Dataset Information

Identification and Characterization of Novel Compounds Blocking Shiga Toxin Expression in Escherichia coli O157:H7.

ABSTRACT: Infections caused by Shiga toxin (Stx)-producing E. coli strains constitute a health problem, as they are problematic to treat. Stx production is a key virulence factor associated with the pathogenicity of enterohaemorrhagic E. coli (EHEC) and can result in the development of haemolytic uremic syndrome in infected patients. The genes encoding Stx are located on temperate lysogenic phages integrated into the bacterial chromosome and expression of the toxin is generally coupled to phage induction through the SOS response. We aimed to find new compounds capable of blocking expression of Stx type 2 (Stx2) as this subtype of Stx is more strongly associated with human disease. High-throughput screening of a small-molecule library identified a lead compound that reduced Stx2 expression in a dose-dependent manner. We show that the optimized compound interferes with the SOS response by directly affecting the activity and oligomerization of RecA, thus limiting phage activation and Stx2 expression. Our work suggests that RecA is highly susceptible to inhibition and that targeting this protein is a viable approach to limiting production of Stx2 by EHEC. This type of approach has the potential to limit production and transfer of other phage induced and transduced determinants.

SUBMITTER: Huerta-Uribe A

PROVIDER: S-EPMC5127787 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Identification and Characterization of Novel Compounds Blocking Shiga Toxin Expression in Escherichia coli O157:H7.

Huerta-Uribe Alejandro A Marjenberg Zoe R ZR Yamaguchi Nao N Fitzgerald Stephen S Connolly James P R JP Carpena Nuria N Uvell Hanna H Douce Gillian G Elofsson Michael M Byron Olwyn O Marquez Rudi R Gally David L DL Roe Andrew J AJ

Frontiers in microbiology 20161130

Infections caused by Shiga toxin (Stx)-producing E. coli strains constitute a health problem, as they are problematic to treat. Stx production is a key virulence factor associated with the pathogenicity of enterohaemorrhagic E. coli (EHEC) and can result in the development of haemolytic uremic syndrome in infected patients. The genes encoding Stx are located on temperate lysogenic phages integrated into the bacterial chromosome and expression of the toxin is generally coupled to ph ...[more]

PMID: 27965652

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Project description:Escherichia coli O157:H7 is a foodborne pathogen implicated in various multistate outbreaks. It encodes Shiga toxin on a prophage, and Shiga toxin production is linked to phage induction. An E. coli strain, designated 0.1229, that amplified Stx2a production when cocultured with E. coli O157:H7 strain PA2 was identified. Growth of PA2 in 0.1229 cell-free supernatants had a similar effect, even when supernatants were heated to 100°C for 10 min, but not after treatment with proteinase K. The secreted molecule was shown to use TolC for export and the TonB system for import. The genes sufficient for production of this molecule were localized to a 5.2-kb region of a 12.8-kb plasmid. This region was annotated, identifying hypothetical proteins, a predicted ABC transporter, and a cupin superfamily protein. These genes were identified and shown to be functional in two other E. coli strains, and bioinformatic analyses identified related gene clusters in similar and distinct bacterial species. These data collectively suggest that E. coli 0.1229 and other E. coli strains produce a microcin that induces the SOS response in target bacteria. Besides adding to the limited number of microcins known to be produced by E. coli, this study provides an additional mechanism by which stx2a expression is increased in response to the gut microflora.IMPORTANCE How the gut microflora influences the progression of bacterial infections is only beginning to be understood. Antibiotics are counterindicated for E. coli O157:H7 infections, limiting treatment options. An increased understanding of how the gut microflora directs O157:H7 virulence gene expression may lead to additional treatment options. This work identified E. coli strains that enhance the production of Shiga toxin by O157:H7 through the secretion of a proposed microcin. Microcins are natural antimicrobial peptides that target specific species, can act as alternatives to antibiotics, and mediate microbial competition. This work demonstrates another mechanism by which non-O157 E. coli strains may increase Shiga toxin production and adds to our understanding of microcins, a group of antimicrobials less well understood than colicins.

Project description:Cattle are the main reservoir of Enterohemorrhagic Escherichia coli (EHEC), with O157:H7 the distinctive serotype. EHEC is the main causative agent of a severe systemic disease, Hemolytic Uremic Syndrome (HUS). Argentina has the highest pediatric HUS incidence worldwide with 12-14 cases per 100,000 children. Herein, we assessed the genomes of EHEC O157:H7 isolates recovered from cattle in the humid Pampas of Argentina. According to phylogenetic studies, EHEC O157 can be divided into clades. Clade 8 strains that were classified as hypervirulent. Most of the strains of this clade have a Shiga toxin stx2a-stx2c genotype. To better understand the molecular bases related to virulence, pathogenicity and evolution of EHEC O157:H7, we performed a comparative genomic analysis of these isolates through whole genome sequencing. The isolates classified as clade 8 (four strains) and clade 6 (four strains) contained 13 to 16 lambdoid prophages per genome, and the observed variability of prophages was analysed. An inter strain comparison show that while some prophages are highly related and can be grouped into families, other are unique. Prophages encoding for stx2a were highly diverse, while those encoding for stx2c were conserved. A cluster of genes exclusively found in clade 8 contained 13 genes that mostly encoded for DNA binding proteins. In the studied strains, polymorphisms in Q antiterminator, the Q-stx2A intergenic region and the O and P γ alleles of prophage replication proteins are associated with different levels of Stx2a production. As expected, all strains had the pO157 plasmid that was highly conserved, although one strain displayed a transposon interruption in the protease EspP gene. This genomic analysis may contribute to the understanding of the genetic basis of the hypervirulence of EHEC O157:H7 strains circulating in Argentine cattle. This work aligns with other studies of O157 strain variation in other populations that shows key differences in Stx2a-encoding prophages.

Dataset Information

Identification and Characterization of Novel Compounds Blocking Shiga Toxin Expression in Escherichia coli O157:H7.

Publications

Identification and Characterization of Novel Compounds Blocking Shiga Toxin Expression in <i>Escherichia coli</i> O157:H7.

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