Protein translocation into host epithelial cells by infecting enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) causes diarrhoea in young children. EPEC induces the formation of actin pedestal in infected epithelial cells. A type III protein secretion system and several proteins that are secreted by this system, including EspB, are involved in inducing the formation of the actin pedestals. We have demonstrated that contact of EPEC with HeLa cells is associated with the induction of production and secretion of EspB. Shortly after infection, EPEC initiates translocation of EspB, and EspB fused to the CyaA reporter protein (EspB–CyaA), into the host cell. The translocated EspB was distributed between the membrane and the cytoplasm of the host cell. Translocation was strongly promoted by attachment of EPEC to the host cell, and both attachment factors of EPEC, intimin and the bundle-forming pili, were needed for full translocation efficiency. Translocation and secretion of EspB and EspB–CyaA were abolished in mutants deficient in components of the type III protein secretion system, including sepA and sepB mutants. EspB–CyaA was secreted but not translocated by an espB mutant. These results indicate that EspB is both translocated and required for protein translocation by EPEC.     

Interactions and predicted host membrane topology of the enteropathogenic Escherichia coli translocator protein EspB

Type 3 secretion systems (T3SSs) are critical for the virulence of numerous deadly Gram-negative pathogens. T3SS translocator proteins are required for effector proteins to traverse the host cell membrane and perturb cell function. Translocator proteins include two hydrophobic proteins, represented in enteropathogenic Escherichia coli (EPEC) by EspB and EspD, which are thought to interact and form a pore in the host membrane. Here we adapted a sequence motif recognized by a host kinase to demonstrate that residues on the carboxyl-terminal side of the EspB transmembrane domain are localized to the host cell cytoplasm. Using functional internal polyhistidine tags, we confirm an interaction between EspD and EspB, and we demonstrate, for the first time, an interaction between EspD and the hydrophilic translocator protein EspA. Using a panel of espB insertion mutations, we describe two regions on either side of a putative transmembrane domain that are required for the binding of EspB to EspD. Finally, we demonstrate that EspB variants incapable of binding EspD fail to adopt the proper host cell membrane topology. These results provide new insights into interactions between translocator proteins critical for virulence.     

Genetically engineered enteropathogenic Escherichia coli strain elicits a specific immune response and protects against a virulent challenge

Enteropathogenic Escherichia coli (EPEC), a major cause of severe disease with diarrhea in infants, is also involved in weaned rabbit colibacillosis. EPEC O103 is frequent in rabbit-fattening units of Western Europe. It causes high mortality and growth retardation, leading to substantial economic losses. We report here the construction by allelic exchange of an EPEC O103 strain mutated in espB and tir, two essential virulence genes. Upon live oral administration to weaned rabbits, the E22DeltaTir/EspB mutant strain efficiently colonized the intestinal tract without any adverse consequences. The rabbits were challenged with the highly pathogenic parental strain E22. The mutant provided complete protection to rabbits and total resistance to intestinal colonization by E22. In addition, E22DeltaTir/EspB strain induced a specific humoral response against the bacterial adhesin AF/R2. These Abs prevent bacterial attachment to epithelial cells in vitro. These results open the way for the development of an efficient vaccine strategy against rabbit EPEC infections.     

Balance of bacterial pro- and anti-inflammatory mediators dictates net effect of enteropathogenic Escherichia coli on intestinal epithelial cells

Enteropathogenic Escherichia coli (EPEC) virulence requires a type III secretion system (TTSS) to deliver effector molecules in host cells. Although the TTSS is crucial to EPEC pathogenesis, its function in EPEC-induced inflammation is not known. The aim of this study was to investigate the role of the TTSS in EPEC-induced inflammation. HT-29 intestinal epithelial cells were infected with wild-type (WT) EPEC or select mutant strains or exposed to corresponding filter-sterilized supernatants (SN), and interleukin-8 (IL-8) secretion was determined by ELISA. EPEC SN stimulated significantly greater IL-8 production than EPEC organisms. Flagellin, as well as a TTSS-independent >50-kDa nonflagellin protein, was found to significantly contribute to this response. Dose-response studies showed that increasing concentrations of WT SN proportionally increased IL-8, whereas increasing multiplicity of infection of EPEC inversely correlated with IL-8 secretion, suggesting that EPEC dampens this host response. Infection with DeltaescN (nonfunctional TTSS) markedly increased IL-8 compared with WT, indicating that a functional TTSS is required for this anti-inflammatory property; complementation of escN restored the attenuated response. Mutation of espB also enhanced the IL-8 response, and complementation returned IL-8 to near WT levels, suggesting involvement of this effector. The anti-inflammatory effect extends to both bacterial and host-derived proinflammatory stimuli, since prior infection with EPEC suppressed the IL-8 response to tumor necrosis factor-alpha, IL-1beta, and enterohemorrhagic E. coli flagellin. These findings indicate that EPEC-induced inflammation is a balance between pro- and anti-inflammatory proteins; extracellular factors, including flagellin and an unidentified TTSS-independent, >50-kDa protein, trigger inflammation while intracellular TTSS-dependent factors, including EspB, attenuate this response.     

The enteropathogenic E. coli effector EspB facilitates microvillus effacing and antiphagocytosis by inhibiting myosin function

Enteropathogenic Escherichia coli (EPEC) destroys intestinal microvilli and suppresses phagocytosis by injecting effectors into infected cells through a type III secretion system (TTSS). EspB, a component of the TTSS, is also injected into the cytoplasm of host cells. However, the physiological functions of EspB within the host cell cytoplasm remain unclear. We show that EspB binds to myosins, which are a superfamily of proteins that interact with actin filaments and mediate essential cellular processes, including microvillus formation and phagocytosis. EspB inhibits the interaction of myosins with actin, and an EspB mutant that lacks the myosin-binding region maintained its TTSS function but could not induce microvillus effacing or suppress phagocytosis. Moreover, the myosin-binding region of EspB is essential for Citrobacter rodentium, an EPEC-related murine pathogen, to efficiently infect mice. These results suggest that EspB inhibits myosin functions and thereby facilitates efficient infection by EPEC.     

The type III protein translocation system of enteropathogenic Escherichia coli involves EspA-EspB protein interactions

Enteropathogenic Escherichia coli (EPEC), like many bacterial pathogens, use a type III secretion system to deliver effector proteins across the bacterial cell wall. In EPEC, four proteins, EspA, EspB, EspD and Tir are known to be exported by a type III secretion system and to be essential for 'attaching and effacing' (A/E) lesion formation, the hallmark of EPEC pathogenicity. EspA was recently shown to be a structural protein and a major component of a large, transiently expressed, filamentous surface organelle which forms a direct link between the bacterium and the host cell. In contrast, EspB is translocated into the host cell where it is localized to both membrane and cytosolic cell fractions. EspA and EspB are required for translocation of Tir to the host cell membrane suggesting that they may both be components of the translocation apparatus. In this study, we show that EspB co-immunoprecipitates with the EspA filaments and that, during EPEC infection of HEp-2 cells, EspB localizes closely with EspA. Using a number of binding assays, we also show that EspB can bind and be copurified with EspA. Nevertheless, binding of EspA filaments to the host cell membranes occurred even in the absence of EspB. These results suggest that following initial attachment of the EspA filaments to the target cells, EspB is delivered into the host cell membrane and that the interaction between EspA and EspB may be important for protein translocation.     

EspB and EspD require a specific chaperone for proper secretion from enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli uses a type III secretion apparatus to deliver proteins essential for pathogenesis to the host epithelium. Several proteins have been detected in culture supernatants of the prototype EPEC strain E2348/69 and three of these, EspA, EspB, and EspD, use type III machinery for export. Here, we report the identification and characterization of CesD, a protein required for proper EspB and EspD secretion. CesD shows sequence homology to chaperone proteins from other type III secretion pathways. Based on this, we hypothesize that CesD may function as a secretion chaperone in EPEC. A mutation in cesD abolished EspD secretion into culture supernatants and reduced the amount of secreted EspB, but had little effect on the amount of secreted EspA. The mutant strain was negative for both FAS and Tir phosphorylation, consistent with the previously described roles for EspB and EspD in EPEC pathogenesis. CesD was shown to interact with EspD but not EspB or EspA. CesD was detected in the bacterial cytosol, and, surprisingly, a substantial amount of the protein was also found to be associated with the inner membrane. Thus, although CesD has some attributes that are similar to other type III secretion chaperones, its membrane localization separates it from previously described members of this family.     

Characterization of EspC, a 110-kilodalton protein secreted by enteropathogenic Escherichia coli which is homologous to members of the immunoglobulin A protease-like family of secreted proteins.

Enteropathogenic Escherichia coli (EPEC) secretes at least five proteins. Two of these proteins, EspA and EspB (previously called EaeB), activate signal transduction pathways in host epithelial cells. While the role of the other three proteins (39, 40, and 110 kDa) remains undetermined, secretion of all five proteins is under the control of perA, a known positive regulator of several EPEC virulence factors. On the basis of amino-terminal protein sequence data, we cloned and sequenced the gene which encodes the 110-kDa secreted protein and examined its possible role in EPEC signaling and interaction with epithelial cells. In accordance with the terminology used for espA and espB, we called this gene espC, for EPEC-secreted protein C. We found significant homology between the predicted EspC protein sequence and a family of immunoglobulin A (IgA) protease-like proteins which are widespread among pathogenic bacteria. Members of this protein family are found in avian pathogenic Escherichia coli (Tsh), Haemophilus influenzae (Hap), and Shigella flexneri (SepA). Although these proteins and EspC do not encode IgA protease activity, they have considerable homology with IgA protease from Neisseria gonorrhoeae and H. influenzae and appear to use a export system for secretion. We found that genes homologous to espC also exist in other pathogenic bacteria which cause attaching and effacing lesions, including Hafnia alvei biotype 19982, Citrobacter freundii biotype 4280, and rabbit diarrheagenic E. coli (RDEC-1). Although these strains secrete various proteins similar in molecular size to the proteins secreted by EPEC, we did not detect secretion of a 110-kDa protein by these strains. To examine the possible role of EspC in EPEC interactions with epithelial cells, we constructed a deletion mutant in espC by allelic exchange and characterized the mutant by standard tissue culture assays. We found that EspC is not necessary for mediating EPEC-induced signal transduction in HeLa epithelial cells and does not play a role in adherence or invasion of tissue culture cells.     

肠出血性大肠杆菌O157∶H7 EspA和EspB蛋白的重组表达及其免疫效果

目的：通过DNA重组技术表达肠出血性大肠杆菌（EHEC）0157：H7的EspA和EspB蛋白,并分析它们的免疫保护性。方法：采用PCR技术从EHEC0157：H7基因组中扩增espA和espB基因,连接至pET-22b（4-）载体上,转化至宿主细胞大肠杆菌BL21（DE3）,经IPTG诱导表达,用亲和层析纯化目的蛋白,SDS-PAGE测定其相对分子质量,免疫小鼠分析其免疫保护性。结果：重组espA和espB基因片段的测序结果与GenBank中的相应基因序列完全一致,一致性均为100％;得到了纯度为95％以上的重组EspA和EspB蛋白,免疫小鼠所得到的抗体效价均为10^6。结论：重组EspA和EspB蛋白获得了可溶性表达,表达的蛋白具有良好的免疫保护性,为进一步制备疫苗奠定了基础。     

Lactoferrin disruption of bacterial type III secretion systems

Many gram-negative bacteria share a closely related mechanism for secretion of virulence proteins. This complex machine, the type III secretion system, secretes virulence proteins in response to sensing the presence of target mammalian cells. We have found that recombinant human lactoferrin impairs the function of this system in two model organisms: Shigella and Enteropathogenic E. coli (EPEC). In the case of Shigella, there is loss and degradation of two proteins secreted by the type III mechanism, invasion plasmid antigens B and C (IpaB and IpaC); these proteins normally form a complex that causes Shigella to be taken up by host mammalian cells. In the case of EPEC, lactoferrin causes loss and degradation of E. coli secreted proteins A, B and D (EspABD) particularly EspB. These proteins are components of type III machinery and are known to be key elements of EPEC pathogenesis. Studies using purified EspB demonstrated that lactoferrin has a direct proteolytic effect on EspB that can be prevented by serine protease inhibitors. A synthetic peptide of the N-terminal 33 amino acids of lactoferrin caused loss of cell associated EspB but, unlike the whole lactoferrin molecule, did not caused degradation of EspB. Thus, in both model systems, brief exposure to lactoferrin causes loss and degradation of type III secretion system virulence proteins.     

Species-specific cell adhesion of enteropathogenic Escherichia coli is mediated by type IV bundle-forming pili

Enteropathogenic Escherichia coli (EPEC) is a causative agent of diarrhoea in humans. Localized adherence of EPEC onto intestinal mucosa was reproduced in an in vitro adherence assay with cultured human epithelial cells. We found that the efficiency of EPEC adherence to a mouse-derived colonic epithelial cell line, CMT-93, was remarkably lower than its adherence to human-derived intestinal cell lines, such as Intestine-407 or Caco-2. Although EPEC did adhere to some cell lines derived from non-human species, fixing the cells with formalin to inactivate one or more formalin-sensitive factors allowed us to observe species-specific differences in EPEC adherence. In contrast to these results, an EPEC mutant that is defective in bundle-forming pili (BFP) production adhered as efficiently to CMT-93 cells as to Caco-2 cells. Furthermore, Citrobacter rodentium expressing BFP adhered to Caco-2 cells much more efficiently than to CMT-93 cells. Finally, a purified BfpA-His6 fusion protein showed higher affinity for Caco-2 cells than for CMT-93 cells, and inhibited EPEC adherence. Following BFP-mediated adherence, secretion of EspB from adherent bacteria and reorganization of F-actin in the host cells was observed. EPEC adhering to CMT-93 cells induced far less secretion of EspB, or reorganization of F-actin in the host CMT-93 cells, than did EPEC adhering to Caco-2 cells. These results indicated that BFP plays an important role in the cell-type-dependent adherence of EPEC and in the progression to the later steps in EPEC adherence.     

EspA filament-mediated protein translocation into red blood cells

Type III secretion allows bacteria to inject effector proteins into host cells. In enteropathogenic Escherichia coli (EPEC), three type III secreted proteins, EspA, EspB and EspD, have been shown to be required for translocation of the Tir effector protein into host cells. EspB and EspD have been proposed to form a pore in the host cell membrane, whereas EspA, which forms a large filamentous structure bridging bacterial and host cell surfaces, is thought to provide a conduit for translocation of effector proteins between pores in the bacterial and host cell membranes. Type III secretion has been correlated with an ability to cause contact-dependent haemolysis of red blood cells (RBCs) in vitro . As EspA filaments link bacteria and the host cell, we predicted that intimate bacteria–RBC contact would not be required for EPEC-induced haemolysis and, therefore, in this study we investigated the interaction of EPEC with monolayers of RBCs attached to polylysine-coated cell culture dishes. EPEC caused total RBC haemolysis in the absence of centrifugation and osmoprotection studies were consistent with the insertion of a hydrophilic pore into the RBC membrane. Cell attachment and haemolysis involved interaction between EspA filaments and the RBC membrane and was dependent upon a functional type III secretion system and on EspD, whereas EPEC lacking EspB still caused some haemolysis. Following haemolysis, only EspD was consistently detected in the RBC membrane. This study shows that intimate bacteria–RBC membrane contact is not a requirement for EPEC-induced haemolysis; it also provides further evidence that EspA filaments are a conduit for protein translocation and that EspD may be the major component of a translocation pore in the host cell membrane.     

Characterization of translocation pores inserted into plasma membranes by type III-secreted Esp proteins of enteropathogenic Escherichia coli

Many mucosal pathogens use type III secretion systems for the injection of effector proteins into target cells. The type III-secreted proteins EspB and EspD of enteropathogenic Escherichia coli (EPEC) are inserted into the target cell membrane. Together with EspA, these proteins are supposed to constitute a molecular syringe, channelling other effector proteins into the host cell. In this model, EspB and EspD would represent the tip of the needle forming a pore into target cell membranes. Although contact-dependent and Esp-mediated haemolytic activity by EPEC has already been described, the formation of a putative pore resulting in haemolysis has not been demonstrated so far. Here, we show that (i) diffusely adhering (DA)-EPEC strains exhibit a type III-dependent haemolytic activity too; (ii) this activity resides in the secreted proteins and, for DA-EPEC strains, in contrast to EPEC strains, does not require bacterial contact; and (iii) pores are introduced into the target cell membrane. Osmoprotection revealed a minimal pore size of 3–5 nm. The pores induced by type III-secreted proteins of DA-EPEC were characterized by electron microscopy techniques. Analysis by atomic force microscopy demonstrated the pores to be composed of six to eight subunits with a lateral extension of 55–65 nm and to be raised 15–20 nm above the membrane plane. We could also demonstrate an association of EspB and EspD with erythrocyte membranes and an interaction of both proteins with each other in vitro . These results, together with the homologies of EspB and EspD to proposed functional domains of other pore-forming proteins (Yop/Ipa), strongly support the idea that both proteins are directly involved in pore formation, which might represent the type III secretion system translocon.     

Development of a Rapid Agglutination Latex Test for Diagnosis of Enteropathogenic and Enterohemorrhagic Escherichia coli Infection in Developing World: Defining the Biomarker,Antibody and Method

Background

Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC/EHEC) are human intestinal pathogens responsible for diarrhea in both developing and industrialized countries. In research laboratories, EPEC and EHEC are defined on the basis of their pathogenic features; nevertheless, their identification in routine laboratories is expensive and laborious. Therefore, the aim of the present work was to develop a rapid and simple assay for EPEC/EHEC detection. Accordingly, the EPEC/EHEC-secreted proteins EspA and EspB were chosen as target antigens.

Methodology

First, we investigated the ideal conditions for EspA/EspB production/secretion by ELISA in a collection of EPEC/EHEC strains after cultivating bacterial isolates in Dulbecco’s modified Eagle’s medium (DMEM) or DMEM containing 1% tryptone or HEp-2 cells-preconditioned DMEM, employing either anti-EspA/anti-EspB polyclonal or monoclonal antibodies developed and characterized herein. Subsequently, a rapid agglutination latex test (RALT) was developed and tested with the same collection of bacterial isolates.

Principal findings

EspB was defined as a biomarker and its corresponding monoclonal antibody as the tool for EPEC/EHEC diagnosis; the production of EspB was better in DMEM medium. RALT assay has the sensitivity and specificity required for high-impact diagnosis of neglected diseases in the developing world.

Conclusion

RALT assay described herein can be considered an alternative assay for diarrhea diagnosis in low-income countries since it achieved 97% sensitivity, 98% specificity and 97% efficiency.     

Labeling of specific lysine residues at the active site of glutamine synthetase

Glutamine synthetase (Escherichia coli) was incubated with three different reagents that react with lysine residues, viz. pyridoxal phosphate, 5'-p-fluorosulfonylbenzoyladenosine, and thiourea dioxide. The latter reagent reacts with the epsilon-nitrogen of lysine to produce homoarginine as shown by amino acid analysis, nmr, and mass spectral analysis of the products. A variety of differential labeling experiments were conducted with the above three reagents to label specific lysine residues. Thus pyridoxal phosphate was found to modify 2 lysine residues leading to an alteration of catalytic activity. At least 1 lysine residue has been reported previously to be modified by pyridoxal phosphate at the active site of glutamine synthetase (Whitley, E. J., and Ginsburg, A. (1978) J. Biol. Chem. 253, 7017-7025). By varying the pH and buffer, one or both residues could be modified. One of these lysine residues was associated with approximately 81% loss in activity after modification while modification of the second lysine residue led to complete inactivation of the enzyme. This second lysine was found to be the residue which reacted specifically with the ATP affinity label 5'-p-fluorosulfonylbenzoyladenosine. Lys-47 has been previously identified as the residue that reacts with this reagent (Pinkofsky, H. B., Ginsburg, A., Reardon, I., Heinrikson, R. L. (1984) J. Biol. Chem. 259, 9616-9622; Foster, W. B., Griffith, M. J., and Kingdon, H. S. (1981) J. Biol. Chem. 256, 882-886). Thiourea dioxide inactivated glutamine synthetase with total loss of activity and concomitant modification of a single lysine residue. The modified amino acid was identified as homoarginine by amino acid analysis. The lysine residue modified by thiourea dioxide was established by differential labeling experiments to be the same residue associated with the 81% partial loss of activity upon pyridoxal phosphate inactivation. Inactivation with either thiourea dioxide or pyridoxal phosphate did not affect ATP binding but glutamate binding was weakened. The glutamate site was implicated as the site of thiourea dioxide modification based on protection against inactivation by saturating levels of glutamate. Glutamate also protected against pyridoxal phosphate labeling of the lysine consistent with this residue being the common site of reaction with thiourea dioxide and pyridoxal phosphate.     

Effects of Psidium guajava leaf extract on secretion systems of gram‐negative enteropathogenic bacteria

In this study, 672 plant‐tissue extracts were screened for phytochemicals that inhibit the function of the type III secretion system (T3SS) of enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC). Among candidates examined, an extract from the leaves of Psidium guajava (guava) was found to inhibit secretion of EPEC‐secreted protein B (EspB) from EPEC and EHEC without affecting bacterial growth. Guava extract (GE) also inhibited EPEC and EHEC from adhering to, and injecting EspB into, HEp‐2 cells. GE seemed to block translocation of EspB from the bacterial cells to the culture medium. In addition, GE also inhibited the T3SS of Yersinia pseudotuberculosis and Salmonella enterica serovar Typhimurium. After exposure to GE, Y. pseudotuberculosis stopped secreting Yersinia outer proteins and was unable to induce apoptosis of mouse bone marrow‐derived macrophages. S. typhimurium exposed to GE stopped secreting Sip proteins and was unable to invade HEp‐2 cells. GE inhibited secretion of EspC, the type V secretion protein of EPEC, but not secretion of Shiga toxin 2 from EHEC. Thus, our results suggest that guava leaves contain a novel type of antimicrobial compound that could be used to treat and prevent gram‐negative enteropathogenic bacterial infections.

     

Chemical modification of the lysine residues of bacterial formate dehydrogenase

Inactivation of formate dehydrogenase by formaldehyde, pyridoxal and pyridoxal phosphate was studied. The effects of concentrations of the modifying agents, substrates, products and inhibitors on the extent of the enzyme inactivation were examined. A complete formate dehydrogenase inactivation by pyridoxal, pyridoxal, phosphate and formaldehyde is achieved by the blocking of 2, 5 and 13 lysine residues per enzyme subunit, respectively. The coenzymes do not protect formate dehydrogenase against inactivation. In the case of modification by pyridoxal and pyridoxal phosphate a complete maintenance of the enzyme activity and specific protection of one lysine residue per enzyme subunit is observed during formation of a binary formate-enzyme complex, or a ternary enzyme--NAD--azide complex. One lysine residue is supposed to be located at the formate-binding site of the formate dehydrogenase active center.     

Two Ser/Thr protein kinases essential for efficient aggregation and spore morphogenesis in Myxococcus xanthus

Myxococcus xanthus has a complex life cycle that involves vegetative growth and development. Previously, we described the espAB locus that is involved in timing events during the initial stages of fruiting body formation. Deletion of espA caused early aggregation and sporulation, whereas deletion of espB caused delayed aggregation and sporulation resulting in reduced spore yields. In this study, we describe two genes, pktA5 and pktB8, that flank the espAB locus and encode Ser/Thr protein kinase (STPK) homologues. Cells deficient in pktA5 or pktB8 formed translucent mounds and produced low spore yields, similar in many respects to espB mutants. Double mutant analysis revealed that espA was epistatic to pktA5 and pktB8 with respect to aggregation and fruiting body morphology, but that pktA5 and pktB8 were epistatic to espA with respect to sporulation efficiency. Expression profiles of pktA5-lacZ and pktB8-lacZ fusions and Western blot analysis showed that the STPKs are expressed under vegetative and developmental conditions. In vitro kinase assays demonstrated that the RD kinase, PktA5, autophosphorylated on threonine residue(s) and phosphorylated the artificial substrate, myelin basic protein. In contrast, autophosphorylation of the non-RD kinase, PktB8, was not observed in vitro; however, the phenotype of a pktB8 kinase-dead point mutant resembled the pktB8 deletion mutant, indicating that this residue was important for function and that it likely functions as a kinase in vivo. Immunoprecipitation of Tap-tagged PktA5 and PktB8 revealed an interaction with EspA during development in M. xanthus. These results, taken together, suggest that PktA5 and PktB8 are STPKs that function during development by interacting with EspA and EspB to regulate M. xanthus development.     

The coiled-coil domain of EspA is essential for the assembly of the type III secretion translocon on the surface of enteropathogenic Escherichia coli

Enteropathogenic E. coli (EPEC) utilize a type III secretion system to deliver virulence-associated effector proteins to the host cell. Four proteins, EspA, EspB, EspD, and Tir, which are integral to the formation of characteristic "attaching and effacing" (A/E) intestinal lesions, are known to be exported via the EPEC type III secretion system. Recent work demonstrated that EspA is a major component of a filamentous structure, elaborated on the surface of EPEC, which is required for translocation of EspB and Tir. The carboxyl terminus of EspA is predicted to comprise an alpha-helical region, which demonstrates heptad periodicity whereby positions a and d in the heptad repeat unit abcdefg are occupied by hydrophobic residues, indicating a propensity for coiled-coil interactions. Here we demonstrate multimeric EspA isoforms in EPEC culture supernatants and EspA:EspA interaction on solid phase. Non-conservative amino acid substitution of specific EspA heptad residues generated EPEC mutants defective in filament assembly but which retained the ability to induce A/E lesions; additional mutation totally abolished EspA filament assembly and A/E lesion formation. These results demonstrate a similarity to flagellar biosynthesis and indicate that the coiled-coil domain of EspA is required for assembly of the EspA filament-associated type III secretion translocon.