Limits...
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

Shiga toxin production in E. coli O157:H7 EDL933 strains. (A) Relative mRNA expression; (B) Relative protein content. EDL933: wild-type E. coli O157:H7 strain; EDL933 Δpnp: EDL933 pnp deletion mutant strain; pBBR1: EDL933 Δpnp strain carrying an empty vector pBBR1; pBBR1::pnp: EDL933 Δpnp strain complemented with pBBR1::pnp. □: EDL933; ■: EDL933Δpnp; ***P < 0.001 (Mean ± SEM; n = 4).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4538292&req=5

Figure 1: Shiga toxin production in E. coli O157:H7 EDL933 strains. (A) Relative mRNA expression; (B) Relative protein content. EDL933: wild-type E. coli O157:H7 strain; EDL933 Δpnp: EDL933 pnp deletion mutant strain; pBBR1: EDL933 Δpnp strain carrying an empty vector pBBR1; pBBR1::pnp: EDL933 Δpnp strain complemented with pBBR1::pnp. □: EDL933; ■: EDL933Δpnp; ***P < 0.001 (Mean ± SEM; n = 4).

Mentions: In transcriptional level, deletion of pnp completely eliminated stx2 mRNA expression in E. coli O157:H7 EDL933 strain without affecting stx1 expression (Figure 1A). We further compared the Stx2 production. In wild-type strain, a 32 kDa band Stx2 A subunit was detected in both supernatant fraction and whole cell lysis (Figure 1B). Deletion of pnp resulted in no detection of Stx2A (Figure 1B). This defective in Stx2 protein production was not restored by complementation of pBBR1::pnp (Figure 1B).


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

Hu J, Zhu MJ - Front Microbiol (2015)

Shiga toxin production in E. coli O157:H7 EDL933 strains. (A) Relative mRNA expression; (B) Relative protein content. EDL933: wild-type E. coli O157:H7 strain; EDL933 Δpnp: EDL933 pnp deletion mutant strain; pBBR1: EDL933 Δpnp strain carrying an empty vector pBBR1; pBBR1::pnp: EDL933 Δpnp strain complemented with pBBR1::pnp. □: EDL933; ■: EDL933Δpnp; ***P < 0.001 (Mean ± SEM; n = 4).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Shiga toxin production in E. coli O157:H7 EDL933 strains. (A) Relative mRNA expression; (B) Relative protein content. EDL933: wild-type E. coli O157:H7 strain; EDL933 Δpnp: EDL933 pnp deletion mutant strain; pBBR1: EDL933 Δpnp strain carrying an empty vector pBBR1; pBBR1::pnp: EDL933 Δpnp strain complemented with pBBR1::pnp. □: EDL933; ■: EDL933Δpnp; ***P < 0.001 (Mean ± SEM; n = 4).
Mentions: In transcriptional level, deletion of pnp completely eliminated stx2 mRNA expression in E. coli O157:H7 EDL933 strain without affecting stx1 expression (Figure 1A). We further compared the Stx2 production. In wild-type strain, a 32 kDa band Stx2 A subunit was detected in both supernatant fraction and whole cell lysis (Figure 1B). Deletion of pnp resulted in no detection of Stx2A (Figure 1B). This defective in Stx2 protein production was not restored by complementation of pBBR1::pnp (Figure 1B).

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