ExoU expression by Pseudomonas aeruginosa correlates with acute cytotoxicity and epithelial injury
ExoU expression by Pseudomonas aeruginosa correlates with acute cytotoxicity and epithelial injury
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DOI：
10.1046/j.1365-2958.1997.4891851.x
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发表时间：
1997-08-01
影响因子：
3.6
通讯作者：
Frank, DW
中科院分区：
文献类型：
--
作者：
FinckBarbancon, V;Goranson, J;Frank, DW
外切酶S的产生与铜绿假单胞菌从上皮定殖位点传播并在烧伤和急性肺感染模型中引起致命败血症的能力相关。外切酶S作为两种多肽ExoS和ExoT的非共价聚集体从培养物上清液中纯化。ExoS和ExoT由独立但高度相似的基因exoS和exoT编码。在体内损伤肺上皮和在体外具有细胞毒性的临床分离株具有exoT但缺乏exoS，表明ExoS不是导致这些测定中测量的病理和细胞死亡的细胞毒素。我们在exoT中构建了一个特定的突变，并表明该菌株PA 103 exoT：：Tc在体外具有细胞毒性，并在体内引起上皮损伤，这表明另一种细胞毒素是所观察到的病理学的原因。为了鉴定与急性细胞毒性相关的蛋白质，我们比较了PA 103、其同基因非细胞毒性衍生物PA 103 exsA：：Omega和几种细胞毒性和非细胞毒性铜绿假单胞菌临床分离株的细胞外蛋白质谱。该分析表明，除了ExoT的表达外，70-kDa蛋白质的表达与细胞毒性表型相关。70 kDa蛋白的特异性抗体结合细胞毒性分离株的细胞外蛋白，但未能结合非细胞毒性菌株或PA 103 exsA：：Omega的类似抗原制剂。为了克隆编码这种潜在的细胞毒素的基因，我们使用Tn 5 Tc诱变和免疫印迹筛选分离插入突变体PA 103 exoU：：Tn 5 Tc，其不再表达70-kDa胞外蛋白，但保持ExoT的表达。PA 103 exoU：：Tn 5 Tc在急性肺感染模型中是无细胞毒性的，并且未能损伤上皮。PA 103 exoU：：Tn 5 Tc与exoU的互补恢复了细胞毒性和上皮损伤。ExoU、ExoS和ExoT具有相似的启动子结构和相同的转录激活因子ExsA结合位点，数据与它们的协同调节一致。此外，所有三种蛋白质的前六个氨基酸几乎相同，表明可能参与铜绿假单胞菌III型分泌器识别的共同氨基末端基序。
The production of exoenzyme S is correlated with the ability of Pseudomonas aeruginosa to disseminate from epithelial colonization sites and cause a fatal sepsis in burn injury and acute lung infection models, Exoenzyme S is purified from culture supernatants as a non-covalent aggregate of two polypeptides, ExoS and ExoT. ExoS and ExoT are encoded by separate but highly similar genes, exoS and exoT. Clinical isolates that injure lung epithelium in vivo and that are cytotoxic in vitro possess exoT but lack exoS, suggesting that ExoS is not the cytotoxin responsible for the pathology and cell death measured in these assays. We constructed a specific mutation in exoT and showed that this strain, PA103 exoT::Tc, was cytotoxic in vitro and caused epithelial injury in vivo, indicating that another cytotoxin was responsible for the observed pathology. To identify the protein associated with acute cytotoxicity, we compared extracellular protein profiles of PA103, its isogenic non-cytotoxic derivative PA103 exsA::Omega and several cytotoxic and non-cytotoxic P. aeruginosa clinical isolates, This analysis indicated that, in addition to expression of ExoT, expression of a 70-kDa protein correlated with the cytotoxic phenotype. Specific antibodies to the 70-kDa protein bound to extracellular proteins from cytotoxic isolates but failed to bind to similar antigen preparations from non-cytotoxic strains or PA103 exsA::Omega. To clone the gene encoding this potential cytotoxin we used Tn5Tc mutagenesis and immunoblot screening to isolate an insertional mutant, PA103exoU:: Tn5Tc, which no longer expressed the 70-kDa extracellular protein but maintained expression of ExoT. PA103 exoU::Tn5Tc was non-cytotoxic and failed to injure the epithelium in an acute lung infection model. Complementation of PA103exoU::Tn5Tc with exoU restored cytotoxicity and epithelial injury. ExoU, ExoS and ExoT share similar promoter structures and an identical binding site for the transcriptional activator, ExsA, data consistent with their co-ordinate regulation, In addition, all three proteins are nearly identical in the first six amino acids, suggesting a common amino terminal motif that may be involved in the recognition of the type III secretory apparatus of P. aeruginosa.