Characterisation of the effects of salicylidene acylhydrazide compounds on type three secretion in Escherichia coli O157:H7

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 <i>Escherichia coli</i> O157:H7 in liquid culture and when the bacteria are attached to bovine epithelial cells. The inhibition of T3SS expression resulted in a reduction in the capacity of the bacteria to form attaching and effacing lesions. Our results show a marked variation in the ability of four structurally-related compounds to inhibit the T3SS of a panel of isolates. Using transcriptomics, we provide a comprehensive analysis of the conserved- and inhibitor-specific transcriptional responses to the 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 between the compounds. Overall, we highlight the importance of assessing the effect of such "anti-virulence" agents on a range of isolates and discuss the possible mechanisms which may lead to the co-ordinate down-regulation of horizontally acquired virulence genes

Pathogenic Potential to Humans of Bovine Escherichia coli O26, Scotland

Escherichia coli O26 and O157 have similar overall prevalences in cattle in Scotland, but in humans, Shiga toxin–producing E. coli O26 infections are fewer and clinically less severe than E. coli O157 infections. To investigate this discrepancy, we genotyped E. coli O26 isolates from cattle and humans in Scotland and continental Europe. The genetic background of some strains from Scotland was closely related to that of strains causing severe infections in Europe. Nonmetric multidimensional scaling found an association between hemolytic uremic syndrome (HUS) and multilocus sequence type 21 strains and confirmed the role of stx<sub>2</sub> in severe human disease. Although the prevalences of E. coli O26 and O157 on cattle farms in Scotland are equivalent, prevalence of more virulent strains is low, reducing human infection risk. However, new data on E. coli O26–associated HUS in humans highlight the need for surveillance of non-O157 enterohemorrhagic E. coli and for understanding stx<sub>2</sub> phage acquisition

Cell wall assembly in gram positive bacteria

SIGLEAvailable from British Library Document Supply Centre-DSC:DXN026619 / BLDSC - British Library Document Supply CentreGBUnited Kingdo

Regulatory interplay between pap operons in uropathogenic Escherichia coli

Pathogenic bacteria have a large repertoire of surface organelles involved in adherence, motility and protein export, but how individual bacteria co-ordinate surface organelle expression to prevent interference and excessive immune stimulation is unclear. Phase variation is a mechanism by which expression of surface factors is limited to a fraction of the bacterial population; however, the presence of multiple homologous surface structures controlled by related mechanisms and regulators antagonizes the independent expression achieved by phase variation. To investigate whether other mechanisms have evolved to sort out the bacterial cell surface, we examined regulatory cross-talk between multiple phase-variable pyelonephritis-associated pili (pap) operons in Escherichia coli isolates associated with urinary tract infections. Allelic variation identified in the regulatory regions and regulators acts synergistically to limit coexpression of homologous fimbrial operons. In particular, there is evidence that papI is under positive selection and PapI variants displayed differences in their capacity to activate related pap operons. Alleles of the high-affinity binding site for PapB were shown to contain a variable number of (T/A)(3) repeats occurring every 9 bp that altered the sensitivity of pap operon activation. Taken together with other examples of surface organelle cross-talk, we illustrate how this regulation could promote sequential expression

Bovine intelectins: cDNA sequencing and expression in the bovine intestine

Intelectins (Itlns) are lectins with potential roles in innate immunity, capable of binding bacteria via galactofuranose residues. Itlns also function as intestinal receptors for the antimicrobial glycoprotein lactoferrin (Lf). Since Lf binds strongly to enterohemorrhagic Escherichia coli O157:H7 (EHEC), we aimed to determine the expression of Lf receptor in terminal rectum, the site of predilection of EHEC in cattle. We sequenced two bovine intelectins (Itln1 and Itln2) and showed that both were expressed in abomasum and rectum, but expression appeared minimal in the jejunum. There was significantly higher expression of Itln2 in terminal rather than proximal rectum. Lactoferrin was expressed in all samples examined. Thus, we have demonstrated two novel bovine Itlns and shown that they are expressed along with Lf in the gastrointestinal tract, where they may interact with microbial pathogens

Controlling injection: regulation of type III secretion in enterohaemorrhagic <i>Escherichia coli</i>

Type III secretion (T3S) systems enable the injection of bacterial proteins through membrane barriers into host cells, either from outside the host cell or from within a vacuole. This system is required for colonization of their ruminant reservoir hosts by enterohaemorrhagic &lt;i&gt;Escherichia coli&lt;/i&gt; (EHEC) and might also be important for the etiology of disease in the incidental human host. T3S systems of &lt;i&gt;E. coli&lt;/i&gt; inject a cocktail of proteins into epithelial cells that enables bacterial attachment and promotes longer-term colonization in the animal. Here, we review recent progress in our understanding of the regulation of T3S in EHEC, focusing on the induction and assembly of the T3S system, the co-ordination of effector protein expression, and the timing of effector protein export through the apparatus. Strain variation is often associated with differences in bacteriophages encoding the production of Shiga toxin and in multiple cryptic prophage elements that can encode effector proteins and T3S regulators. It is evident that this repertoire of phage-related sequences results in the different levels of T3S demonstrated between strains, with implications for EHEC epidemiology and strain evolution

Alternate thermoregulation and functional binding of Escherichia coli Type 1 fimbriae in environmental and animal isolates

Type 1 fimbriae (T1F) are well characterised cell surface organelles expressed by Escherichia coli and required for adherence to mannosylated host tissue. They satisfy molecular Koch's postulates as a virulence determinant and a host-adapted role has been reinforced by reports that T1F expression is repressed at sub-mammalian temperatures. Analysis of a group of 136 environmental and animal E. coli isolates that express T1F at 37 °C showed that 28% are also capable of expression at 20 °C, in a phase variable manner. The heterogeneous proportions varied widely, and although growth temperature impacted the total proportion expressing T1F, there was no direct correlation between growth at 37 and 20 °C, indicative of differences in thermoregulation of the genetic switch (fimS) that controls phase variation. Specificities of the adhesin (FimH) also varied between the isolates: most bound to α-(1-3) mannan and yeast extracts as expected, but some recognised β-(1-4)-mannans and N-linked glycoproteins from plants, and T1F from two of the isolates mediated binding to plant roots. The results expand our view of a well described adherence factor to show alternative expression profiles and adhesin specificities, which in turn may confer an advantage for certain isolates in alternative hosts and habitats

Quantification of selected antimicrobial resistance genes in pig faeces on a British commercial pig farm during a typical production cycle

The data presented are quantitative polymerase chain reaction (qPCR) read outs from antimicrobial resistance gene (AMRG) assays and associated metadata from this project. In this dataset, the mean gene copy numbers per microlitre of DNA extract are shown. The data were collected from faecal and environmental samples which were obtained from a single British commercial pig unit. The former were collected from the sow housing barn, pig growing houses and slurry tanks within the farm unit and the latter were obtained through random stratified sampling of the farm and the surrounding land. These samples were taken from what will be referred to as the 'main study'. A further study was carried out to obtain samples after a partial depopulation which took place on this farm. Faecal samples were obtained from the sow housing barn, pig growing houses and slurry tanks and will be referred to as the 'depopulation (depop) study'. For the main study, the samples were collected between October 19th 2016 and April 5th 2017. For the depop study, the samples were collected between 19th June 2017 and 13th November 2017. The data associated with all samples were generated between August 1st 2017 and May 1st 2018.,Faecal and soil samples were collected from the study farm weekly using spooned universal tubes and were stored at -20°C, batch transported on dry ice to the laboratory where they were stored at -80°C until DNA extraction was carried out. DNA extracts were quantified and assessed using a NanoDrop spectrophotometer and split into aliquots for storage. DNA extracts were then screened in triplicate by qPCR and the number of gene copies assessed through the use of a standard curve generated by the inclusion of standards with known copy numbers of each target gene. Non-template controls were also included to ensure that non-specific binding or contamination were not occurring. The qPCR machine software (i.e. MxPro) was then used to calculate the number of gene copies in each of the collected samples and the mean was calculated using the three replicates. These values are included in the data set provided.

Immunization of cattle with a combination of purified intimin-531, EspA and Tir significantly reduces shedding of Escherichia coli O157:H7 following oral challenge

Enterohemorrhagic Escherichia call (EHEC) O157:H7 is a human pathogen that can cause gastrointestinal disease with potentially fatal consequences as a result of systemic Shiga toxin activity. Cattle are the main reservoir host of EHEC O157 and interventions need to be developed that prevent cattle colonization or limit shedding of the organism from this host. EHEC 0157 predominately colonizes the bovine terminal rectum and requires a type III secretion system (T3SS) for adherence and persistence at this site. A vaccine based on concentrated bacterial supernatant that contains T3S proteins has shown some efficacy. Here we have demonstrated that vaccination with a combination of antigens associated with T3S-mediated adherence; the translocon filament protein, EspA, the extracellular region of the outer membrane adhesin, intimin, and the translocated intimin receptor (Tir) significantly reduced shedding of EHEC 0157 from experimentally infected animals. Furthermore, this protection may be augmented by addition of H7 flagellin to the vaccine preparation that has been previously demonstrated to be partially protective in cattle. Protection correlates with systemic and mucosal antibody responses to the defined antigens and validates the targeting of these colonization factors