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REVIEW ARTICLE |
https://doi.org/10.5005/jp-journals-11002-0056
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Pathophysiology of Enteropathogenic Escherichia coli-induced Diarrhea
1Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois at Chicago, Illinois, United States of America
2Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois; Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois, United States of America
Corresponding Author: Pradeep K Dudeja, Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois; Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois, USA, Phone: +(312) 996-6651, e-mail: pkdudeja@uic.edu
How to cite this article: Kaur P, Dudeja PK. Pathophysiology of Enteropathogenic Escherichia coli-induced Diarrhea. Newborn 2023;2(1):102–113.
Source of support: Supported by work done as part of the NIH projects DK054016, DK92441, the Veterans Affairs Senior Research Career Scientist Award 1IK6BX005242, VA-BCCMA award IO1BX005862 and the VA Merit Award BX002011 (to Pradeep K. Dudeja).
Conflict of interest: None
Received on: 04 March 2023; Accepted on: 25 March 2023; Published on: 06 April 2023
ABSTRACT
Enteropathogenic Escherichia coli (EPEC) are important diarrheal pathogens of infants and young children. Since the availability of molecular diagnosis methods, we now have new insights into the incidence and prevalence of these infections. Recent epidemiological studies indicate that atypical EPEC (aEPEC) are seen more frequently than typical EPEC (tEPEC) worldwide, including in both endemic diarrhea and diarrhea outbreaks. Therefore, it is important to further characterize the pathogenicity of these emerging strains. The virulence mechanisms and pathophysiology of the attaching and effacing lesion (A/E) and the type-three-secretion-system (T3SS) are complex but well-studied. A/E strains use their pool of locus of enterocyte effacement (LEE)-encoded and non-LEE-encoded effector proteins to subvert and modulate cellular and barrier properties of the host. However, the exact mechanisms of diarrhea in EPEC infection are not completely understood. From the clinical perspective, there is a need for fast, easy, and inexpensive diagnostic methods to define optimal treatment and prevention for children in endemic areas. In this article, we present a review of the classification of EPEC, epidemiology, pathogenesis of the disease caused by these bacteria, determinants of virulence, alterations in signaling, determinants of colonization vs. those of disease, and the limited information we have on the pathophysiology of EPEC-induced diarrhea. This article combines peer-reviewed evidence from our own studies and the results of an extensive literature search in the databases PubMed, EMBASE, and Scopus.
Keywords: Attaching and effacing lesion (A/E), Epidemiology, Ion transporters, LEE pathogenicity island, Type III secretion system (T3SS), Tight junctions.
KEY POINTS
Enteropathogenic Escherichia coli (EPEC) is a leading cause of infantile diarrhea worldwide and particularly in developing countries.
Global prevalence of atypical EPEC (aEPEC) is higher than typical EPEC (tEPEC).
Enteropathogenic E. coli strains adhere to intestinal epithelial cells (IECs) in two patterns; the first one is of localized adherence (LA), where bacteria adhere in discrete microcolonies, and the second one is of diffuse adherence in which bacteria adhere uniformly over the cell surface.
Enteropathogenic E. coli employs its type III secretion system and effector proteins to modulate cellular and barrier properties of the host intestinal milieu.
Enteropathogenic E. coli infection leads to extensive disruption of microvilli on IECs and consequent loss of absorptive surfaces and altered electrolyte transport that may be secondary to both altered expression of ion/solute transporters and the loss of mucosal surface area.
INTRODUCTION
Escherichia coli (E. coli) is the predominant facultative anaerobic species in the intestine. Most strains are non-pathogenic and play an important role in maintaining intestinal physiology.1,2 This organism was first described by German pediatrician Theobald Escherich in 1885, under the name “Bacterium coli commune” as a short rod that had initially been isolated from normal infant feces.3 The current classification systems of E. coli consider many strains (Flowchart 1).4
Flowchart 1: Categorization of pathogenic E. coli
Enteropathogenic E. coli (EPEC) is a major cause of infantile diarrhea in developing countries.5 EPEC strains were epidemiologically associated with outbreaks in 1940s and 1950s and were first described in 1955.6 These strains currently account for 1.3 million deaths every year.7 The incidence is now being noted more accurately since the development of molecular diagnostic methods. In this study, we have reviewed the epidemiology of EPEC infections in infants and children and our studies in animal models to understand the pathophysiology of EPEC-associated diarrhea.
Epidemiology of EPEC Infections
Although most strains of E. coli are avirulent commensals in the gastrointestinal tract, many can cause diarrhea, urinary tract infection, and sepsis/meningitis. Several E. coli pathogens have been implicated in public health problems worldwide.8 The incidence of EPEC-related disease seems to have decreased over the last several decades. It is unclear if this reduced incidence is due to interventions such as the promotion of breastfeeding, or whether earlier studies based on O:H-serotyping overestimated the relative contribution of these organisms compared to newer molecular methods and/or adherence assays.7
Enteropathogenic E. coli was the first strain of E. coli identified as the cause of infantile diarrhea in the 1940s and 1950s. These outbreaks of “summer diarrhea” were frequent in developed countries until the 1950s and had high mortality.6 EPEC strains were first shown to be pathogenic in human volunteer studies carried out by Levine et al.9 in 1978. They tested classic EPEC strains (O127 and O142) associated with infant diarrhea that had been stored for 7–9 years. These isolated strains did not express LT and ST enterotoxins or show invasiveness. Enteral administration to healthy young adult volunteers caused a notable diarrheal illness.
In a systematic review of 266 studies published between 1990 and 2002, EPEC was identified with a median prevalence of 8.8% (inter-quartile range, IQR of 6.6–13.2) in the community setting, 9.1% (IQR 4.5–19.4) in the outpatient setting, and 15.6% (IQR 8.3–27.5) in the inpatient setting. Enteropathogenic E. coli may be the second most frequently seen cause of diarrhea after rotavirus (25.4%) in the inpatient setting. However, there are important regional and temporal variations.10 Investigators from Peru combined data on six different diarrheagenic strains of E. coli from eight different studies of children <3 years of age. Multiplex real-time PCR showed that the average EPEC prevalence in diarrheal stool samples (n = 4,243) was 8.5% (95% CI: 7.6–9.3), second only to enteroaggregative E. coli (EAEC; 9.9%). Enteropathogenic E. coli prevalence increased with age; these strains were found in 3% of diarrheal samples in children <6 months, in 11% of children 6–12 months, and in 16% of children 13–24 months. In these cohorts, exclusive breastfeeding was more frequent than in other studies (>80% for infants younger than 6 months), and hence young infants may have been protected from symptomatic infection. Among asymptomatic controls (n = 3,760), EPEC was detected in 10.9% (95% CI: 9.4–11.4).11–13 Similarly, in a recent study in India, EPECs were identified in 3.2% of 648 children <5 years of age who were hospitalized for diarrhea.14 In another study, EPEC has been noted to be the most prevalent pathotype with an average prevalence of 10.9% (95% CI: 9.4–11.4), followed by EAEC (10.4%).7 A study reported that more than 20% of all episodes of persistent diarrhea in the pediatric population; aged >14 days are mainly caused by diarrheagenic E. coli such as aEPEC. Another study identified specific E. coli strains from patients of infantile gastroenteritis. This study reported that serogroups O111 and/or O55 were more putative in causing diarrhea in recipients, and disease outcome in terms of severity of symptoms was largely dependent on the size of the dose.15–17
Enteropathogenic E. coli is known to be an important cause of infantile diarrhea in Brazil, Chile, Peru, and Iran.18 Studies in Brazil, Mexico, South Africa, and Bangladesh have shown that EPEC infections cause 30–40% of infant diarrhea with high mortality rates.19–23 In several studies conducted in Latin America, tEPEC was found to be the main cause of endemic diarrhea in children < 1 year of age. The frequency of tEPEC infection drops with an increase in age group, and adults rarely experience tEPEC episodes.18 This may be due to development of immunity or the loss of receptors interacting with some specific adhesins. Although tEPEC were major agents of acute diarrhea in infants until the 1990s, a clear decline in many of these countries was seen in the global enteric multicenter study, a population-based case–control study including seven countries in Africa and Asia with the goal to identify the etiology, burden, and mortality of acute moderate-to-severe pediatric diarrhea.24,25 The reasons for the decline are unclear but may be linked to improved public health with active interventions, therapy, sanitary conditions, and control of hospital infections.24,26 However, tEPEC infections remain associated with a 2.8-fold higher risk of death among infants aged 0–11 months.25
Atypical EPEC continues to be frequently detected in various parts of the world.7 Thirteen studies from peri-equatorial/tropical countries showed aEPEC isolates in 78% (131/169) of all EPEC cases in children.18 Wheeler et al.27,28 reported the identification of 142 aEPEC strains with only one tEPEC in 2774 samples from symptomatic children from the UK. A study from Australia identified 61 EPEC strains from a stool samples of symptomatic patients and highlighted the higher frequency of aEPEC at 95.1% (58/61).29 In 2009, the aEPEC strain O76 was reportedly responsible for a nursery outbreak in Finland.30
In another study, Sakkejha et al.31 detected 109 EPEC isolates in England from 2010 to 2012, with 93% of the patients with diarrhea; aEPEC were seen more frequently than tEPEC. Overall, according to 266 studies published between 1990 and 2002, EPEC remains major pediatric pathogen.7 As such, in 2014 a European, multicenter, prospective quarterly point-prevalence study of community-acquired diarrhea (EUCODI) showed a high frequency of EPEC.32
For unknown reasons, EPEC disease is becoming less frequent in infants in developed countries in developed/temperate climate zones of the world. However, day care centers and pediatric wards of hospitals are still prime breeding grounds for EPEC outbreaks.33–35 Globally, EPEC is responsible for infantile diarrhea in under-developed nations with nearly 30% mortality.36
Even though EPEC is strongly associated with infant diarrhea, many studies have also found EPEC, particularly aEPEC, in asymptomatic controls.18 There may be multiple possible reasons for this apparent anomaly: (a) host susceptibility.37 There may be genetic variability in specific mucosal receptors, including proteins and carbohydrate moieties; (b) individual variability in non-specific host barriers such as the gut microbiome, mucus layer, and epithelium. The variability in the strength of these barriers may influence bacterial overgrowth and susceptibility to disease.38 (c) Immune status of the host, which may limit bacterial flora to colonization but not cross numerical thresholds needed to cause disease.39 In addition, secretory immunoglobulin A (sIgA) in the intestine and in human milk can limit/prevent enterocyte colonization/mucosal invasion by enteropathogens.40 Human milk also contains other non-specific defense factors such as lactoferrin and enterotoxin-binding oligosaccharides. In endemic areas, colostrum contains specific sIgA against EPEC.41 In addition, children may acquire natural immunity with age. Opintan et al.42 showed that EPEC carriage, not disease, is frequently seen in healthy children in endemic areas after 2 years of age. Bacterial factors are also important in asymptomatic carriage of EPEC. Some strains are more likely to not cause symptoms, such as those with the phylogenetic marker gene yhaA. Children without diarrhea frequently carried aEPEC strains that were OI-122 efa1/lifA-negative and yhaA-positive. There is considerable variability in the severity of disease between individual strains.43
The variability in diagnostic tests also needs attention. In this regard, the bacterial load is an important consideration. Barletta et al.44 compared children with diarrhea vs. asymptomatic controls. When a quantitative real-time PCR assay was used, the bacterial load was significantly higher in the symptomatic infants than in age-matched controls. Other factors may also need consideration. For instance, the collection of control samples and sample size are pivotal factors.45 The transmission of EPEC from controls to other patients needs further consideration. Finally, environmental factors such as poor hygiene and fecal contamination may also increase the bacterial load in control groups.7
EPEC Definition and Classification
Escherichia coli serotypes were first classified based on the Kauffmann system in the 1940s. The three antigen systems included the somatic O, flagellar H, and the capsular K surface antigens.46,47 In 1955, the term EPEC was coined to describe strains that were primary intestinal pathogens but were rarely encountered in the feces of healthy individuals and in infections other than diarrheal diseases.4 Formally, 187 O serotypes were documented, but currently 176 are considered as true O serotypes. Six (O31, O47, O67, O72, O94, and O122) are no longer considered as O serotypes, some being duplicate names for an O antigen and others were in organisms that were reclassified into other genera and three (O34, O89, and O144) strains are also removed from this classification which are incapable of producing O antigens and are removed from these O serotypes. For O serotypes, the most variable cell component is O antigen because of existence of variations in sugar moieties and the linkages present within as well as between O units. Due to the existence of these variations, there is diversity of various clones in the species. Each expresses different surface antigen on the cell surface which offers selective advantages in varied environments. The O antigen is one of main virulence factors and its loss can severely impair the pathogenicity and virulence. O antigens play vital roles, including protection against phagocytosis and clearance via neutrophils and monocytes, as well as have inhibitory effects on the bactericidal activity of lysozyme, a key player in host innate immunity.48 The major O serogroups, including O55, O86, O111, O119, O125, O126, O127, O128ab, and O142, are considered to contain EPEC serotypes.49,50 The variability in O surface antigen provides basis for typing of the bacterial species for taxonomical as well as epidemiological purposes. It is most widely utilized to signify the presence of enteropathogens and considered as a basic tool for bacterial outbreak investigations and surveillance.48 O55 serotype is most rarely found in healthy individuals. However, varied pathogenicity levels are exhibited within O serotypes as all serotypes are not equally pathogenic, and only a limited number of H serotypes are incriminated within O serotypes.49 Another antigen, H (flagellar) is also expressed by EPEC strains. H2 and H6 are predominantly expressed flagellar antigens, and the least frequent ones include H7, H8, H9, H12, H21, H27, H25, and H34. However, some EPEC strains lack H flagellar antigens and are, therefore, classified as H-negative. These strains are non-motile.51
Several EPEC serogroups may share characteristics with the Shiga toxin-producing E. coli (STEC).52,53 Both can induce attaching-and-effacing (A/E) lesions on intestinal epithelial cells (IECs), and the bacteria attach to IECs and efface the microvilli on the cell surface (Fig. 1).54 There is a need to identify specific virulence genes to distinguish between the two bacterial genera as these differ in pathogenicity. EPEC pathotypes do not produce the Shiga toxin (stx–), but some aEPEC strains such as the O55:H7 resemble the LEE-positive Shiga toxin-producing E. coli such as the STEC O157:H7 in their genetic and virulence characteristics.4 Most tEPEC and aEPEC strains may differ in adherence patterns; tEPEC strains show localized adherence (LA) patterns, but the aEPEC can produce a localized-like adherence, a diffuse adherence (DA), or an aggregative adherence (AA) pattern.24
Fig. 1: Attachment of EPEC to the host epithelial cells results in the formation of cup and pedestal structures in the A/E lesion. A/E lesions exhibit intimate bacterial adherence to intestinal epithelial cells, extensive disruption of microvilli and enterocyte borders, and alterations in F-actin arrangement with accumulation of cytoskeletal proteins beneath adherent microcolonies resulting in the formation of a typical cup and pedestal-like structure
Enteropathogenic E. coli binds IECs by an outer membrane protein called intimin, which is encoded by the gene eae. The genetic elements needed to produce the A/E lesions are encoded on a genomic pathogenicity island, the locus of enterocyte effacement (LEE).55,56 Another pathogenicity factor is the plasmid E. coli adherence factor (pEAF).4,57 Enteropathogenic E. coli is classified as typical or atypical based on the presence of pEAF, which contains two important operons.24 These include a type IV bundle-forming pilus (bfp) and a plasmid-encoded regulator (per). The bfp promotes bacterial adherence and formation of compact microcolonies. Bfp and per are important transcriptional activators for LEE pathogenicity island.4,58
The plasmid pEAF imparts important characteristics to EPECs. Three subgroups can be seen:
(1) Typical EPECs (tEPECs) are eae+ bfpA+ stx−. Most belong to classical O:H serotypes and express bfp to show the localized adherence (LA) phenotype.59 The expression of EPEC virulence genes on classical EPEC serogroups is not universal. However, tEPEC strains are more homogeneous in their virulence traits than aEPEC. Most of the typical strains produce the virulence factors encoded by the LEE region and EAF plasmid.24
(2) Atypical E. coli (aEPEC) strains lack the EAF plasmid and hence are bfpA negative and are defined as eae+ bfpA− stx−. The lack of Bfp, makes atypical EPEC strains exhibit localized-like (LAL) pattern, which is mainly characterized by the presence of bacterial microcolonies. LAL is the most common pattern, but atypical EPEC strains also exhibit diffuse (DA) or aggregative adherence (AA) patterns.58,60 LAL+ aEPECs show pili and other known adhesins. Some aEPECs express the enteroaggregative heat-stable toxin (EAST1) and other potential virulence factors not encoded in the LEE, such as a hemolysin.60–62
(3) Non-typeable EPECs, which are identified among aEPECs and do not belong to classical EPEC serogroups. There are >200 of these strains.63,64
Virulence Factors and Signaling
For successful infection and formation of an A/E lesion, two major virulence factors are needed, the type IV bundle-forming pilus (BFP) and LEE.
Type IV Bundle-forming Pilus (BFP)
Type IV BFP is a dynamic fibrillar organelle responsible for the initiation of initial non-intimate attachment of EPEC to the host IECs. Further, BFP recruits individual EPEC together as aggregates and leads to the formation of microcolony on the host cell membranes, typically known as a localized adherence (LA) phenotype. The ~80 kb plasmid (pEAF) encodes 14 genes, which are required for the biogenesis of BFP and consequently in the formation of the EPEC adherence factor (EAF). The strains lacking pEAF are incapable of forming typical LA phenotype.57,65,66 Activation of BfpA is mediated by the plasmid-encoded regulator A (PerA). The activated form is a major pilus subunit and is called pre-bundlin. Further, pre-bundlin is acted upon by the prepilin peptidase, BfpP, and is then converted to the mature forms.67,68
Two nucleotide-binding proteins, BfpD and BfpF, further mediate the extension of the pilus and retraction, respectively. Aggregation of EPEC is promoted by BfpD, whereas BfpF facilitates the separation of EPEC from cellular aggregates that are maintaining a constant supply of bacterial cells for further infectious steps. BfpF-mediated dissociation of bacterial cell aggregates permits the intimate attachment of individual EPEC to the gut epithelium, resulting in efficient activation of T3SS and successful translocation of effector molecules into the host cells (Fig. 2).65 In addition to filamentous actin, cytoskeletal proteins such as α-actinin, talin, ezrin, myosin-light chain, vasodilator-stimulated phosphoprotein (VASP), the Wiskott–Aldrich syndrome protein (WASP), and the actin-related protein 2/3 (Arp2/3) complex are also observed in EPEC-induced A/E lesions. Additionally, many proteins involved in focal adhesion such as α-actinin and vinculin were found to be recruited to sites of A/E lesions.69–73 After EPEC attachment to the host surface, kinases encoded by Ab1/Arg, Src, and Tec families lead to phosphorylation of tyrosine residues in the cytoplasmic domain of translocated intimin receptor (Tir). Phosphorylated Tir interacts with two adaptor proteins (Nck1 and Nck2). This interaction results in the recruitment of actin nucleation-promoting factor, N-WASP, which further activates the Arp2/3 complex that assembles actin beneath EPEC (Fig. 2). These signaling events lead to the formation of actin-rich pedestals on host cell luminal membrane, along with inflammatory response and diarrhea.65
Fig. 2: Schematic representation of localization of virulence factors type IV BFP and LEE of EPEC on the small intestine and other interacting proteins involved during A/E lesion formation: Stage 1: Initial adherence and microcolony formation of EPEC on intestinal epithelial cells induced via type IV BFP and its activator Per. Stage 2: Effacement of microvilli mediated by activation of LEE operons via EspABD complex and translocation of T3SS effector proteins into intestinal epithelial cells. EPEC utilizes a type III secretion system (T3SS) to inject bacterial virulence factors directly into host cells. The T3SS apparatus is composed of several key protein components, including EspA, EspB, and EspD. EspA forms a needle-like channel and EspB and EspD cap this structure to form a pore that allows direct translocation of secreted effector molecules known as E. coli secreted proteins, EspF, EspG, EspH, Tir, and Map into the host cytosol. Translocated intimin receptor (Tir) is inserted into the plasma membrane, where it serves as a receptor for intimin, with Tir-intimin interaction triggering signaling events leading to pedestal formation. Stage 3: Intimate attachment of EPEC on the surface of host epithelial cells mediated by the interaction of adhesin intimin with Tir. This is followed by phosphorylation of Tir and recruitment of host cellular proteins and other adaptor proteins (Nck, N-WASP, and Arp2/3 complex) resulting in induction of actin polymerization beneath attached EPEC (BFP, bundle forming pilus; LEE, locus of enterocyte effacement; Tir, translocated receptor; EPEC, Enteropathogenic Escherichia coli; EAF, EPEC adherence factor; PER, plasmid-encoded regulator; A/E, attaching and effacing; Nck, Non-catalytic tyrosine kinase; WASP, Wiskott–Aldrich syndrome protein and Arp2/3, actin-related protein 2/3 complex)
Locus of Enterocyte Effacement
Once the bacterial aggregates dissociate from the host cell membranes via BfpF, EPEC expresses the LEE for further intimate attachment to intestinal epithelial cells (Fig. 2). Enteropathogenic Escherichia coli contains a 35,624 base pair LEE pathogenicity island (LPI), which contains 41 open reading frames (ORFs) of more than 50 amino acids arranged in five major polycistronic operons (LEE1 to LEE5).74,75 Locus of enterocyte effacement pathogenicity island encodes for the majority of EPEC effector proteins. Locus of enterocyte effacement encodes for the T3SS machinery (Esc and Sep proteins), outer membrane adhesin (intimin), translocators (EspA, EspB, and EspD), chaperones (Ces proteins), effector proteins (EspF, EspG, EspH, Map, and EspZ), translocated intimin receptor (Tir), regulatory proteins Ler (LEE-encoded regulator), repressors including GrlR (global regulator of LEE proteins), and activators such as GrlA (global regulator of LEE proteins).76 Various factors influence the regulation of LEE, including Ler, GrlR, and GrlA; and E. coli global regulators such as the H+NS, IHF, and FIS.77,78 These genes are separated into three functional domains – a region encoding intimate adherence (Tir and intimin), a region encoding the EPEC-secreted proteins (including espA, espB, espD, and espF) and their putative chaperones, and the region encoding a type III secretion system.79
LEE1, LEE2, and LEE3 encode for the genes involved in the production, assembly, and regulation of T3SS. Locus of enterocyte effacement-encoded structures are comprised of three vital components: (a) outer membrane needle complex (EscC, EscD, EscF, EscI, and EscJ); (b) inner membrane, which contains an export apparatus (EscRST, EscU, and EscV); and (c) a cytoplasmic sorting platform (EscA, EscK, EscL, EscN, and EscQ). The gene of translocation apparatus, the extracellularly secreted proteins of T3SS are encoded via LEE4 genes (EspA, EspB, and EspD). The role of EspB is implicated in the effacement of microvilli on the intestinal surface. The EspABD translocon apparatus of T3SS is responsible for the translocation of six LEE-encoded effectors (Tir, Map, EspF, EspG, EspZ, and EspH). These effectors are involved in the sequential events during EPEC infection which include disruption of tight junctions, mitochondrial dysfunction, and formation of filopodia in host intestinal epithelial cells. The genes for adhesin (intimin), 94 kDa outer membrane protein of EPEC, and its translocation receptor (Tir) are encoded via LEE5.65 The gene encoding for intimin, eae (E. coli attaching-and-effacing), is comprised of four distinct intimin subtypes (α, β, γ, and δ).80 Different intimin subtypes are expressed in different tissues; the small intestinal mucosal layer expresses intimin-α clones, and the Peyer’s patches exhibit expression of intimin-γ.81 Different intimin types could bind to the host cell protein nucleolin, which then colocalizes with adherent bacteria.82 Chaperone proteins have also been discovered in T3SS in EPEC and are essential for secretion of espD, espA, and espB.76
Effectors encoded outside the LEE pathogenicity island have been described in all A/E-producing pathogens.24 Scattered across the whole genome, six pathogenicity islands harbor the clusters of non-LEE-encoded (Nle) effectors.75,83 These Nle effectors include NleA-H, EspG2/Orf3, Cif, EspJ, and EspL. NleA (also called EspI) suppresses protein secretion; EspJ inhibits phagocytosis; and NleE and NleH activate innate immune responses. A/E E. coli strains utilize both LEE-encoded and non-LEE-encoded effector proteins to subvert and modulate cellular and barrier properties of the host for successful infection in a well-controlled manner.7
Pathogenesis
Enteropathogenic E. coli is generally considered to be a noninvasive pathogen but can cause subclinical to fatal diarrhea.4 Studies with adult volunteers reported that 12–24 hours post infection with tEPEC (109–1010 of bacterial inoculum) can induce diarrhea.9 As discussed before, EPEC strains attach to IECs in two different patterns – localized adherence (LA) in which bacteria adhere in discrete microcolonies and diffuse adherence (DA) in which bacteria adhere uniformly over the cell surface. Localized adherence was highly correlated with specific EPEC serogroups in strains isolated from patients with diarrhea.66 The BFP is usually seen as the initial EPEC attachment factor.84 The major pilin subunit of BFP is identified as the bfpA. Bundle-forming pilus is encoded by a cluster of 14 genes on the EAF plasmid and mediates LA phenotype, which is further responsible for antigenicity, biofilm formation, autoaggregation, and compact microcolony formation.51,57,85 Genes external to the bfp gene cluster were also necessary for full expression of BFP. This included the global regulator element of EPEC pathogenesis perABC (bfpTVW) and the chromosomal dsbA gene encoding for a disulfide isomerase.86
The BFP-mediated interbacterial interactions may allow the dispersal of individual bacteria from autoaggregates and colonization to other epithelial sites, contributing to the spread of infection within the gut. In addition to BFP, additional fimbrial structures have also been characterized and could have roles in EPEC-host cell adhesion. There may be rod-like fimbriae and fibrillae, suggesting that the bacterial–host cell interaction is a multifactorial process. More recently, flagella have been implicated in EPEC adherence to IECs.87 However, there is some uncertainty because a flagellated strain that lacked BFP, intimin and EspA failed to adhere to IECs in ex vivo studies. The term EPEC adherence factor (EAF) refers to the plasmid-mediated adhesion. Escherichia coli strains isolated from outbreaks of infantile gastroenteritis almost invariably possess the EAF plasmid.88 EPEC adherence factor plasmid generally promotes non-intimate cell adhesion. For A/E lesion formation, chromosomally encoded factors were required for the A/E phenotype, and the genes on the plasmid may play a secondary role.89 Localized adherence (LA) pattern is exhibited by various EPEC serogroups including O55, O86, O111ab, O119, O125, O128ab, and O142.90 The existence of 60 MDa plasmid (denoted as pMAR2) is responsible for localized adherence pattern exhibited by EPEC strain E2348/69 (O127:H6).91
Mucosal adhesion by EPEC may involve two distinct stages: (a) initial attachment of EPEC promoted by plasmid-encoded adhesins; and (b) effacement of brush border microvilli leading to intimate EPEC attachment. Although the second stage could occur without the first, the presence of plasmid-encoded adhesin enhanced mucosal colonization.89 A/E lesions exhibit association of bacterial cells to IECs followed by extensive disruption, loss of brush borders and microvilli, alterations in F actin rearrangements, and ultimately cup and pedestal formations.7 These structures may provide a strong attachment of EPEC to the cell surface, preventing dislodgement in the ensuing diarrheal response. Many affected bowel segments show depletion of glycocalyx. Some areas show a mucous pseudomembrane coating on the mucosal surface. There are characteristic cytoskeletal alterations with disruption of the brush border cytoskeleton and proliferation of filamentous actin beneath the foci where bacteria adhered to the host cell surface. There are at least three prominent changes: (a) adherence to IECs; (b) delivery of 25–50 virulence factors into the host cell using a type III secretion system (T3SS)51; and finally, (c) firm adherence to the cell surface with the formation of pedestals (Fig. 2).86 The T3SS is one of the five most important secretion systems utilized by Gram-negative bacteria, besides the T4SS, T5SS, T6SS, and T7SS, to inject effector proteins into the host cells to promote colonization and virulence. It is important because it is exclusively involved in virulence.92,93
The T3SS, intimin, and the translocated intimin receptor (tir) are all essential virulence determinants of the intimate adherence, a process that requires the T3SS to inject tir into the host cell. Tir acts as a receptor for bacterial binding via tir–intimin interaction. These trigger many signaling cascades such as phosphorylation of a host phospholipase and recruitment of cytoskeletal proteins beneath the adherent bacteria. Intimin can also subvert cellular processes independently of tir.86 Mitochondrial-associated protein (Map) targets host cell mitochondria and also contributes to the disruption of the epithelial barrier.94
The hallmark of EPEC infection is A/E lesion which marks the intimate attachment of the bacteria to the host enterocytes and results in the effacement of the microvilli. The IEC membrane in these foci can also be raised locally in a characteristic pedestal shape that may extend up to 10 μm outwards from the cell to form pseudopod-like structures.95 This near-complete destruction and extensive loss of intestinal epithelial surface with villus atrophy and thinning of the mucosal layer is frequently seen during severe EPEC infections.96 The extensive loss of microvilli on the infected IECs alters the expression and function of ion transporters, channels, and tight junctions. The pathogenesis of microvillus effacement is seen as a 2-step process that requires synergistic action of three effectors (Map, EspF, and Tir) on intimin, and retention of the detached microvillar material. Other studies have focused on the type III secretion system and its effectors including tir, map, espF, and espG.65 Enteropathogenic E. coli also rapidly inactivates the sodium-D-glucose cotransporter (SGLT-1) by multiple mechanisms. SGLT-1 plays a crucial role in the daily uptake of fluids from the intestinal lumen.97 Calcium signaling may also be important; it may activate actin-severing proteins, resulting in cytoskeletal rearrangement and brush border effacement. However, all these possibilities need further confirmation.98,99 Some aEPEC is strongly associated with acute disease, whereas others have been noted in persistent diarrhea.100 Clinically, aEPEC outbreaks may cause mild but prolonged non-dehydrating, non-inflammatory diarrhea. There is usually no fever, vomiting, or abdominal pain.
Pathophysiology of Diarrhea
We now understand EPEC pathogenesis at cellular and genetic levels, but the pathophysiology of the resulting diarrhea remains elusive. The extensive loss of microvillus and subsequent reduction in absorptive surface certainly contributes to diarrhea. However, the rapid onset of diarrhea remains unexplained and appears to be multifactorial in nature. Enteropathogenic E. coli can alter epithelial permeability by activating signaling cascades that phosphorylate Ser/Thr residues on the myosin light chains.101 This might contribute to diarrhea through increased permeability and disruption of tight junction integrity (Fig. 3). Recently, another EPEC effector molecule, the espF, was shown to be translocated by the T3SS into host cells, where it disrupts host IEC tight junctions and could contribute to diarrhea.102 Enteropathogenic E. coli can also activate NF-κB in host cells and induce host inflammatory responses, which, in turn, could increase paracellular permeability and cause tissue damage.103 The stimulatory effects of EPEC infection have been implicated on NF-κB activation and downstream enhancement of Cl secretion and fluid accumulation in the colon.104,105
Fig. 3: EPEC infection induces inflammation and disrupts the epithelial barrier resulting in leaky tight junctions. Bacterial overgrowth, cytokine expression, biofilms, and leukocyte infiltration all create positive-feedback loops of inflammatory changes. Cytokines such as interleukin (IL)-1β and chemokines such as IL-8 activate regulatory factors such as the nuclear factor-κB and progressively enhance the inflammatory changes and dysfunction of the epithelial barrier
Prolonged EPEC infection leads to inflammation and disruption of structure and barrier function of tight junctions (Fig. 3).5,106,107 Enhanced paracellular permeability, inflammation, and disruption of tight junctions have been implicated in EPEC-mediated chronic diarrhea.108 Studies have highlighted the downstream effects of prolonged inflammation in terms of increased influx of neutrophils, resulting in the release of 5-AMP that is further converted into secretagogue adenosine.109,110
Enteropathogens such as EPEC likely cause diarrhea by altering electrolyte transport.111 Impairment of ion and solute transport may directly or indirectly influence the fluid transport processes and barrier integrity in gut epithelial cells.112 Recent advances indicate that EPEC infection can directly influence ion transport mechanisms involving Cl−/HCO3 −/OH− exchange, Na+/H+ exchange, serotonin transporter, and short-chain fatty acids transporters. The following section will review the potential mechanism(s) involved in the regulation/alteration of ion and nutrient transporters on the gut epithelial cells during EPEC-induced diarrhea.
(a) Effect of EPEC Infection on Na+/H+ Exchanger Type 3 (NHE3)
Diarrhea caused by enteric pathogens may involve decreased NaCl absorption, enhanced Cl– secretion, or both.5 In early onset diarrhea, decreased intestinal NaCl may be pathophysiologically more important than the rise in Cl− secretion.113,114 The effector proteins of EPEC namely, NleA and Map, interact with Na+/H+ exchanger regulatory factor 2 (NHERF2) and alter its function and ultimately leading to decreased Na+ uptake.115 In intestinal epithelial cells, the expression of NHE2 and NHE3 is restricted to the apical surface, whereas NHE1 is expressed on the basolateral membranes. Our group has shown that EPEC infection in in vitro models activated NHE2 but inhibited the NHE3, the key Na+ absorbing transporter (Fig. 4).116 In in vitro models, EPEC infection leads to inhibition of the Cl−/HCO3 −/OH−/exchange activity critical for intestinal chloride absorption.116 Also, as stated above the activity of NHE3, which is a major Na+ absorbing isoform, is inhibited.114 These findings may be a source of uncertainty in the relative pathophysiological importance of NHE2 vs. NHE3; NHE3 here could very well be the more important of these two as a regulator of Na+ absorption and determinant of the onset of diarrhea.114 Also, prolonged EPEC infection contributes to inflammation and disruption of the structure and barrier function integrity of tight junctions and could contribute to diarrhea.117
Fig. 4: Schematic representation of transporters affected during EPEC infection. EPEC infection affects intestinal epithelial barrier and leads to reduced expression/function of ion and solute transporters and results in the development of diarrhea. Type III secretion system of EPEC is responsible for the release of E. coli-secreted proteins (Esps) into the infected host cells. EspF exhibits inhibitory impact on Na+/H+ exchange isoform 3 and EspG disrupts the microtubules, which further leads to decreased apical expression of DRA resulting in reduction of apical Cl−/OH− (HCO3−) exchange activity and inhibition of electroneutral NaCl absorption in the intestinal milieu. EPEC inhibits butyrate absorption by reducing the plasma membrane expression of monocarboxylate transporter 1 (MCT-1). EPEC also inhibits the function of serotonin transporter (SERT) and increases 5-HT availability by activating protein tyrosine phosphatases (PTPases), which can further modulate the ion absorption and contribute to the onset of diarrhea. EPEC-induced inhibition of SGLT-1 also promotes fluid accumulation with similar effects. (NHE3, Na+/H+ exchanger type 3; DRA, downregulated in adenoma; MCT-1, Monocarboxylate transporter 1; ASBT, Apical sodium-dependent bile acid transporter; BA, Bile acid; SERT, Serotonin transporter; SGLT-1, D-glucose transporter)
(b) Effect of EPEC Infection on Downregulated in Adenoma (DRA/SLC26A3)
Intestinal epithelial cells express an integral membrane Cl−/HCO3 − transporter, the downregulated in adenoma (DRA/SLC26A3).115 EPEC suppresses the function and apical expression of DRA/SLC26A3, and may thus contribute to the pathophysiology of diarrhea. Studies from our group demonstrated an increased endocytosis and decreased apical expression of DRA/SLC26A3 in EPEC-infected cells (Fig. 4).118 Other studies suggest that reduced exocytosis may also play a role. The virulence factors EspG1 and EspG2 may alter DRA/SLC26A3 expression on epithelial cells via mechanisms involving microtubule disruption.118
(c) Effect of EPEC Infection on Absorption of Short-chain Fatty Acids (SCFAs)
Short-chain fatty acids play a significant role in sustaining colonocyte health and metabolism, integrity of epithelial lining, and in the maintenance of colonic fluid and electrolyte balance. Butyrate, a key SCFA, has been shown to play an important role in fluid balance by enhancing electroneutral NaCl absorption119 and reducing Cl− secretion.120 Our group has shown that EPEC infection can significantly reduce butyrate uptake by intestinal epithelial cell lines (Fig. 4).117 EPEC infection reduced the expression of monocarboxylate transporter 1 (MCT1), the primary SCFA transporter in gut epithelial cells. Butyrate also plays an anti-inflammatory role,121 and decreased availability of butyrate has been noted both in acute and chronic inflammatory conditions.122
(d) Effect of EPEC Infection on Apical Sodium-dependent Bile Acid Transporter (ASBT)
Apical sodium-dependent bile acid transporter (ASBT) is a putative transporter responsible for stimulating the intestinal absorption of bile acids. Reduced ASBT expression/function has been implicated in the pathogenesis of diarrhea. Annaba et al.123 have shown the negative impact of EPEC infection on ileal ASBT expression/function in various in vitro models.
(e) Effect of EPEC Infection on the Serotonin Transporter (SERT)
Serotonin transporter is a key regulator of the extracellular availability of serotonin (5-HT), and its function was inhibited in response to EPEC infection in intestinal epithelial cells. Serotonin transporter activity is reduced via activation of the Src-homology-2 (SH2) domain containing protein tyrosine phosphatase (PTPase). In the absence of SERT, 5-HT circulates in the extracellular milieu resulting in the activation/sensitization of its cognate receptors.124 In this study, SHP2 is associated with SERT during EPEC infection due to dephosphorylation at tyrosine residues and thereby inhibiting its function and activity. High luminal serotonin levels (due to inhibition of SERT) have been linked to fluid accumulation in the gut lumen.
(f) Effect of EPEC Infection on Sodium D-glucose Transporter (SGLT-1)
In addition to SERT and other transporters outlined above, EPEC has also been shown to inhibit the function of the sodium-D-glucose transporter (SGLT1), which is a major contributor of fluid uptake in the small intestine112 and hence could contribute to diarrhea.
Effect of EPEC Infection on Tight Junctions
Enteropathogenic E. coli-mediated disruption of the gut epithelial barrier also contributes to the onset of diarrhea. Epithelial cells are normally bound together by a network of tight junctions. The membrane barrier is selectively permeable for the passage of ions and solutes across the paracellular space. It also serves as a boundary that prevents the coalescence of apical and basal plasma membrane proteins to maintain the polarity of the epithelial cells and prevents the backflow of fluids into the lumen.115 The cell–cell adhesion is maintained by the transmembrane proteins which are associated with the cytoskeleton via the adaptor proteins. The members of claudin family and the transmembrane proteins of the marvel-domain containing protein families, such as occludin, tricellulin/marvelD2, and marvelD3, are key regulators of paracellular permeability. Tight junction-associated exchange factors for Rho GTPases also modulate the actin cytoskeleton and membrane permeability.115,125–127 During EPEC-induced diarrhea, leakages are observed in the tight junctions; studies suggest the potential role(s) of effector proteins EspF, Map, EspG1/G2, and NleA in disrupting the host cell tight junctions.128–130 The N-terminus of EspF contains mitochondrial- and nucleolus-targeting sequences that can alter the function of these organelles. The C-terminus of EspF contains three proline-rich repeats that interact with the eukaryotic sorting nexin 9 (SNX9) and neuronal Wiskott–Aldrich syndrome protein (N-WASP), and are ultimately involved in the activation of the Arp2/3 complex and regulation of actin polymerization.128–132 EspF may recruit zonula occludens (ZO-1 and ZO-2) into actin pedestals.133 In murine models, EspF can disrupt tight junctions via internalization of claudin-1, 3, and 5.115
Another EPEC effector protein, Map, interacts with EspF and is involved in the disruption of tight junctions. Similar to EspF, Map is recruited to mitochondria where it modulates the mitochondrial processes and functions. Map acts as a guanine-nucleotide exchange factor (GEF) for Cdc42 GTPase and promotes its activation leading to the formation of transient filopodia. A Thr-Arg-Leu motif is present at the C-terminus of Map, which interacts with the Na+/H+ exchanger regulatory factor 1 (NHERF1). This complex links with ezrin and then promotes the interaction between Map and actin cytoskeleton.128,134,135 The tight junction proteins, the zonula occludens-1 and occludin, are disrupted by NleA leading to increased paracellular permeability.130
CONCLUSIONS
Studies show aEPEC to be more prevalent than tEPEC worldwide. Therefore, it is important to further characterize the pathogenicity of these strains, virulence mechanisms, and the pathophysiology of these infections. While there is strong evidence showing that EPEC-induced diarrhea is multifactorial in nature and involves compromised gut barrier integrity and decreased absorption of fluid, which is contributed by decreased NaCl and solute absorption. However, the exact mechanisms of diarrhea in EPEC infection are still evolving. From the clinical perspective, there is a need for fast, easy, and inexpensive diagnostic methods to define optimal treatment and prevention for children in endemic areas.
REFERENCES
1. Drasar BS and Hill MJ. Human Intestinal Flora. London: Academic Press; 1974.
2. Siitonen A. Escherichia coli in faecal flora of healthy adults: Serotypes, P and type IC fimbriae, non-P mannose resistant adhesions and haemolytic activity. J Infect Dis 1992;116:10581065.
3. Escherich T. Die Darmbacterien des Neugeborenen und Sauglings. Fortschritte der Medizin 1885;3:515–522.
4. Hu J, Torres AG. Enteropathogenic Escherichia coli: Foe or innocent bystander? Clin Microbiol Infect 2015; 21(8):729–734.DOI. 10.1016/J.CMI.2015.01.015.
5. Borthakur A, Gill RK, Hodges K, et al. Enteropathogenic Escherichia coli inhibits butyrate uptake in Caco-2 cells by altering the apical membrane MCT1 level. Am J Physiol Gastrointest Liver Physiol 2006;290(1):30–35. DOI: 10.1152/ajpgi.00302.2005.
6. Neter E, Westphal O, Luderitz O, et al. Demonstration of antibodies against enteropathogenic Escherichia coli in sera of children of various ages. Pediatrics 1955;16(6):801–808. PMID: 13273119.
7. Ochoa TJ, Contreras CA. Enteropathogenic E. coli (EPEC) infection in children. Curr Opin Infect Dis 2011;24(5):478–483. DOI: 10.1097/QCO.0b013e32834a8b8b.
8. World Health Organization (WHO). https://www.who.int/.
9. Levine MM, Bergquist EJ, Nalin DR, et al. Escherichia coli strains that cause diarrhoea but do not produce heat-labile or heat-stable enterotoxins and are non-invasive. Lancet 1978;1(8074):1119–1122. DOI: 10.1016/s0140-6736(78)90299-4.
10. Lanata CF, Walter M, BR E. Improving diarrhoea estimates. WHO; 2002. http://www.who.int/child_adolescent_health/documents/pdfs/improving_diarrhoea_estimates.pdf.
11. Ochoa TJ, Mercado EH, Durand D, et al. Frequency and pathotypes of diarrheagenic Escherichia coli in Peruvian children with and without diarrhea. Rev Peru Med Exp Salud Publica 2011;28(1):13–20. DOI: 10.1590/s1726-46342011000100003.
12. Guion CE, Ochoa TJ, Walker CM, et al. Detection of diarrheagenic Escherichia coli by use of melting-curve analysis and real-time multiplex PCR. J Clin Microbiol 2008;46(5):1752–1757. DOI: 10.1128/JCM.02341-07.
13. Barletta F, Ochoa TJ, Ecker L, et al. Validation of five-colony pool analysis using multiplex real-time PCR for detection of diarrheagenic Escherichia coli. J Clin Microbiol 2009;47(6):1915–1919. DOI: 10.1128/JCM.00608-09.
14. Nair GB, Ramamurthy T, Bhattacharya MK, et al. Emerging trends in the etiology of enteric pathogens as evidenced from an active surveillance of hospitalized diarrhoeal patients in Kolkata, India. Gut Pathog 2010;2:4. DOI: 10.1186/1757-4749-2-4.
15. Neter E, Shumway CN. E. coli serotype D433: Occurrence in intestinal and respiratory tracts, cultural characteristics, pathogenicity, sensitivity to antibiotics. Proc Soc Exp Biol Med 1950; 75(2):504–507.DOI: 10.3181/00379727-75-18246.
16. Ferguson WW, June RC. Experiments on feeding adult volunteers with Escherichia coli 111, B4, a coliform organism associated with infant diarrhea. Am J Hyg 1952;55(2):155–69. DOI: 10.1093/oxfordjournals.aje.a119510.
17. June RC, Ferguson WW, Worfel M. Experiments in feeding adult volunteers with Escherichia coli 55, B5, a coliform organism associated with infant diarrhea. Am J Hyg 1953;57(2):222–36. DOI: 10.1093/oxfordjournals.aje.a119570.
18. Ochoa TJ, Francesca B, Carmen C, et al. New insights into the epidemiology of enteropathogenic Escherichia coli infection. Trans R Soc Trop Med Hyg 2008;102(9):852–856. DOI: 10.1016/j.trstmh.2008.03.017.
19. Gomes TA, Rassi V, MacDonald KL, et al. Enteropathogens associated with acute diarrheal disease in urban infants in São Paulo, Brazil. J Infect Dis 1991;164(2):331–337. DOI: 10.1093/infdis/164.2.331.
20. Cravioto A, Reyes RE, Ortega R, et al. Prospective study of diarrhoeal disease in a cohort of rural Mexican children: Incidence and isolated pathogens during the first two years of life. Epidemiol Infect 1988;101(1):123–134. DOI: 10.1017/s0950268800029289.
21. Cravioto A, Reyes RE, Trujillo F, et al. Risk of diarrhea during the first year of life associated with initial and subsequent colonization by specific enteropathogens. Am J Epidemiol 1990;131(5):886–904. DOI: 10.1093/oxfordjournals.aje.a115579.
22. Robins-Browne RM, Levine, MM, Rowe B GE. Failure to detect conventional enterotoxins in classical enteropathogenic (serotyped) Escherichia coli strains of proven pathogenicity. Infect Immun 1982; 38(2):798–801.DOI: 10.1128/iai.38.2.798-801.1982.
23. Albert MJ. Epidemiology of enteropathogenic Escherichia coli infection in Bangladesh. Rev Microbiol São Paulo 1996;27(Suppl 1):17–20. ISSN: 0001-3714.
24. Trabulsi LR, Keller R, Tardelli Gomes TA. Typical and atypical enteropathogenic Escherichia coli. Emerg Infect Dis 2002;8(5):508–513. DOI: 10.3201/eid0805.010385.
25. Kotloff KL, Nataro JP, Blackwelder WC, et al. Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): A prospective, case-control study. Lancet 2013;382(9888):209–222. DOI: 10.1016/S0140-6736(13)60844-2.
26. Tozzoli SR and Scheutz F.Pathogenic Escherichia coli: Molecular and Cellular Microbiology. Caister Academic Press; 2014. https://www.caister.com/ecoli.
27. Wheeler JG, Sethi D, Cowden JM, et al. Study of infectious intestinal disease in England: Rates in the community, presenting to general practice, and reported to national surveillance. BMJ 1999;318(7190):1046–1050. DOI: 10.1136/bmj.318.7190.1046.
28. Jenkins C, Smith HR, Lawson AJ, et al. Serotypes, intimin subtypes, and antimicrobial resistance patterns of atypical enteropathogenic Escherichia coli isolated in England from 1993 to 1996. Eur J Clin Microbiol Infect Dis 2006;25(1):19–24. DOI: 10.1007/s10096-005-0075-x.
29. Staples M, Doyle CJ, Graham RMA, et al. Molecular epidemiological typing of enteropathogenic Escherichia coli strains from Australian patients. Diagn Microbiol Infect Dis 2013;75(3):320–324. DOI: 10.1016/j.diagmicrobio.2012.11.010.
30. Møller-Stray J, Eriksen HM, Bruheim T, et al. Two outbreaks of diarrhoea in nurseries in norway after farm visits, April to May 2009. Eurosurveillance 2012;17(47):20321. DOI: 10.2807/ese.17.47.20321-en.
31. Sakkejha H, Byrne L, Lawson AJ, et al. An update on the microbiology and epidemiology of enteropathogenic Escherichia coli in England 2010-2012. J Med Microbiol 2013;62(Pt 10):1531–1534. DOI: 10.1099/jmm.0.062380-0.
32. Spina A, Kerr KG, Cormican M, et al. Spectrum of enteropathogens detected by the FilmArray GI panel in a multicentre study of community-acquired gastroenteritis. Clin Microbiol Infect 2015;21(8):719–728. DOI: 10.1016/j.cmi.2015.04.007.
33. Bower JR, Congeni BL, Cleary TG, et al. Escherichia coli O114:nonmotile as a pathogen in an outbreak of severe diarrhea associated with a day care center. J Infect Dis 1989;160(2):243–247. DOI: 10.1093/infdis/160.2.243.
34. Paulozzi LJ, Johnson KE, Kamahele LM, et al. Diarrhea associated with adherent enteropathogenic Escherichia coli in an infant and toddler center, Seattle, Washington. Pediatrics 1986;77(3):296–300. PMID: 3513114.
35. Snehaa K, Singh T, Dar SA, et al. Typical and atypical enteropathogenic Escherichia coli in children with acute diarrhoea: Changing trend in East Delhi. Biomed J 2021;44(4):471–478. DOI: 10.1016/j.bj.2020.03.011.
36. Senerwa D, Olsvik O, Mutanda LN, et al. Enteropathogenic Escherichia coli serotype O111:HNT isolated from preterm neonates in Nairobi, Kenya. J Clin Microbiol 1989;27(6):1307–1311. DOI: 10.1128/jcm.27.6.1307-1311.1989
37. Donnenberg MS and Finlay BB. Combating enteropathogenic Escherichia coli (EPEC) infections: The way forward. Trends Microbiol 2013;21(7):317–319. DOI: 10.1016/j.tim.2013.05.00338.
38. Levine MM and Robins-Browne RM. Factors that explain excretion of enteric pathogens by persons without diarrhea. Clin Infect Dis 2012;55(Suppl 4):S303–S311. DOI: 10.1093/cid/cis789.
39. Kotloff KL, Nataro JP, Losonsky GA, et al. A modified Shigella volunteer challenge model in which the inoculum is administered with bicarbonate buffer: Clinical experience and implications for Shigella infectivity. Vaccine 1995;13(16):1488–1494. DOI: 10.1016/0264-410x(95)00102-7.
40. Manthey CF, Autran CA, Eckmann L, et al. Human milk oligosaccharides protect against enteropathogenic E. coli (EPEC) attachment in vitro and EPEC colonization in suckling mice. J Pediatr Gastroenterol Nutr 2014;58(2):165–168. DOI: 10.1097/MPG.0000000000000172.
41. Parissi-Crivelli A, Parissi-Crivelli JM, Girón JA. Recognition of enteropathogenic Escherichia coli virulence determinants by human colostrum and serum antibodies. J Clin Microbiol 2000;38(7):2696–2700. DOI: 10.1128/JCM.38.7.2696-2700.2000.
42. Opintan JA, Bishar RA, Newman MJ, et al. Carriage of diarrhoeagenic Escherichia coli by older children and adults in Accra, Ghana. Trans R Soc Trop Med Hyg 2010;104(7):504–506. DOI: 10.1016/j.trstmh.2010.02.011.
43. Afset JE, Bruant G, Brousseau R, et al. Identification of virulence genes linked with diarrhea due to atypical enteropathogenic Escherichia coli by DNA microarray analysis and PCR. J Clin Microbiol 2006;44(10):3703–3711. DOI: 10.1128/JCM.00429-06.
44. Barletta F, Ochoa TJ, Mercado E, et al. Quantitative real-time polymerase chain reaction for enteropathogenic Escherichia coli: A tool for investigation of asymptomatic versus symptomatic infections. Clin Infect Dis 2011;53(12):1223–1229. DOI: 10.1093/cid/cir730.
45. Enserink R, Scholts R, Bruijning-Verhagen P, et al. High detection rates of enteropathogens in asymptomatic children attending day care. PLoS One 2014;9(2):e89496. DOI: 10.1371/journal.pone.0089496.
46. Kauffmann F. The serology of the coli group. J Immunol 1947;57(1):71–100. PMID: 20264689
47. Fratamico PM, DebRoy C, Liu Y, et al. Advances in molecular serotyping and subtyping of Escherichia coli. Front Microbiol 2016;7:644. DOI: 10.3389/fmicb.2016.00644.
48. Liu B, Furevi A, Perepelov AV, et al. Structure and genetics of Escherichia coli O antigens. FEMS microbiol Rev 2020;44(6):655–683. DOI: 10.1093/femsre/fuz028.
49. Ewirig WH, Davis BR, Montague TS. Studies on the Occurrence of Escherichia coli Serotypes Associated with Diarrheal Disease. Atlanta, Georgia: Communicable Disease Center, U.S. Department of Health, Education and Welfare; 1963. pp. 38.
50. Taylor J. Host specificity and enteropathogenicity of Escherichia coli. J Appl Bacteriol 1961;24(3):316–325. DOI: 10.1111/j.1365-2672.1961.tb00264.x.
51. Mare AD, Ciurea CN, Man A, et al. Enteropathogenic Escherichia coli—A summary of the literature. Gastroenterol Insights 2021;12(1):28–40. DOI: 10.3390/gastroent12010004.
52. Bryan A, Youngster I, McAdam AJ. Shiga toxin producing Escherichia coli. Clin Lab Med 2015;35(2):247–272. DOI: 10.1016/j.cll.2015.02.004.
53. Fierz L, Cernela N, Hauser E, et al. Characteristics of Shigatoxin-producing Escherichia coli strains isolated during 2010-2014 from human infections in Switzerland. Front Microbiol 2017;8:1471. DOI: 10.3389/fmicb.2017.01471.
54. Gaytán MO, Martínez-Santos VI, Soto E, et al. Type three secretion system in attaching and effacing pathogens. Front Cell Infect Microbiol 2016;6:129. DOI: 10.3389/fcimb.2016.00129.
55. McDaniel TK, Kaper JB. A cloned pathogenicity island from enteropathogenic Escherichia coli confers the attaching and effacing phenotype on E. coli K-12. Mol Microbiol 1997;23(2):399–407. DOI: 10.1046/j.1365-2958.1997.2311591.x.
56. Vallance BA, Finlay BB. Exploitation of host cells by enteropathogenic Escherichia coli. Proc Natl Acad Sci USA 2000;97(16):8799–806. DOI: 10.1073/pnas.97.16.8799.
57. Stone KD, Zhang HZ, Carlson LK, et al. A cluster of fourteen genes from enteropathogenic Escherichia coli is sufficient for the biogenesis of a type IV pilus. Mol Microbiol 1996;20(2):325–337. DOI: 10.1111/j.1365-2958.1996.tb02620.x.
58. Silva SS, Monfardini MV, Scaletsky ICA. Large plasmids encoding antibiotic resistance and localized-like adherence in atypical enteropathogenic Escherichia coli strains. BMC Microbiol 2020;20(1):138. DOI: 10.1186/s12866-020-01809-4.
59. Ochoa TJ, Barletta F, Contreras C, et al. New insights into the epidemiology of enteropathogenic Escherichia coli infection. Trans R Soc Trop Med Hyg 2008;102(9):852–856. DOI: 10.1016/j.trstmh.2008.03.017.
60. Rodrigues J, Scaletsky IC, Campos LC, et al. Clonal structure and virulence factors in strains of Escherichia coli of the classic serogroup O55. Infect Immun 1996;64(7):2680–2686. DOI: 10.1128/iai.64.7.2680-2686.1996.
61. Campos LC, Whittam TS, Gomes TA, et al. Escherichia coli serogroup 0111 includes several clones of diarrheagenic strains with different virulence properties. Infect Immun 1994;62(8):3282–3288. DOI: 10.1128/iai.62.8.3282-3288.1994.
62. Gonçalves AG, Campos LC, Gomes TA, et al. Virulence properties and clonal structures of strains of Escherichia coli O119 serotypes. Infect Dis 1997;65(6):2034–2040. DOI: 10.1128/iai.65.6.2034-2040.1997.
63. Schmidt MA. LEEways: Tales of EPEC, ATEC and EHEC. Cell Microbiol 2010;12(11):1544–1552. DOI: 10.1111/j.1462-5822.2010.01518.x
64. Jafari A, Aslani MM, and Bouzari S. Escherichia coli: A brief review of diarrheagenic pathotypes and their role in diarrheal diseases in Iran. Iran J Microbiol 2012;4(3):102–117. PMID: 23066484.
65. Lee JB, Kim SK, Yoon JW. Pathophysiology of enteropathogenic Escherichia coli during a host infection. J Vet Sci 2022;23(2):e28. DOI: 10.4142/jvs.21160.
66. Scaletsky IC, Silva ML, Trabulsi LR. Distinctive patterns of adherence of enteropathogenic Escherichia coli to HeLa cells. Infect Immun 1984;45(2):534–536. DOI: 10.1128/iai.45.2.534-536.1984.
67. Bieber D, Ramer SW, Wu CY, et al. Type IV pili, transient bacterial aggregates, and virulence of enteropathogenic Escherichia coli. Science 1998;280(5372):2114–2118. DOI: 10.1126/science.280.5372.2114.
68. Zhang HZ, Lory S and Donnenberg MS. A plasmid-encoded prepilin peptidase gene from enteropathogenic Escherichia coli. J Bacteriol 1994;176(22):6885–6891. DOI: 10.1128/jb.176.22.6885-6891.1994.
69. Adam T, Arpin M, Prevost MC, et al. Cytoskeletal rearrangements and the functional role of T-Plastin during entry of Shiglla flexneri into HeLa cells. J Cell Biol 1995;129(2):367–381. DOI: 10.1083/jcb.129.2.367.
70. Finlay BB, Rosenshine I, Donnenberg MS, et al. Cytoskeletal composition of attaching and effacing lesions associated with enteropathogenic Escherichia coli adherence to HeLa cells. Infect Immun 1992;60(6):2541–2543. DOI: 10.1128/iai.60.6.2541-2543.1992.
71. Goosney DL, DeVinney R, Pfuetzner RA. Enteropathogenic E. coli translocated intimin receptor, Tir, interacts directly with α-actinin. Curr Biol 2000;10(12):735–738. DOI: 10.1016/s0960-9822(00)00543-1.
72. Kalman D, Weiner OD, Goosney DL, et al. Enteropathogenic E. coli acts through WASP and Arp2/3 complex to form actin pedestals. Nat Cell Biol 1999;1(6):389–391. DOI: 10.1038/14087.
73. Sanger JM, Chang R, Ashton F, et al. Novel form of actin-based motility transports bacteria on the surfaces of infected cells. Cell Motil Cytoskelet 1996;34(4):279–287. DOI: 10.1002/(SICI)1097-0169(1996)34:4<279::AID-CM3>3.0.CO;2-3.
74. Perna NT, Mayhew GF, Pósfai G, et al. Molecular evolution of a pathogenicity island from enterohemorrhagic Escherichia coli O157:H7. Infect Immun 1998;66(8):3810–3817. DOI: 10.1128/IAI.66.8.3810-3817.1998.
75. Deng W, Puente JL, Gruenheid S, et al. Dissecting virulence: Systematic and functional analyses of a pathogenicity island. Proc Natl Acad Sci USA 2004;101(10):3597–3602. DOI: 10.1073/pnas.0400326101.
76. Luo W, Donnenberg MS. Analysis of the function of enteropathogenic Escherichia coli EspB by random mutagenesis. Infect Immun 2006;74(2):810–820. DOI: 10.1128/IAI.74.2.810-820.2006.
77. Mellies JL, Barron AMS, Carmona AM. Enteropathogenic and rnterohemorrhagic Escherichia coli virulence gene regulation. Infect Immun 2007;75(9):4199–4210. DOI: 10.1128/IAI.01927-06.
78. Yang J, Tauschek M, Hart E. Virulence regulation in Citrobacter rodentium: The art of timing. Microb Biotechnol 2010;3(3):259–268. DOI: 10.1111/j.1751-7915.2009.00114.x
79. Elliott SJ, Wainwright LA, McDaniel TK, et al. The complete sequence of the locus of enterocyte effacement (LEE) from enteropathogenic Escherichia coli E2348/69. Mol Microbiol 1998;28(1):1–4. DOI: 10.1046/j.1365-2958.1998.00783.x.
80. Hernandes RT, Elias WP, Vieira MAM, et al. An overview of atypical enteropathogenic Escherichia coli. FEMS Microbiol Lett 2009;297(2):137–149. DOI: 10.1111/j.1574-6968.2009.01664.x.
81. Fitzhenry RJ, Pickard DJ, Hartland EL, et al. Intimin type influences the site of human intestinal mucosal colonisation by enterohaemorrhagic Escherichia coli O157:H7. Gut 2002;50(2):180–185. DOI: 10.1136/gut.50.2.180.
82. Sinclair JF, O’Brien AD. Intimin types α, β, and γ bind to nucleolin with equivalent affinity but lower avidity than to the translocated intimin receptor. J Biol Chem 2004;279(32):33751–33758. DOI: 10.1074/jbc.M401616200.
83. Dean P, Kenny B. The effector repertoire of enteropathogenic E. coli: Ganging up on the host cell. Curr Opin Microbiol 2009;12(1–3):101–109. DOI: 10.1016/j.mib.2008.11.006.
84. Girón JA, Ho AS, Schoolnik GK. Characterization of fimbriae produced by enteropathogenic Escherichia coli. J Bacteriol 1993; 175(22):7391–7403.DOI: 10.1128/jb.175.22.7391-7403.1993.
85. Sohel I, Puente JL, Ramer SW, et al. Enteropathogenic Escherichia coli: Identification of a gene cluster coding for bundle-forming pilus morphogenesis. J Bacteriol 1996;178(9):2613–2628. DOI: 10.1128/jb.178.9.2613-2628.1996.
86. Clarke SC, Haigh RD, Freestone PPE, et al. Virulence of enteropathogenic Escherichia coli, a global pathogen. Clin Microbiol Rev 2003;16(3):365–378. DOI: 10.1128/CMR.16.3.365-378.2003.
87. Girón JA, Torres AG, Freer E, et al. The flagella of enteropathogenic Escherichia coli mediate adherence to epithelial cells. Mol Microbiol 2002;44(2):361–379. DOI: 10.1046/j.1365-2958.2002.02899.x.
88. Nataro JP, Baldini MM, Kaper JB, et al. Detection of an adherence factor of enteropathogenic Escherichia coli with a DNA probe. J Infect Dis 1985;152(3):560–565. DOI: 10.1093/infdis/152.3.560.
89. Chen HD, Frankel G. Enteropathogenic Escherichia coli: Unravelling pathogenesis. FEMS Microbiol Rev 2005;29(1):83–98. DOI: 10.1016/j.femsre.2004.07.002.
90. Scaletsky IC, Silva ML, Toledo MR, et al. Correlation between adherence to HeLa cells and serogroups, serotypes, and bioserotypes of Escherichia coli. Infect Immun 1985;4(3)9:528–532. DOI: 10.1128/iai.49.3.528-532.1985.
91. McConnell MM, Chart H, Scotland SM, et al. Properties of adherence factor plasmids of enteropathogenic Escherichia coli and the effect of host strain on expression of adherence to HEp-2 cells. J Gen Microbiol 1989;135(5):1123–1124. DOI: 10.1099/00221287-135-5-1123.
92. Coburn B, Sekirov I, Finlay BB. Type III secretion systems and disease. Clin Microbiol Rev 2007;20(4):535–549. DOI: 10.1128/CMR.00013-07.
93. Garmendia J, Frankel G, Crepin VF. Enteropathogenic and enterohemorrhagic Escherichia coli infections: Translocation, translocation, translocation. Infect Immun 2005;73(5):2573–2585. DOI: 10.1128/IAI.73.5.2573-2585.2005.
94. Kenny B, Jepson M. Targeting of an enteropathogenic Escherichia coli (EPEC) effector protein to host mitochondria. Cell Microbiol 2000;2(6):579–590. DOI: 10.1046/j.1462-5822.2000.00082.x.
95. Moon HW, Whipp SC, Argenzio RA, et al. Attaching and effacing activities of rabbit and human enteropathogenic Escherichia coli in pig and rabbit intestines. Infect Immun 1983;41(3):1340–1351. DOI: 10.1128/iai.41.3.1340-1351.1983.
96. Goosney DL, Gruenheid S, Finlay BB. Gut feelings: Enteropathogenic E. coli (EPEC) interactions with the host. Annu Rev Cell Dev Biol 2000;16:173–189. DOI: 10.1146/annurev.cellbio.16.1.173.
97. Dean P, Maresca M, Schüller S. Potent diarrheagenic mechanism mediated by the cooperative action of three enteropathogenic Escherichia coli-injected effector proteins. Proc Natl Acad Sci USA 2006;103(6):1876–1881. DOI: 10.1073/pnas.0509451103.
98. Nzegwu HC, Levin RJ. Neurally maintained hypersecretion in undernourished rat intestine activated by E. coli STa enterotoxin and cyclic nucleotides in vitro. J Physiol 1994;479(Pt 1):159–69. DOI: 10.1113/jphysiol.1994.sp020285.
99. Nzegwu HC, Levin RJ. Luminal capsaicin inhibits fluid secretion induced by enterotoxin E. coli STa, but not by carbachol, in vivo in rat small and large intestine. Exp Physiol 1996;81(2):313–315. DOI: 10.1113/expphysiol.1996.sp003935.
100. Abba K, Sinfield R, Hart CA, et al. Pathogens associated with persistent diarrhoea in children in low and middle income countries: Systematic review. BMC Infect Dis 2009;9:88. DOI: 10.1186/1471-2334-9-88.
101. Manjarrez-Hernandez HA, Baldwin TJ, Aitken A, et al. Intestinal epithelial cell protein phosphorylation in enteropathogenic Escherichia coli diarrhoea. Lancet 1992;339(8792):521–523. DOI: 10.1016/0140-6736(92)90340-9.
102. McNamara BP, Koutsouris A, O’Connell CB, et al. Translocated EspF protein from enteropathogenic Escherichia coli disrupts host intestinal barrier function. J Clin Invest 2001;107(5):621–629. DOI: 10.1172/JCI11138.
103. Choi HJ, Kim J, Do KH, et al. Prolonged NF-B activation by a macrophage inhibitory cytokine 1-linked signal in enteropathogenic Escherichia coli-infected epithelial cells. Infect Immun 2013; 81(6):1860–1869.DOI: 10.1128/IAI.00162-13.
104. Hecht G, Marrero JA, Danilkovich A, et al. Pathogenic Escherichia coli increase Cl- secretion from intestinal epithelia by upregulating galanin-1 receptor expression. J Clin Invest 1999;104(3):253–262. DOI: 10.1172/JCI6373.
105. Matkowskyj KA, Danilkovich A, Marrero J, et al. Galanin-1 receptor up-regulation mediates the excess colonic fluid production caused by infection with enteric pathogens. Nat Med 2000;6(9):1048–1051. DOI: 10.1038/79563.
106. Muza-Moons MM, Schneeberger EE, Hecht GA. Enteropathogenic Escherichia coli infection leads to appearance of aberrant tight junctions strands in the lateral membrane of intestinal epithelial cells. Cell Microbiol 2004;(8)6:783–793. DOI: 10.1111/j.1462-5822.2004.00404.x.
107. Savkovic SD, Koutsouris A, Hecht G. Activation of NF-κB in intestinal epithelial cells by enteropathogenic Escherichia coli. Am J Physiol 1997;273(4):C1160–C1167. DOI: 10.1152/ajpcell.1997.273.4.C1160.
108. Spitz J, Yuhan R, Koutsouris A, et al. Enteropathogenic Escherichia coli adherence to intestinal epithelial monolayers diminishes barrier function. Am J Physiol Gastrointest Liver 1995;268(2 Pt 1):G374–G379. DOI: 10.1152/ajpgi.1995.268.2.G374.
109. Savkovic SD, Koutsouris A, and Hecht G. Attachment of a noninvasive enteric pathogen, enteropathogenic Escherichia coli, to cultured human intestinal epithelial monolayers induces transmigration of neutrophils. Infect Immun 1996;64(11):4480–4487. DOI: 10.1128/iai.64.11.4480-4487.1996.
110. Madara JL, Patapoff TW, Gillece-Castro B, et al. 5’-Adenosine monophosphate is the neutrophil-derived paracrine factor that elicits chloride secretion from T84 intestinal epithelial cell monolayers. J Clin Invest 1993;91(5):2320–2325. DOI: 10.1172/JCI116462.
111. Das S, Jayaratne R, Barrett KE. The role of ion transporters in the pathophysiology of infectious diarrhea. Cell Mol Gastroenterol Hepatol 2018;6(1):33–45. DOI: 10.1016/j.jcmgh.2018.02.009. eCollection 2018.
112. Hodges K, Gill R. Infectious diarrhea cellular and molecular mechanisms. Gut Microbes 2010;1(1):4–21. DOI: 10.4161/gmic.1.1.11036.
113. Gill RK, Borthakur A, Hodges K, et al. Mechanism underlying inhibition of intestinal apical Cl–/OH– exchange following infection with enteropathogenic E. coli. J Clin Invest 2007;117(2):428–437. DOI: 10.1172/JCI29625.
114. Hecht G, Hodges H, Gill RK, et al. Differential regulation of Na+/H+ exchange isoform activities by enteropathogenic E. coli in human intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 2004;287(2):370–378. DOI: 10.1152/ajpgi.00432.2003.
115. Singh AP and Aijaza S. Enteropathogenic E. coli: Breaking the intestinal tight junction barrier. F1000Res 2015;4:231. DOI: 10.12688/f1000research.6778.2.
116. Hecht G, Gill R, Saksena S, et al. Enteropathogenic E. coli inhibits Cl/OH exchange activity in Caco2 cells (Abstract).Gastroenterology 2003;124:A482. DOI: 10.1152/ajpgi.00302.2005.
117. Borthakur A, Gill RK, Hodges K. Enteropathogenic Escherichia coli inhibits butyrate uptake in Caco-2 cells by altering the apical membrane MCT1 level. Am J Physiol Gastrointest Liver Physiol 2006;290(1):30–35. DOI: 10.1152/ajpgi.00302.2005.
118. Gujral T, Kumar A, Priyamvada S, et al. Mechanisms of DRA recycling in intestinal epithelial cells: Effect of enteropathogenic E. coli. Am J Physiol Gastrointest Liver Physiol 2015;309(12):C835–C846. DOI: 10.1152/ajpcell.00107.2015.
119. Alrefai WA, Tyagi S, Gill R, et al. Regulation of butyrate uptake in Caco-2 cells by phorbol 12-myristate 13-acetate. Am J Physiol Gastrointest Liver Physiol 2004;286(2):G197–G203. DOI: 10.1152/ajpgi.00144.2003.
120. Resta-Lenert S, Truong F, Barrett KE, et al. Inhibition of epithelial chloride secretion by butyrate: Role of reduced adenylyl cyclase expression and activity. Am J Physiol Cell Physiol 2001;281(6):C1837–C1849. DOI: 10.1152/ajpcell.2001.281.6.C1837.
121. Inan MS, Rasoulpour RJ, Yin L. The luminal short-chain fatty acid butyrate modulates NF-κB activity in a human colonic epithelial cell line. Gastroenterology 2000;118(4):724–734. DOI: 10.1016/s0016-5085(00)70142-9.
122. Cook SI, Sellin JH. Review article: Short chain fatty acids in health and disease. Aliment Pharmacol Ther 1998;12(6):499–507. DOI: 10.1046/j.1365-2036.1998.00337.x.
123. Annaba F, Sarwar Z, Gill RK, et al. Enteropathogenic Escherichia coli inhibits ileal sodium-dependent bile acid transporter ASBT. Am J Physiol Gastrointest Liver Physiol 2012;302(10):G1216–G1222. DOI: 10.1152/ajpgi.00017.2012.
124. Singhal M, Manzella C, Soni V, et al. Role of SHP2 protein tyrosine phosphatase in SERT inhibition by enteropathogenic E. coli (EPEC).Am J Physiol Gastrointest Liver Physiol 2017;312(5):G443–G449. DOI: 10.1152/ajpgi.00011.2017.
125. Aijaz S, Balda MS, Matter K. Tight junctions: Molecular architecture and function. Int Rev Cytol 2006;248:261–298. DOI: 10.1016/S0074-7696(06)48005-0.
126. Van Itallie CM, Anderson JM. Architecture of tight junctions and principles of molecular composition. Semin Cell Dev Biol 2014;0:157–165. DOI: 10.1016/j.semcdb.2014.08.011.
127. Krug SM, Schulzke JD, Fromm M. Tight junction, selective permeability, and related diseases. Semin Cell Dev Biol 2014;36:166–176. DOI: 10.1016/j.semcdb.2014.09.002.
128. Dean P, Kenny B. Intestinal barrier dysfunction by enteropathogenic Escherichia coli is mediated by two effector molecules and a bacterial surface protein. Mol Microbiol 2004;54(3):665–675. DOI: 10.1111/j.1365-2958.2004.04308.x.
129. Matsuzawa T, Kuwae A, Abe A. Enteropathogenic Escherichia coli type III effectors EspG and EspG2 alter epithelial paracellular permeability. Infect Immun 2005;73(10):6283–6289. DOI: 10.1128/IAI.73.10.6283-6289.2005.
130. Thanabalasuriar A, Koutsouris A, Weflen A, et al. The bacterial virulence factor NleA is required for the disruption of intestinal tight junctions by enteropathogenic E. coli. Cell Microbiol 2010;12(1):31–41. DOI: 10.1111/j.1462-5822.2009.01376.x.
131. Holmes A, Mühlen S, Roe AJ, et al. The EspF effector, a bacterial pathogen’s Swiss army knife. Infect Immun 2010;78(11):4445–4453. DOI: 10.1128/IAI.00635-10.
132. Alto NM, Weflen AW, Rardin MJ, et al. The type III effector EspF coordinates membrane trafficking by the spatiotemporal activation of two eukaryotic signaling pathways. J Cell Biol 2007;178(7):1265–1278.DOI: 10.1083/jcb.200705021.
133. Peralta-Ramírez J, Hernandez JM, Manning-Cela R, et al. EspF Interacts with nucleation-promoting factors to recruit junctional proteins into pedestals for pedestal maturation and disruption of paracellular permeability. Infect Immun 2008;76(9):3854–3868. DOI: 10.1128/IAI.00072-08.
134. Huang Z, Sutton SE, Wallenfang AJ, et al. Structural insights into host GTPase isoform selection by a family of bacterial GEF mimics. Nat Struct Mol Biol 2009;16(8):853–860. DOI: 10.1038/nsmb.1647.
135. Simpson N, Shaw R, Crepin VF, et al. The enteropathogenic Escherichia coli type III secretion system effector Map binds EBP50/NHERF1: Implication for cell signalling and diarrhoea. Mol Microbiol 2006;60(2):349–363. DOI: 10.1111/j.1365-2958.2006.05109.x.
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