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

Chronopotentiometric study of the transport of phosphoric acid anions through an anion-exchange membrane under different pH values

[EN] Phosphate is the main cause of eutrophication in many water bodies. Its presence in waters is associated to the fact that is not completely removed in conventional wastewater treatment plants. On the other side, phosphate rocks are a non-renewable resource and considered as a critical raw material. A membrane separation process, able to recover phosphate from wastewater, is a promising process to avoid pollution and to reuse phosphate. This paper investigates the transport of salts of phosphoric acid through an anion-exchange membrane (AEM) by means of chronopotentiograms and polarization curves (CVCs). The presence of multiple transition times in the chronopotentiograms and the corresponding limiting current densities in the CVCs indicate a change in the species being transported in the membrane/diffusion boundary layer system, due to the hydrolysis reactions that take place when the concentration polarization is reached. Under the experimental conditions tested, coupled convection (gravitational and elctroconvection) occurs when a certain threshold in the membrane voltage drop is surpassed independently of the electrolyte concentration. However, at high pH values, only one transition time in the chronopotentiograms, due to the transfer of OH- ions with greater concentration and mobility. This fact is reflected in the CVCs by the large plateaus obtained, which hinders the occurrence of coupled convection phenomena, and consequently, water splitting can be considered as the main mechanism responsible for the overlimiting regime.The authors wish to thank the financial support from FINEP, FAPERGS, CAPES and CNPq (Brazil), from the BRICS-STI/CNPq (BRICS STI Framework Programme), from the European Union through the Erasmus Mundus Program (EBW +) and from the CYTED (Network 318RT0551).Gally, C.; García Gabaldón, M.; Ortega Navarro, EM.; Bernardes, A.; Pérez-Herranz, V. (2020). 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The leucine-responsive regulatory protein binds to the fim switch to control phase variation of type 1 fimbrial expression in Escherichia coli K-12

Phase variation of type 1 fimbriation in Escherichia coli is associated with the site-specific recombination of a 314-bp DNA invertible element. The fim switch directs transcription of fimA, the major fimbrial subunit gene, in one orientation (on) but not the other (off). Switching requires either fimB (on-to-off or off-to-on inversion) or fimE (on-to-off inversion only) and is reduced sharply in strains containing lrp::Tn10 mutations. Both fimE-promoted switching and fimB-promoted switching are stimulated by the amino acids alanine, isoleucine, leucine, and valine, and this regulation requires lrp. Here it is shown that the leucine-responsive regulatory protein (Lrp) binds in and adjacent to the fim switch. Mutations in fim that lower Lrp binding in vitro have corresponding effects on both fimB-promoted switching and fimE-promoted switching in vivo. Lrp initiates binding at one of two sites within the fim switch. Additional cooperative binding results in an extensive region of protection from both DNase I and 1,10-phenanthroline-copper complex-activated DNA cleavage. The region of protection can extend to within 12 bp of the right inverted repeat (switch off) and occupies over one-third of the switch. It is proposed that wrapping of fim DNA around an Lrp complex is required to form a recombination-proficient structure

Phylogenetic relationship and virulence composition of Escherichia coli O26:H11 cattle and human strain collections in Scotland; 2002-2020

O26 is the commonest non-O157 Shiga toxin (stx)-producing Escherichia coli serogroup reported in human infections worldwide. Ruminants, particularly cattle, are the primary reservoir source for human infection. In this study, we compared the whole genomes and virulence profiles of O26:H11 strains (n = 99) isolated from Scottish cattle with strains from human infections (n = 96) held by the Scottish Escherichia coli O157/STEC Reference Laboratory, isolated between 2002 and 2020. Bovine strains were from two national cross-sectional cattle surveys conducted between 2002–2004 and 2014–2015. A maximum likelihood phylogeny was constructed from a core-genome alignment with the O26:H11 strain 11368 reference genome. Genomes were screened against a panel of 2,710 virulence genes using the Virulence Finder Database. All stx-positive bovine O26:H11 strains belonged to the ST21 lineage and were grouped into three main clades. Bovine and human source strains were interspersed, and the stx subtype was relatively clade-specific. Highly pathogenic stx2a-only ST21 strains were identified in two herds sampled in the second cattle survey and in human clinical infections from 2010 onwards. The closest pairwise distance was 9 single-nucleotide polymorphisms (SNPs) between Scottish bovine and human strains and 69 SNPs between the two cattle surveys. Bovine O26:H11 was compared to public EnteroBase ST29 complex genomes and found to have the greatest commonality with O26:H11 strains from the rest of the UK, followed by France, Italy, and Belgium. Virulence profiles of stx-positive bovine and human strains were similar but more conserved for the stx2a subtype. O26:H11 stx-negative ST29 (n = 17) and ST396 strains (n = 5) were isolated from 19 cattle herds; all were eae-positive, and 10 of these herds yielded strains positive for ehxA, espK, and Z2098, gene markers suggestive of enterohaemorrhagic potential. There was a significant association (p < 0.001) between nucleotide sequence percent identity and stx status for the bacteriophage insertion site genes yecE for stx2 and yehV for stx1. Acquired antimicrobial resistance genes were identified in silico in 12.1% of bovine and 17.7% of human O26:H11 strains, with sul2, tet, aph(3″), and aph(6″) being most common. This study describes the diversity among Scottish bovine O26:H11 strains and investigates their relationship to human STEC infections

Predicting the public health benefit of vaccinating cattle against Escherichia coli O157

Identifying the major sources of risk in disease transmission is key to designing effective controls. However, understanding of transmission dynamics across species boundaries is typically poor, making the design and evaluation of controls particularly challenging for zoonotic pathogens. One such global pathogen is Escherichia coli O157, which causes a serious and sometimes fatal gastrointestinal illness. Cattle are the main reservoir for E. coli O157, and vaccines for cattle now exist. However, adoption of vaccines is being delayed by conflicting responsibilities of veterinary and public health agencies, economic drivers, and because clinical trials cannot easily test interventions across species boundaries, lack of information on the public health benefits. Here, we examine transmission risk across the cattle–human species boundary and show three key results. First, supershedding of the pathogen by cattle is associated with the genetic marker stx2. Second, by quantifying the link between shedding density in cattle and human risk, we show that only the relatively rare supershedding events contribute significantly to human risk. Third, we show that this finding has profound consequences for the public health benefits of the cattle vaccine. A naïve evaluation based on efficacy in cattle would suggest a 50% reduction in risk; however, because the vaccine targets the major source of human risk, we predict a reduction in human cases of nearly 85%. By accounting for nonlinearities in transmission across the human–animal interface, we show that adoption of these vaccines by the livestock industry could prevent substantial numbers of human E. coli O157 cases

Comparison between classical potentials and ab initio for silicon under large shear

The homogeneous shear of the {111} planes along the direction of bulk silicon has been investigated using ab initio techniques, to better understand the strain properties of both shuffle and glide set planes. Similar calculations have been done with three empirical potentials, Stillinger-Weber, Tersoff and EDIP, in order to find the one giving the best results under large shear strains. The generalized stacking fault energies have also been calculated with these potentials to complement this study. It turns out that the Stillinger-Weber potential better reproduces the ab initio results, for the smoothness and the amplitude of the energy variation as well as the localization of shear in the shuffle set

RfaH Suppresses Small RNA MicA Inhibition of fimB Expression in Escherichia coli K-12

The phase variation (reversible on-off switching) of the type 1 fimbrial adhesin of Escherichia coli involves a DNA inversion catalyzed by FimB (switching in either direction) or FimE (on-to-off switching). Here, we demonstrate that RfaH activates expression of a FimB-LacZ protein fusion while having a modest inhibitory effect on a comparable fimB-lacZ operon construct and on a FimE-LacZ protein fusion, indicating that RfaH selectively controls fimB expression at the posttranscriptional level. Further work demonstrates that loss of RfaH enables small RNA (sRNA) MicA inhibition of fimB expression even in the absence of exogenous inducing stress. This effect is explained by induction of σE , and hence MicA, in the absence of RfaH. Additional work con- firms that the procaine-dependent induction of micA requires OmpR, as reported previously (A. Coornaert et al., Mol. Microbiol. 76:467–479, 2010, doi:10.1111/j.1365-2958.2010.07115.x), but also demonstrates that RfaH inhibition of fimB transcription is enhanced by procaine independently of OmpR. While the effect of procaine on fimB transcription is shown to be independent of RcsB, it was found to require SlyA, another known regulator of fimB transcription. These results demonstrate a complex role for RfaH as a regulator of fimB expression

Genome structural variation in Escherichia coli O157:H7

The human zoonotic pathogen Escherichia coli O157:H7 is defined by its extensive prophage repertoire including those that encode Shiga toxin, the factor responsible for inducing life-threatening pathology in humans. As well as introducing genes that can contribute to the virulence of a strain, prophage can enable the generation of large-chromosomal rearrangements (LCRs) by homologous recombination. This work examines the types and frequencies of LCRs across the major lineages of the O157:H7 serotype. We demonstrate that LCRs are a major source of genomic variation across all lineages of E. coli O157:H7 and by using both optical mapping and Oxford Nanopore long-read sequencing prove that LCRs are generated in laboratory cultures started from a single colony and that these variants can be recovered from colonized cattle. LCRs are biased towards the terminus region of the genome and are bounded by specific prophages that share large regions of sequence homology associated with the recombinational activity. RNA transcriptional profiling and phenotyping of specific structural variants indicated that important virulence phenotypes such as Shiga-toxin production, type-3 secretion and motility can be affected by LCRs. In summary, E. coli O157:H7 has acquired multiple prophage regions over time that act to continually produce structural variants of the genome. These findings raise important questions about the significance of this prophage-mediated genome contingency to enhance adaptability between environments

The actin-myosin regulatory MRCK kinases: regulation, biological functions and associations with human cancer

The contractile actin-myosin cytoskeleton provides much of the force required for numerous cellular activities such as motility, adhesion, cytokinesis and changes in morphology. Key elements that respond to various signal pathways are the myosin II regulatory light chains (MLC), which participate in actin-myosin contraction by modulating the ATPase activity and consequent contractile force generation mediated by myosin heavy chain heads. Considerable effort has focussed on the role of MLC kinases, and yet the contributions of the myotonic dystrophy-related Cdc42-binding kinases (MRCK) proteins in MLC phosphorylation and cytoskeleton regulation have not been well characterized. In contrast to the closely related ROCK1 and ROCK2 kinases that are regulated by the RhoA and RhoC GTPases, there is relatively little information about the CDC42-regulated MRCKα, MRCKβ and MRCKγ members of the AGC (PKA, PKG and PKC) kinase family. As well as differences in upstream activation pathways, MRCK and ROCK kinases apparently differ in the way that they spatially regulate MLC phosphorylation, which ultimately affects their influence on the organization and dynamics of the actin-myosin cytoskeleton. In this review, we will summarize the MRCK protein structures, expression patterns, small molecule inhibitors, biological functions and associations with human diseases such as cancer