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Defects in polynucleotide phosphorylase impairs virulence in Escherichia coli O157:H7.

Hu J, Zhu MJ - Front Microbiol (2015)

Bottom Line: 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.Collectively, data indicate that PNPase has pleiotropic effects on the virulence of E. coli O157:H7.

View Article: PubMed Central - PubMed

Affiliation: School of Food Science, Washington State University, Pullman, WA USA ; Department of Animal Science, University of Wyoming, Laramie, WY USA.

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.

No MeSH data available.


Related in: MedlinePlus

Prophage enumeration in E. coli O157:H7 EDL933 wild type (□) and pnp deletion strains (■). (A) Spontaneous lambdoid prophage enumeration; (B) Stx2 phage PCR; (C) Phage lysis related gene expressions. ***P < 0.001, **P < 0.01, *P < 0.05 (Mean ± SEM, n = 4).
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Figure 2: Prophage enumeration in E. coli O157:H7 EDL933 wild type (□) and pnp deletion strains (■). (A) Spontaneous lambdoid prophage enumeration; (B) Stx2 phage PCR; (C) Phage lysis related gene expressions. ***P < 0.001, **P < 0.01, *P < 0.05 (Mean ± SEM, n = 4).

Mentions: Shiga toxin production is mediated by Stx2 phage activation, thus we further compared phage activation in E. coli O157:H7 EDL933 and its pnp mutant strains. Deletion of pnp completely abolished lambdoid phage progeny production in E. coli O157:H7 (Figure 2A). Quantitative PCR further confirmed that the stx2 phage DNA accumulation was repressed in PNPase defective E. coli O157:H7 strain (Figure 2B). To gain insights into molecular mechanisms of PNPase mediated impairment in Stx phage activation, we further analyzed expression of phage genes regulating prophage activation. Deletion of pnp did not affect recA and rcsA expression (Figure 2C). We could not detect cro mRNA expressions and only detected a very low level of cI expression in Δpnp strain (Figure 2C). In addition, the transcriptional levels of cII and cIII, phage lysis S gene and phage replication gene O were all diminished in Δpnp strain (Figure 2C). Altogether, these data indicated that PNPase in E. coli O157:H7 plays an indispensable role in Stx2 prophage lysis.


Defects in polynucleotide phosphorylase impairs virulence in Escherichia coli O157:H7.

Hu J, Zhu MJ - Front Microbiol (2015)

Prophage enumeration in E. coli O157:H7 EDL933 wild type (□) and pnp deletion strains (■). (A) Spontaneous lambdoid prophage enumeration; (B) Stx2 phage PCR; (C) Phage lysis related gene expressions. ***P < 0.001, **P < 0.01, *P < 0.05 (Mean ± SEM, n = 4).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4538292&req=5

Figure 2: Prophage enumeration in E. coli O157:H7 EDL933 wild type (□) and pnp deletion strains (■). (A) Spontaneous lambdoid prophage enumeration; (B) Stx2 phage PCR; (C) Phage lysis related gene expressions. ***P < 0.001, **P < 0.01, *P < 0.05 (Mean ± SEM, n = 4).
Mentions: Shiga toxin production is mediated by Stx2 phage activation, thus we further compared phage activation in E. coli O157:H7 EDL933 and its pnp mutant strains. Deletion of pnp completely abolished lambdoid phage progeny production in E. coli O157:H7 (Figure 2A). Quantitative PCR further confirmed that the stx2 phage DNA accumulation was repressed in PNPase defective E. coli O157:H7 strain (Figure 2B). To gain insights into molecular mechanisms of PNPase mediated impairment in Stx phage activation, we further analyzed expression of phage genes regulating prophage activation. Deletion of pnp did not affect recA and rcsA expression (Figure 2C). We could not detect cro mRNA expressions and only detected a very low level of cI expression in Δpnp strain (Figure 2C). In addition, the transcriptional levels of cII and cIII, phage lysis S gene and phage replication gene O were all diminished in Δpnp strain (Figure 2C). Altogether, these data indicated that PNPase in E. coli O157:H7 plays an indispensable role in Stx2 prophage lysis.

Bottom Line: 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.Collectively, data indicate that PNPase has pleiotropic effects on the virulence of E. coli O157:H7.

View Article: PubMed Central - PubMed

Affiliation: School of Food Science, Washington State University, Pullman, WA USA ; Department of Animal Science, University of Wyoming, Laramie, WY USA.

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.

No MeSH data available.


Related in: MedlinePlus