Toru Tobe

The regulated expression of virulence genes is critical for successful infection by an intestinal pathogen. Bacteria rely on sensing environmental signals to find preferable niches and reach the infectious state. Orally ingested enterohemorrhagic Escherichia coli (EHEC) travels through the gastrointestinal tract and encounters a variety of environmental factors, some of which act as triggering signals for the induction of virulence genes. Butyrate, one of the main short-chain fatty acids (SCFAs), is such a signal, enhancing the expression of genes for intimate attachment and type III secretion. We further explored the role of SCFAs and found a positive effect of SCFAs on flagellar expression. Although EHEC did not produce flagella when grown in Dulbecco's modified Eagle's medium (DMEM), a tissue culture medium that enhances virulence gene expression, the addition of SCFAs to the medium induced the production of flagella, and the EHEC bacteria became motile. Among SCFAs, butyrate simultaneously activates both virulence and flagellar genes. Flagella did not affect initial adherence, and they were not expressed in adherent bacteria during microcolony formation. SCFAs activated flagellar genes via two regulatory steps. Butyrate activated the flhDC regulatory genes through leucine-responsive regulatory protein (Lrp), which is also a regulator of virulence genes. However, butyrate, acetate, and propionate also activated downstream genes independently of flhDC activation. Consequently, when encountering increased concentrations of SCFAs, which are abundant in acetate, in the intestine, EHEC first activates flagellar production and motility, followed by genes involved in adherence and type III secretion, which leads to efficient adherence in a preferable niche.

The NF-κB signaling pathway is central to the innate and adaptive immune responses. Upon their detection of pathogen-associated molecular patterns, Toll-like receptors on the cell surface initiate signal transduction and activate the NF-κB pathway, leading to the production of a wide array of inflammatory cytokines, in attempt to eradicate the invaders. As a countermeasure, pathogens have evolved ways to subvert and manipulate this system to their advantage. Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) are closely related bacteria responsible for major food-borne diseases worldwide. Via a needle-like protein complex called the type three secretion system (T3SS), these pathogens deliver virulence factors directly to host cells and modify cellular functions, including by suppressing the inflammatory response. Using gain- and loss-of-function screenings, we identified two bacterial effectors, NleC and NleE, that down-regulate the NF-κB signal upon being injected into a host cell via the T3SS. A recent report showed that NleE inhibits NF-κB activation, although an NleE-deficient pathogen was still immune-suppressive, indicating that other anti-inflammatory effectors are involved. In agreement, our present results showed that NleC was also required to inhibit inflammation. We found that NleC is a zinc protease that disrupts NF-κB activation by the direct cleavage of NF-κB's p65 subunit in the cytoplasm, thereby decreasing the available p65 and reducing the total nuclear entry of active p65. More importantly, we showed that a mutant EPEC/EHEC lacking both NleC and NleE (ΔnleC ΔnleE) caused greater inflammatory response than bacteria carrying ΔnleC or ΔnleE alone. This effect was similar to that of a T3SS-defective mutant. In conclusion, we found that NleC is an anti-inflammatory bacterial zinc protease, and that the cooperative function of NleE and NleC disrupts the NF-κB pathway and accounts for most of the immune suppression caused by EHEC/EPEC. Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) cause food-borne diseases, including watery diarrhea or severe bloody diarrhea and life-threatening kidney disease (hemolytic uremic syndrome). Upon ingestion, EPEC/EHEC colonize the cells of the epithelial lining in the intestinal tract. In response, the affected cells initiate an immune response by secreting cytokines that attract immune cells. To prevent their early elimination by the host, these bacteria have developed strategies to prevent the host immune response. They do this by injecting bacterial effectors into the host cells to disrupt the NF-κB pathway, an essential effector of the host cell immune response. In the current study, we report the discovery of an NF-κB suppressive effector in EPEC/EHEC called NleC, and its novel mechanism. We found that NleC is a zinc protease that can digest p65, a critical component of the NF-κB pathway, thus dampening the host inflammatory response. NleE is another recently identified anti-inflammatory effector. We show here that an EPEC/EHEC mutant deficient in both NleC and NleE loses most of its ability to suppress the host inflammatory response. Our findings show how two different bacterial effectors can function in cooperation to modify the host immune response.

Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic Escherichia coli (EPEC) are attaching/effacing pathogens that possess a type III secretion system and deliver a variety of effectors into host cells for successful infection. EHEC produces at least 20 effector families with various functions. Reorganization of the cellular cytoskeleton at the adherent site is a hallmark of these pathogens. EspL2 of EHEC is a novel effector class that can modulate the cellular cytoskeleton. By interacting with and activating Annexin A2, EspL2 contributes to the formation of a condensed microcolony and may adhere to host cells in a translocated intimin receptor-independent manner. The interaction of EspL2 with Annexin A2 increases F-actin bundling activity and strengthens the membrane-cytoskeleton linkage, resulting in the condensation of actin fibers and the induction of a pseudopod-like structure. Membrane microdomains, namely the lipid raft, which is rich in Annexin A2, may be a platform by which EHEC/EPEC infection modulates cellular signaling and the cytoskeleton.

Enterohaemorrhagic Escherichia coli (EHEC) colonizes and proliferates at the mucosal surface, inducing severe diarrhoea. Short-chain fatty acids (SCFAs) are abundant in the intestine owing to the metabolic activity of microflora, and are important for colonic health. We found that, although a high concentration of SCFAs inhibited the growth of EHEC, at low concentrations, the SCFAs markedly enhanced the expression of the virulence genes required for cell adherence and the induction of attaching and effacing (A/E) lesions. Of the SCFAs tested, butyrate markedly enhanced the expression of these virulence-associated genes, even at the low concentration of 1.25 mM, but acetate and propionate showed only a small effect at concentrations higher than 40 mM. Butyrate enhanced the promoter activity of the LEE1 operon, which encodes a global regulator of the LEE genes, Ler. This enhancement was dependent on a regulator, PchA. Butyrate sensing was completely abrogated by the deletion of lrp, the gene for the leucine-responsive regulatory protein, Lrp. Expression of a constitutively active mutant of Lrp enhanced the expression of the LEE genes in the absence of butyrate, and a response-defective Lrp derivative reduced the response to butyrate. Thus, upon entering the distal ileum, EHEC may respond to the higher butyrate level via Lrp by increasing its virulence expression, leading to efficient colonization of the target niche.

Enterohemorrhagic Escherichia coli (EHEC) delivers virulence factors into host cells through the type III secretion system (T3SS) to exert the bacterial pathogenicity. EHEC encodes more than 20 type III secretion system‐delivered families of effectors that have different functions at different infectious stages and enable a successful infection. One of them, EspL2, is encoded on the SpLE3 phage‐like element in EHEC O157:H7 Sakai and is well conserved among various EHEC strains. Here we show that, after delivery into host cells, EspL2 accumulated under adherent bacteria, as did polymerized F‐actin. EspL2‐expressing EHEC formed three‐dimensional, condensed microcolonies, into which the host cell extended plasma membrane protrusions on an F‐actin‐rich cytoskeleton. EspL2 bound F‐actin‐aggregating annexin 2 directly, increasing its activity. In addition, annexin 2 depletion abolished the EspL2‐dependent formation of condensed microcolonies and F‐actin aggregation. The EspL2‐induced pseudopod‐like protrusion of the host plasma membrane interacted with and supported colonization by the bacteria, independent of Tir‐mediated actin polymerization. Thus, EspL2 supports efficient colonization by increasing annexin 2's ability to aggregate Tir‐induced F‐actin and by modifying the morphology of the host cell membrane.

Enterohemorrhagic Escherichia coli is an emerging pathogen that causes diarrhea and hemolytic uremic syndrome. Much of the genomic information that affects virulence is acquired by horizontal transfer. Genes necessary for attaching and effacing lesions are located in the locus for enterocyte effacement (LEE) pathogenicity island. LEE gene transcription is positively regulated by Ler, which is also encoded by the LEE, and by Pch regulators, which are encoded at other loci. Here we identified genes whose transcription profiles were similar to those of the LEE genes, by comparing the effects of altering ler and pch transcript levels. We assigned these genes into two classes, according to their transcription profiles. By determining the binding profiles for Ler and Pch, we showed that both were involved in regulating one class of genes, but only Pch was involved in regulating the other class. Binding sites were found in the coding region as well as the promoter region of regulated genes, which include genes common to K12 strains as well as 0157-specific genes, suggesting that both act as a global regulator. These results indicate that Ler and Pch orchestrate the transcription of virulence genes, which are captured by horizontal transfer and scattered throughout the chromosome.

Two-component systems (TCSs) are well conserved among E. coli strains, including pathogenic E. coli and also closely related Shigella spp. Although 25% of the genome of pathogenic E. coli is strain-specific, only small number of strain-specific TCSs is found. Regulation of virulence genes in response to environmental stimuli is partly dependett on TCSs commonly present in nonpathogenic E. coli strains. Some virulence genes are directly regulated by response regulator of TCS but some are affected at posttranscriptional steps of production or assembly of macromolecule by TCS-induced products. In the process of acquiring virulence traits, regulatory systems for virulence genes expression seem to be built by integrating E. coli backbone TCSs with the virulence regulatory network via transcription regulatory gene.

Background: Enterohemorrhagic Escherichia coli (EHEC) O157 causes severe food-borne illness in humans. The chromosome of O157 consists of 4.1 Mb backbone sequences shared by benign E. coli K-12, and 1.4 Mb O157-specific sequences encoding many virulence determinants, such as Shiga toxin genes (stx genes) and the locus of enterocyte effacement (LEE). Non-O157 EHECs belonging to distinct clonal lineages from O157 also cause similar illness in humans. According to the 'parallel' evolution model, they have independently acquired the major virulence determinants, the stx genes and LEE. However, the genomic differences between O157 and non-O157 EHECs have not yet been systematically analyzed. Results: Using microarray and whole genome PCR scanning analyses, we performed a whole genome comparison of 20 EHEC strains of O26, O111, and O103 serotypes with O157. In non-O157 EHEC strains, although genome sizes were similar with or rather larger than O157 and the backbone regions were well conserved, O157-specific regions were very poorly conserved. Around only 20% of the O157-specific genes were fully conserved in each non-O157 serotype. However, the non-O157 EHECs contained a significant number of virulence genes that are found on prophages and plasmids in O157, and also multiple prophages similar to, but significantly divergent from, those in O157. Conclusion: Although O157 and non-O157 EHECs have independently acquired a huge amount of serotype- or strain-specific genes by lateral gene transfer, they share an unexpectedly large number of virulence genes. Independent infections of similar but distinct bacteriophages carrying these virulence determinants are deeply involved in the evolution of O157 and non-O157 EHECs.

Enterohaemorrhagic Escherichia coli (EHEC) is a gastrointestinal pathogen that causes diarrhoea and more severe diseases in humans. A key feature of EHEC is the type III secretion system (TTSS), which translocates virulence factors (effectors) directly into host cells. In this study, the expression and secretion of effectors in EHEC grown under anaerobic conditions were examined. The secretion of effectors was greatly enhanced, without an increase in their expression levels, when EHEC was grown in the presence of specific electron acceptors, such as trimethylamine N-oxide (TMAO) and nitrate, for anaerobic respiration. The activation of the TTSS was dependent on the activity of respiratory systems, including electron-acceptor-specific signalling systems and reductases. Although de novo protein synthesis was not required for TTSS activation, the inhibition of respiratory activity abolished secretion. EHEC grown with either TMAO or nitrate possessed a more intact type III secretion (TTS) apparatus, including the needle protein EscF and the translocator protein EspA, than EHEC grown without an electron acceptor. These observations suggest that activation of either the TMAO- or the nitrate-specific respiratory system accelerates the maturation of functional TTS apparatus under anaerobic growth conditions.

Several pathogenic strains of Escherichia coli exploit type III secretion to inject “effector proteins” into human cells, which then subvert eukaryotic cell biology to the bacterium's advantage. We have exploited bioinformatics and experimental approaches to establish that the effector repertoire in the Sakai strain of enterohemorrhagic E. coli (EHEC) O157:H7 is much larger than previously thought. Homology searches led to the identification of >60 putative effector genes. Thirteen of these were judged to be likely pseudogenes, whereas 49 were judged to be potentially functional. In total, 39 proteins were confirmed experimentally as effectors: 31 through proteomics and 28 through translocation assays. At the protein level, the EHEC effector sequences fall into >20 families. The largest family, the NleG family, contains 14 members in the Sakai strain alone. EHEC also harbors functional homologs of effectors from plant pathogens (HopPtoH, HopW, AvrA) and from Shigella (OspD, OspE, OspG), and two additional members of the Map/IpgB family. Genes encoding proven or predicted effectors occur in >20 exchangeable effector loci scattered throughout the chromosome. Crucially, the majority of functional effector genes are encoded by nine exchangeable effector loci that lie within lambdoid prophages. Thus, type III secretion in E. coli is linked to a vast phage “metagenome,” acting as a crucible for the evolution of pathogenicity.