The mechanism by which methionine relieves the growth inhibition of Escherichia coli K-12 that is caused by organic weak acid food preservatives was investigated. In the presence of 8 mM acetate the specific growth rate of E. coli Frag1 (in MacIlvaine's minimal medium pH 6 0) is reduced by 50 %.

Macrophages in the healthy intestine are highly specialized and usually respond to the gut microbiota without provoking an inflammatory response. A breakdown in this tolerance leads to inflammatory bowel disease (IBD), but the mechanisms by which intestinal macrophages normally become conditioned to promote microbial tolerance are unclear. Strong epidemiological evidence linking disruption of the gut microbiota by antibiotic use early in life to IBD indicates an important role for the gut microbiota in modulating intestinal immunity. Here, we show that antibiotic use causes intestinal macrophages to become hyperresponsive to bacterial stimulation, producing excess inflammatory cytokines. Re-exposure of antibiotic-treated mice to conventional microbiota induced a long-term, macrophage-dependent increase in inflammatory T helper 1 (TH1) responses in the colon and sustained dysbiosis. The consequences of this dysregulated macrophage activity for T cell function were demonstrated by increased susceptibility to infections requiring TH17 and TH2 responses for clearance (bacterial Citrobacter rodentium and helminth Trichuris muris infections), corresponding with increased inflammation. Short-chain fatty acids (SCFAs) were depleted during antibiotic administration; supplementation of antibiotics with the SCFA butyrate restored the characteristic hyporesponsiveness of intestinal macrophages and prevented T cell dysfunction. Butyrate altered the metabolic behavior of macrophages to increase oxidative phosphorylation and also promoted alternative macrophage activation. In summary, the gut microbiota is essential to maintain macrophage-dependent intestinal immune homeostasis, mediated by SCFA-dependent pathways. Oral antibiotics disrupt this process to promote sustained T cell–mediated dysfunction and increased susceptibility to infections, highlighting important implications of repeated broad-spectrum antibiotic use.

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/β-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.

The sequence of two enterohaemorrhagic Escherichia coli (EHEC) O157:H7 strains reveals the possession of at least 16 fimbrial gene clusters, many of the chaperone/usher class. The first part of this study examined the distribution of these clusters in a selection of EHEC/EPEC (enteropathogenic E. coli) serotypes to determine if any were likely to be unique to E. coli O157:H7. Six of the clusters, as determined by the presence of amplified main subunit or usher gene sequences, were detected only in the E. coli O157 and O145 serotypes tested. With the exception of one serotype O103 strain that contained an lpf2 cluster, lpf sequences were only detected in E. coli O157 of the serotypes tested. Expression from each cluster was measured by the construction of chromosomally integrated lacZ promoter fusions and plasmid-based eGFP fusions in E. coli O157:H7. This analysis demonstrated that the majority (11/15) of main fimbrial subunit genes were not expressed under the majority of conditions tested in vitro. One of the clusters showing promoter activity, loc8, has a temperature expression optimum indicating a possible role outside the host. From the presence of pseudogenes in three of the clusters, the lack of FimH-like minor adhesins in the clusters and their limited expression in vitro, it would appear that E. coli O157:H7 has a limited repertoire of expressed functional fimbriae. This restricted selection of fimbriae may be important in bringing about the tropism E. coli O157:H7 demonstrates for the terminal rectum of cattle.

Type III secretion systems of enteric bacteria enable translocation of effector proteins into host cells. Secreted proteins of verotoxigenic Escherichia coli O157 strains include components of a translocation apparatus, EspA, -B, and -D, as well as "effectors" such as the translocated intimin receptor (Tir) and the mitochondrion-associated protein (Map). This research has investigated the regulation of LEE4 translocon proteins, in particular EspA. EspA filaments could not be detected on the bacterial cell surface when E. coli O157:H7 was cultured in M9 minimal medium but were expressed from only a proportion of the bacterial population when cultured in minimal essential medium modified with 25 mM HEPES. The highest proportions of EspA-filamented bacteria were detected in late exponential phase, after which filaments were lost rapidly from the bacterial cell surface. Our previous research had shown that human and bovine E. coli O157:H7 strains exhibit marked differences in EspD secretion levels. Here it is demonstrated that the proportion of the bacterial population expressing EspA filaments was associated with the level of EspD secretion. The ability of individual bacteria to express EspA filaments was not controlled at the level of LEE1-4 operon transcription, as demonstrated by using both ␤-galactosidase and green fluorescent protein (GFP) promoter fusions. All bacteria, whether expressing EspA filaments or not, showed equivalent levels of GFP expression when LEE1-4 translational fusions were used. Despite this, the LEE4-espADB mRNA was more abundant from populations with a high proportion of nonsecreting bacteria (low secretors) than from populations with a high proportion of secreting and therefore filamented bacteria (high secretors). This research demonstrates that while specific environmental conditions are required to induce LEE1-4 expression, a further checkpoint exists before EspA filaments are produced on the bacterial surface and secretion of effector proteins occurs. This checkpoint in E. coli O157:H7 translocon expression is controlled by a posttranscriptional mechanism acting on LEE4-espADB mRNA. The heterogeneity in EspA filamentation could arise from phase-variable expression of regulators that control this posttranscriptional mechanism.

Enterohaemorrhagic Escherichia coli O157 : H7 is a human pathogen that causes no apparent disease in cattle, its primary reservoir host. Recent research has demonstrated that E. coli O157 : H7 predominately colonizes the distal few centimetres of the bovine rectum, and in this study, the LEE4 operon encoding a type III secretion system translocon and associated proteins was shown to be essential for colonization. A deletion mutant of LEE4 failed to colonize cattle, in contrast to a co-inoculated strain containing a chromosomal complement of the operon, therefore fulfilling 'molecular' Koch's postulates for this virulence determinant. In addition, attaching and effacing (A/E) lesions were detectable in E. coli O157 : H7 microcolonies from the terminal rectum of both naturally and experimentally colonized cattle when examined by transmission electron microscopy. This study proves that type III secretion is required for colonization of cattle by E. coli O157 : H7, and that A/E lesion formation occurs at the bovine terminal rectum within E. coli O157 : H7 microcolonies. The research confirms the value of using type III secreted proteins as vaccine candidates in cattle. INTRODUCTIONEnterohaemorrhagic Escherichia coli (EHEC) has emerged in developed countries over the past 20 years as an important cause of human intestinal disease. In addition to bloody diarrhoea, intestinal infection can lead to potentially fatal systemic sequelae resulting from the activity of Shiga toxins. The majority of these infections in the USA, Canada, UK and Japan are caused by E. coli O157 : H7 (Nataro & Kaper, 1998). This serotype has been frequently isolated from cattle faeces, and most human E. coli O157 : H7 infections originate, either directly or indirectly, from this source (Besser et al., 1999;Borczyk et al., 1987). It is widely acknowledged that controlling E. coli O157 : H7 within the bovine population would be an effective method of reducing transmission to humans (Stevens et al., 2002).In common with other EHEC and EPEC (enteropathogenic E. coli), E. coli O157 : H7 contains a pathogenicity island, known as the locus of enterocyte effacement (LEE), that confers the attaching and effacing (A/E) phenotype McDaniel & Kaper, 1997). The LEE encodes a type III secretion system (TTSS) (Hueck, 1998), various translocators and effectors, the outer-membrane protein intimin (Jerse et al., 1990) and its receptor, termed Tir (translocated intimin receptor) (Kenny et al., 1997). The LEE is arranged into several polycistronic operons termed LEE1 to LEE5 (Elliott et al., 1998). The LEE4 operon encodes several proteins essential for the A/E phenotype. These include SepL (Kresse et al., 2000) and EscF (Wilson et al., 2001), both essential components of the LEE TTSS, and Esps (EPEC secreted proteins) A, B, D and F (Knutton et al., 1998;Taylor et al., 1998;Wachter et al., 1999; and the non-LEE-encoded Cif, EspI/NleA and TccP (Marches et al., 2003;Mundy et al., 2004;Gruenheid et al., 2004;Garmendia et al., 2004). Other than EspF, these are all likel...

A class of anti-virulence compounds, the salicylidene acylhydrazides, has been widely reported to block the function of the type three secretion system of several Gram-negative pathogens by a previously unknown mechanism. In this work we provide the first identification of bacterial proteins that are targeted by this group of compounds. We provide evidence that their mode of action is likely to result from a synergistic effect arising from a perturbation of the function of several conserved proteins. We also examine the contribution of selected target proteins to the pathogenicity of Yersinia pseudotuberculosis and to expression of virulence genes in Escherichia coli O157.

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.