cAMP and Vfr Control Exolysin Expression and Cytotoxicity of Pseudomonas aeruginosa Taxonomic Outliers
cAMP and Vfr Control Exolysin Expression and Cytotoxicity of Pseudomonas aeruginosa Taxonomic Outliers
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DOI：
10.1128/jb.00135-18
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发表时间：
2018-06-01
影响因子：
3.2
通讯作者：
Elsen, Sylvie
中科院分区：
文献类型：
--
作者：
Berry, Alice;Han, Kook;Elsen, Sylvie
由属于PA7组的铜绿假单胞菌菌株表达的双伴侣分泌系统ExIBA由于宿主细胞膜的破坏而诱导肺出血。在这里，我们表明，exIBA基因的控制由cAMP和毒力因子调节器（VFR）的途径。与cAMP相互作用后，Vfr以高亲和力直接结合exIBA启动子（平衡结合常数[K-eq]约为2.5 nM）。在Vfr阴性突变体中，exIB和exIA表达减少，并且随着细胞内cAMP水平的增加而上调。在exIBA启动子中的Vfr结合序列原位突变，导致突变体的细胞毒性降低，表明Vfr是上皮细胞中毒期间exIBA表达所需的。Vfr还调节先前显示促进上皮细胞上的ExIA活性的4型pill的功能，这表明cAMP/Vfr途径协调完全细胞毒性所需的这两个因子。如在大多数铜绿假单胞菌菌株中，腺苷酸环化酶CyaB是在体外生长和真核细胞感染期间用于Vfr调节的cAMP的主要提供者。我们发现，在参考菌株PA7的功能性Vfr的情况下是由基因的移码引起的，并占其细胞毒性降低，揭示了保护ExIBA控制的CyaB-cAMP/Vfr途径在铜绿假单胞菌分类outliers.IMPORTANCE人类机会致病菌铜绿假单胞菌引起严重的急性和慢性人类感染与定义的毒力因子。铜绿假单胞菌分类异常值的主要毒力决定因素是外溶素，这是一种属于双伴侣分泌系统ExIBA的破坏膜孔形成毒素。在这项工作中，我们证明了保守的CyaBcAMP/Vfr途径通过直接转录激活exIBA操纵子来控制异常临床菌株的细胞毒性。因此，尽管III型分泌系统和外泌溶素在经典菌株和离群菌株中分别是相互排斥的，但这两个主要毒力决定因子在其调节机制方面具有相似性。
The two-partner secretion system ExIBA, expressed by strains of Pseudomonas aeruginosa belonging to the PA7 group, induces hemorrhage in lungs due to disruption of host cellular membranes. Here we demonstrate that the exIBA genes are controlled by a pathway consisting of cAMP and the virulence factor regulator (Vfr). Upon interaction with cAMP, Vfr binds directly to the exIBA promoter with high affinity (equilibrium binding constant [K-eq] of approximate to 2.5 nM). The exIB and exIA expression was diminished in the Vfr-negative mutant and upregulated with increased intracellular cAMP levels. The Vfr binding sequence in the exIBA promoter was mutated in situ, resulting in reduced cytotoxicity of the mutant, showing that Vfr is required for the exIBA expression during intoxication of epithelial cells. Vfr also regulates function of type 4 pill previously shown to facilitate ExIA activity on epithelial cells, which indicates that the cAMP/Vfr pathway coordinates these two factors needed for full cytotoxicity. As in most P. aeruginosa strains, the adenylate cyclase CyaB is the main provider of cAMP for Vfr regulation during both in vitro 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 ExIBA control by the CyaB-cAMP/Vfr pathway in P. aeruginosa taxonomic outliers.IMPORTANCE The human opportunistic pathogen Pseudomonas aeruginosa provokes severe acute and chronic human infections associated with defined sets of virulence factors. The main virulence determinant of P. aeruginosa taxonomic outliers is exolysin, a membrane-disrupting pore-forming toxin belonging to the two-partner secretion system ExIBA. In this work, we demonstrate that the conserved CyaBcAMP/Vfr pathway controls cytotoxicity of outlier clinical strains through direct transcriptional activation of the exIBA 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.