The enteric pathogen, Salmonella enterica serovar Typhimurium, causes food poisoning resulting in gastroenteritis. The S. Typhimurium effector protein, SipA, promotes gastroenteritis by functional motifs that either trigger mechanisms of inflammation or bacterial entry. During infection of intestinal epithelial cells, SipA was found to be responsible for the early activation of caspase-3, an enzyme that is required for SipA cleavage at a specific recognition motif that divided the protein into its two functional domains, and activated SipA in a manner necessary for pathogenicity. Other caspase-3 cleavage sites identified in S. Typhimurium appeared to be restricted to secreted effector proteins, indicating this may be a general strategy employed by this pathogen for processing of its secreted effectors.

SummaryIn human intestinal disease induced by Salmonella enterica serotype Typhimurium (S. typhimurium) transepithelial migration of polymorphonuclear leukocytes (PMNs) rapidly follows attachment of the bacteria to the epithelial apical membrane. Previously, we have shown that the S. typhimurium effector protein, SipA, plays a pivotal role in signalling epithelial cell responses that lead to the transepithelial migration of PMNs. Thus, the objective of this study was to determine the functional domain of SipA that regulates this signalling event. SipA was divided into two fragments: the SipAb C-terminal fragment (259 AA), which binds actin, and the SipAa fragment 2-425 (424 AA), which a role has yet to be described. In both in vitro and in vivo models of S. typhimuriuminduced intestinal inflammation the SipAa fragment exhibited a profound ability to induce PMN transmigration, whereas the SipAb actin-binding domain failed to induce PMN transmigration. Subsequent mapping of the SipAa domain identified a 131-amino-acid region (SipAa3 ) responsible for modulating PMN transepithelial migration. Interestingly, neither intracellular translocation nor actin association of SipA was necessary for its ability to induce PMN transepithelial migration. As these results indicate SipA has at least two separate functional domains, we speculate that during infection S. typhimurium requires delivery of SipA to both extracellular and intracellular spaces to maximize pro-inflammatory responses and mechanisms of bacterial invasion.

Background The predominance of specific bacteria such as adherent-invasive Escherichia coli (AIEC) within the Crohn's disease (CD) intestine remains poorly understood with little evidence uncovered to support a selective pressure underlying their presence. Intestinal ethanolamine is however readily accessible during periods of intestinal inflammation, and enables pathogens to outcompete the host microbiota under such circumstances. Methods Quantitative RT-PCR (qRT-PCR) to determine expression of genes central to ethanolamine metabolism; transmission electron microscopy to detect presence of bacterial microcompartments (MCPs); in vitro infections of both murine and human macrophage cell lines examining intracellular replication of the AIEC-type strain LF82 and clinical E. coli isolates in the presence of ethanolamine; determination of E. coli ethanolamine utilization ( eut ) operon transcription in faecal samples from healthy patients, patients with active CD and the same patients in remission following treatment. Results Growth on the intestinal short chain fatty acid propionic acid (PA) stimulates significantly increased transcription of the eut operon (fold change relative to glucose: >16.9; p -value <.01). Additionally ethanolamine was accessible to intra-macrophage AIEC and stimulated significant increases in growth intracellularly when it was added extracellularly at concentrations comparable to those in the human intestine. Finally, qRT-PCR indicated that expression of the E. coli eut operon was increased in children with active CD compared to healthy controls (fold change increase: >4.72; P < .02). After clinical remission post-exclusive enteral nutrition treatment, the same CD patients exhibited significantly reduced eut expression (Pre vs Post fold change decrease: >15.64; P < .01). Interpretation Our data indicates a role for ethanolamine metabolism in selecting for AIEC that are consistently overrepresented in the CD intestine. The increased E. coli metabolism of ethanolamine seen in the intestine during active CD, and its decrease during remission, indicates ethanolamine use may be a key factor in shaping the intestinal microbiome in CD patients, particularly during times of inflammation. Fund This work was funded by Biotechnology and Biological Sciences Research Council (BBSRC) grants BB/K008005/1 & BB/P003281/1 to DMW; by a Tenovus Scotland grant to MJO; by Glasgow Children's Hospital Charity, Nestle Health Sciences, Engineering and Physical Sciences Research Council (EPSRC) and Catherine McEwan Foundation grants awarded to ...

Alterations to the gut microbiome are associated with various neurological diseases, yet evidence of causality and identity of microbiome-derived compounds that mediate gut-brain axis interaction remain elusive. Here, we identify two previously unknown bacterial metabolites 3-methyl-4-(trimethylammonio)butanoate and 4-(trimethylammonio)pentanoate, structural analogs of carnitine that are present in both gut and brain of specific pathogen–free mice but absent in germ-free mice. We demonstrate that these compounds are produced by anaerobic commensal bacteria from the family Lachnospiraceae (Clostridiales) family, colocalize with carnitine in brain white matter, and inhibit carnitine-mediated fatty acid oxidation in a murine cell culture model of central nervous system white matter. This is the first description of direct molecular inter-kingdom exchange between gut prokaryotes and mammalian brain cells, leading to inhibition of brain cell function.

Highlights d PA induces virulence-associated changes in CD-associated AIEC d PA-induced phenotype is reproducible in recently isolated clinical strains d Phenotypic changes are transcriptional in nature and reversible d Strains exposed to PA outcompete wild-type strains in a ''humanized'' murine model

Rhodococcus equi is an important pathogen of foals, causing severe pyogranulomatous pneumonia. Virulent R. equi strains grow within macrophages, a process which remains poorly characterized. A potential source of carbon for intramacrophage R. equi is membrane lipid-derived fatty acids, which following ␤ oxidation are assimilated via the glyoxylate bypass. To assess the importance of isocitrate lyase, the first enzyme of the glyoxylate bypass, in virulence of a foal isolate of R. equi, a mutant was constructed by a strategy of single homologous recombination using a suicide plasmid containing an internal fragment of the R. equi aceA gene encoding isocitrate lyase. Complementation of the resulting mutant with aceA showed that the mutant was specific for this gene. Assessment of virulence in a mouse macrophage cell line showed that the mutant was killed, in contrast to the parent strain. Studies in the liver of intravenously infected mice showed enhanced clearance of the mutant. When four 3-week-old foals were infected intrabronchially, the aceA mutant was completely attenuated, in contrast to the parent strain. In conclusion, the aceA gene was shown to be essential for virulence of R. equi, suggesting that membrane lipids may be an important source of carbon for phagocytosed R. equi.

Studies over the last decade have shown that Salmonella enterica serovar Typhimurium (S. typhimurium) is able to preferentially locate to sites of tumor growth and modulate (shrink) the growth of many cancers. Given this unique association between S. typhimurium and cancer cells, the objective of this study was to investigate the capacity of this microorganism to modulate the plasma membrane multidrug resistance (MDR) protein P-glycoprotein (P-gp), an ATP-binding cassette transporter responsible for effluxing many cancer drugs. Using an in vitro model of S. typhimurium infection of polarized human cancer intestinal cell lines, we have found that this enteric pathogen functionally downregulates the efflux capabilities of P-gp. Specifically, we show that S. typhimurium infection of human intestinal cancer cells results in the enhanced intracellular accumulation of a number of P-gp substrates that corresponds to the posttranscriptional downregulation of P-gp expression. Furthermore, cells expressing small interfering RNAs against MDR1, the gene encoding P-gp, were significantly more susceptible to the cytotoxic effects of bacterial infection. This result is consistent with our observation that S. typhimurium was significantly less able to invade cells overexpressing MDR1. Taken together, these results reveal a novel role for P-gp in the maintenance of homeostasis in the gastrointestinal tract in regard to bacterial infection. Thus the regulation of P-gp by S. typhimurium has important implications not only for the development of new cancer therapeutics aimed at reversing drug resistance but also in the understanding of how microbes have evolved diverse strategies to interact with their host.

Isocitrate lyase is the first enzyme of the glyoxylate shunt which is required for the assimilation of fatty acids and acetate. The intracellular pathogen Rhodococcus equi contains high activities of this enzyme following growth on acetate and lactate, indicating that it plays an important role in the metabolism of these substrates. The gene encoding isocitrate lyase (aceA) was cloned and sequenced. It specifies a 46 846 Da protein, which was shown to be functional by expressing it in Escherichia coli. A gene similar to fadB, encoding 3-hydroxyacyl-CoA dehydrogenase, was located 90 bp downstream from aceA. Northern hybridization and RT-PCR experiments showed that aceA and fadB are cotranscribed into a 28 kb transcript. A smaller 16 kb aceA transcript was also observed which was 25-fold more abundant than the aceA-fadB transcript. It is proposed that a stable hairpin structure with a free energy (∆G) of N285 kcal mol N1 and located in the 90 bp aceA-fadB intergenic region is involved in stabilizing the aceA transcript.