Immunity to Enteropathogenic Escherichia coli

Immunity to Enteropathogenic Escherichia coli Jaclyn S Pearson, University of Melbourne at The Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia Gad Frankel, Imperial College, MRC Centre for Molecular Bacteriology and Infection, London, UK Ó 2016 Elsevier Ltd. All rights reserved.

Abstract Enteropathogenic Escherichia coli (EPEC) is a highly evolved pathogen that has adapted to survive and cause disease in diverse environmental niches. The inflammatory response is an integral part of host defense against gut pathogens but also contributes to disease pathology. In this article, we explore the factors leading to inflammation during EPEC infection and the mechanisms EPEC and other attaching and effacing (A/E) pathogens have evolved to suppress inflammatory signaling. Infection with EPEC stimulates a rapid host inflammatory response via recognition of flagellin and LPS. Infection models of EPEC have revealed many of the immune factors that mediate this response. In particular, the outcome of infection is greatly influenced by the ability of an infected epithelial cell to mount an effective host inflammatory response. In order to counterbalance the inflammatory response, the bacteria inject effector proteins that inhibit inflammatory cytokine production, allowing the bacteria command exquisite control over the host to coordinate a successful infection strategy and cause disease. Overall, innate mucosal immune responses in the gastrointestinal tract during infection with EPEC are highly complex and ultimate clearance of the pathogen depends on multiple factors including inflammatory mediators, bacterial burden, and the function and integrity of resident intestinal epithelial cells.

Introduction Enteropathogenic Escherichia coli (EPEC) is a pathogenic gramnegative bacillus that causes disease in humans, particularly in children under 2 years of age. Illness presents as nonspecific gastroenteritis, with acute diarrhea and mucous production, vomiting, fever, and malaise. Transmission of EPEC occurs via the fecal–oral route, carried on hands, contaminated fomites, weaning foods, and infant formula. The main reservoir of EPEC is symptomatic and asymptomatic infants, asymptomatic adults and adults that are in high contact with infants (Levine and Edelman, 1984). EPEC belongs to the attaching and effacing (A/E) family of enteric pathogens which comprises enterohemorrhagic E. coli (EHEC), including the well-known Shiga-toxin producing O157 strain, rabbit-specific E. coli (REPEC) and the natural mouse pathogen Citrobacter rodentium. During infection these pathogens remain extracellular and form characteristic A/E lesions on the intestinal epithelium (Figure 1). In particular, EPEC interacts with Peyer’s patch follicle-associated epithelium (FAE) and enterocytes of the small intestine (Phillips and Frankel, 2000). The A/E histopathology is a distinct feature of infection, and is characterized by the effacement of the local brush border microvilli on enterocytes, intimate bacterial attachment to the apical membrane, and formation of actin-rich pedestal-like structures beneath the adherent bacteria (Moon et al., 1983). All A/E pathogens share core virulence factors, which allow them to attach intimately to enterocytes and cause diarrheal disease. The virulence factors have been acquired via evolutionary events, primarily horizontal gene transfer. One of the most important and highly studied virulence factors of A/E pathogens is encoded by the locus of enterocyte effacement (LEE) pathogenicity island (PAI). The LEE is a 35 kb chromosomal locus encoding: (1) structural proteins for a type III secretion system (T3SS), (2) secreted translocator proteins,

Encyclopedia of Immunobiology, Volume 4

(3) translocated effector proteins, including the translocated intimin receptor (Tir), (4) the outer membrane adhesion intimin, (5) chaperones, and (6) gene regulators (Elliott et al., 1998, 2000; Deng et al., 2001, 2004, 2005). Intimate attachment of EPEC to host cells is a hallmark of infection and is essential for virulence. A number of factors contribute to EPEC adherence; these include (1) the T3SS structural protein EspA (Knutton et al., 1998; Cleary et al., 2004), which polymerizes to form a filamentous extension at the tip of the

Figure 1 Attaching and effacing lesions. Electron micrographs showing A/E lesion formation by an A/E pathogen. Inset details intimate bacterial attachment to host intestinal epithelium (arrow), destruction of local brush border microvilli (star), and a pedestal-like structure formed beneath the adherent bacteria.

http://dx.doi.org/10.1016/B978-0-12-374279-7.13010-3

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Immunity to Bacterial, Parasitic and Fungal Infections j Immunity to Enteropathogenic Escherichia coli

T3SS needle complex, (2) the interaction of the EPEC outer membrane adhesin intimin with its translocated receptor Tir (Kenny et al., 1997; de Grado et al., 1999; Hartland et al., 1999; Kenny, 1999; Luo et al., 2000; Goosney et al., 2001; Campellone and Leong, 2003), (3) flagella (Girón et al., 2002), and (4) in typical EPEC strains, the EAF plasmid encoding for bundle forming pili (BFP) (Girón et al., 1991; Anantha et al., 2000). T3SS are utilized by many bacterial pathogens of humans, animals, and plants to transport virulence (effector) proteins directly into host cells where they can manipulate host cell processes and antimicrobial responses to facilitate survival and replication of the pathogen (Hueck, 1998; Galan et al., 2014). The T3SS of EPEC secretes proteins that are both LEE- and non-LEE–encoded (Wong et al., 2011). A group of 21 core LEE- and non-LEE–encoded effectors are shared by all A/E pathogens (Iguchi et al., 2009); however, the repertoire of non-LEE– encoded effectors differs significantly among clinical EPEC and EHEC isolates, suggesting that these pathogens employ different infection strategies. This article will primarily focus on EPEC, with reference to other A/E pathogens for the purpose of demonstrating model in vivo experiments.

EPEC and the Inflammatory Response Many of the early studies on EPEC-mediated inflammation focused on the proinflammatory response characterized by marked infiltration and transmigration of PMNs in the intestinal epithelium. However, some 10 years ago, researchers made the remarkable observation that EPEC strains could inhibit nuclear factor-kappa B (NF-kB) and mitogen-activated protein kinase (MAPK) activation as well as inhibitor of NF-kB (IkB) degradation and the production of proinflammatory cytokines including IL-8 and IL-6, early in infection of cultured epithelial cells (Hauf and Charkraborty, 2003; Ruchaud-Sparagano et al., 2007). Furthermore, this inhibitory mechanism was dependent upon the presence of a functional T3SS (Hauf and Charkraborty, 2003). This suggested that EPEC had evolved specialized mechanisms to dampen the early inflammatory response during infection, possibly in order to persist for longer periods before the overall immune response would inevitably clear the bacteria. The early discovery of the LEE and its association with A/E lesion formation led to the intensive study of LEE-encoded effectors and their involvement in cytoskeletal reorganization and disruption of tight junctions in the epithelium (Wong et al., 2011). However, the activity of these effectors could not explain how EPEC suppressed the production of inflammatory cytokines. Characterization of several non-LEE–encoded effectors over the last 6 years has revealed that EPEC injects multiple effector proteins into host cells that specifically target various innate immune factors. These effectors have highly specific and diverse functions that interfere with a range of innate signaling pathways, including NF-kB and MAPK activation (Wong et al., 2011; Giogha et al., 2014).

Host Recognition of EPEC The mammalian host initially senses and responds to bacterial pathogens via activation of the innate immune response. This

involves the recognition of pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS), flagellin, peptidoglycan (PG), and CpG DNA by specialized germ line– encoded pattern recognition receptors (PRRs) expressed by host cells (Akira et al., 2006). PRR families described to date include the Toll-like receptors (TLRs), retinoic acid–inducible gene (RIG)-I-like receptors (RLRs), and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) (Latz et al., 2013; O’Neill et al., 2013). Upon PRR stimulation, the host rapidly produces a subset of proinflammatory cytokines, which in turn activate the innate response (Latz et al., 2013; O’Neill et al., 2013). This innate response ultimately leads to the development of a more specific and long-term adaptive response to a specific pathogen, mediated by B cells and T cells. EPEC primarily infects the epithelium lining the mucosal surface of the gastrointestinal tract (Robins-Browne and Hartland, 2002). Gastrointestinal epithelial cells play a pivotal role in ion transport, fluid uptake, and secretion that are critical to the homeostatic state of the digestive system. They also coordinate the expression and upregulation of specific antimicrobial products in response to infection, including cytokines with proinflammatory (TNF and IL-1) and chemoattractant (IL-8, MIP1-a, MCP-1) functions (Kagnoff and Eckmann, 1997). EPEC infection studies in vitro indicate that the initial and most potent activation of the host inflammatory response is mediated by TLR5 recognition of flagellin and also, although not as pronounced, by TLR4 recognition of LPS (Miyamoto et al., 2006; Badea et al., 2009; Schuller et al., 2009). IL-8 is a potent chemoattractant for neutrophils and a number of early studies showed that EPEC (Savkovic et al., 1997) and other bacterial enteric pathogens stimulate epithelium-derived IL-8 expression during infection (McCormick et al., 1993; Jung et al., 1995; Philpott et al., 2000). However, it is not entirely clear to what extent epithelium-derived IL-8 contributes to mucosal defense and the inflammatory response generated against A/E pathogens. Infection of gnotobiotic piglets with either EPEC or EHEC induces extensive inflammatory cell infiltration in the lamina propria as well as transmigration of inflammatory cells across the intestinal epithelium into the intestinal lumen (Moon et al., 1983; Tzipori et al., 1985, 1989). Depletion of neutrophils in mice infected with the mouse A/E pathogen, C. rodentium, results in elevated bacterial loads in the liver and spleen, suggesting an important role for neutrophils in controlling infection with A/E pathogens (Lebeis et al., 2007).

Animal Models of EPEC Infection: Opportunities to Study Immunity Studying the in vivo inflammatory response in the human host is not a viable option for EPEC infection. Therefore, a number of animal models have been utilized to understand the nature of the immune response to infection. The most common and relevant model is the infection of mice by oral gavage with the A/E pathogen C. rodentium. This model represents a natural host–pathogen infection with an organism that has evolved convergently with EPEC and, therefore, shares most of the key virulence factors as the human pathogen, including the

Immunity to Bacterial, Parasitic and Fungal Infections j Immunity to Enteropathogenic Escherichia coli LEE PAI (Deng et al., 2001, 2010; Petty et al., 2010). Furthermore, C. rodentium has been used as a model to study a number of human intestinal disorders, including inflammatory bowel disease, Crohn’s disease, ulcerative colitis, and colon tumorigenesis (Higgins et al., 1999; Chandrakesan et al., 2014). Other animal models include infection of rabbits with REPEC and infection of gnotobiotic piglets and calves with EPEC or EHEC.

Citrobacter rodentium and Immune Responses in Mice Infection with C. rodentium causes colitis characterized by inflammatory cell infiltration, hyperplasia of crypt cells, loss of goblet cells, and significant intestinal barrier disruption (Luperchio and Schauer, 2001; Ma et al., 2006). Both innate and adaptive responses contribute to the control of C. rodentium infection, dissemination, and elimination. For instance, mice lacking B cells, T cells, or both have greater pathogen loads in their colonic and peripheral tissues, and despite a severe disease phenotype in these mice, a significant percentage of mice survive, suggesting that more than an adaptive response is involved (Simmons et al., 2003; Maaser et al., 2004). For example, in the absence of neutrophils, mice infected with C. rodentium also suffer a high bacterial load, which highlights the importance of the inflammatory response in the early stages of infection (Lebeis et al., 2007). Activation of NF-kB has been demonstrated in vivo during C. rodentium infection (Wang et al., 2006); however, unlike EHEC and EPEC, C. rodentium is nonflagellated and does not activate TLR5 (Khan et al., 2008). Therefore it is likely that activation is either T3SS-dependent and/or as a result of TLR4 recognition of LPS. Mice with an intact adaptive system but deficient in mast cells display increased colonic inflammation and increased production of proinflammatory cytokines (Wei et al., 2005). These mice also suffer systemic infection and most die within 4–7 days of C. rodentium infection (Wei et al., 2005), which suggests that mast cells play a role in control and clearance of C. rodentium rather than regulating inflammation. TLRs have been strongly implicated in mediating inflammation during C. rodentium. For instance, TLR4, although not protective, mediates chemokine induction and subsequent neutrophil and macrophage recruitment and is partially responsible for tissue pathology during C. rodentium infection (Khan et al., 2006). Conversely, TLR2 is not critical in mediating proinflammatory responses associated with C. rodentium–induced colitis but helps maintain mucosal integrity during C. rodentium infection (Gibson et al., 2008). The signaling adapter MyD88 (myeloid differentiation primary response protein 88) is essential in the control of mucosal infection by a number of pathogens (Skerrett et al., 2004; Weiss et al., 2005; Hawn et al., 2006; Watson et al., 2007). MyD88 activation initiates NF-kB activation, and in the intestine, plays an important role in maintaining tissue homeostasis (Akira and Takeda, 2004). During C. rodentium infection, MyD88 protects against bacteremia and severe pathology in C57BL/6 mice (Lebeis et al., 2007) and MyD88-deficient mice experience elevated bacterial load in the colon and peripheral tissues, which correlates with a decrease in neutrophil infiltration into the colonic tissue. MyD88-deficient mice also suffer from severe colonic ulceration and bleeding, resulting in high

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levels of morbidity and mortality. This severe disease phenotype is due to impaired epithelial barrier function and defective cellular proliferation, followed by an inability to repair mucosal damage. MyD88 association with the IL-1R but not the IL-18R affords protection against increased mortality and pathology during infection with C. rodentium (Lebeis et al., 2009). IL1R-deficient mice infected with C. rodentium suffer disease similar to that seen in MyD88-deficient mice; however, they do not suffer higher bacterial loads, or an inability to recruit neutrophils and mediate tissue damage repair. The mice are unable to induce production of proinflammatory cytokines, IL-6, and IFN-g during infection, suggesting that the bacteria are mediating severe tissue pathology rather than a dysregulated immune response, and that IL-1R signaling regulates susceptibility to C. rodentium infection (Lebeis et al., 2009). The importance of IL-1R signaling is supported by work showing that mice deficient for the inflammasome components Nlrc4, Nlrp3, and caspase-1 that lead to IL-1b and IL-18 secretion were highly susceptible to C. rodentium suffering exacerbated intestinal inflammation and increased bacterial load after day 10 of infection (Liu et al., 2012). Similar observations were made in IL-1b- and IL-18-deficient mice. Despite the fact that Nlrc4-deficient mice were highly susceptible to C. rodentium infection, only Nlrp3 and not Nlrc4 induced caspase-1 activation (Liu et al., 2012). This makes sense given that C. rodentium lacks flagella, which are a major stimulus of Nlrc4, and suggests that C. rodentium-induced damage during infection allows the intestinal flora to activate Nlrc4 and induce an inflammatory response. A strong Th1/Th17 response, characterized by an increase in the expression of IL-1b, TNF, IL-12, IFN-g, IL-17, and IL-22, is elicited in the colon of C. rodentium–infected mice and produces pathology similar to that seen in mouse models of inflammatory bowel disease (Higgins et al., 1999; Zheng et al., 2008; Geddes et al., 2011). Citrobacter rodentium infection induces a Th17 response within 2 weeks of infection and this is needed for clearance of the pathogen (Zheng et al., 2008). The induction of this response requires Nod1/Nod2 signaling and the proinflammatory cytokine IL-6 (Geddes et al., 2011). IL6-deficient mice develop severe mucosal ulcerations and increased crypt cell apoptosis, suggesting a protective role for the cytokine during gut infection (Gibson et al., 2008). Although it is not clear if all these immune and inflammatory factors are required for mucosal defense during human infection with EPEC, some studies have highlighted the importance of IL-6 in clearance of EPEC in children (Eckmann, 2006; Long et al., 2010). Studies have suggested that TNF plays an important role in clearance of EPEC in children (Long et al., 2010) and control of bacterial load in animal models of infection (Gonçalves et al., 2001; Ramirez et al., 2005). Mice that are TNF deficient (TNFRp55/) demonstrate increased tissue pathology during C. rodentium infection, including pronounced hyperplasia, increased T cell infiltrate, and higher levels of mucosal IFN-g and IL-12 production (Gonçalves et al., 2001). However, TNFRp55/ mice are not compromised in their ability to clear infection compared to wild-type C57BL/6 mice and despite the compensatory increase in cytokine production, they experience significantly higher bacterial loads than wild-type mice

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(Gonçalves et al., 2001). Overall, it is plausible that the increased bacterial burden may be a major contributing factor to increased tissue pathology in the absence of TNF and that TNF plays a major role in limiting bacterial replication. IL-22 plays a major role in maintaining mucosal homeostasis in the gastrointestinal tract (Sonnenberg et al., 2011). During infection with C. rodentium, Il-22 is highly upregulated, leading to the production of a number of antimicrobial and inflammatory mediators (Zheng et al., 2008). Furthermore, Il22/ mice are highly susceptible to infection with C. rodentium (Zheng et al., 2008), highlighting the importance of IL-22 in controlling infection. Group 3 ILCs and Th22 cells are the major source of IL-22 in C. rodentium infection (Basu et al., 2012; Guo et al., 2015). A recent study showed that mice deficient in Il22ra suffer a high systemic burden of C. rodentium, which is related to the systemic dissemination of the resident gut bacterium Enterococcus faecalis and systemic inflammation (Pham et al., 2014). IL-22RA1 is a component of the IL-22 receptor that is only expressed in subset of organs and tissues, including the gastrointestinal tract. This receptor component is thought to mediate the effects of IL-22 to maintain barrier homeostasis in the gut (Wolk et al., 2004; Zheng et al., 2008). IL-10 plays an important role in modulating the initial immune response in C. rodentium infection, as mice deficient for IL-10 or IL-10R suffer severe colonic inflammation (Krause et al., 2015). The intestinal damage was attributed to an increase in IL-23 synthesis by macrophages. IL-10-producing macrophages protect against excessive inflammation during C. rodentium infection by restricting macrophage IL-23 production (Krause et al., 2015), production of which is known to promote innate immune pathology in the intestine (Hue et al., 2006; Uhlig et al., 2006). Another recent study has suggested that an increase in IL-7 production by IECs in C. rodentium infection also promotes IL-23 production (Zhang et al., 2015). CD4þ-mediated humoral immunity plays a critical role in controlling C. rodentium infection and limiting systemic spread of the pathogen (Simmons et al., 2003; Bry and Brenner, 2004). IgG specifically recognizes pathogen virulence factors and selectively binds these bacteria in the lumen of the gut, promoting neutrophil engulfment (Bry and Brenner, 2004; Maaser et al., 2004; Kamada et al., 2015).

Proinflammatory T3SS Effectors Some studies have suggested that a subset of EPEC effectors induce a proinflammatory effect during infection. Evidence for this was shown when an EPEC mutant lacking genomic islands PP4 and IE6 (encoding seven effectors) (Iguchi et al., 2009) induced p65 nuclear translocation and pronounced IL8 secretion independently of TNF stimulation (Newton et al., 2010a; Pearson et al., 2011). Nuclear translocation of p65 induced by the PP4/IE6 double mutant was greater than for a T3SS mutant (escN), suggesting that other translocated effectors stimulated NF-kB activity (Newton et al.). Recent in vitro work on the effector NleF supports the idea that some effectors activate NF-kB signaling (Pallett et al., 2014). Transient expression of NleF in HeLa cells resulted in an increase in NF-kB activation and IL-8 expression, in addition, NleF promoted early p65 nuclear translocation during EPEC infection of HeLa cells

(Pallett et al., 2014). Another study showed that NleF binds inflammatory caspases-4, -8, as well as the executioner caspase-9 (Figure 2); however, the effect of this in EPEC infection is yet to be investigated (Blasche et al., 2013). The WxxxE effector, EspT, has also been implicated in NF-kB activation (Raymond et al., 2011). EspT induced IL-8 and IL-1b secretion, which required the small GTPase Rac1 (Raymond et al., 2011). A similar phenomenon was evident during Salmonella infection where activation of Rac1 and Cdc42 by SopE also induced NF-kB activation. NF-kB activation by Rac1 occurred via NOD1 and RIPK2 signaling and was also applicable to the sensing of PG by NOD1 (Keestra et al., 2013). This recent study concluded that NOD1 senses microbial infection by monitoring the activation state of small GTPases, such as Rac1 and Cdc42. Since SopE and the WxxxE effectors have known roles in cytoskeletal rearrangement and bacterial invasion, this illustrates the point that the interaction of an effector protein with a host protein may inadvertently stimulate an inflammatory response.

EPEC Effector Proteins that Inhibit Inflammatory Signaling Multiple effectors, including NleE, NleC, NleD, NleH, and Tir, have been shown to have anti-inflammatory activity, targeting different stages in host immune signaling cascades (Figure 2). This suggests that dampening innate immune responses is central to the EPEC infection strategy.

NleE Non-LEE–encoded effector E (NleE) is conserved across all A/E pathogens and was first discovered as a type III secreted effector of C. rodentium (Deng et al., 2004; Kelly et al., 2006). Furthermore, all Shigella species carry a homolog of NleE known as OspZ, sharing around 74% identity with NleE from EPEC (Zurawski et al., 2008). NleE is consistently associated with the virulence profile of EPEC strains, as well as O157 and non-O157 EHEC (Bugarel et al., 2011; Buvens and Piérard, 2012). A number of in vitro studies have shown that NleE potently silences the production of inflammatory cytokines during EPEC infection by specifically blocking NF-kB activation in epithelial cells and dendritic cells (Nadler et al., 2010; Newton et al., 2010b; Vossenkämper et al., 2010; Zhang et al., 2011). NleE displays unique S-adenosyl-L-methionine (SAM)-dependent methyltransferase activity against the TAK1-binding proteins 2 and 3 (TAB2 and TAB3) and specifically modifies the zinc finger domains of TAB2/3 with a cysteine residue which renders them unable to bind polyubiquitin chains on TRAF2/6 following activation of TNF or TLR/IL-1-R signaling (Zhang et al., 2011). This activity ultimately blocks the downstream signaling cascade of the NF-kB pathway, preventing nuclear translocation of transcription factors required for the production of inflammatory cytokines such as IL-8 and IL-6 (Figure 2).

NleC and NleD NleC and NleD are zinc metalloprotease effectors that target key innate signaling networks during EPEC infection. While

Immunity to Bacterial, Parasitic and Fungal Infections j Immunity to Enteropathogenic Escherichia coli

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Figure 2 Inflammatory signaling pathways inhibited by EPEC. EPEC T3SS effector proteins are injected by the LEE-encoded translocon and inhibit inflammatory signaling at different points in the various pathways. The signaling factors and T3SS effector proteins, as well as their mechanisms of action, are described in detail in the main text. Ub, ubiquitin; P, phosphorylation; GlcNAc, N-acetylglucosamine.

NleC cleaves NF-kB Rel proteins, NleD cleaves the MAPK enzymes, JNK and p38 (Baruch et al., 2011; Mühlen et al., 2011; Pearson et al., 2011; Sham et al., 2011). Multiple studies have shown that NleC directly cleaves the NF-kB subunit, p65 (Figure 2; Yen et al., 2010; Baruch et al., 2011; Mühlen et al., 2011; Pearson et al., 2011; Sham et al., 2011) and that mutation of the histidines or the glutamate within the metalloprotease motif (183HEIIH) renders the protein inactive (Yen et al., 2010; Baruch et al., 2011; Mühlen et al., 2011; Pearson et al., 2011; Sham et al., 2011; Giogha et al., 2015; Hodgson et al., 2015). Although the exact location of the cleavage site is still under debate, three independent groups have identified sites within the N-terminal Rel homology domain (RHD) of p65 (Yen et al., 2010; Baruch et al., 2011; Li et al., 2014). The RHD of NF-kB proteins is required for binding to NF-kB consensus sites, dimerization, and nuclear localization (Gilmore and Wolenski, 2012) and cleavage within this site by NleC would render p65 unable to bind target genes and activate transcription of inflammatory cytokines. A number of studies have indicated that NleC cleaves other NF-kB proteins, in addition to p65, including p50

and IkB (Yen et al., 2010; Mühlen et al., 2011; Pearson et al., 2011); however, conflicting results suggest this requires more rigorous investigation. Another study suggested that NleC cleaves the acetyltransferase p300, a transcriptional coactivator of many host cell genes including p65 in a metalloprotease-dependent manner (Hayden and Ghosh, 2008; Shames et al., 2011). Overexpression of p300 in host cells resulted in a significant increase in IL-8 production during wild-type EPEC infection and NleC-dependent cleavage of p300 assisted in suppression of IL-8 during infection (Shames et al., 2011). In addition, NleC was observed to target MAPK signaling by inhibiting phosphorylation of p38 during EPEC infection, although this was independent of the zinc metalloprotease motif (Sham et al., 2011). Although there is apparent redundancy in the functions of NleE and NleC, secreted IL-8 levels are significantly higher upon infection with a double nleC/nleE mutant than single nleE or nleC mutants, suggesting that NleC and NleE act synergistically to inhibit IL-8 production (Yen et al., 2010; Baruch et al., 2011; Pearson et al., 2011; Sham et al., 2011; Shames et al., 2011).

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NleD directly cleaves JNK and p38 within a conserved activation loop present in both signaling kinases (Figure 2). ERK, however, is unaffected by the metalloprotease which is likely explained by the fact that it does not contain the activation loop present in p38 and JNK. NleD specifically cleaves JNK after residue P184, within a TPY motif, the phosphorylation of which is required for its activation (Baruch et al., 2011). As with NleC, mutation of the histidines or the glutamate within the metalloprotease motif (141HELLH) renders NleD inactive. Despite these activities, there is a lack of evidence that NleD significantly contributes to the suppression of inflammatory cytokine production in EPEC infection in vitro (Baruch et al., 2011); however, one recent study has shown that NleD inhibits IFN-b expression and secretion in EPEC-infected Caco-2 monolayers, resulting in an increase in barrier disruption (Long et al., 2014).

NleB NleB was first identified as a secreted effector of C. rodentium, and early studies suggested the effector played a significant role in virulence in vivo as a C. rodentium nleB deletion mutant is highly attenuated (Kelly et al., 2006; Deng et al., 2010). Furthermore, a study on the transmissibility of C. rodentium showed that NleB contributes significantly to the fitness of the pathogen (Wickham et al., 2007). EPEC and EHEC carry two copies of nleB (nleB1 and nleB2, 80% amino acid similarity) whereas C. rodentium carries only one that is most similar to NleB1 (96%). Homologs of NleB (termed SseK1, SseK2, and SseK3) exist in a number of Salmonella spp., where their function and contribution to virulence are still under investigation (Brown et al., 2011). Early in vitro studies suggested that transient expression of NleB1 in cultured epithelial cells could block IkB degradation and NF-kB activation in response to TNF (Newton et al., 2010b; Baruch et al., 2011), however, during EPEC infection, NleB1 had no significant effect on IL-8 production (Pearson et al., 2013). More recent studies have revealed that NleB1 resembles the GT-8 family of glycosyltransferases and exhibits GlcNAc transferase activity (Gao et al., 2013; Li et al., 2013; Pearson et al., 2013). NleB1 contains a DxD catalytic motif that is essential for its enzymatic activity and utilizes the donor substrate UDP-GlcNAc to catalyze the transfer of a single sugar to a conserved arginine residue in the host death domain (DD) proteins FADD, TRADD, and RIPK1 (Figure 2; Li et al., 2013; Pearson et al., 2013). The addition of the GlcNAc renders the DD proteins unable to bind their cognate death receptors upon stimulation and, thereby, blocks apoptosis and necroptosis during EPEC infection (Li et al., 2013; Pearson et al., 2013). To highlight the in vivo significance of these findings, a C. rodentium nleB mutant complemented with a catalytic mutant of NleB was attenuated to similar levels as the nleB mutant, indicating that the DxD motif contributes to the virulence of A/E pathogens (Gao et al., 2013; Li et al., 2013). Furthermore, Fas receptor (Faslpr/lpr) or FasL-deficient (Fasgld/gld) mice were compromised in their ability to clear C. rodentium and suffered more severe disease than wild-type mice (Pearson et al., 2013), indicating that Fas signaling plays an important role in controlling bacterial gut infection.

NleH1 and NleH2 NleH1 and NleH2 are homologous effectors of EPEC, EHEC, and C. rodentium (84% amino acid identity). To date, researchers are still debating the role of the NleH effectors in EPEC infection; however, a number of studies suggest that they interfere with NFkB signaling. Initial work showed that EHEC nleH1 and nleH2 bind to a non-Rel NF-kB subunit, ribosomal protein S3 (RPS3), a KH domain protein that regulates NF-kB–dependent transcription (Figure 2; Wan et al., 2007; Gao et al., 2009). NleH1 reduces the nuclear abundance of RPS3 via inhibition of IKKb-mediated phosphorylation of RPS3 at serine residue 209, with no effect on other NF-kB signaling factors (Gao et al., 2009; Wan et al., 2011). NleH2 does not inhibit RPS3 nuclear translocation; however, it does increase expression from an AP-1-dependent luciferase reporter, hence it may affect a different signaling pathway (Gao et al., 2009). NleH1 and NleH2 were also found to suppress TNF-induced IkBa degradation in cultured epithelial cells by interfering with phosphoIkBa ubiquitylation. This was dependent on conserved lysine residues, K159 and K169, in NleH1 and NleH2, respectively, which are implicated in kinase activity (Royan et al., 2010). A number of studies conducted using animal models have tried to dissect the contribution of NleH to pathogenesis and inflammatory responses in the host; however, they have been somewhat inconsistent (Mundy et al., 2006; Garcia-Angulo et al., 2008; Hemrajani et al., 2008; Gao et al., 2009; Royan et al., 2010; Pham et al., 2012). Despite intense interest in the mechanism of action and function of NleH, neither NleH1 nor NleH2 inhibit NF-kB activity to the same extent as NleE or NleC in vitro (Newton et al., 2010a).

Tir Tir is the most abundant effector protein delivered into host cells during EPEC infection, and is absolutely essential for intimate attachment of the bacteria. In addition to the primary role of this effector, a number of recent studies have identified a role for Tir in inhibiting host innate signaling mechanisms. For example, one study showed that transient expression of Tir from EPEC in HeLa cells could inhibit TNF-induced NF-kB activation by binding and degrading the cytoplasmic TNF receptor– associated protein (TRAF2) in a proteasome-independent manner (Ruchaud-Sparagano et al., 2011). Consequently, a slight yet significant increase in IL-8 production in HeLa cells infected with an EPEC tir deletion mutant was observed (Ruchaud-Sparagano et al., 2011). A subsequent study showed that Tir shares sequence similarity with host cellular immunoreceptor tyrosine–based inhibition motifs (ITIMs) and inhibits TLR signaling (Yan et al., 2012). ITIMs are critical negative regulators of eukaryotic immunoreceptor signaling pathways (Barrow and Trowsdale, 2006; Daeron et al., 2008). Two studies have provided evidence that Tir binds directly to SHP-1 and SHP-2 (Yan et al., 2012, 2013), phosphatases that suppresses cellular immune responses by dephosphorylation of NF-kB and MAPKs (Figure 2; Nandan et al., 1999). Downregulation or mutagenesis of SHP-1/SHP-2 increased TNF and IL-6 production in EPEC-infected cells. Furthermore, Tir was required for the inhibition of proinflammatory cytokine (TNF and IL-6) expression during EPEC and C. rodentium

Immunity to Bacterial, Parasitic and Fungal Infections j Immunity to Enteropathogenic Escherichia coli infection in vitro and in vivo, respectively (Yan et al., 2012). However, these studies utilized the EPEC strain JPN15, which has been cured of the plasmid that encodes BFP and adheres less efficiently to cells than wild-type EPEC, thereby, complicating the interpretation of above experiment.

Future Directions The gastrointestinal tract is the largest inflammatory organ in the mammalian body; therefore, it is not surprising that EPEC has evolved numerous mechanisms for diverting or arresting inflammatory processes in order to persist and cause disease. The diversity of effector protein function exemplifies how A/E pathogens have evolved to evade numerous host defense mechanisms, particularly innate immunity. Current research efforts to increase our understanding of the specific mechanisms of immunity to EPEC infection will continue to be bolstered by the use of C. rodentium infection of mice combined with such techniques such as RNA sequencing and two-photon imaging. Finally, a number of recent studies are addressing the importance of the host microbiota and nutrition in infection with gut pathogens (Collins et al., 2014). This will provide the research community with critical yet often overlooked information on host–pathogen interaction.

See also: Anatomy and Microanatomy of the Immune System: Anatomy and Function of the Gut Immune System; Roles of Chemokines in Immune Cell Trafficking to Lymphoid Tissues. B Cell Activation: Cytokine Regulation of B Cell Activation and Differentiation; TNF and TNFR Family Members and B Cell Activation. Cells of the Innate Immune System: Macrophage Activation and Polarization; Mast Cells in Allergy, Host Defense, and Immune Regulation; Natural Killer Cells; Neutrophils – Role in Innate Immunity. Cytokines and Their Receptors: Function of Chemokines and Their Receptors in Immunity; Inflammasomes; Interferon g: An Overview of Its Functions in Health and Disease; Interleukin-17 Family; Roles of TNF and Other Members of the TNF Family in the Regulation of Innate Immunity; The Biology of Interleukin-6, a Major Target in Anti-Inflammatory Therapies; The IL-12/IL-23 Cytokine Family; The Interleukin-1 Family. Development of T Cells and Innate Lymphoid Cells: Innate Lymphoid Cells Type 3. Immunity to Bacterial, Parasitic and Fungal Infections: Immune-Driven Pathogen Evolution; Immunity to Salmonella; Immunology of Bacterial and Parasitic Diseases: An Overview. Immunity to Viral Infections: Innate Cytokine Responses and Their Functions during Viral Infections. Molecular Aspects of Innate Immunity: Inflammatory Products of the Complement Pathway; The NOD-Like Receptors. Physiology of the Immune System: Role of the Microbiota in Immune Development. Signal Transduction: Adapter Molecules in Immune Receptor Signaling; Chemokine Receptor Signaling; Jak-STAT Signaling Pathways; Signaling Pathways Downstream of TLRs and IL-1 Family Receptors; TNF Receptor Superfamily Signaling Pathways in Immune Cells; Ubiquitin Signaling to NF-kB. Structure and Function of Diversifying Receptors: IgG Structure and Function; Structure, Function, and Spatial Organization of the B Cell Receptor. T Cell Activation: Th17 and Th22 Cells.

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