Phosphorylation of the flagellar regulatory protein FlrC is necessary for Vibrio cholerae motility and enhanced colonization
Phosphorylation of the flagellar regulatory protein FlrC is necessary for Vibrio cholerae motility and enhanced colonization
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DOI：
10.1046/j.1365-2958.2000.01745.x
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发表时间：
2000-02-01
影响因子：
3.6
通讯作者：
Klose, KE
中科院分区：
文献类型：
--
作者：
Correa, NE;Lauriano, CM;Klose, KE
人类病原体霍乱弧菌在宿主体内特异性表达毒力因子，包括霍乱毒素（CT）和毒素共调节菌毛（TCP），这使其能够定殖肠道并引起疾病。霍乱弧菌是一种具有极鞭毛的高度能动性生物，运动性被认为是毒力的一个重要方面，但运动性在发病机制中的确切作用仍不明确。极性鞭毛合成需要双组分调控系统FlrB/FlrC; FlrC是sigma（54）依赖性转录激活因子。我们证明FlrC的转录活性影响霍乱弧菌的运动性和定殖。在纯化的体外反应中，FlrB将磷酸转移至野生型FlrC蛋白，但不转移至突变体形式，其中氨基酸位置54处的天冬氨酸残基已改变为丙氨酸（D54 A），这与FlrC的磷酸化位点一致。野生型FlrC蛋白而非D54 A蛋白在异源系统中激活σ（54）依赖性转录，表明磷酸-FlrC是转录活性形式。含有染色体flrCD 54 A等位基因的霍乱弧菌菌株不合成鞭毛，并且没有可检测水平的关键σ（54）依赖性鞭毛蛋白基因flaA的转录。霍乱弧菌flrCD 54 A突变株在其定殖幼鼠小肠的能力方面也有缺陷，比同基因野生型菌株差约50倍。FlrC的另一种突变（蛋氨酸114至异亮氨酸; M114 I）在不存在磷酸化的情况下赋予组成型转录活性，但是霍乱弧菌flrCM 114 I突变株，尽管有鞭毛且能动，但其定殖能力也有缺陷。携带D54 A或M114 I突变FlrC蛋白的菌株在体外诱导条件下表达正常水平的CT和TCP。我们的研究结果表明，FlrC“锁定”成非活性（D54 A）或活性（M114 I）状态导致定殖缺陷，从而证明在霍乱弧菌发病过程中需要调节FlrC活性。因此，鞭毛调节蛋白FlrC的σ（54）依赖性转录活性不仅有助于运动，而且有助于霍乱弧菌的定殖。
The human pathogen Vibrio cholerae specifically expresses virulence factors within the host, including cholera toxin (CT) and the toxin co-regulated pilus (TCP), which allow it to colonize the intestine and cause disease. V. cholerae is a highly motile organism by virtue of a polar flagellum, and motility has been inferred to be an important aspect of virulence, yet the exact role of motility in pathogenesis has remained undefined. The two-component regulatory system FlrB/FlrC is required for polar flagellar synthesis; FlrC is a sigma(54)-dependent transcriptional activator. We demonstrate that the transcriptional activity of FlrC affects both motility and colonization of V. cholerae. In a purified in vitro reaction, FlrB transfers phosphate to the wild-type FlrC protein, but not to a mutant form in which the aspartate residue at amino acid position 54 has been changed to alanine (D54A), consistent with this being the site of phosphorylation of FlrC. The wild-type FlrC protein, but not the D54A protein, activates sigma(54)-dependent transcription in a heterologous system, demonstrating that phospho-FlrC is the transcriptionally active form. A V. cholerae strain containing a chromosomal flrCD54A allele did not synthesize a flagellum and had no detectable levels of transcription of the critical sigma(54)-dependent flagellin gene flaA. The V. cholerae flrCD54A mutant strain was also defective in its ability to colonize the infant mouse small intestine, approximately 50-fold worse than an isogenic wild-type strain. Another mutation of FlrC (methionine 114 to isoleucine; M114I) confers constitutive transcriptional activity in the absence of phosphorylation, but a V. cholerae flrCM114I mutant strain, although flagellated and motile, was also defective in its ability to colonize. The strains carrying D54A or M114I mutant FlrC proteins expressed normal levels of CT and TCP under in vitro inducing conditions. Our results show that FlrC 'locked' into either an inactive (D54A) or an active (M114I) state results in colonization defects, thereby demonstrating a requirement for modulation of FlrC activity during V. cholerae pathogenesis. Thus, the sigma(54)-dependent transcriptional activity of the flagellar regulatory protein FlrC contributes not only to motility, but also to colonization of V. cholerae.