From ingestion to colonization: the influence of the host environment on regulation of the LEE encoded type III secretion system in enterohaemorrhagic Escherichia coli

Enterohaemorrhagic Escherichia coli (EHEC) binds to host tissue and intimately attaches to intestinal cells using a dedicated type III secretion system (T3SS). This complex multi-protein organelle is encoded within a large pathogenicity island called the locus of enterocyte effacement (LEE), which is subject to extensive regulatory control. Over the past 15 years we have gained a wealth of knowledge concerning how the LEE is regulated transcriptionally by specific, global and phage encoded regulators. More recently, significant advances have been made in our understanding of how specific signals, including host or microbiota derived metabolic products and various nutrient sources, can affect how the LEE-encoded T3SS is regulated. In this review we discuss regulation of the LEE, focusing on how these physiologically relevant signals are sensed and how they affect the expression of this major virulence factor. The implications for understanding the disease process by specific regulatory mechanisms are also discussed

Subversion of actin dynamics by EspM effectors of attaching and effacing bacterial pathogens

Rho GTPases are common targets of bacterial toxins and type III secretion system effectors. IpgB1 and IpgB2 of Shigella and Map of enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli were recently grouped together on the basis that they share a conserved WxxxE motif. In this study, we characterized six WxxxE effectors from attaching and effacing pathogens: TrcA and EspM1 of EPEC strain B171, EspM1 and EspM2 of EHEC strain Sakai and EspM2 and EspM3 of Citrobacter rodentium. We show that EspM2 triggers formation of global parallel stress fibres, TrcA and EspM1 induce formation of localized parallel stress fibres and EspM3 triggers formation of localized radial stress fibres. Using EspM2 and EspM3 as model effectors, we report that while substituting the conserved Trp with Ala abolished activity, conservative Trp to Tyr or Glu to Asp substitutions did not affect stress-fibre formation. We show, using dominant negative constructs and chemical inhibitors, that the activity of EspM2 and EspM3 is RhoA and ROCK-dependent. Using Rhotekin pull-downs, we have shown that EspM2 and EspM3 activate RhoA; translocation of EspM2 and EspM3 triggered phosphorylation of cofilin. These results suggest that the EspM effectors modulate actin dynamics by activating the RhoA signalling pathway

Differences in the fecal microbiota of dairy calves reared with differing sources of milk and levels of maternal contact

The practice of rearing cows and calves together is gaining popularity on dairy farms, with different systems currently under assessment in mainland Europe, the United Kingdom, and Oceania. Research into the effects of cow–calf rearing has primarily focused on direct health and welfare implications, and little work has examined the role of different rearing paradigms on calf microbiota. We trialed a cow–calf rearing system on a Canadian dairy farm and compared fecal microbiota of these calves with the microbiota of calves reared according to the conventional practice of the same farm (separated from the dam and fed waste milk). At 4 wk, the conventionally reared calves had reduced relative abundance of Lactobacillus and higher relative abundance of other taxa, including Sutterella, Prevotella, and Bacteroides. We also detected predicted functional differences, such as reduced l-tryptophan biosynthesis in conventionally reared calves. These results suggest that maternal contact may influence the calf microbiota, but the observed differences are also likely related to other aspects of the rearing environment independent of maternal contact (e.g., potential exposure to antibiotic residues in waste milk). These findings provide preliminary evidence of the effects of early rearing environments on the establishment of the dairy calf fecal microbiota. This research is needed, given the critical role of the bovine gut microbiome in behavioral, metabolic, and immune development

Master Sculptor at Work: Enteropathogenic Escherichia coli Infection Uniquely Modifies Mitochondrial Proteolysis during Its Control of Human Cell Death

Enteropathogenic Escherichia coli (EPEC) causes severe diarrheal disease and is present globally. EPEC virulence requires a bacterial type III secretion system to inject 20 effector proteins into human intestinal cells. Three effectors travel to mitochondria and modulate apoptosis; however, the mechanisms by which effectors control apoptosis from within mitochondria are unknown. To identify and quantify global changes in mitochondrial proteolysis during infection, we applied the mitochondrial terminal proteomics technique mitochondrial stable isotope labeling by amino acids in cell culture-terminal amine isotopic labeling of substrates (MS-TAILS). MS-TAILS identified 1,695 amino N-terminal peptides from 1,060 unique proteins and 390 N-terminal peptides from 215 mitochondrial proteins at a false discovery rate of 0.01. Infection modified 230 cellular and 40 mitochondrial proteins, generating 27 cleaved mitochondrial neo-N termini, demonstrating altered proteolytic processing within mitochondria. To distinguish proteolytic events specific to EPEC from those of canonical apoptosis, we compared mitochondrial changes during infection with those reported from chemically induced apoptosis. During infection, fewer than half of all mitochondrial cleavages were previously described for canonical apoptosis, and we identified nine mitochondrial proteolytic sites not previously reported, including several in proteins with an annotated role in apoptosis, although none occurred at canonical Asp-Glu-Val-Asp (DEVD) sites associated with caspase cleavage. The identification and quantification of novel neo-N termini evidences the involvement of noncaspase human or EPEC protease(s) resulting from mitochondrialtargeting effectors that modulate cell death upon infection. All proteomics data are available via ProteomeXchange with identifier PXD016994. IMPORTANCE To our knowledge, this is the first study of the mitochondrial proteome or N-terminome during bacterial infection. Identified cleavage sites that had not been previously reported in the mitochondrial N-terminome and that were not generated in canonical apoptosis revealed a pathogen-specific strategy to control human cell apoptosis. These data inform new mechanisms of virulence factors targeting mitochondria and apoptosis during infection and highlight how enteropathogenic Escherichia coli (EPEC) manipulates human cell death pathways during infection, including candidate substrates of an EPEC protease within mitochondria. This understanding informs the development of new antivirulence strategies against the many human pathogens that targe

Towards standards for human fecal sample processing in metagenomic studies

Technical variation in metagenomic analysis must be minimized to confidently assess the contributions of microbiota to human health. Here we tested 21 representative DNA extraction protocols on the same fecal samples and quantified differences in observed microbial community composition. We compared them with differences due to library preparation and sample storage, which we contrasted with observed biological variation within the same specimen or within an individual over time. We found that DNA extraction had the largest effect on the outcome of metagenomic analysis. To rank DNA extraction protocols, we considered resulting DNA quantity and quality, and we ascertained biases in estimates of community diversity and the ratio between Gram-positive and Gram-negative bacteria. We recommend a standardized DNA extraction method for human fecal samples, for which transferability across labs was established and which was further benchmarked using a mock community of known composition. Its adoption will improve comparability of human gut microbiome studies and facilitate meta-analyses

Size Doesn't Matter: Towards a More Inclusive Philosophy of Biology

notes: As the primary author, O’Malley drafted the paper, and gathered and analysed data (scientific papers and talks). Conceptual analysis was conducted by both authors.publication-status: Publishedtypes: ArticlePhilosophers of biology, along with everyone else, generally perceive life to fall into two broad categories, the microbes and macrobes, and then pay most of their attention to the latter. ‘Macrobe’ is the word we propose for larger life forms, and we use it as part of an argument for microbial equality. We suggest that taking more notice of microbes – the dominant life form on the planet, both now and throughout evolutionary history – will transform some of the philosophy of biology’s standard ideas on ontology, evolution, taxonomy and biodiversity. We set out a number of recent developments in microbiology – including biofilm formation, chemotaxis, quorum sensing and gene transfer – that highlight microbial capacities for cooperation and communication and break down conventional thinking that microbes are solely or primarily single-celled organisms. These insights also bring new perspectives to the levels of selection debate, as well as to discussions of the evolution and nature of multicellularity, and to neo-Darwinian understandings of evolutionary mechanisms. We show how these revisions lead to further complications for microbial classification and the philosophies of systematics and biodiversity. Incorporating microbial insights into the philosophy of biology will challenge many of its assumptions, but also give greater scope and depth to its investigations

Microbial Pathogenesis: New Niches for Salmonella

AbstractSalmonella occupies a vacuolar compartment inside cells of its host. Recent studies have shown that the fate of this vacuole is different in various cell types, and that the outcome of colonization is determined by both the infecting bacterium and defense mechanisms of the host cell

Identification of a Salmonella virulence gene required for formation of filamentous structures containing lysosomal membrane glycoproteins within epithelial cells

10.1111/j.1365-2958.1996.tb02497.xMolecular Microbiology201151-164MOMI