Mitotic Arrest-Deficient 2 Like 2 (MAD2L2) Interacts with Escherichia coli Effector Protein EspF.

Enteropathogenic (EPEC) and Enterohemorrhagic (EHEC) Escherichia coli are considered emerging zoonotic pathogens of worldwide distribution. The pathogenicity of the bacteria is conferred by multiple virulence determinants, including the locus of enterocyte effacement (LEE) pathogenicity island, which encodes a type III secretion system (T3SS) and effector proteins, including the multifunctional secreted effector protein (EspF). EspF sequences differ between EPEC and EHEC serotypes in terms of the number and residues of SH3-binding polyproline-rich repeats and N-terminal localization sequence. The aim of this study was to discover additional cellular interactions of EspF that may play important roles in E. coli colonization using the Yeast two-hybrid screening system (Y2H). Y2H screening identified the anaphase-promoting complex inhibitor Mitotic Arrest-Deficient 2 Like 2 (MAD2L2) as a host protein that interacts with EspF. Using LUMIER assays, MAD2L2 was shown to interact with EspF variants from EHEC O157:H7 and O26:H11 as well as EPEC O127:H6. MAD2L2 is targeted by the non-homologous Shigella effector protein invasion plasmid antigen B (IpaB) to halt the cell cycle and limit epithelial cell turnover. Therefore, we postulate that interactions between EspF and MAD2L2 serve a similar function in promoting EPEC and EHEC colonization, since cellular turnover is a key method for bacteria removal from the epithelium. Future work should investigate the biological importance of this interaction that could promote the colonization of EPEC and EHEC E. coli in the host.

Shiga toxin sub-type 2a increases the efficiency of Escherichia coli O157 transmission between animals and restricts epithelial regeneration in bovine enteroids.

Specific Escherichia coli isolates lysogenised with prophages that express Shiga toxin (Stx) can be a threat to human health, with cattle being an important natural reservoir. In many countries the most severe pathology is associated with enterohaemorrhagic E. coli (EHEC) serogroups that express Stx subtype 2a. In the United Kingdom, phage type (PT) 21/28 O157 strains have emerged as the predominant cause of life-threatening EHEC infections and this phage type commonly encodes both Stx2a and Stx2c toxin types. PT21/28 is also epidemiologically linked to super-shedding (>103 cfu/g of faeces) which is significant for inter-animal transmission and human infection as demonstrated using modelling studies. We demonstrate that Stx2a is the main toxin produced by stx2a+/stx2c+ PT21/28 strains induced with mitomycin C and this is associated with more rapid induction of gene expression from the Stx2a-encoding prophage compared to that from the Stx2c-encoding prophage. Bacterial supernatants containing either Stx2a and/or Stx2c were demonstrated to restrict growth of bovine gastrointestinal organoids with no restriction when toxin production was not induced or prevented by mutation. Isogenic strains that differed in their capacity to produce Stx2a were selected for experimental oral colonisation of calves to assess the significance of Stx2a for both super-shedding and transmission between animals. Restoration of Stx2a expression in a PT21/28 background significantly increased animal-to-animal transmission and the number of sentinel animals that became super-shedders. We propose that while both Stx2a and Stx2c can restrict regeneration of the epithelium, it is the relatively rapid and higher levels of Stx2a induction, compared to Stx2c, that have contributed to the successful emergence of Stx2a+ E. coli isolates in cattle in the last 40 years. We propose a model in which Stx2a enhances E. coli O157 colonisation of in-contact animals by restricting regeneration and turnover of the colonised gastrointestinal epithelium.

Type III Secretion-Dependent Sensitivity of Escherichia coli O157 to Specific Ketolides.

A subset of Gram-negative bacterial pathogens uses a type III secretion system (T3SS) to open up a conduit into eukaryotic cells in order to inject effector proteins. These modulate pathways to enhance bacterial colonization. In this study, we screened established bioactive compounds for any that could repress T3SS expression in enterohemorrhagic Escherichia coli (EHEC) O157. The ketolides telithromycin and, subsequently, solithromycin both demonstrated repressive effects on expression of the bacterial T3SS at sub-MICs, leading to significant reductions in bacterial binding and actin-rich pedestal formation on epithelial cells. Preincubation of epithelial cells with solithromycin resulted in significantly less attachment of E. coli O157. Moreover, bacteria expressing the T3SS were more susceptible to solithromycin, and there was significant preferential killing of E. coli O157 bacteria when they were added to epithelial cells that had been preexposed to the ketolide. This killing was dependent on expression of the T3SS. Taken together, this research indicates that the ketolide that has accumulated in epithelial cells may traffic back into the bacteria via the T3SS. Considering that neither ketolide induces the SOS response, nontoxic members of this class of antibiotics, such as solithromycin, should be considered for future testing and trials evaluating their use for treatment of EHEC infections. These antibiotics may also have broader significance for treating infections caused by other pathogenic bacteria, including intracellular bacteria, that express a T3SS.

Salmonella transforms follicle-associated epithelial cells into M cells to promote intestinal invasion.

Salmonella Typhimurium specifically targets antigen-sampling microfold (M) cells to translocate across the gut epithelium. Although M cells represent a small proportion of the specialized follicular-associated epithelium (FAE) overlying mucosa-associated lymphoid tissues, their density increases during Salmonella infection, but the underlying molecular mechanism remains unclear. Using in vitro and in vivo infection models, we demonstrate that the S. Typhimurium type III effector protein SopB induces an epithelial-mesenchymal transition (EMT) of FAE enterocytes into M cells. This cellular transdifferentiation is a result of SopB-dependent activation of Wnt/ß-catenin signaling leading to induction of both receptor activator of NF-κB ligand (RANKL) and its receptor RANK. The autocrine activation of RelB-expressing FAE enterocytes by RANKL/RANK induces the EMT-regulating transcription factor Slug that marks epithelial transdifferentiation into M cells. Thus, via the activity of a single secreted effector, S. Typhimurium transforms primed epithelial cells into M cells to promote host colonization and invasion.

Comparative analysis of EspF variants in inhibition of Escherichia coli phagocytosis by macrophages and inhibition of E. coli translocation through human- and bovine-derived M cells.

The EspF protein is secreted by the type III secretion system of enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively). EspF sequences differ between EHEC O157:H7, EHEC O26:H11, and EPEC O127:H6 in terms of the number of SH3-binding polyproline-rich repeats and specific residues in these regions, as well as residues in the amino domain involved in cellular localization. EspF(O127) is important for the inhibition of phagocytosis by EPEC and also limits EPEC translocation through antigen-sampling cells (M cells). EspF(O127) has been shown to have effects on cellular organelle function and interacts with several host proteins, including N-WASP and sorting nexin 9 (SNX9). In this study, we compared the capacities of different espF alleles to inhibit (i) bacterial phagocytosis by macrophages, (ii) translocation through an M-cell coculture system, and (iii) uptake by and translocation through cultured bovine epithelial cells. The espF gene from E. coli serotype O157 (espF(O157)) allele was significantly less effective at inhibiting phagocytosis and also had reduced capacity to inhibit E. coli translocation through a human-derived in vitro M-cell coculture system in comparison to espF(O127) and espF(O26). In contrast, espF(O157) was the most effective allele at restricting bacterial uptake into and translocation through primary epithelial cells cultured from the bovine terminal rectum, the predominant colonization site of EHEC O157 in cattle and a site containing M-like cells. Although LUMIER binding assays demonstrated differences in the interactions of the EspF variants with SNX9 and N-WASP, we propose that other, as-yet-uncharacterized interactions contribute to the host-based variation in EspF activity demonstrated here.

Characterization of the effects of salicylidene acylhydrazide compounds on type III secretion in Escherichia coli O157:H7.

Recent work has highlighted a number of compounds that target bacterial virulence by affecting gene regulation. In this work, we show that small-molecule inhibitors affect the expression of the type III secretion system (T3SS) of Escherichia coli O157:H7 in liquid culture and when this bacterium is attached to bovine epithelial cells. Inhibition of T3SS expression resulted in a reduction in the capacity of the bacteria to form attaching and effacing lesions. Our results show that there is marked variation in the abilities of four structurally related compounds to inhibit the T3SS of a panel of isolates. Using transcriptomics, we performed a comprehensive analysis of the conserved and inhibitor-specific transcriptional responses to these four compounds. These analyses of gene expression show that numerous virulence genes, located on horizontally acquired DNA elements, are affected by the compounds, but the number of genes significantly affected varied markedly for the different compounds. Overall, we highlight the importance of assessing the effect of such "antivirulence" agents on a range of isolates and discuss the possible mechanisms which may lead to the coordinate downregulation of horizontally acquired virulence genes.

The Escherichia coli O157:H7 EhaB autotransporter protein binds to laminin and collagen I and induces a serum IgA response in O157:H7 challenged cattle.

Enterohaemorrhagic Escherichia coli (EHEC) are a subgroup of Shiga toxin-producing E. coli that cause gastrointestinal disease with the potential for life-threatening sequelae. Cattle serve as the natural reservoir for EHEC and outbreaks occur sporadically as a result of contaminated beef and other farming products. While certain EHEC virulence mechanisms have been extensively studied, the factors that mediate host colonization are poorly defined. Previously, we identified four proteins (EhaA,B,C,D) from the prototypic EHEC strain EDL933 that belong to the autotransporter (AT) family. Here we characterize the EhaB AT protein. EhaB was shown to be located at the cell surface and overexpression in E. coli K-12 resulted in significant biofilm formation under continuous flow conditions. Overexpression of EhaB in E. coli K12 and EDL933 backgrounds also promoted adhesion to the extracellular matrix proteins collagen I and laminin. An EhaB-specific antibody revealed that EhaB is expressed in E. coli EDL933 following in vitro growth. EhaB also cross-reacted with serum IgA from cattle challenged with E. coli O157:H7, indicating that EhaB is expressed in vivo and elicits a host IgA immune response.

An investigation of the expression and adhesin function of H7 flagella in the interaction of Escherichia coli O157 : H7 with bovine intestinal epithelium.

Enterohaemorrhagic Escherichia coli O157 : H7 is a bacterial pathogen that can cause haemorrhagic colitis and haemolytic uremic syndrome. In the primary reservoir host, cattle, the terminal rectum is the principal site of E. coli O157 colonization. In this study, bovine terminal rectal primary epithelial cells were used to examine the role of H7 flagella in epithelial adherence. Binding of a fliC(H7) mutant O157 strain to rectal epithelium was significantly reduced as was binding of the flagellated wild-type strain following incubation with H7-specific antibodies. Complementation of fliC(H7) mutant O157 strain with fliC(H7) restored the adherence to wild-type levels; however, complementation with fliC(H6) did not restore it. High-resolution ultrastructural and imunofluorescence studies demonstrated the presence of abundant flagella forming physical contact points with the rectal epithelium. Binding to terminal rectal epithelium was specific to H7 by comparison with other flagellin types tested. In-cell Western assays confirmed temporal expression of flagella during O157 interaction with epithelium, early expression was suppressed during the later stages of microcolony and attaching and effacing lesion formation. H7 flagella are expressed in vivo by individual bacteria in contact with rectal mucosa. Our data demonstrate that the H7 flagellum acts as an adhesin to bovine intestinal epithelium and its involvement in this crucial initiating step for colonization indicates that H7 flagella could be an important target in intervention strategies.

Pathogenic potential of emergent sorbitol-fermenting Escherichia coli O157:NM.

Non-sorbitol-fermenting (NSF) Escherichia coli O157:H7 is the primary Shiga toxin-producing E. coli (STEC) serotype associated with human infection. Since 1988, sorbitol-fermenting (SF) STEC O157:NM strains have emerged and have been associated with a higher incidence of progression to hemolytic-uremic syndrome (HUS) than NSF STEC O157:H7. This study investigated bacterial factors that may account for the increased pathogenic potential of SF STEC O157:NM. While no evidence of toxin or toxin expression differences between the two O157 groups was found, the SF STEC O157:NM strains adhered at significantly higher levels to a human colonic cell line. Under the conditions tested, curli were shown to be the main factor responsible for the increased adherence to Caco-2 cells. Notably, 52 of 66 (79%) European SF STEC O157:NM strains tested bound Congo red at 37 degrees C and this correlated with curli expression. In a subset of strains, curli expression was due to increased expression from the csgBAC promoter that was not always a consequence of increased csgD expression. The capacity of SF STEC O157:NM strains to express curli at 37 degrees C may have relevance to the epidemiology of human infections as curliated strains could promote higher levels of colonization and inflammation in the human intestine. In turn, this could lead to increased toxin exposure and an increased likelihood of progression to HUS.

Escherichia coli O157:H7 colonization in cattle following systemic and mucosal immunization with purified H7 flagellin.

Escherichia coli O157:H7 is an important pathogen of humans. Cattle are most frequently identified as the primary source of infection, and therefore, reduction in E. coli O157:H7 prevalence in cattle by vaccination represents an attractive strategy for reducing the incidence of human disease. H7 flagella have been implicated in intestinal-epithelial colonization of E. coli O157:H7 and may represent a useful target for vaccination. In this study, calves were immunized either systemically with H7 flagellin by intramuscular injection or mucosally via the rectum with either H7 or H7 incorporated into poly(DL-lactide-co-glycolide) microparticles (PLG:H7). Systemic immunization resulted in high levels of flagellin-specific immunoglobulin G (IgG) and IgA in both serum and nasal secretions and detectable levels of both antibody isotypes in rectal secretions. Rectal administration of flagellin resulted in levels of rectal IgA similar to those by the intramuscular route but failed to induce any other antibody response, whereas rectal immunization with PLG:H7 failed to induce any H7-specific antibodies. Following subsequent oral challenge with E. coli O157:H7, reduced colonization rates and delayed peak bacterial shedding were observed in the intramuscularly immunized group compared to nonvaccinated calves, but no reduction in total bacterial shedding occurred. Rectal immunization with either H7 or PLG:H7 had no effect on subsequent bacterial colonization or shedding. Furthermore, purified H7-specific IgA and IgG from intramuscularly immunized calves were shown to reduce intestinal-epithelial binding in vitro. These results indicate that H7 flagellin may be a useful component in a systemic vaccine to reduce E. coli O157:H7 colonization in cattle.

Simple methods for measurement of bovine mucosal antibody responses in vivo.

The mucosal immune response serves as the first line of defence against many bacterial and viral diseases. Therefore, measurement of mucosal immune responses is important in evaluating mucosal immunisation protocols and understanding initial host/pathogen interactions. In this study we compare two methods for repeated sampling of bovine rectal mucosal secretions, namely rectal swabbing and rectal biopsies, and evaluate a simple swabbing method for sampling bovine nasal secretions. Both rectal swabs and rectal biopsies yielded similar quantities of total IgA (TIgA)/ml. However, rectal biopsies yielded five times more total IgG (TIgG)/ml than rectal swabs. Blood contamination was estimated to contribute approximately 7% of TIgG and <0.05% TIgA in rectal swab samples compared to 40% of TIgG and 4.5% of TIgA in rectal biopsy samples, indicating that rectal swabbing was more effective at sampling rectal mucosal secretions. Nasal swabs were effective at obtaining nasal secretion samples with only 1% of TIgG and <0.05% TIgA estimated to be blood derived. Furthermore, H7 flagellin-specific antibodies were detected in both nasal and rectal swab samples following either rectal immunisation with purified H7 flagellin or oral challenge with live E. coli O157:H7, indicating that both techniques are effective methods for monitoring mucosal antibody responses in cattle.

Verotoxin 1 binding to intestinal crypt epithelial cells results in localization to lysosomes and abrogation of toxicity.

Verotoxins (VTs) are important virulence factors of enterohaemorrhagic Escherichia coli (EHEC), a group of bacteria associated with severe disease sequelae in humans. The potent cytotoxic activity of VTs is important in pathogenicity, resulting in the death of cells expressing receptor Gb3 (globotriaosylceramide). EHEC, particularly serotype O157:H7, frequently colonize reservoir hosts (such as cattle) in the absence of disease, however, the basis to avirulence in this host has been unclear. The objective of this study was assessment of interaction between VT and intestinal epithelium, which represents the major interface between the host and enteric organisms. Bovine intestinal epithelial cells expressed Gb3 in vitro in primary cell cultures, localizing specifically to proliferating crypt cells in corroboration with in situ immunohistological observations on intestinal mucosa. Expression of receptor by these cells contrasts with the absence of Gb3 on human intestinal epithelium in vivo. Despite receptor expression, VT exhibited no cytotoxic activity against bovine epithelial cells. Sub-cellular localization of VT indicated that this toxin was excluded from endoplasmic reticulum but localized to lysosomes, corresponding with abrogation of cytotoxicity. VT intracellular trafficking was unaffected by treatment of primary cell cultures with methyl-beta-cyclodextrin, indicating that Gb3 in these cells is not associated with lipid rafts but is randomly distributed in the membrane. The combination of Gb3 isoform, membrane distribution and VT trafficking correlate with observations of other receptor-positive cells that resist verocytotoxicity. These studies demonstrate that intestinal epithelium is an important determinant in VT interaction with major implications for the differential consequences of EHEC infection in reservoir hosts and humans.