Open access peer-reviewed chapter

Systematic Deletion of Type III Secretion System Effectors in Enteropathogenic E. coli Unveils the Role of Non-LEE Effectors in A/E Lesion Formation

Written By

Massiel Cepeda-Molero, Stephanie Schüller, Gad Frankel and Luis Ángel Fernández

Submitted: 17 October 2019 Reviewed: 10 February 2020 Published: 09 March 2020

DOI: 10.5772/intechopen.91677

Chapter metrics overview

1,011 Chapter Downloads

View Full Metrics


Enteropathogenic E. coli (EPEC) is a diarrheagenic human pathogen. The hallmark of EPEC infection is the formation of the attaching and effacing (A/E) lesion in the intestinal epithelial cells, characterized by the effacement of brush border microvilli and the intimate bacterial attachment to the enterocyte in actin-rich pedestal-like structures. The locus of enterocyte effacement (LEE) in the EPEC genome encodes a type III protein secretion system (T3SS) that translocates multiple effector proteins into the host cell to subvert cellular functions for the benefit of the pathogen. These effectors are encoded both within and outside the LEE. In vitro cell culture infections have shown that LEE effectors are required for intimate bacterial attachment to the epithelial cells, whereas non-LEE effectors mostly play a role in modulating inflammation and cell apoptosis in the gut epithelium. We constructed a set of EPEC mutant strains harboring deletions in the complete repertoire of genes encoding T3SS effectors. Infection of human intestinal in vitro organ cultures (IVOC) with these mutant strains surprisingly revealed that non-LEE effectors are also needed to induce efficient A/E lesion formation in the intestinal mucosal tissue.


  • A/E lesion
  • EPEC
  • effectors
  • infection
  • IVOC
  • T3SS

1. Introduction

1.1 Enteropathogenic E. coli

Enteropathogenic E. coli (EPEC) was the first pathotype of E. coli to be associated with human disease and is a major cause of acute and chronic diarrhea in infants [1, 2]. The low microbial density of the small bowel caused by the forceful peristalsis in this part of the intestine is overcome by EPEC, which can successfully colonize the small intestine of humans [3, 4]. EPEC primarily affects children younger than 2 years old; however some outbreaks of EPEC infection in healthy adults have been associated with large inoculum ingestion [5]. The mechanism of transmission of EPEC is the fecal-oral route. In the 1940s and 1950s, EPEC was an important cause of diarrhea in developed countries with a mortality of 50% during outbreaks, but nowadays the infection by EPEC in industrial countries has a limited importance. In contrast, in low-income countries, EPEC is still an major cause of infant diarrhea [5, 6].

1.2 Hallmark of EPEC gastrointestinal infection

The phenotype that defines EPEC infection is the attaching and effacing (A/E) lesion [2, 7]. By adhering to intestinal epithelial cells, EPEC subverts cytoskeletal processes of the host cell and produces the histopathological feature of the A/E lesion. This lesion, which was first described in 1980 [8], is characterized by the intimate attachment of the bacteria to the intestinal epithelial cells and elongation and effacement of the brush border microvilli. Later on it was shown that infection is also associated with cytoskeletal rearrangements, including the accumulation of polymerized F-actin in pedestal-like structures underneath the attached bacteria [9] (Figure 1). EPEC together with enterohemorrhagic E. coli (EHEC) and Citrobacter rodentium (CR) is a member of the A/E family of bacterial pathogens that colonize the gastrointestinal tract via the A/E lesion. EPEC and EHEC are important human pathogens, while CR is a mouse-restricted pathogen [10, 11, 12, 13].

Figure 1.

Localized adherence (LA), intimate attachment, and EPEC A/E lesion formation in the intestinal epithelial surface. At an early stage, EPEC interacts in a non-intimate manner with the intestinal surface mainly through the BFP and EspA filament. After assembly of the translocation pore, EPEC injects translocated intimin receptor (Tir). Ser/Thr phosphorylation of Tir induces its anchoring in the enterocyte plasma membrane, leaving the TirM region exposed for the interaction with intimin. Subsequent Tir-intimin interaction triggers actin polymerization and pedestal formation underneath the attached bacterium. Tir phosphorylation of residue Y474 engages the host adaptor NcK, which later recruits N-WASP and WIP. N-WASP recruits the ARP2/3 complex, which induces actin nucleation and polymerization.


2. EPEC virulence factors

2.1 A pathogenicity island called LEE

The ability of EPEC to induce A/E lesions is related to a pathogenicity island (PAI) of 35 kb called the locus of enterocyte effacement (LEE) [14]. The LEE comprises 41 genes organized in 5 principal operons (LEE1-LEE5) and several smaller transcriptional units (Figure 2) [15, 16]. Orthologues of LEE are also found in other members of A/E pathogens [11]. The LEE encodes all the structural proteins necessary for the assembly of a filamentous type III secretion system (T3SS) injectisome on the bacterial cell envelope [17, 18]. The LEE also encodes transcriptional regulators (Ler, GrlR, and GrlA), translocator proteins (EspA, EspB, and EspD), six secreted effector proteins (including the translocated intimin receptor), the outer membrane protein intimin, molecular chaperones, and a lytic transglycosylase (EtgA) [19]. The mechanism of LEE regulation is complex and depends on environmental conditions, quorum sensing (QS), and several transcriptional regulators encoded within and outside the LEE [20, 21].

2.2 The type III secretion system

The type III secretion system is a macromolecular transport apparatus that is used by many gram-negative bacterial pathogens (e.g., Shigella, Yersinia, Salmonella) to translocate virulence proteins, called effectors, into the cytosol of infected cells, thereby subverting host cellular functions for the benefit of the pathogen [22]. Since pathogens use this transport apparatus to inject proteins into the host cells, this structure is also known as the injectisome. The EPEC T3SS mediates the translocation of multiple effector proteins during infection. Some of them are encoded in the LEE, whereas others are encoded outside of the LEE being generally referred to as non-LEE effectors (Nle) [23, 24]. EspA filaments link the tip of the injectisome in the bacterial cell wall to a 3–5 nm translocation pore, formed in the plasma membrane of infected cells by the translocator proteins EspB and EspD (Figure 1) [25, 26].

Figure 2.

Effectors of EPEC E2348/69. (A) Representation of the LEE island and effector genes espG, espZ, espH, map, tir, and espF. (B) Non-LEE effectors located outside the LEE are localized in integrative elements (IEs) and prophages (PPs). Effector genes are labeled in red. Pseudogenes are specified with asterisk. Scale of 5 kb is indicated at the bottom. Figure from [32].

2.3 Bundle-forming pilus (BFP)

Typical EPEC is endowed with a plasmid called pMAR2 which contains a 14-gene operon encoding the type IV pilus BFP [27, 28]. The BFP is a rope-like bundle, which allows EPEC to form microcolonies in a pattern called localized adherence and also mediates the initial interaction of bacteria with host cell surfaces (Figure 1) [29, 30, 31].


3. EPEC pathogenesis

EPEC tightly regulates its virulence genes in response to environmental conditions such as temperature [16], the increase of the pH of the small intestine [33, 34], and some hormones which are released during stress conditions [20]. Upon EPEC interaction with enterocytes, EspB and EspD proteins are inserted into the host cell membrane and assemble to form a translocation pore [25, 26]. EPEC then injects its own receptor called Tir, which is integrated into the plasma membrane in a hairpin loop topology, with the loop facing the outside of the cell where it serves as a receptor for the bacterial adhesin intimin [35, 36, 37]. Tir-intimin interaction induces clustering and dimerization of Tir, and this activates a signal cascade that starts with the phosphorylation of serine/threonine residues and leads to actin polymerization and pedestal formation underneath the attached bacterium [10, 38]. The most critical event for actin polymerization is the phosphorylation of the cytoplasmic Tir residue Y474 [39]. This induces a signal cascade which recruits the host cell adaptor Nck and N-WASP required to engage and activate the actin-nucleating ARP2/3 complex, which produces the actin nucleation and polymerization. Actin polymerization drives membrane protrusion and pedestal formation [10, 40] (Figure 1). Through the T3SS injectisome, EPEC translocates LEE-encoded effector proteins and additional effectors localized in mobile genetic element outside the LEE (Nle).


4. LEE effectors

Six effector proteins (EspG, EspZ, EspH, Map, Tir, and EspF) are encoded in the LEE island (Figure 2). Most of these, except EspZ, have important functions destabilizing the physiology of the intestinal epithelium, triggering cytoskeleton reorganization, inducing cytotoxicity and electrolyte imbalance which lead to diarrhea [11, 41]. The rapid onset of EPEC-induced diarrhea is likely induced by the cooperative action of Tir, Map, and EspF, which inhibits the sodium-D-glucose transporter (SGLT-1), the major water pump of the small intestine responsible for about 70% of the total fluid uptake [42]. In addition, Map and EspF reduce Na + absorption by the sodium-hydrogen exchanger (NHE3) [43], and EspG1/2 proteins alter the membrane targeting of the Cl-/OH-exchanger (DRA), resulting in reduced Cl-uptake. These processes result in the accumulation of salts in the gut lumen, which drives water loss from the mucosa [44].

Inhibition of endosomal trafficking by EspG1/2 reduces the level of cell surface receptors [45]. In addition, EspF and EspG induce mislocalization of aquaporins (AQP), thereby reducing epithelial water absorption [46]. Furthermore, EspB, Tir, EspF, and Map induce microvillus effacement, and this reduction of absorptive surface likely exacerbates EPEC diarrhea [47]. While EspF and Map synergistically disrupt TJs [48], EspG1/2 induces microtubule disruption contributing to TJ disruption [49]. The effector protein NleA also disrupts TJs by blocking the delivery of new TJ proteins [49, 50, 51]. The disruption of TJs increases intestinal permeability and thereby likely contributes to EPEC-induced diarrhea [52] (Figure 3).

Figure 3.

EPEC effector proteins altering epithelial cell function and inducing water loss and diarrhea. Tir, map, and EspF inhibit the sodium-D-glucose transporter. EspF reduces expression of the sodium-hydrogen exchanger NHE3. EspG and EspF induce mislocalization of aquaporins (AqP). EspG1/EspG2 alters membrane targeting of the Cl-/OH-exchanger. EspF, map, NleA, EspG1, and EspG2 disrupt tight junction complexes (TJ). EspB, Tir, EspF, and map induce microvilli effacement.


5. Non-LEE effectors

In EPEC prototype strain E2348/69, 17 functional Nle effectors are encoded in different integrative elements and prophages, frequently associated in gene clusters, with some effectors having duplicated gene copies and/or paralogs in different clusters [53] (Figure 2). EPEC infection is characterized by a weak inflammatory response [54]. Previous studies have shown that most Nle effectors and some LEE effectors inhibit the host immune response, which favors bacterial survival (Figure 4). Although NleF and NleH2 activate the NF-κB inflammatory pathway during early infection (ref), EPEC translocates several effectors that dampen the proinflammatory pathways of the cell [11]. Thus, a large number of Nle effectors inhibit host inflammation by different mechanisms, such as inhibition of the NF-κB (NleB, C, E, and H) and MAPK proinflammatory pathways (NleC and D) [55, 56, 57, 58], inhibition of the canonical (NleA) and noncanonical (NleF) inflammasomes [59], and inhibition of proliferation of lymphocytes and interleukin production (LifA) [60, 61].

Figure 4.

Schematic representation of multifunctional and overlapping effectors to control host immune response. The NF-κB proinflammatory pathway is activated by NleF and NleH2 and is inhibited by NleE, NleB, NleH1, Tir, and NleC. NleC and NleD inhibit the MAPK proinflammatory pathway. EspF, EspJ, EspH, and EspB prevent macrophage phagocytosis. NleA disrupts inflammasome activation, and LifA inhibits IL-2 and IL-4 production and lymphocyte proliferation. While EspF and map induce intrinsic apoptosis, EspZ counteracts these effects by stabilizing mitochondrial membrane potential. NleH1/NleH2 and NleF inhibit intrinsic apoptosis, and NleF, NleD, and NleB counteract extrinsic apoptosis.

The control of the epithelial cell death response to microbial infection is pivotal for pathogens and the host. Pathogens that are colonizing the epithelium need to prevent cell death to preserve their replicative foothold; by contrast, the host needs to eliminate infected cells in order to minimize tissue damage [62]. During infection of the intestinal epithelial cells, surface properties of EPEC are recognized by cell surface death receptors and induce extrinsic apoptotic pathways, while T3SS effectors (Map and EspF) trigger cytochrome c release, activation of caspases, and downstream intrinsic apoptotic pathways [11, 24]. Interestingly, early stages of apoptosis can be observed during EPEC infection, but late stages are not evident because EPEC translocates effector proteins that antagonize these pro-apoptotic effects. NleD and NleB interfere with the pro-apoptotic death receptor signaling and disrupt the downstream extrinsic apoptosis [63, 64]. NLeH1/2 and EspZ also inhibit intrinsic apoptosis and promote host cell survival [65, 66, 67] (Figure 4). NleF directly inhibits caspases involved in both intrinsic and extrinsic apoptosis pathways, including caspases 4, 8, and 9 [68]. In addition, EspZ localizes to the cytoplasmic side of the plasma membrane at the site of bacterial attachment and interacts with the translocator protein EspD. It has been proposed that EspZ indirectly prevents cell death by downregulating protein translocation and protecting cells from an overdose of effector proteins. Consistently, a ΔespZ mutant was found to be highly cytotoxic [69]. EPEC effectors are injected in a regulated manner to guarantee the success of infection. While the pro-survival effector EspZ is translocated at the early stages of infection, the pro-apoptotic effectors EspF and Map follow later [70].


6. Classical methodologies to study effector functions

Most research on EPEC effectors has been conducted by generating deletion mutants in a single or a few effector genes that are later complemented with multicopy plasmids overexpressing the effector(s). In addition, ectopic expression of individual effectors by plasmid transfection of the host cell has been applied. Both situations are prone to effector overexpression resulting in nonphysiological levels of effectors inside the host cell, which could alter effector activities. In addition, effectors often have synergistic and overlapping functions that cannot be fully appreciated by single mutations and individual transfection experiments [11, 54]. In order to overcome these limitations, we employed a marker-less gene deletion strategy to delete the whole repertoire of known effector genes found in the genome of the prototypical EPEC strain E2348/69 [32]. The genome engineering method for sequential deletion of EPEC effectors was based on the marker-less gene deletion technique described by Posfai et al. [71] and is illustrated in Figure 5.

Figure 5.

Marker-less gene deletion strategy of EPEC effector genes. Deletions using pGE-suicide plasmids with I-SceI sites and mutant alleles assembled by fusing homology regions (HRs) flanking the targeted effector gene(s). Co-integrants are identified by the Kanamycin resistance phenotype. Expression of the I-SceI in vivo from helper plasmid induces double-strand brakes that are repaired by homologous recombination. Depending on the HRs involved in this second recombination, either the WT or the mutant allele can be obtained. Figure from [32].

Using this strategy, a set of EPEC mutants with sequential deletions of effectors was generated (Table 1), ultimately resulting in strains expressing only Tir and EspZ (EPEC2), Tir (EPEC1), and the effector-less strain EPEC0 (Table 1). This approach proved to be effective to specifically modify the genome of EPEC E2348/69, avoiding the introduction of unintended alterations in the genome and leaving no sequence “scars” or antibiotic resistance genes in the chromosome as demonstrated by whole-genome sequencing [32]. Besides, the deletion mutant strains showed normal growth and maintained functional T3SS injectisomes. In addition, they allowed the translocation of individual effectors from single-copy chromosomal genes under endogenous regulation, showing the expected phenotypes without the background of the other effectors [32]. Hence these mutant strains are an excellent tool to investigate the role of individual effectors and specific combinations maintaining physiological protein levels in the context of infection.

StrainEffector genes remaining*
EPEC2espZ and tir
EPEC9espZ + tir + IE2 + IE5 + IE6 + PP2 + PP3 + PP4 + PP6
EPEC2-LEE+espZ + tir + map + espH + espF + espG

Table 1.

EPEC mutant strains generated with the marker-less deletion strategy.

Encoded effectors in the indicated IEs and PPs.


7. LEE effectors are sufficient for intimate adhesion of EPEC to the epithelial cells in vitro

When EPEC bacteria adhere in vitro to cultured cells, there is an accumulation of actin filaments in the cytoplasm beneath the adherent bacteria, due to a signal cascade triggered by intimin-Tir interaction [35, 38]. Using the effector deletion mutants of EPEC, we demonstrated that the LEE effector Tir along with intimin is necessary and sufficient to induce these cytoskeletal rearrangements during in vitro infection of HeLa cells. Strains EPEC2 (bearing EspZ and Tir) and EPEC1 (bearing only Tir) were able to induce actin-pedestal formation underneath attached bacteria similar to the EPEC wild type (WT) (Figure 6). As expected because of the essential role of Tir in this process, infection of HeLa cells with the effector-less mutant EPEC0 did not induce any actin-pedestal formation (Figure 6). These data demonstrate that the individual translocation of Tir by EPEC1 is sufficient to trigger actin pedestals in HeLa cells and that non-LEE effectors are dispensable for this phenotype during in vitro infection of cultured cells.

Figure 6.

Infection of HeLa cells with EPEC WT and effector mutant strains. Immunofluorescence confocal microscopy of HeLa cells infected with EPEC WT, EPEC2, EPEC1, and EPEC0 for 1.5 h using a MOI of 200. EPEC is labeled with anti-intimin-280 serum (green), actin is stained with TRITC phalloidin (red), and cell nuclei are labeled with DAPI (gray). Actin polymerization beneath adherent bacteria is observed in EPEC WT, EPEC2, and EPEC1. Scale bar 5 μm. Figure from [32].


8. Non-LEE effectors are required for efficient A/E lesion formation in intestinal tissue

EPEC pathogenic mechanisms have been widely investigated by in vitro infection of cultured epithelial cell lines, albeit in most cases these cells are non-polarized and are not from intestinal origin. In addition, EPEC infection studies in vivo are hindered because EPEC is a human-restricted pathogen [72]. A surrogate model established to investigate A/E pathogenesis in vivo is the mouse pathogen Citrobacter rodentium (CR) [12, 13]. Although Citrobacter infection in vitro requires Tir phosphorylation for actin-pedestal formation in cell lines, Tir phosphorylation-deficient mutants still colonize the mouse gut and induce A/E lesion formation and crypt hyperplasia typical of CR infection [73]. This result highlights the necessity of a model for EPEC infection closer to the in vivo conditions in the human gut. A good established model to study EPEC-host interactions is the infection of in vitro cultured human intestinal biopsies, which allows the formation of A/E lesions undistinguishable from those observed in vivo in biopsies of patients with EPEC-induced diarrhea [4, 36, 74]. Similar to results obtained in CR-infected mice, Tir phosphorylation was not necessary for EPEC A/E lesion formation in human intestinal biopsies [75]. Surprisingly, when EPEC2 and EPEC1 deletion mutants were used to infect human duodenal biopsies, none of the infected biopsies showed A/E lesions (Table 2 and Figure 7), which contrasts with the pedestal formation observed in HeLa cells. Thus, intimin and Tir are not sufficient to induce A/E lesions in the intestinal tissue, and the IVOC model was used to identify additional LEE or non-LEE effector(s) required for A/E lesion formation. For this purpose, two additional effector mutant strains were tested: EPEC2-LEE+ (carrying all LEE effectors) and EPEC9 (carrying EspZ, Tir, and all non-LEE effectors). Whereas infection with EPEC2-LEE+ did not reveal A/E lesions, infection with EPEC9 induced A/E lesions to a similar level as the wild-type strain (Table 2 and Figure 7). It was previously reported that the LEE island is sufficient to confer the A/E phenotype to E. coli K-12 in the infection of colon carcinoma cell lines [76]. However, our results indicate that the LEE is not sufficient for A/E lesion formation in human mucosal tissue and that non-LEE effectors are required [32].

StrainEffector genes remaining*Biopsies with A/E lesions positive/total (%)
WTAll13/17 (76)
EPEC2espZ and tir0/6 (0)
EPEC1tir0/6 (0)
EPEC0None0/5 (0)
EPEC9espZ + tir + IE2 + IE5 + IE6 + PP2 + PP3 + PP4 + PP65/6 (83)
EPEC2-LEE+espZ + tir + map + espH + espF + espG0/5 (0)

Table 2.

Human duodenal biopsies infected by EPEC WT and EPEC effector mutants.

Encoded effectors in the indicated IEs and PPs.

Figure 7.

Scanning electron microscopy of human duodenal biopsies infected with EPEC WT and mutant strains EPEC2, EPEC1, EPEC0, EPEC9, and EPEC2-LEE+. EPEC WT and EPEC9 induce characteristic A/E lesions with bacterial microcolony formation (asterisk) and microvilli elongation around bacterial colonies (arrowheads). In contrast, biopsies infected with EPEC2, EPEC1, EPEC0, and EPEC2-LEE+ lack adherent bacteria and A/E lesions and show a normal microvillous brush border. Scale bar 2 μm. Figure from [32].


9. Conclusions and future perspectives

The marker-less gene deletion strategy enabled the generation of effector-less strains of EPEC O127:H6 using the prototypical strain E2348/69 [32]. Given the conservation of the recombination machinery among E. coli strains, it is likely that this strategy could be applied to other A/E pathogens, E. coli pathogens, and other bacteria. The effector mutant strains can be useful to study the role of individual effectors and of combinations of effectors in pathogenesis. An individual effector or a defined combination can be inserted in the effector-less strains in their endogenous genomic loci to obtain physiological expression levels and regulation. In cell culture infections, all EPEC effector mutant strains carrying intimin and Tir were able to trigger actin-rich pedestal-like structures underneath attached bacteria. On the other hand, when the infection was performed in human intestinal tissues, translocation of Tir alone was insufficient to induce A/E lesions. Furthermore, an EPEC deletion mutant maintaining all LEE effectors and devoid of all non-LEE effectors (EPEC2-LEE+) was still unable to induce A/E lesions in human intestinal biopsies. In contrast, an EPEC strain producing the complete repertoire of non-LEE effectors and devoid of LEE effectors, except Tir and EspZ, formed A/E lesions in intestinal tissue at wild-type levels [32]. Thus, these experiments revealed that non-LEE effectors are needed for A/E lesion formation in human intestinal tissue.

In addition to their potential for basic studies, the EPEC effector mutant strains may have different applications. For instance, EPEC (and other pathogenic) strains lacking multiple effectors are likely to be strongly attenuated, but they maintain the external antigenicity of the wild-type strain. Thus, an EPEC mutant strain with a functional T3SS and the minimum set of effectors necessary to colonize the intestinal surface could be a good vaccine candidate. Further, EPEC mutant strains with the ability to attach to the human intestine could also be engineered to translocate heterologous protein antigens to generate protection against other enteric pathogens causing diarrhea, including EHEC strains [77, 78, 79, 80]. Lastly, the EPEC effector mutant strains may also have the potential to deliver therapeutic proteins to the intestinal epithelium, for instance, to combat inflammation and autoimmune disorders in the gastrointestinal tract [81].



We acknowledge support for the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).


  1. 1. Kaper JB, Nataro JP, Mobley HL. Pathogenic Escherichia coli. Nature Reviews Microbiology. 2004;2:123-139
  2. 2. Chen HD, Frankel G. Enteropathogenic Escherichia coli: Unravelling pathogenesis. FEMS Microbiology Reviews. 2005;29(1):83-98
  3. 3. Tlaskalova-Hogenova H et al. Commensal bacteria (normal microflora), mucosal immunity and chronic inflammatory and autoimmune diseases. Immunology Letters. 2004;93(2-3):97-108
  4. 4. Hicks S et al. Role of intimin and bundle-forming pili in enteropathogenic Escherichia coli adhesion to pediatric intestinal tissue in vitro. Infection and Immunity. 1998;66(4):1570-1578
  5. 5. Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clinical Microbiology Reviews. 1998;11(1):142-201
  6. 6. Ochoa TJ et al. New insights into the epidemiology of enteropathogenic Escherichia coli infection. Transactions of the Royal Society of Tropical Medicine and Hygiene. 2008;102(9):852-856
  7. 7. Garmendia J, Frankel G, Crepin VF. Enteropathogenic and enterohemorrhagic Escherichia coli infections: Translocation, translocation, translocation. Infection and Immunity. 2005;73(5):2573-2585
  8. 8. Ulshen MH, Rollo JL. Pathogenesis of Escherichia coli gastroenteritis in man--another mechanism. The New England Journal of Medicine. 1980;302(2):99-101
  9. 9. Knutton S et al. Actin accumulation at sites of bacterial adhesion to tissue culture cells: Basis of a new diagnostic test for enteropathogenic and enterohemorrhagic Escherichia coli. Infection and Immunity. 1989;57(4):1290-1298
  10. 10. Lai Y et al. Intimate host attachment: Enteropathogenic and enterohaemorrhagic Escherichia coli. Cellular Microbiology. 2013;15(11):1796-1808
  11. 11. Wong AR et al. Enteropathogenic and enterohaemorrhagic Escherichia coli: Even more subversive elements. Molecular Microbiology. 2011;80(6):1420-1438
  12. 12. Collins JW et al. Citrobacter rodentium: Infection, inflammation and the microbiota. Nature Reviews. Microbiology. 2014;12(9):612-623
  13. 13. Mundy R et al. Citrobacter rodentium of mice and man. Cellular Microbiology. 2005;7(12):1697-1706
  14. 14. McDaniel TK et al. A genetic locus of enterocyte effacement conserved among diverse enterobacterial pathogens. Proceedings of the National Academy of Sciences of the United States of America. 1995;92(5):1664-1668
  15. 15. Elliott SJ et al. The locus of enterocyte effacement (LEE)-encoded regulator controls expression of both LEE- and non-LEE-encoded virulence factors in enteropathogenic and enterohemorrhagic Escherichia coli. Infection and Immunity. 2000;68(11):6115-6126
  16. 16. Yerushalmi G et al. Dynamics of expression and maturation of the type III secretion system of enteropathogenic Escherichia coli. Journal of Bacteriology. 2014;196(15):2798-2806
  17. 17. Daniell SJ et al. The filamentous type III secretion translocon of enteropathogenic Escherichia coli. Cellular Microbiology. 2001;3(12):865-871
  18. 18. Gaytán MO et al. Type three secretion system in attaching and effacing pathogens. Frontiers in Cellular and Infection Microbiology. 2016;6:129
  19. 19. García-Gómez E et al. The muramidase EtgA from enteropathogenic Escherichia coli is required for efficient type III secretion. Microbiology. 2011;157(Pt 4):1145-1160
  20. 20. Franzin FM, Sircili MP. Locus of enterocyte effacement: A pathogenicity island involved in the virulence of enteropathogenic and enterohemorrhagic subjected to a complex network of gene regulation. BioMed Research International. 2015;2015:534738
  21. 21. Furniss RCD, Clements A. Regulation of the locus of enterocyte effacement in attaching and effacing pathogens. Journal of Bacteriology. 2018;200(2):e00336-17
  22. 22. Portaliou AG et al. Type III secretion: Building and operating a remarkable nanomachine. Trends in Biochemical Sciences. 2016;41(2):175-189
  23. 23. Raymond B et al. Subversion of trafficking, apoptosis, and innate immunity by type III secretion system effectors. Trends in Microbiology. 2013;21(8):430-441
  24. 24. Santos AS, Finlay BB. Bringing down the host: Enteropathogenic and enterohaemorrhagic Escherichia coli effector-mediated subversion of host innate immune pathways. Cellular Microbiology. 2015;17(3):318-332
  25. 25. Ide T et al. Characterization of translocation pores inserted into plasma membranes by type III-secreted Esp proteins of enteropathogenic Escherichia coli. Cellular Microbiology. 2001;3(10):669-679
  26. 26. Luo W, Donnenberg MS. Interactions and predicted host membrane topology of the enteropathogenic Escherichia coli translocator protein EspB. Journal of Bacteriology. 2011;193(12):2972-2980
  27. 27. Stone KD et al. A cluster of fourteen genes from enteropathogenic Escherichia coli is sufficient for the biogenesis of a type IV pilus. Molecular Microbiology. 1996;20(2):325-337
  28. 28. Brinkley C et al. Nucleotide sequence analysis of the enteropathogenic Escherichia coli adherence factor plasmid pMAR7. Infection and Immunity. 2006;74(9):5408-5413
  29. 29. Ramboarina S et al. Structure of the bundle-forming pilus from enteropathogenic Escherichia coli. The Journal of Biological Chemistry. 2005;280(48):40252-40260
  30. 30. Hyland RM et al. The bundlin pilin protein of enteropathogenic Escherichia coli is an N-acetyllactosamine-specific lectin. Cellular Microbiology. 2008;10(1):177-187
  31. 31. Saldana Z et al. The Escherichia coli common Pilus and the bundle-forming Pilus act in concert during the formation of localized adherence by Enteropathogenic E. coli. Journal of Bacteriology. 2009;191(11):3451-3461
  32. 32. Cepeda-Molero M et al. Attaching and effacing (A/E) lesion formation by enteropathogenic E. coli on human intestinal mucosa is dependent on non-LEE effectors. PLoS Pathogens. 2017;13(10):e1006706
  33. 33. Fallingborg J. Intraluminal pH of the human gastrointestinal tract. Danish Medical Bulletin. 1999;46(3):183-196
  34. 34. Shin S et al. An activator of glutamate decarboxylase genes regulates the expression of enteropathogenic Escherichia coli virulence genes through control of the plasmid-encoded regulator, Per. Molecular Microbiology. 2001;41(5):1133-1150
  35. 35. Kenny B et al. Enteropathogenic E. coli (EPEC) transfers its receptor for intimate adherence into mammalian cells. Cell. 1997;91(4):511-520
  36. 36. Frankel G et al. Generation of Escherichia coli intimin derivatives with differing biological activities using site-directed mutagenesis of the intimin C-terminus domain. Molecular Microbiology. 1998;29(2):559-570
  37. 37. Luo Y et al. Crystal structure of enteropathogenic Escherichia coli intimin-receptor complex. Nature. 2000;405(6790):1073-1077
  38. 38. Frankel G, Phillips AD. Attaching effacing Escherichia coli and paradigms of Tir-triggered actin polymerization: Getting off the pedestal. Cellular Microbiology. 2008;10(3):549-556
  39. 39. Devinney R et al. Tir tyrosine phosphorylation and pedestal formation are delayed in enteropathogenic Escherichia coli sepZ::TnphoA mutant 30-5-1(3). Infection and Immunity. 2001;69(1):559-563
  40. 40. Goosney DL, de Grado M, Finlay BB. Putting E. coli on a pedestal: A unique system to study signal transduction and the actin cytoskeleton. Trends in Cell Biology. 1999;9(1):11-14
  41. 41. Guttman JA, Finlay BB. Subcellular alterations that lead to diarrhea during bacterial pathogenesis. Trends in Microbiology. 2008;16(11):535-542
  42. 42. Dean P et al. Potent diarrheagenic mechanism mediated by the cooperative action of three enteropathogenic Escherichia coli-injected effector proteins. Proceedings of the National Academy of Sciences of the United States of America. 2006;103(6):1876-1881
  43. 43. Simpson N et al. The enteropathogenic Escherichia coli type III secretion system effector map binds EBP50/NHERF1: Implication for cell signalling and diarrhoea. Molecular Microbiology. 2006;60(2):349-363
  44. 44. Gill RK et al. Mechanism underlying inhibition of intestinal apical Cl/OH exchange following infection with enteropathogenic E. coli. The Journal of Clinical Investigation. 2007;117(2):428-437
  45. 45. Clements A et al. Enterohaemorrhagic Escherichia coli inhibits recycling endosome function and trafficking of surface receptors. Cellular Microbiology. 2014;16(11):1693-1705
  46. 46. Guttman JA et al. Aquaporins contribute to diarrhoea caused by attaching and effacing bacterial pathogens. Cellular Microbiology. 2007;9(1):131-141
  47. 47. Iizumi Y et al. The enteropathogenic E. coli effector EspB facilitates microvillus effacing and antiphagocytosis by inhibiting myosin function. Cell Host & Microbe. 2007;2(6):383-392
  48. 48. Dean P, Kenny B. Intestinal barrier dysfunction by enteropathogenic Escherichia coli is mediated by two effector molecules and a bacterial surface protein. Molecular Microbiology. 2004;54(3):665-675
  49. 49. Glotfelty LG et al. Enteropathogenic E. coli effectors EspG1/G2 disrupt microtubules, contribute to tight junction perturbation and inhibit restoration. Cellular Microbiology. 2014;16(12):1767-1783
  50. 50. Thanabalasuriar A et al. The bacterial virulence factor NleA is required for the disruption of intestinal tight junctions by enteropathogenic Escherichia coli. Cellular Microbiology. 2010;12(1):31-41
  51. 51. Kim J et al. The bacterial virulence factor NleA inhibits cellular protein secretion by disrupting mammalian COPII function. Cell Host & Microbe. 2007;2(3):160-171
  52. 52. Croxen MA, Finlay BB. Molecular mechanisms of Escherichia coli pathogenicity. Nature Reviews. Microbiology. 2010;8(1):26-38
  53. 53. Iguchi A et al. Complete genome sequence and comparative genome analysis of enteropathogenic Escherichia coli O127:H6 strain E2348/69. Journal of Bacteriology. 2009;191(1):347-354
  54. 54. Dean P, Kenny B. The effector repertoire of enteropathogenic E. coli: Ganging up on the host cell. Current Opinion in Microbiology. 2009;12(1):101-109
  55. 55. Baruch K et al. Metalloprotease type III effectors that specifically cleave JNK and NF-kappaB. The EMBO Journal. 2011;30(1):221-231
  56. 56. Nadler C et al. The type III secretion effector NleE inhibits NF-kappaB activation. PLoS Pathogens. 2010;6(1):e1000743
  57. 57. Sham HP et al. Attaching and effacing bacterial effector NleC suppresses epithelial inflammatory responses by inhibiting NF-{kappa}B and p38 mitogen-activated protein kinase activation. Infection and Immunity. 2011;79(9):3552-3562
  58. 58. Gao X et al. NleB, a bacterial effector with glycosyltransferase activity, targets GADPH function to inhibit NF-κB activation. Cell Host & Microbe. 2013;13(1):87-99
  59. 59. Yen H, Sugimoto N, Tobe T. Enteropathogenic Escherichia coli uses NleA to inhibit NLRP3 inflammasome activation. PLoS Pathogens. 2015;11(9):e1005121
  60. 60. Abu-Median AB et al. Functional analysis of lymphostatin homologues in enterohaemorrhagic Escherichia coli. FEMS Microbiology Letters. 2006;258(1):43-49
  61. 61. Klapproth JM et al. A large toxin from pathogenic Escherichia coli strains that inhibits lymphocyte activation. Infection and Immunity. 2000;68(4):2148-2155
  62. 62. Kim M et al. Bacterial interactions with the host epithelium. Cell Host & Microbe. 2010;8(1):20-35
  63. 63. Pearson JS et al. A type III effector antagonizes death receptor signalling during bacterial gut infection. Nature. 2013;501(7466):247-251
  64. 64. Wong Fok Lung T et al. The cell death response to enteropathogenic Escherichia coli infection. Cellular Microbiology. 2014;16(12):1736-1745
  65. 65. Roxas JL et al. The enteropathogenic Escherichia coli-secreted protein EspZ inhibits host cell apoptosis. Infection and Immunity. 2012;80(11):3850-3857
  66. 66. Hemrajani C et al. NleH effectors interact with Bax inhibitor-1 to block apoptosis during enteropathogenic Escherichia coli infection. Proceedings of the National Academy of Sciences. 2010;107(7):3129-3134
  67. 67. Royan SV et al. Enteropathogenic E. coli non-LEE encoded effectors NleH1 and NleH2 attenuate NF-kappaB activation. Molecular Microbiology. 2010;78(5):1232-1245
  68. 68. Blasche S et al. The E. coli effector protein NleF is a Caspase inhibitor. PLoS One. 2013;8(3):e58937
  69. 69. Berger CN et al. EspZ of enteropathogenic and enterohemorrhagic Escherichia coli regulates type III secretion system protein translocation. MBio. 2012;3(5):e00317-12
  70. 70. Mills E et al. Real-time analysis of effector translocation by the type III secretion system of enteropathogenic Escherichia coli. Cell Host & Microbe. 2008;3(2):104-113
  71. 71. Posfai G et al. Markerless gene replacement in Escherichia coli stimulated by a double-strand break in the chromosome. Nucleic Acids Research. 1999;27(22):4409-4415
  72. 72. Hill SM, Phillips AD, Walker-Smith JA. Enteropathogenic Escherichia coli and life threatening chronic diarrhoea. Gut. 1991;32(2):154-158
  73. 73. Deng W et al. Citrobacter rodentium translocated intimin receptor (Tir) is an essential virulence factor needed for actin condensation, intestinal colonization and colonic hyperplasia in mice. Molecular Microbiology. 2003;48(1):95-115
  74. 74. Knutton S, Lloyd DR, McNeish AS. Adhesion of enteropathogenic Escherichia coli to human intestinal enterocytes and cultured human intestinal mucosa. Infection and Immunity. 1987;55(1):69-77
  75. 75. Schüller S et al. Tir phosphorylation and Nck/N-WASP recruitment by enteropathogenic and enterohaemorrhagic Escherichia coli during ex vivo colonization of human intestinal mucosa is different to cell culture models. Cellular Microbiology. 2007;9(5):1352-1364
  76. 76. McDaniel TK, Kaper JB. A cloned pathogenicity island from enteropathogenic Escherichia coli confers the attaching and effacing phenotype on E. coli K-12. Molecular Microbiology. 1997;23(2):399-407
  77. 77. Riquelme-Neira R et al. Vaccination with DNA encoding truncated enterohemorrhagic Escherichia coli (EHEC) factor for adherence-1 gene (efa-1′) confers protective immunity to mice infected with E. coli O157:H7. Frontiers in Cellular and Infection Microbiology. 2016;5:104-104
  78. 78. Szu SC, Ahmed A. Clinical studies of Escherichia coli O157:H7 conjugate vaccines in adults and young children. Microbiology Spectrum. 2014;2(6):1-7
  79. 79. Rabinovitz BC et al. The intranasal vaccination of pregnant dams with Intimin and EspB confers protection in neonatal mice from Escherichia coli (EHEC) O157:H7 infection. Vaccine. 2016;34(25):2793-2797
  80. 80. Marcato P et al. Recombinant Shiga toxin B-subunit-keyhole limpet hemocyanin conjugate vaccine protects mice from Shigatoxemia. Infection and Immunity. 2005;73(10):6523-6529
  81. 81. Piñero-Lambea C, Ruano-Gallego D, Fernández LÁ. Engineered bacteria as therapeutic agents. Current Opinion in Biotechnology. 2015;35:94-102

Written By

Massiel Cepeda-Molero, Stephanie Schüller, Gad Frankel and Luis Ángel Fernández

Submitted: 17 October 2019 Reviewed: 10 February 2020 Published: 09 March 2020