Role of bundle-forming pilus of enteropathogenic Escherichia coli in host cell adherence and in microcolony development

Enteropathogenic Escherichia coli (EPEC) adheres to epithelial cells and forms microcolonies in localized areas. Bundle-forming pili (BFP) are necessary for autoaggregation and the formation of microcolonies. In this study, we show that BFP, expressed by EPEC on epithelial cells, disappeared with the expansion of the microcolony. Bacterial dispersal and the release of BFP from the EPEC aggregates were induced by contact with host cellular membrane extract. In addition, BFP-expressing EPEC adhered directly to cell surfaces, in preference to attaching to pre-formed microcolonies on the cells. These results suggested that BFP mediate the initial attachment of EPEC through direct interaction with the host cell rather than through the recruitment of unattached bacteria to microcolonies on the cell.     

Feedback effects of host-derived adenosine on enteropathogenic Escherichia coli

Enteropathogenic E. coli (EPEC) is a common cause of diarrhea in children in developing countries. After adhering to intestinal cells, EPEC secretes effector proteins into host cells, causing cell damage and eventually death. We previously showed that EPEC infection triggers the release of ATP from host cells and that ATP is broken down to ADP, AMP, and adenosine. Adenosine produced from the breakdown of extracellular ATP triggers fluid secretion in intestinal monolayers and may be an important mediator of EPEC-induced diarrhea. Here we examined whether adenosine has any effects on EPEC bacteria. Adenosine stimulated EPEC growth in several types of media in vitro . Adenosine also altered the pattern of EPEC adherence to cultured cells from a localized adherence pattern to a more diffuse pattern. Adenosine changed the expression of virulence factors in EPEC, inhibiting the expression of the bundle-forming pilus (BFP) and enhancing expression of the EPEC secreted proteins (Esps). In vivo , experimental manipulations of adenosine levels had strong effects on the outcome of EPEC infection in rabbit intestinal loops. In addition to its previously reported effects on host tissues, adenosine has strong effects on EPEC bacteria, stimulating EPEC growth, altering its adherence pattern, and changing the expression of several important virulence genes. Adenosine, like noradrenaline, is a small, host-derived molecule that is utilized as a signal by EPEC.     

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.     

Enteropathogenic Escherichia coli Tir translocation and pedestal formation requires membrane cholesterol in the absence of bundle-forming pili

Enteropathogenic Escherichia coli (EPEC) is a significant cause of paediatric diarrhoea worldwide. Virulence requires adherence to intestinal epithelial cells, mediated in part through type IV bundle-forming pili (BFP), and the EPEC protein Tir. Tir is inserted into the enterocyte plasma membrane (PM), resulting in the formation of actin-rich pedestals. Tir is translocated by the type III secretion system (TTSS), through a pore comprised of EPEC proteins inserted into the PM. Here, we demonstrate that in the absence of BFP, EPEC adherence, effector translocation and pedestal formation are dependent on lipid rafts. Lipid raft disruption using methyl-beta-cyclodextrin (MbetaCD) decreased adherence by an EPEC BFP-deficient strain from 85% to 1%. Translocation of the effectors Tir and EspF was blocked by MbetaCD treatment, although the TTSS pore still formed. MbetaCD treatment after Tir delivery decreased pedestal formation by EPEC from 40% to 5%, but not by the related pathogen E. coli O157:H7 which uses a different Tir-based mechanism. In contrast, EPEC expressing the BFP can circumvent the requirement for membrane cholesterol. This suggests that lipid rafts play a role in virulence of this medically important pathogen.     

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.     

LamB-mediated adherence of enteropathogenic Escherichia coli to HEp-2 cells

Aims:  To establish the role of maltoporin (LamB) in adherence of enteropathogenic Escherichia coli (EPEC) to epithelial cells in vitro.
Methods and Results:  Three strains, wild type (WT) EPEC, a maltoporin (LamB) mutant ΔlamB , and DH5α were used to study adherence to cultured HEp-2 cells. Mutant ΔlamB was found to be deficient in adherence compared to WT EPEC. Adherence of ΔlamB was restored to wild type levels when complemented with the cloned lamB gene. The non–adherent strain DH5α also adhered to HEp-2 cells when it harboured the cloned lamB gene. The LamB protein was isolated from WT EPEC by electroelution and antibodies were raised in rabbits. The specificity of the antibodies was analysed by Western blotting. Anti-LamB antiserum reduced adherence of WT EPEC to HEp-2 cells. The LamB protein was coated on latex beads and the beads adhered to HEp-2 cells. Anti-LamB antiserum prevented bead adherence to HEp-2 cells. Multiple sequence alignment showed that the L9 loop of EPEC LamB had four amino acids different from the L9 loop of LamB from several other related pathogens.
Conclusions:  LamB serves as an alternative or additional adherence factor for EPEC.
Significance and Impact of the Study:  Adherence is an important component of the pathogenesis of noninvasive pathogens like EPEC. A putative adhesin such as LamB, which has already been found to be co-expressed with virulence factor EspB may be a potential vaccine candidate for control of EPEC and related pathogens.     

Inhibition of adhesion of enteroinvasive pathogens to human intestinal Caco-2 cells by Lactobacillus acidophilus strain LB decreases bacterial invasion

Abstract Salmonella typhimurium and enteropathogenic Escherichia coli (EPEC) were found to adhere to the brush border of differentiated human intestinal epithelial Caco-2 cells in culture, whereas Yersinia pseudotuberculosis and Listeria monocytogenes adhered to the periphery of undifferentiated Caco-2 cells. All these enterovirulent strains invaded the Caco-2 cells. Using a heat-killed human Lactobacillus acidophilus (strain LB) which strongly adheres both to undifferentiated and differentiated Caco-2 cells, we have studied inhibition of cell association with and invasion within Caco-2 cells by enterovirulent bacteria. Living and heat-killed Lactobacillus acidophilus strain LB inhibited both cell association and invasion of Caco-2 cells by enterovirulent bacteria in a concentration-dependent manner. The mechanism of inhibition of both adhesion and invasion appears to be due to steric hindrance of human enterocytic pathogen receptors by whole-cell lactobacilli rather than to a specific blockade of receptors.     

The bundlin pilin protein of enteropathogenic Escherichia coli is an N-acetyllactosamine-specific lectin

Synthetic N -acetyllactosamine (LacNAc) glycoside sequences coupled to BSA competitively inhibit enteropathogenic Escherichia coli (EPEC) localized adherence (LA) to human intestinal biopsy specimens and tissue culture cell monolayers. The LacNAc-specific adhesin appears to be associated with the bundle-forming pili (BFP) expressed by EPEC during the early stages of colonization. Herein, we report that recombinant bundlin inhibits EPEC LA to HEp-2 cells and binds to HEp-2 cells. Recombinant bundlin also binds, with millimolar association constants ( K _assoc), to synthetic LacNAc-Benzene and LacNAc-O(CH₂)₈CONH₂ glycosides as assessed in the gas phase by nanoelectrospray ionization mass spectrometry. Furthermore, LacNAc-BSA inhibits LA only of EPEC strains that express α bundlin alleles, suggesting putative locations for the LacNAc-binding pocket in the α bundlin monomer. Collectively, these results suggest that α bundlin possesses lectin-like properties that are responsible for LacNAc-specific initial adherence of α bundlin-expressing EPEC strains to host intestinal epithelial cells.     

Characterization of fimbriae produced by enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) express rope-like bundles of filaments, termed bundle-forming pili (BFP) (J. A. Girón, A. S. Y. Ho, and G. K. Schoolnik, Science 254:710-713, 1991). Expression of BFP is associated with localized adherence to HEp-2 cells and the presence of the EPEC adherence factor plasmid. In this study, we describe the identification of rod-like fimbriae and fibrillae expressed simultaneously on the bacterial surface of three prototype EPEC strains. Upon fimbrial extraction from EPEC B171 (O111:NM), three fimbrial subunits with masses of 16.5, 15.5, and 14.7 kDa were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Their N-terminal amino acid sequence showed homology with F9 and F7(2) fimbriae of uropathogenic E. coli and F1845 of diffuse-adhering E. coli, respectively. The mixture of fimbrial subunits (called FB171) exhibited mannose-resistant agglutination of human erythrocytes only, and this activity was not inhibited by alpha-D-Gal(1-4)-beta-Gal disaccharide or any other described receptor analogs for P, S, F, M, G, and Dr hemagglutinins of uropathogenic E. coli, which suggests a different receptor specificity. Hemagglutination was inhibited by extracellular matrix glycoproteins, i.e., collagen type IV, laminin, and fibronectin, and to a lesser extent by gangliosides, fetuin, and asialofetuin. Scanning electron microscopic studies performed on clusters of bacteria adhering to HEp-2 cells revealed the presence of structures resembling BFP and rod-like fimbriae linking bacteria to bacteria and bacteria to the eukaryotic cell membrane. We suggest a role of these surface appendages in the interaction of EPEC with eukaryotic cells as well as in the overall pathogenesis of intestinal disease caused by EPEC.     

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.     

Translocation of enteropathogenic Escherichia coli across an in vitro M cell model is regulated by its type III secretion system

Enteropathogenic Escherichia coli (EPEC) is an extracellular pathogen that utilizes a type III secretion system (TTSS) to modulate diverse host cell processes including cytoskeletal dynamics, tight junction permeability and macrophage phagocytosis. Some EPEC strains exhibit selective tropism for the specialized follicle-associated epithelium (FAE) overlying lymphoid follicles in the gut, which is a major site of uptake of inert particulates and pathogens, but do not translocate from the intestinal lumen in significant numbers. We have investigated the interaction of EPEC with FAE using an established in vitro model of the specialized FAE in which polarized enterocyte-like Caco-2 cells cocultured with the Raji B cell line undergo a phenotypic switch to a form that morphologically and functionally resembles the specialized antigen-transporting M cells found within FAE. Having confirmed that coculture with Raji B cells induces brush border reorganization and enhances particle transport across Caco-2 cells, we investigated translocation of bacteria across the M cell model. While Salmonella translocation was markedly upregulated by Raji coculture, transport of wild-type EPEC occurred at similarly low levels across both native Caco-2 and Caco-2/Raji-cocultured layers. Translocation rates were markedly higher for EPEC strains lacking either functional TTSS or the effector protein EspF. These observations resemble previously reported data on the inhibition of macrophage phagocytosis by EPEC, which has also been reported to be dependent on TTSS and EspF. Furthermore, as with macrophage phagocytosis, enhanced translocation of a TTSS mutant was blocked by wortmannin, implicating inhibition of phosphatidyl inositol 3-kinase-mediated signalling in the regulation of M cell translocation by EPEC.     

Numb modulates the paracellular permeability of intestinal epithelial cells through regulating apical junctional complex assembly and myosin light chain phosphorylation

Numb is highly expressed throughout the crypt-villus axis of intestinal mucosa and functions as cell fate determinant and integrator of cell-to-cell adhesion. Increased paracellular permeability of intestinal epithelial cells is associated with the epithelial barrier dysfunction of inflammatory bowel diseases (IBDs). The apical junctional complex (AJC) assembly and myosin light chain (MLC) phosphorylation regulate adherens junctions (AJ) and tight junctions (TJ). We determined whether and how Numb modulate the paracellular permeability of intestinal epithelial cells. Caco-2 intestinal epithelial cells and their Numb-interfered counterparts were used in the study for physiological, morphological and biological analyses. Numb, expressed in intestinal epithelial cells and located at the plasma membrane of Caco-2 cells in a basolateral to apical distribution, increased in the intestinal epithelial cells with the formation of the intestinal epithelial barrier. Numb expression decreased and accumulated in the cytoplasm of intestinal epithelial cells in a DSS-induced colitis mouse model. Numb co-localized with E-cadherin, ZO-1 and Par3 at the plasma membrane and interacted with E-cadherin and Par3. Knockdown of Numb in Caco-2 cells altered the F-actin structure during the Ca²⁺ switch assay, enhanced TNFα-/INF-γ-induced intestinal epithelial barrier dysfunction and TJ destruction, and increased the Claudin-2 protein level. Immunofluorescence experiments revealed that NMIIA and F-actin co-localized at the cell surface of Caco-2 cells. Numb knockdown in Caco-2 cells increased F-actin contraction and the abundance of phosphorylated MLC. Numb modulated the intestinal epithelial barrier in a Notch signaling-independent manner. These findings suggest that Numb modulates the paracellular permeability by affecting AJC assembly and MLC phosphorylation.     

Interactions of Escherichia coli strains of non-EPEC serogroups that carry eae and lack the EAF and stx gene sequences with undifferentiated and differentiated intestinal human Caco-2 cells

Escherichia coli strains of non-EPEC serotypes that carry eae and lack the EAF and the Shiga toxin (stx) gene sequences have been found in acute diarrhea. Both the cell association and the cell entry of these strains in human intestinal epithelial cells were studied as a function of cell differentiation and polarization. The eae+/EAF-/stx- non-EPEC E. coli strains invaded undifferentiated Caco-2 cells more efficiently than differentiated cells. In contrast, prototype EPEC strain E2348/69 did not show significative differences from invasion rates of undifferentiated and differentiated cells. The uptake of these strains was greatly enhanced by pretreatment of differentiated Caco-2 cells with EGTA. These results suggest that the eae+/EAF-/stx- non-EPEC E. coli invasion of intestinal cells may be dependent on receptors expressed on the surface of undifferentiated cells and the basolateral pole of differentiated cells.     

From alpha to beta: identification of amino acids required for the N-acetyllactosamine-specific lectin-like activity of bundlin

Bundle-forming pili (BFP) promote the adherence of typical enteropathogenic Escherichia coli (EPEC) to human intestinal epithelial cells. BFP are polymers of bundlin and nine bundlin alleles have been identified in EPEC isolated from diverse sources. These alleles are divided into two main groups, α and β, based on their amino acid sequences. Alpha bundlins are also N -acetyllactosamine- (LacNAc) specific lectins and bind to HEp-2 cells, whereas β bundlins do not display these characteristics. The four surface-exposed regions of amino acid sequence heterogeneity between α and β bundlin were therefore investigated as potential LacNAc-specific carbohydrate-binding domains in a bundlin. Mutation of one of these domains, 137-GENNI-141, in α₁ bundlin to that of β bundlin (136-SPDST-140) resulted in BFP that no longer bound to LacNAc or HEp-2 cells. Conversely, mutating the β₃ bundlin gene to encode the α bundlin sequence at this domain resulted in the gain of HEp-2 cell adherence. The importance of this domain in carbohydrate binding is supported by the finding that introducing the mutation GENNI→GENNT altered the α₁ bundlin carbohydrate-binding specificity from LacNAc to the Lewis X glycan sequence.     

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.     

Bundle-forming pilus retraction enhances enteropathogenic Escherichia coli infectivity

Enteropathogenic Escherichia coli (EPEC) is an important human pathogen that causes acute infantile diarrhea. The type IV bundle-forming pili (BFP) of typical EPEC strains are dynamic fibrillar organelles that can extend out and retract into the bacterium. The bfpF gene encodes for BfpF, a protein that promotes pili retraction. The BFP are involved in bacterial autoaggregation and in mediating the initial adherence of the bacterium with its host cell. Importantly, BFP retraction is implicated in virulence in experimental human infection. How pili retraction contributes to EPEC pathogenesis at the cellular level remains largely obscure, however. In this study, an effort has been made to address this question using engineered EPEC strains with induced BFP retraction capacity. We show that the retraction is important for tight-junction disruption and, to a lesser extent, actin-rich pedestal formation by promoting efficient translocation of bacterial protein effectors into the host cells. A model is proposed whereby BFP retraction permits closer apposition between the bacterial and the host cell surfaces, thus enabling timely and effective introduction of bacterial effectors into the host cell via the type III secretion apparatus. Our studies hence suggest novel insights into the involvement of pili retraction in EPEC pathogenesis.