa et al., n.d.). LPS is the only known ligand of Toll-like receptor 4 (TLR4), which upregulates the expression of NF-κB. NF-κB is a major transcriptional regulator in innate immunity, and the resulting increased production of proinflammatory chemokines and cytokines results in significant exacerbation of cellular and tissue pathology. This outcome is the primary reason antibiotics are not recommended for E. coli infection, as increased levels of LPS contribute to thrombosis, apoptosis, and tissue necrosis; antibiotic treatment is associated with higher risk for developing HUS and other serious sequelae (Melton-celsa et al., n.d.). Additionally, cases of permanent neurological damage and an association with increased risk for long-term complications, such as diabetes have also been reported (Caprioli, Scavia, & Morabito, 2013).

1.2.2. Outbreaks

To estimate the public health burden, it’s important to note that not all cases are reported. In the United States, E. coli O157:H7 has been responsible for more than 350 outbreaks between 1982 and 2002, and 255 outbreaks between 2003 and 2012 (Rangel, Sparling, Crowe, Griffin, & Swerdlow, 2005) (Heiman, Mody, Johnson, Griffin, & Gould, 2015). Between 17-30% of the reported illnesses resulted in hospitalizations, with 1-2% progressing to HUS, and 0.4-0.7% ended in death. Additionally, the National Outbreak Reporting System estimated over 100 EHEC outbreaks in the United States in 2009 to 2010 alone, indicating that the true number of EHEC illnesses and its public health burden is extremely underestimated (Heiman et al., 2015) (Hegde et al., 2012)(Hall et al., 2013). By itself, E. coli O157:H7 poses a significant health burden worldwide and considering the recent estimates have stated that up to 70-80% of EHEC infections are non-O157 isolates, all current EHEC cases combined constitute an underestimated and significant source of morbidity and mortality.

E. coli O157:H7 was first recognized as an outbreak-causing pathogen in 1982, after an outbreak occurred in the United States due to undercooked hamburger meat (Rangel et al., 2005), but was not made a nationally reportable pathogen until after a large multi-state outbreak in 1993 (Rangel et al., 2005). As was the 1982 outbreak, the 1993 source was undercooked beef from fast-food chain restaurants, which was linked to cases in four Midwestern states and sickened more than 700 people, with over 150 people requiring hospitalization. Approximately 7.5% of cases developed HUS, with some cases resulting in permanent kidney or brain damage, and there were four deaths (Rangel et al., 2005). As a result of this outbreak, E. coli O157:H7 became a nationally reportable food contaminant by the USDA, and also upgraded the recommended internal temperature for cooked hamburgers from 140 °F (60 °C) to 155 °F (68 °C). By the year 2000, 48 states required mandatory reporting of contamination with E. coli O157:H7.

In 2006, a highly publicized outbreak involving fresh spinach occurred, affecting 26 states, and was likely due to the proximity of the spinach fields to a cattle ranch which tested positive for O157:H7 during the investigation. Over 200 cases were reported, with three confirmed deaths linked to the outbreak.

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In 2011, a unique and highly virulent non-O157 strain of STEC caused a large outbreak in Germany. The E. coli serotype O104:H4, which involved in this outbreak infected almost 4,000 people and had a significantly higher proportion of cases developing HUS as well as a significantly higher rate of mortality. The identified strain of E. coli O104:H4 was a unique isolate due to high virulence and the lack of the intimin/tir adherence system, which led to categorize it as an EAEC instead of an EHEC strain. However, the ability of this isolate to produce Shiga toxin, a trait not characteristic of EAEC, contributed to its virulence. The appearance of this seemingly hybrid strain of pathogen raised many concerns and questions that are still being investigated.

At the time of this publication in July 2018, (at the time of this publication in July 2018)?? there is an ongoing multi-state outbreak of E. coli O157:H7 from contaminated Romaine lettuce. The Centers for Disease Control and Prevention (CDC), the U.S. Food and Drug Administration (FDA), several state organizations announced on April 10^th, 2018 that there were reports from multiple states of confirmed E. coli O157:H7 infection linked to Romaine lettuce harvested in the Yuma region of Arizona [CDC]. As of early May, this outbreak has caused reported illnesses of 172 people in 32 states, with 75 hospitalizations and one death. Of the hospitalized cases, 20 people developed HUS.

1.3. Molecular Pathogenesis

1.3.1. Ingestion and Acid Resistance

One of the most important factors in E. coli O157:H7 virulence is its acid tolerance. The primary acid resistance (AR) system used by E. coli O157:H7 (Gad system) is a major player in virulence and adherence gene regulation (see section 1.6. on acid resistance and gene regulation), and the AR functions are thought to be primarily responsible for the impressively low infectious dose required for successful infection. While other enteric bacterial pathogens, such as non-typhoidal Salmonella and Vibrio cholerae have infectious doses between 10⁵ to 10⁹ cells, respectively; E. coli O157:H7 and some Shigella species have very effective mechanisms of acid tolerance and have infectious doses as low as ~100 bacterial cells or colony-forming units (Lin et al., 1996). The human stomach has an average pH between 1.5 to 2.5, which is below the threshold required for protons to leak through the membrane into a bacterial cell (Zhao & Houry, 2010). When the internal pH reaches approximately 4.5, the cell loses its transmembrane electrical potential, resulting in a disastrous inability to import or export anything across the cell membrane, eventually causing death. This pH range also causes damage to cellular components, such as proteins and enzymes  (Tramonti, De Canio, Delany, Scarlato, & De Biase, 2006). In E. coli O157:H7, acid stress is mainly handled by one of three AR systems (AR1-AR3). The AR2 (Gad) and AR3 (Adi) are more crucial and consisted of highly effective amino acid decarboxylase/antiporter systems (Foster, 2004) (Zhao & Houry, 2010).

AR1, also known as the glucose-repressed or oxidative AR system, is not well characterized but appears to be less critical than the decarboxylase systems. AR1 study is limited but has been described as active in bacteria that have been cultured in media buffered to pH 5.5, then challenged with pH 2.5. AR1 allows the adapted bacteria to survive an acid challenge that would kill un-adapted bacterial populations (Foster, 2004). Both the cAMP receptor protein (CRP) and alternative sigma factor S (σS) are required for its activation (Foster, 2004). However, its relationship to virulence or adherence is currently unknown.

AR2, more commonly known as the Gad system, is a glutamate decarboxylase/antiporter system whose function is to use up an intracellular proton in each decarboxylase reaction and export it from the bacterial cell, ultimately resulting in increased internal pH. The Gad system has three functional components: glutamate decarboxylases, antiporters, and transcription regulation. GadA and GadB are both glutamate decarboxylase enzymes, GadC is the glutamate/ γ-amino butyric acid (GABA) antiporter, and GadE is the system expression regulator (Foster, 2004). The reaction occurs as follows (Zhao & Houry, 2010):

(COO^–)–CH–(NH₃⁺)–CH₂–CH₂–(COO^–)  +  H⁺                             (NH₃⁺)–CH₂–CH₂–CH₂–(COO^–)  +  CO₂

AR3, the Adi system, is an arginine decarboxylase/antiporter system that functions by the same mechanism shown above. It is not as thoroughly studied, as it seems less critical than the Gad system. AdiA is the arginine decarboxylase, AdiC is the arginine/agmatine antiporter, and AdiY is a system regulator (Foster, 2004).

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There are other putative AR systems in E. coli, but are not well characterized, and their role in E. coli O157:H7 pathogenesis is unknown.

1.3.2. Adherence

In order to produce the fully virulent classic presentation of severe intestinal pathology that EHEC is known for, several robust virulence factors, namely the LEE encoded adherence and virulence effectors and Stx, are utilized by EHEC. EHEC forms a particularly strong bond with the host cell using its adhesin intimin (encoded by the gene eae) and the translocated intimin receptor, Tir. The genes encoding intimate adherence components are located on the LEE pathogenicity island. Intimate adherence is only possible after the bacterium is able to establish an initial attachment allowing it to stay stably adjacent to the host cell.

To initiate and establish intimate adherence, and also to translocate other virulence effectors, the EHEC bacterial cell assembles a type III secretion system (T3SS). The T3SS functions by directly injecting factors through the host cell membrane into the cytosol (Schmidt, 2010). The proteins necessary to assemble the T3SS are located on the LEE operon. The main components of the assembly itself include the proteins EscR, S, T, U, and V (the base spanning the bacterial inner membrane); Esc J, C, and F (which span the periplasmic space and the bacterial outer membrane); EspA (the ‘needle’ filament); and EspD/B (which spans the epithelial cell membrane). The system is powered through EscN, an cytoplasmic ATPase (Garmendia, 2005). Once the cell membrane of the host intestinal epithelial cell (IEC) has been breached, EHEC injects effector proteins into the IEC. E. coli secreted effector proteins (Esps) have a variety of functions involved in manipulating eukaryotic cell signaling pathways to further facilitate bacterial adherence and colonization (Brady et al., 2011). Many effector proteins have overlapping and redundant functions, and EHEC translocates about twice as many effectors as the LEE-positive pathotype EPEC does (Croxen & Finlay, 2010). The main LEE-encoded effectors are: Tir, Map, EspF, EspG, EspH, EspB, and Intimin (Wong et al., 2011). Tir and intimin are crucial for intimate adherence because they are the adhesin/ligand that allow the EHEC to bind tightly to the IEC surface. Tir, the translocated intimin receptor, is the most well-characterized adhesion factor of EHEC and is the receptor for the bacterial cell-surface adhesin intimin. Tir is injected into the host epithelial cell through the T3SS, where it then localizes to the epithelial cell membrane. It assembles and functions as a dimer, and each dimer is able to bind to other Tir dimers, which allows for tight clustering of receptors, resulting in for multiple strong adhesion points between the bacterium and the epithelial cell (Garmendia, 2005). The intracellular portion of Tir also interacts with the host cytoskeleton, resulting in actin accumulation directly below the bacterium, contributing to the characteristic pathology seen in EHEC infections (Garmendia, 2005).

1.3.3. Attaching and Effacing Lesions and Shiga Toxins

Presentation of EHEC infection involves the formation of attaching and effacing (AE) lesions, which is characterized by the loss of normal, healthy microvilli structures on the IEC, caused by microvilli effacement and pedestal formation by virulence effectors. The reorganization of host actin cytoskeletal structure destroys the brush-border microvilli, and replaces it with a pedestal on which the adhered bacterium takes up residence (Nguyen, Sperandio, Padola, & Starai, 2012). Another effector protein, Map (mitochondrion-associated protein), functions in three ways: first, it causes mitochondrial damage and dysfunction by interfering with the maintenance of mitochondrial membrane potential; second, it forms transient filopodium-like structures, and third, it is required for disruption of tight junctions (TJs) resulting in compromised intestinal barrier functions (Garmendia, 2005). Map mutants showed attenuated virulence and decreased ability to compete with wild type strains in vivo. EspF is involved in many different pathways, including parallel functions of mitochondrial dysfunction displayed by Map. EspF triggers actin assembly, promotes degradation of anti-apoptotic proteins, inhibition of non-opsonized phagocytosis, inhibition of ion transport leading to compromised intestinal barrier function (thought to be a direct cause of diarrhea), and apoptosis (Wong et al., 2011). EspG is involved with the degradation of microtubules and the formation and accumulation of actin stress fibers, thus aiding the formation of pedestals. EspH also inhibits phagocytosis and aids in formation of actin pedestals, and functions by inhibiting Rho GTPases, which in turn inhibits actin cytoskeletal rearrangement necessary to engulf the bacterium. EspB is involved in actin cytoskeletal rearrangement and promotion of a hospitable environment for the attaching bacterium, by binding host factors involved in cytoskeletal modulation and inhibition of proteins with the potential to reduce T3SS effectiveness (Wong et al., 2011).

Many effector proteins get translocated by theT3SS; however not all of them are encoded in the LEE. Effector proteins encoded elsewhere in the genome include Esp1 (NleA- non-lee encoded effector A), EspJ, EspM, EspT, EspV, an assortment of Nle proteins, and Tccp/EspFu (Wong et al., 2011). One of the first identified non-LEE encoded effector proteins was Orf3, which is an EspG homologue (Dean, Maresca, & Kenny, 2005). Esp1/NleA is responsible for inhibition of protein exportation from the host cell’s endoplasmic reticulum (ER), and aids in TJ dysfunction. EspJ inhibits bacterial phagocytosis, EspM increases epithelial stress fiber formation, and EspT and EspV both aid in the modulation of the actin cytoskeleton. Nle proteins including but not limited to NleD, E, F, H, and K are involved in inhibition of NF-κB activation and other cell signaling pathways (Wong et al., 2011). Tccp/EspFu (Tir-cytoskeletion coupling protein) is the main factor for producing pedestal formation and serves as the link between Tir and the actin cytoskeleton. It functions by inhibiting the auto-inhibition of pathways that inhibit actin polymerization, thus allowing rearrangement and polymerization into a pedestal. It also aids in colonization and AE lesion formation, but is not required for either (Wong et al., 2011).

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AE lesions and other pathology caused by EHEC are significantly enhanced by Shiga toxins. EHEC strains can contain variants of one or both Stx types (Stx1 and Stx2) in any combination and the following description applies generally to all variants. Shiga toxins are structured as AB5 toxins, consisting of one A subunit with enzymatic toxic activity, plus five identical B subunits in a circular pentamer. The B subunit ring binds to cell-surface receptors and creates a pore in the epithelial cell membrane, which then allows the A subunit to enter the cytosol and execute its enzymatic functions. The Stxa subunit (referring to both Stx1 and Stx2 variants) is an RNA N-glycosidase, which functions by cleaving an adenine residue from domain VI of 28S ribosomal RNA in eukaryotic cells. Cleavage at this site inhibits amino-acyl tRNA binding, thus preventing amino acid chain elongation, resulting in an inhibition or halt of protein synthesis in the cell leading to apoptosis (Johannes & Römer, 2010). Stx binds the cell surface receptor   3 (Gb3), which is expressed on Paneth cells in the large intestine and on epithelial cells in the kidneys (Johannes & Römer, 2010). The exact mechanism(s) by which Stx enters the bloodstream is not entirely clear, though it is generally accepted that the main mechanism involves being taken up into the cell, followed by retrograde transport and enzymatic activation. When the Stx AB5 complex binds to the surface of the epithelial cell, it will induce one of several mechanisms in order to be taken up by the cell (Johannes & Römer, 2010). Stx induces endocytosis by clathrin-coated pits, however clathrin-coated pits are not required and the absence of them does not inhibit Stx uptake. Stx will engage in retrograde transport by localizing to early endosome, then transferring to the Trans-Golgi Network (TGN), then to the ER. While Stx is in the early endosome, it undergoes an enzymatic cleavage resulting in two components: a catalytic A1 subunit with RNA N-glycosidase function, and a complex of B5 subunits plus the A2 subunit. These fragments are not separated until reaching the ER, where the A1 subunit undergoes retrograde transport into the host cell cytosol where it will execute its functions (Johannes & Römer, 2010).

Stx has more diverse effects than protein synthesis inhibition in epithelial cells. Stx-mediated damage to the vasculature leads to hemorrhagic colitis, which then exposes bacteria in the intestine to the rich nutrients and growth promoters available from the blood. Stx also causes an increase in epithelial cell expression of nucleolin, a cell surface protein that can bind to intimin, which gives an advantage of increased intimate adherence during infection (Robinson, Sinclair, Smith, & O’Brien, 2006). The effect of Stx on immune function is less clear, with conflicting evidence of both pro- and anti-inflammatory effects. Some reports state that Stx induces the release of pro-inflammatory cytokines from macrophages and monocytes, which then leads to an increase of Gb3 expression on epithelial cells in the intestine. Other postulated pro-inflammatory effects include cytokine secretion and increased expression of IL-8 (Pacheco & Sperandio, 2012). Other reports have speculated that either Stx or other effectors downregulate factors involved in inflammation (Pacheco & Sperandio, 2012).

1.3.4. Treatments and Resolution of Infection

In cases of EHEC infection that do not progress to HUS, symptoms generally resolve in five to seven days. Treatment for EHEC infection is limited to supportive/rehydration therapy, due to the adverse effects of antibiotic treatment. Many alternative therapies have been explored, but none have shown to be significantly effective in humans, and no human vaccines currently exist. Some treatments have been effective to varying degrees in animal models of infection: for example, Stx2 antibodies increased the rate of survival in experimentally infected gnotobiotic piglets, and the administration of the probiotic species Lactobacillus casei demonstrated some protection against toxins in experimentally infected infant rabbits (Rahal, Kazzi, Nassar, & Matar, 2012), but no significant advances have been made in safe, effective treatments in human infection.

1.4. Genetics

1.4.1. E. coli O157:H7 Genome

The E. coli O157:H7 genome is approximately 5.5 Mb in size; large in comparison to nonpathogenic E. coli strain K12 MG1655 at only 4.6 Mb. E. coli O157:H7 contains a highly conserved 4.1 Mb backbone sequence observed in all E. coli strains, with its additional sequences shown to be specific to E. coli O157:H7 or EHEC strains and largely contain genes likely to have been obtained by horizontal gene transfer or are of phage origin. Many of these acquired sequences are pathogenicity islands and contain virulence genes and operons. E. coli O157:H7 contains many different defined and putative genes contributing to its pathogenicity through survival and fitness, adherence, and virulence. Highlighted here is an overview of several of the most characteristic and important genes responsible for EHEC’s high virulence and unique pathogenesis.

1.4.2. The Locus of Enterocyte Effacement (LEE) Operon

The Locus of Enterocyte Effacement (LEE) operon is one of the most notorious features of EHEC and EPEC pathovars. The genomic pathogenicity island (PI), found in both EHEC and EPEC pathovars, is approximately 43 kb and contains more than 40 genes organized into five operons (Ji Youn Lim, Jang W. Yoon, 2013). Although EPEC carries this PI, the LEE operon in E. coli O157:H7 has an additional 7.5 kb of uncharacterized sequence that EPEC strains do not have (Lim, Yoon, & Hovde, 2010). The LEE is critical for the formation of severe attaching and effacing (A/E) lesions and the microvilli effacement associated with pedestal formation; hallmarks of EHEC infection (Dean et al., 2005). The gene products can be divided into categories- the type three secretion system (T3SS), intimate adherence factors intimin (adhesin) and its receptor Tir (the translocated intimin receptor), and other secreted virulence effectors (Gyles, 2007). In the first operon LEE1, the ler gene encodes the LEE-encoded regulator Ler, which is considered the master regulator of the entire PI. Although many other regulatory factors have been shown to effect LEE, Ler remains the primary control, with many of the LEE-effecting regulators acting through it indirectly (Nguyen et al., 2012).

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1.4.3. Plasmid pO157

The pO157 plasmid is a 92-kb plasmid, with 100 open reading frames (ORFs) encoding many of the virulence genes utilized by E. coli O157:H7  (Landstorfer et al., 2014) (Ji Youn Lim, Jang W. Yoon, 2013). Several ORFs of note include hemolysin, a type 2 secretion system (T2SS), and a putative virulence factor ToxB. Hemolysin is a pore-forming cytolysin toxin responsible for lysing erythrocytes; encoded by the hly genes (Burgos & Beutin, 2010). The T2SS, encoded by etp genes, secretes a variety of virulence effectors and is required for full virulence and adherence to HeLa cells in vitro (Ho, Davis, Ritchie, & Waldor, 2008). The toxB is a gene of interest in the study of adherence and virulence. It has some sequence similarity to toxin B in Clostridium difficile, and efa-1/lifA found in other pathogenic E. coli strains. It is proposed to contribute to Caco-2 cell adherence through increased activity of the T2SS, but may also have some function in inhibiting lymphocytes (Ji Youn Lim, Jang W. Yoon, 2013). The pO157 plasmid contains many other ORFs relevant to E. coli O157:H7 virulence and its full role in pathogenesis is not yet known.

Large plasmids similar to the pO157 have been observed in many non-O157 EHEC strains, varying in size from about 70 to 200 kb. Most of these reported plasmids carry the genes for hemolysin (hly), but other known pO157 encoded virulence factors are not similarly conserved. Although the presence of hemolysin is associated with a higher risk of developing HUS, the uncharacterized non-O157 plasmids otherwise have unknown contributions to virulence and adherence (Lim et al., 2010).

1.4.4. Shiga Toxins

Shiga toxins (Stx) are produced by Shigella dysenteriae and EHEC. The stx genes were acquired by horizontal gene transfer, and the phage genome was integrated into the bacterial chromosome. The stx genes are encoded by two lambda phage genomes, 933W and 933J (Mora et al., 2004).  There are multiple subtypes: the two main classes are Stx1 and Stx2. Stx1 is nearly 100% homologous to Stx from S. dysenteriae, while being only 55% homologous to Stx2 though they share the same mechanism of action (Pacheco & Sperandio, 2012).  Stx1 has three variants: Stx1, Stx1c, and Stx1d. Stx2 has a larger number of variants: Stx2c, Stx2c2, Stx2e, and Stx2f. These variants can be found in any combination in any EHEC strain; however, strains containing only Stx2 variants are associated with higher virulence than strains containing only Stx1 or Stx1 and Stx2 variants (Johannes & Römer, 2010). These genes can be found together or separately and can also be differentially regulated. The stx genes are upregulated in response to a variety of environmental and host signals, including iron concentration, stress response, pH, host hormones, and antibiotics (Kimmitt, Harwood, & Barer, 2000).

1.5. Expression of Virulence and Adherence Factors

1.5.1. Overview of Virulence Gene Regulatory Signals

Molecular pathogenesis in E. coli O157:H7 is a tightly controlled series of steps, which is regulated by a series of host and environmental signaling factors encountered during its passage through the GI tract. Brief highlights of signals and their effects are shown in Table 1.1.

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Table 1.1. Select Signals Affecting Virulence Gene Expression in E. coli O157:H7 Within the Host