Silva Anisia J, Sultan Syed Zafar, Liang Weili, Benitez Jorge A
Department of Microbiology, Biochemistry, and Immunology, Morehouse School of Medicine, 720 Westview Dr., SW, Atlanta, GA 30310, USA.
J Bacteriol. 2008 Nov;190(22):7335-45. doi: 10.1128/JB.00360-08. Epub 2008 Sep 12.
Production of the Zn-metalloprotease hemagglutinin (HA)/protease by Vibrio cholerae has been reported to enhance enterotoxicity in rabbit ileal loops and the reactogenicity of live cholera vaccine candidates. Expression of HA/protease requires the alternate sigma factor sigma(S) (RpoS), encoded by rpoS. The histone-like nucleoid structuring protein (H-NS) has been shown to repress rpoS expression in Escherichia coli. In V. cholerae strains of the classical biotype, H-NS has been reported to silence virulence gene expression. In this study we examined the role of H-NS in the expression of HA/protease and motility in an El Tor biotype strain by constructing a Deltahns mutant. The Deltahns mutant exhibited multiple phenotypes, such as production of cholera toxin in nonpermissive LB medium, reduced resistance to high osmolarity, enhanced resistance to low pH and hydrogen peroxide, and reduced motility. Depletion of H-NS by overexpression of a dominant-negative allele or by deletion of hns resulted in diminished expression of HA/protease. Epistasis analysis of HA/protease expression in Deltahns, DeltarpoS, and Deltahns DeltarpoS mutants, analysis of RpoS reporter fusions, quantitative reverse transcription-PCR measurements, and ectopic expression of RpoS in DeltarpoS and DeltarpoS Deltahns mutants showed that H-NS posttranscriptionally enhances RpoS expression. The Deltahns mutant exhibited a lower degree of motility and lower levels of expression of flaA, flaC, cheR-2, and motX mRNAs than the wild type. Comparison of the mRNA abundances of these genes in wild-type, Deltahns, DeltarpoS, and Deltahns DeltarpoS strains revealed that deletion of rpoS had a more severe negative effect on their expression. Interestingly, deletion of hns in the rpoS background resulted in higher expression levels of flaA, flaC, and motX, suggesting that H-NS represses the expression of these genes in the absence of sigma(S). Finally, we show that the cyclic AMP receptor protein and H-NS act along the same pathway to positively affect RpoS expression.
据报道，霍乱弧菌产生的锌金属蛋白酶血凝素（HA）/蛋白酶可增强兔回肠袢中的肠毒性以及霍乱活疫苗候选株的反应原性。HA/蛋白酶的表达需要由rpoS编码的替代σ因子σ（S）（RpoS）。已证明组蛋白样核仁结构蛋白（H-NS）可抑制大肠杆菌中rpoS的表达。在古典生物型霍乱弧菌菌株中，据报道H-NS可使毒力基因表达沉默。在本研究中，我们通过构建Δhns突变体，研究了H-NS在埃尔托生物型菌株中HA/蛋白酶表达和运动性中的作用。Δhns突变体表现出多种表型，如在非许可性LB培养基中产生霍乱毒素、对高渗透压的抗性降低、对低pH和过氧化氢的抗性增强以及运动性降低。通过显性负等位基因的过表达或hns的缺失使H-NS耗尽，导致HA/蛋白酶的表达减少。对Δhns、ΔrpoS和ΔhnsΔrpoS突变体中HA/蛋白酶表达的上位性分析、RpoS报告基因融合分析、定量逆转录-PCR测量以及RpoS在ΔrpoS和ΔrpoSΔhns突变体中的异位表达表明，H-NS在转录后增强RpoS表达。与野生型相比，Δhns突变体的运动性较低，flaA、flaC、cheR-2和motX mRNA的表达水平也较低。比较野生型、Δhns、ΔrpoS和ΔhnsΔrpoS菌株中这些基因的mRNA丰度发现，rpoS的缺失对它们的表达有更严重的负面影响。有趣的是，在rpoS背景中hns的缺失导致flaA、flaC和motX的表达水平更高，这表明在没有σ（S）的情况下，H-NS抑制这些基因的表达。最后，我们表明环磷酸腺苷受体蛋白和H-NS沿着相同的途径发挥作用，对RpoS表达产生正向影响。
