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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

Type three secretion system (T3SS) and its regulators in E. coli O157:H7 EDL933 strains. (A) mRNA expressions of T3SS; (B) Tir protein content in E. coli O157:H7; (C) mRNA expression of T3SS regulators. 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, **P < 0.01, *P < 0.05 (Mean ± SEM; n = 4).
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Figure 4: Type three secretion system (T3SS) and its regulators in E. coli O157:H7 EDL933 strains. (A) mRNA expressions of T3SS; (B) Tir protein content in E. coli O157:H7; (C) mRNA expression of T3SS regulators. 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, **P < 0.01, *P < 0.05 (Mean ± SEM; n = 4).

Mentions: To understand possible factors limiting E. coli O157:H7 epithelial colonization in the absence of PNPase, we further analyzed T3SS apparatus and effectors transcripts and proteins in EDL933Δpnp and its wild type strains. Surprisingly, the transcriptional levels of genes located on LEE region including tir, eae, and espB were enhanced in EDL933Δpnp strain (Figure 4A). Tir protein production was also increased in both supernatant fraction and whole cell lysis of EDL933Δpnp strain compared to the wild-type EDL933 strain (Figure 4B). In line with, the transcriptional level of LEE positive regulators, the ler transcript level, was significantly enhanced in EDL933Δpnp strain (Figure 4C), though other regulators of LEE (hns, csrA, and grlA) did not differ between EDL933Δpnp and its isogenic wild type strain.


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

Hu J, Zhu MJ - Front Microbiol (2015)

Type three secretion system (T3SS) and its regulators in E. coli O157:H7 EDL933 strains. (A) mRNA expressions of T3SS; (B) Tir protein content in E. coli O157:H7; (C) mRNA expression of T3SS regulators. 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, **P < 0.01, *P < 0.05 (Mean ± SEM; n = 4).
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Related In: Results  -  Collection

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

Figure 4: Type three secretion system (T3SS) and its regulators in E. coli O157:H7 EDL933 strains. (A) mRNA expressions of T3SS; (B) Tir protein content in E. coli O157:H7; (C) mRNA expression of T3SS regulators. 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, **P < 0.01, *P < 0.05 (Mean ± SEM; n = 4).
Mentions: To understand possible factors limiting E. coli O157:H7 epithelial colonization in the absence of PNPase, we further analyzed T3SS apparatus and effectors transcripts and proteins in EDL933Δpnp and its wild type strains. Surprisingly, the transcriptional levels of genes located on LEE region including tir, eae, and espB were enhanced in EDL933Δpnp strain (Figure 4A). Tir protein production was also increased in both supernatant fraction and whole cell lysis of EDL933Δpnp strain compared to the wild-type EDL933 strain (Figure 4B). In line with, the transcriptional level of LEE positive regulators, the ler transcript level, was significantly enhanced in EDL933Δpnp strain (Figure 4C), though other regulators of LEE (hns, csrA, and grlA) did not differ between EDL933Δpnp and its isogenic wild type strain.

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