Toru Tobe

Bifidobacteria can protect from enteropathogenic infection through production of acetate

The human gut is colonized with a wide variety of microorganisms, including species, such as those belonging to the bacterial genus Bifidobacterium, that have beneficial effects on human physiology and pathology. Among the most distinctive benefits of bifidobacteria are modulation of host defence responses and protection against infectious diseases. Nevertheless, the molecular mechanisms underlying these effects have barely been elucidated. To investigate these mechanisms, we used mice associated with certain bifidobacterial strains and a simplified model of lethal infection with enterohaemorrhagic Escherichia coli O157:H7, together with an integrated ‘omics’ approach. Here we show that genes encoding an ATP-binding-cassette-type carbohydrate transporter present in certain bifidobacteria contribute to protecting mice against death induced by E. coli O157:H7. We found that this effect can be attributed, at least in part, to increased production of acetate and that translocation of the E. coli O157:H7 Shiga toxin from the gut lumen to the blood was inhibited. We propose that acetate produced by protective bifidobacteria improves intestinal defence mediated by epithelial cells and thereby protects the host against lethal infection.

Uptake through glycoprotein 2 of FimH + bacteria by M cells initiates mucosal immune response

The mucosal immune system forms the largest part of the entire immune system, containing about three-quarters of all lymphocytes and producing grams of secretory IgA daily to protect the mucosal surface from pathogens. To evoke the mucosal immune response, antigens on the mucosal surface must be transported across the epithelial barrier into organized lymphoid structures such as Peyer’s patches. This function, called antigen transcytosis, is mediated by specialized epithelial M cells. The molecular mechanisms promoting this antigen uptake, however, are largely unknown. Here we report that glycoprotein 2 (GP2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for mucosal antigens. Recombinant GP2 protein selectively bound a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane. Consistently, these bacteria were colocalized with endogenous GP2 on the apical plasma membrane as well as in cytoplasmic vesicles in M cells. Moreover, deficiency of bacterial FimH or host GP2 led to defects in transcytosis of type-I-piliated bacteria through M cells, resulting in an attenuation of antigen-specific immune responses in Peyer’s patches. GP2 is therefore a previously unrecognized transcytotic receptor on M cells for type-I-piliated bacteria and is a prerequisite for the mucosal immune response to these bacteria. Given that M cells are considered a promising target for oral vaccination against various infectious diseases, the GP2-dependent transcytotic pathway could provide a new target for the development of M-cell-targeted mucosal vaccines.

Comparative genomics reveal the mechanism of the parallel evolution of O157 and non-O157 enterohemorrhagic Escherichia coli

Among the various pathogenic Escherichia coli strains, enterohemorrhagic E. coli (EHEC) is the most devastating. Although serotype O157:H7 strains are the most prevalent, strains of different serotypes also possess similar pathogenic potential. Here, we present the results of a genomic comparison between EHECs of serotype O157, O26, O111, and O103, as well as 21 other, fully sequenced E. coli/Shigella strains. All EHECs have much larger genomes (5.5–5.9 Mb) than the other strains and contain surprisingly large numbers of prophages and integrative elements (IEs). The gene contents of the 4 EHECs do not follow the phylogenetic relationships of the strains, and they share virulence genes for Shiga toxins and many other factors. We found many lambdoid phages, IEs, and virulence plasmids that carry the same or similar virulence genes but have distinct evolutionary histories, indicating that independent acquisition of these mobile genetic elements has driven the evolution of each EHEC. Particularly interesting is the evolution of the type III secretion system (T3SS). We found that the T3SS of EHECs is composed of genes that were introduced by 3 different types of genetic elements: an IE referred to as the locus of enterocyte effacement, which encodes a central part of the T3SS; SpLE3-like IEs; and lambdoid phages carrying numerous T3SS effector genes and other T3SS-related genes. Our data demonstrate how E. coli strains of different phylogenies can independently evolve into EHECs, providing unique insights into the mechanisms underlying the parallel evolution of complex virulence systems in bacteria.

ppGpp with DksA controls gene expression in the locus of enterocyte effacement (LEE) pathogenicity island of enterohaemorrhagic Escherichia coli through activation of two virulence regulatory genes.

For a new pathogen to emerge, it must acquire both virulence genes and a system for responding to changes in environmental conditions. Starvation of nutrients or growth arrest induces the stringent response in Escherichia coli, via increased ppGpp. We found the adherence capacity of enterohaemorrhagic E.u2003coli (EHEC) and gene expression in the locus of enterocyte effacement (LEE) were enhanced by a downshift in nutrients or by entry into the stationary growth phase, both of which increase the ppGpp concentration. The activation was dependent on relA and spoT, which encode enzymes for the synthesis and degradation of ppGpp, and on dksA, which encodes an RNA polymerase accessory protein required for the stringent response. Upon induction of RelA expression, LEE gene transcription was activated within 20u2003min, even without starvation. The expression of two LEE transcriptional regulators, Ler and Pch, was activated by ppGpp and essential for the enhancement of LEE gene expression. In addition, the ler and pch promoters were directly activated by ppGpp in an in vitro transcription system. These findings suggest that the regulation of virulence genes in EHEC is integrated with E.u2003colis stringent response system, through the regulation of virulence regulatory genes.

NleC, a type III secretion protease, compromises NF-κB activation by targeting p65/RelA.

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-κBs 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.

Regulation of virulence by butyrate sensing in enterohaemorrhagic Escherichia coli.

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.

Extensive genomic diversity and selective conservation of virulence-determinants in enterohemorrhagic Escherichia coli strains of O157 and non-O157 serotypes

BackgroundEnterohemorrhagic 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.ResultsUsing 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.ConclusionAlthough 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.

Dual regulatory pathways integrating the RcsC–RcsD–RcsB signalling system control enterohaemorrhagic Escherichia coli pathogenicity

Bacterial pathogenesis is strictly regulated in response to changes in environmental conditions. A His–Asp phosphorelay system consisting of a sensor kinase and response regulator is used by Gram‐positive and Gram‐negative bacteria to control gene expression in response to environmental stimuli. We screened His–Asp phosphorelay systems for their effect on virulence expression in enterohaemorrhagic Escherichia coli (EHEC), and found rcsD or rcsB overexpression enhanced locus for enterocyte effacement (LEE) gene transcription and adherence to Caco‐2 cells through transcriptional activation of the ler regulatory gene. An EHEC‐specific regulator GrvA, encoded by ECs1274, was required for ler transcription activation by RcsB. Furthermore, GrvA activated ler transcription in E. coli K12. Stimulation of the RcsDCB regulatory system by RcsF overexpression slightly increased EspB expression in the wild type but not the ECs1274 mutant. However, EspB expression in an rcsB deletion mutant increased compared with wild type, suggesting that RcsB negatively regulates LEE gene expression and that active RcsB protein is present under normal growth conditions. Deletion of pchA, which encodes a positive regulator for ler, abolished the effect of the rcsB deletion, suggesting that pchA mediated the negative RcsB effect. pchA transcript levels decreased when RcsB expression increased. Thus, LEE gene transcription may be regulated by RcsB through two oppositely regulated O157‐specific regulators, PchA and GrvA.

Activation of Motility by Sensing Short-Chain Fatty Acids via Two Steps in a Flagellar Gene Regulatory Cascade in Enterohemorrhagic Escherichia coli

ABSTRACT 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 Dulbeccos modified Eagles 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.

Enteropathogenic Escherichia coli Uses NleA to Inhibit NLRP3 Inflammasome Activation.

Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) are related strains capable of inducing severe gastrointestinal disease. For optimal infection, these pathogens actively modulate cellular functions through the deployment of effector proteins in a type three secretion system (T3SS)-dependent manner. In response to enteric pathogen invasion, the Nod-like receptor pyrin domain containing (NLRP) inflammasome has been increasingly recognized as an important cytoplasmic sensor against microbial infection by activating caspase-1 and releasing IL-1β. EPEC and EHEC are known to elicit inflammasome activation in macrophages and epithelial cells; however, whether the pathogens actively counteract such innate immune responses is unknown. Using a series of compound effector-gene deletion strains of EPEC, we screened and identified NleA, which could subdue host IL-1β secretion. It was found that the reduction is not because of blocked NF-κB activity; instead, the reduction results from inhibited caspase-1 activation by NleA. Immunostaining of human macrophage-like cells following infection revealed limited formation of inflammasome foci with constituents of total caspase-1, ASC and NLRP3 in the presence of NleA. Pulldown of PMA-induced differentiated THP-1 lysate with purified MBP-NleA reveals that NLRP3 is a target of NleA. The interaction was verified by an immunoprecipitation assay and direct interaction assay in which purified MBP-NleA and GST-NLRP3 were used. We further showed that the effector interacts with regions of NLRP3 containing the PYD and LRR domains. Additionally, NleA was found to associate with non-ubiquitinated and ubiquitinated NLRP3 and to interrupt de-ubiquitination of NLRP3, which is a required process for inflammasome activation. Cumulatively, our findings provide the first example of EPEC-mediated suppression of inflammasome activity in which NieA plays a novel role in controlling the host immune response through targeting of NLRP3.