Abstract
Polynucleotide phosphorylase (PNPase) is reported to regulate virulence in Salmonella, Yersinia sp. and Campylobacter jejuni, yet its role in Escherichia coli O157:H7 has not been investigated. To gain insights into its roles in E. coli O157:H7 virulence, pnp deletion mutants were generated and the major virulence factors were compared to their parental wild type strains. Deletion of pnp in E. coli O157:H7 dramatically decreased stx2 mRNA expression and Stx2 protein production, and impaired lambdoid prophage activation in E. coli O157:H7. Quantitative PCR further confirmed that the Stx2 phage lytic growth was repressed by pnp deletion. Consistent with reduced Stx2 production and Stx2 phage activation, the transcriptional levels of genes involved in phage lysis and replication were down-regulated. In addition, disruption of pnp in E. coli O157:H7 decreased its adhesion to intestinal epithelial cells as well as cattle colonic explant tissues. On the other hand, PNPase inactivation in E. coli O157:H7 enhanced Tir protein content and the transcription of type three secretion system components, including genes encoding intimin, Tir, and EspB as well as locus of enterocyte and effacement positive regulator, Ler. Collectively, data indicate that PNPase has pleiotropic effects on the virulence of E. coli O157:H7.
Highlights
Shiga toxin (Stx) producing Escherichia coli O157:H7 is a major food safety threat that results in significant economic losses, especially in the beef industry
Polynucleotide phosphorylase (PNPase) is Essential for Stx2 Production in E. coli O157:H7 EDL933 Strain In transcriptional level, deletion of pnp completely eliminated stx2 mRNA expression in E. coli O157:H7 EDL933 strain without affecting stx1 expression (Figure 1A)
The transcriptional levels of cII and cIII, phage lysis S gene and phage replication gene O were all diminished in pnp strain (Figure 2C). These data indicated that PNPase in E. coli O157:H7 plays an indispensable role in Stx2 prophage lysis
Summary
Shiga toxin (Stx) producing Escherichia coli O157:H7 is a major food safety threat that results in significant economic losses, especially in the beef industry. Stx is the major virulence factor in E. coli O157:H7, which causes bloody diarrhea and life threatening hemolytic-uremic syndrome (Mainil and Daube, 2005). The mortality associated with E. coli O157:H7 infection is due to the production and release of Stx, which is composed of a single 32-kDa A subunit and five 7.7-kDa B subunits (Paton and Paton, 1998). Stx binds to receptors on cell surface and is internalized through endocytosis, which inhibits protein synthesis and causes complications associated with E. coli O157:H7 infection (Johannes and Romer, 2010). The LEE is composed of five major operons (LEE1-5) that encode type three secretion system (T3SS) apparatus and effector proteins (Deng et al, 2004)
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