Biology of Cancer and Infection, U1036 INSERM, CEA, University of Grenoble Alpes, ERL5261 CNRS, Grenoble, France.
Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA.
J Bacteriol. 2018 May 24;200(12). doi: 10.1128/JB.00135-18. Print 2018 Jun 15.
The two-partner secretion system ExlBA, expressed by strains of belonging to the PA7 group, induces hemorrhage in lungs due to disruption of host cellular membranes. Here we demonstrate that the genes are controlled by a pathway consisting of cAMP and the virulence factor regulator (Vfr). Upon interaction with cAMP, Vfr binds directly to the promoter with high affinity (equilibrium binding constant [] of ≈2.5 nM). The and expression was diminished in the Vfr-negative mutant and upregulated with increased intracellular cAMP levels. The Vfr binding sequence in the promoter was mutated , resulting in reduced cytotoxicity of the mutant, showing that Vfr is required for the expression during intoxication of epithelial cells. Vfr also regulates function of type 4 pili previously shown to facilitate ExlA activity on epithelial cells, which indicates that the cAMP/Vfr pathway coordinates these two factors needed for full cytotoxicity. As in most strains, the adenylate cyclase CyaB is the main provider of cAMP for Vfr regulation during both growth and eukaryotic cell infection. We discovered that the absence of functional Vfr in the reference strain PA7 is caused by a frameshift in the gene and accounts for its reduced cytotoxicity, revealing the conservation of ExlBA control by the CyaB-cAMP/Vfr pathway in taxonomic outliers. The human opportunistic pathogen provokes severe acute and chronic human infections associated with defined sets of virulence factors. The main virulence determinant of taxonomic outliers is exolysin, a membrane-disrupting pore-forming toxin belonging to the two-partner secretion system ExlBA. In this work, we demonstrate that the conserved CyaB-cAMP/Vfr pathway controls cytotoxicity of outlier clinical strains through direct transcriptional activation of the operon. Therefore, despite the fact that the type III secretion system and exolysin are mutually exclusive in classical and outlier strains, respectively, these two major virulence determinants share similarities in their mechanisms of regulation.
由属于 PA7 群的菌株表达的双伙伴分泌系统 ExlBA，由于破坏宿主细胞膜而导致肺部出血。在这里，我们证明 基因受包含环腺苷酸 (cAMP) 和毒力因子调节因子 (Vfr) 的途径控制。当与 cAMP 相互作用时，Vfr 以高亲和力（平衡结合常数 [Kd] 约为 2.5 nM）直接结合到 启动子上。Vfr 阴性突变体中 的表达减少，细胞内 cAMP 水平增加时上调。 启动子中的 Vfr 结合序列发生突变 ，导致突变体的细胞毒性降低，表明 Vfr 是上皮细胞中毒时 表达所必需的。Vfr 还调节先前显示有助于 ExlA 在上皮细胞上活性的 IV 型菌毛的功能，这表明 cAMP/Vfr 途径协调了这两种因子对于完全细胞毒性所必需的。与大多数 菌株一样，腺苷酸环化酶 CyaB 是 Vfr 调节期间细菌生长和真核细胞感染的主要 cAMP 提供者。我们发现，参考菌株 PA7 中功能性 Vfr 的缺失是由于 基因中的移码，导致其细胞毒性降低，这揭示了 CyaB-cAMP/Vfr 途径在 分类学异常中对 ExlBA 控制的保守性。人类机会性病原体 引起与明确毒力因子集相关的严重急性和慢性人类感染。分类学异常的主要毒力决定因素是外毒素，一种属于双伙伴分泌系统 ExlBA 的膜破坏孔形成毒素。在这项工作中，我们证明了保守的 CyaB-cAMP/Vfr 途径通过直接转录激活 操纵子来控制异常临床菌株的细胞毒性。因此，尽管 III 型分泌系统和外毒素在经典和异常菌株中分别是相互排斥的，但这两种主要毒力决定因素在其调节机制上存在相似之处。
