Infectious Diseases and Immunology Division, Indian Institute of Chemical Biology, Council of Scientific and Industrial Research, Kolkata 700 032, India.
Infect Immun. 2011 Jan;79(1):258-66. doi: 10.1128/IAI.00663-10. Epub 2010 Nov 1.
In the enteric pathogen Vibrio cholerae, expression of the major virulence factors is controlled by the hierarchical expression of several regulatory proteins comprising the ToxR regulon. In this study, we demonstrate that disruption of the fadD gene encoding a long-chain fatty acyl coenzyme A ligase has marked effects on expression of the ToxR virulence regulon, motility, and in vivo lethality of V. cholerae. In the V. cholerae fadD mutant, expression of the major virulence genes ctxAB and tcpA, encoding cholera toxin (CT), and the major subunit of the toxin-coregulated pilus (TCP) was drastically repressed and a growth-phase-dependent reduction in the expression of toxT, encoding the transcriptional activator of ctxAB and tcpA, was observed. Expression of toxT from an inducible promoter completely restored CT to wild-type levels in the V. cholerae fadD mutant, suggesting that FadD probably acts upstream of toxT expression. Expression of toxT is activated by the synergistic effect of two transcriptional regulators, TcpP and ToxR. Reverse transcription-PCR and Western blot analysis indicated that although gene expression and production of both TcpP and ToxR are unaffected in the fadD mutant strain, membrane localization of TcpP, but not ToxR, is severely impaired in the fadD mutant strain from the mid-logarithmic phase of growth. Since the decrease in toxT expression occurred concomitantly with the reduction in membrane localization of TcpP, a direct correlation between the defect in membrane localization of TcpP and reduced toxT expression in the fadD mutant strain is suggested.
在肠道病原体霍乱弧菌中，几种调节蛋白的层次表达控制着主要毒力因子的表达，这些调节蛋白构成了 ToxR 调节子。在这项研究中，我们证明了编码长链脂肪酸辅酶 A 连接酶的 fadD 基因的破坏对 ToxR 毒力调节子的表达、运动性和霍乱弧菌的体内致死性有显著影响。在霍乱弧菌 fadD 突变体中，编码霍乱毒素（CT）的主要毒力基因 ctxAB 和 tcpA 以及毒素调节菌毛（TCP）的主要亚单位的表达被严重抑制，并观察到编码 ctxAB 和 tcpA 的转录激活子 toxT 的表达随生长阶段呈依赖性减少。来自诱导型启动子的 toxT 的表达完全恢复了 V. cholerae fadD 突变体中的 CT 至野生型水平，表明 FadD 可能在上游作用于 toxT 的表达。ToxT 的表达受两种转录调节剂 TcpP 和 ToxR 的协同作用激活。反转录-PCR 和 Western blot 分析表明，尽管 fadD 突变体菌株中的 toxT 基因表达和 TcpP 和 ToxR 的产生不受影响，但 fadD 突变体菌株中 TcpP 的膜定位，而不是 ToxR，在对数中期生长阶段严重受损。由于 toxT 表达的减少与 TcpP 膜定位的减少同时发生，因此 fadD 突变体菌株中 TcpP 膜定位的缺陷与 toxT 表达减少之间存在直接相关性。
