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. 2000 Aug 29;97(18):10220-4.
doi: 10.1073/pnas.170219997.

"VSports app下载" Altered expression of the ToxR-regulated porins OmpU and OmpT diminishes Vibrio cholerae bile resistance, virulence factor expression, and intestinal colonization

D Provenzano  1 , K E Klose
Affiliations

Altered expression of the ToxR-regulated porins OmpU and OmpT diminishes Vibrio cholerae bile resistance, virulence factor expression, and intestinal colonization (V体育ios版)

D Provenzano et al. Proc Natl Acad Sci U S A. .

Abstract

The transmembrane transcriptional activators ToxR and TcpP modulate expression of Vibrio cholerae virulence factors by exerting control over toxT, which encodes the cytoplasmic transcriptional activator of the ctx, tcp, and acf virulence genes. However, ToxR, independently of TcpP and ToxT, activates and represses transcription of the genes encoding two outer-membrane porins, OmpU and OmpT. To determine the role of ToxR-dependent porin regulation in V. cholerae pathogenesis, the ToxR-activated ompU promoter was used to drive ompT transcription in a strain lacking OmpU VSports手机版. Likewise, the ToxR-repressed ompT promoter was used to drive ompU transcription in a strain lacking both ToxR and OmpT. This strategy allowed the generation of a toxR(+) strain that expresses OmpT in place of OmpU, and a toxR(-) strain that expresses OmpU in place of OmpT. Growth rates in the presence of bile salts and other anionic detergents were retarded for the toxR(+) V. cholerae expressing OmpT in place of OmpU, but increased in toxR(-) V. cholerae expressing OmpU in place of OmpT. Additionally, the toxR(+) V. cholerae expressing OmpT in place of OmpU expressed less cholera toxin and toxin-coregulated pilus, and this effect was shown to be caused by reduced toxT transcription in this strain. Finally, the toxR(+) V. cholerae expressing OmpT in place of OmpU was approximately 100-fold reduced in its ability to colonize the infant-mouse intestine. Our results indicate that ToxR-dependent modulation of the outer membrane porins OmpU and OmpT is critical for V. cholerae bile resistance, virulence factor expression, and intestinal colonization. .

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Figures

Figure 1
Figure 1
Ectopic expression of OmpU and OmpT. (A) The ompT promoter fragment corresponding to −429 to +104 with respect to the transcription start site was fused to the ompU ORF to form Tp-U (pKEK256), which allows ectopic expression of OmpU in a toxR− strain. The ompU promoter fragment corresponding to −674 to +149 with respect to the transcription start site was fused to the ompT ORF to form Up-T (pKEK257), which allows ectopic expression of OmpT in a toxR+ strain. Translational fusions were facilitated by the incorporation of an NdeI site (CATATG) at the initiating Met codon of each ORF. (B) V. cholerae strains O395 (toxR+; lane 1); KKV61 (ΔtoxR; lane 2); KKV780 (ΔompU) carrying either plasmid pWSK30 (vector, lane 3); pKEK253 (Up-U, lane 4); or pKEK257 (Up-T, lane 5); and KKV 804 (ΔompT ΔtoxR) carrying either plasmid pWSK30 (vector, lane 6), pKEK255 (Tp-T, lane 7), or pKEK256 (Tp-U, lane 8). Whole-cell lysates were matched by OD600 and separated on 10% SDS/PAGE, then stained with Coomassie blue; left-hand lane contains molecular mass standards (in kDa), and OmpT and OmpU bands are designated by arrows. (C) The samples of lanes 1–8 were subjected to Western analysis by using rabbit polyclonal antisera against OmpT (α-OmpT, Upper) and OmpU (α-OmpU, Lower).
Figure 2
Figure 2
V. cholerae strains expressing OmpT display retarded growth rates in anionic detergents compared with strains expressing OmpU, regardless of the presence/absence of ToxR. Growth rates are shown relative to the growth rate in the absence of DC or SDS (note logarithmic scale for DC and SDS concentrations). (A) V. cholerae toxR+ strain KKV780 (ΔompU) carrying either pKEK253 (Up-U, ●) or pKEK257 (Up-T, ○) was grown in LB medium at 37°C containing the DC concentrations indicated. (B) V. cholerae strain KKV804 (ΔtoxR ΔompT) carrying either pKEK256 (Tp-U, ■) or pKEK255 (Tp-T, □) were grown in LB medium at 37°C containing the DC concentrations indicated. (C) V. cholerae toxR+ strain KKV780 (ΔompU) carrying either pKEK253 (Up-U, ●) or pKEK257 (Up-T, ○) was grown in LB medium at 37°C containing the SDS concentrations indicated. (D) V. cholerae strain KKV804 (ΔtoxR ΔompT) carrying either pKEK256 (Tp-U, ■) or pKEK255 (Tp-T, □) was grown in LB medium at 37°C containing the DC concentrations indicated.
Figure 3
Figure 3
The V. cholerae toxR+ strain expressing OmpT in place of OmpU demonstrates a colonization defect. Assay was performed as described in Materials and Methods. Strain KKV780 (ΔompU) carrying either plasmid pKEK253 (Up-U) or pKEK257 (Up-T) was coinoculated with isogenic strain KKV598 (O395 ΔlacZ). The competitive index is the ratio of output mutant/wild type (recovered from small intestine) divided by the ratio of input mutant/wild type (inoculated into mouse); thus, if a mutant strain has no colonization defect, we expect a competitive index close to 1. Data points represent individual mice. Value for colonization of KKV780/Up-T had a P value < 0.01 when compared with the value for colonization of KKV780/Up-U by using the Student's two-tailed t test.
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