Acquisition of expression of the Pseudomonas aeruginosa ExoU cytotoxin leads to increased bacterial virulence in a murine model of acute pneumonia and systemic spread
Acquisition of expression of the Pseudomonas aeruginosa ExoU cytotoxin leads to increased bacterial virulence in a murine model of acute pneumonia and systemic spread
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DOI：
10.1128/iai.68.7.3998-4004.2000
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发表时间：
2000-07-01
影响因子：
3.1
通讯作者：
Pier, GB
中科院分区：
文献类型：
--
作者：
Allewelt, M;Coleman, FT;Pier, GB
铜绿假单胞菌是最常从呼吸道分离到的医院细菌病原体。这种感染的动物模型对于毒力和免疫的研究极其有价值。因此，我们通过将细菌施用于麻醉动物的鼻孔后表征 BALB/c 小鼠的细菌感染过程，评估了简单的急性肺炎模型用于分析铜绿假单胞菌毒力的效用。细菌被快速吸入肺部，几分钟内即可从肺部回收 67% 至 100% 的接种物，感染后不久在细胞内发现了 0.1% 至 1% 的接种物。对受感染肺部的单细胞悬浮液进行庆大霉素排除分析表明，在后来的时间点，多达 10% 的细菌存在于细胞内。铜绿假单胞菌表达的外切酶 U (ExoU) 与体外上皮细胞的细胞毒性作用和动物模型中的毒力相关。 exoU 基因中的插入突变赋予突变株非细胞毒性表型并降低对动物的毒力。我们使用急性肺炎模型来确定将 exoU 基因引入缺乏该基因的非细胞毒性铜绿假单胞菌菌株中是否会影响毒力。用携带 exoU 基因及其相关分子伴侣的 pUCP19exoUspcU 转化 7 个表型非细胞毒性铜绿假单胞菌菌株，其中 3 个菌株对体外培养的上皮细胞具有细胞毒性。这些菌株均分泌ExoU，通过用特异性抗血清检测ExoU蛋白证实，对于携带质粒pUCP19exoUspcU的所有三个铜绿假单胞菌分离株，表达exoU的菌株的50%致死剂量显着低于同基因exoU阴性菌株。在感染了转化的铜绿假单胞菌菌株的动物的肺部中，很容易检测到 ExoU 特异的 mRNA。 exoU基因的引入赋予一些但不是全部非细胞毒性铜绿假单胞菌菌株细胞毒性表型，并且对于能够表达ExoU的重组菌株，在急性肺炎和全身传播的小鼠模型中毒力显着增加。
Pseudomonas aeruginosa is the nosocomial bacterial pathogen most commonly isolated from the respiratory tract. Animal models of this infection are extremely valuable for studies of virulence and immunity. We thus evaluated the utility of a simple model of acute pneumonia for analyzing P. aeruginosa virulence by characterizing the course of bacterial infection in BALB/c mice following application of bacteria to the nares of anesthetized animals. Bacterial aspiration into the lungs was rapid, and 67 to 100% of the inoculum could be recovered within minutes from the lungs, with 0.1 to 1% of the inoculum found intracellularly shortly after infection. At later time points up to 10% of the bacteria were intracellular, as revealed by gentamicin exclusion assays on single-cell suspensions of infected lungs. Expression of exoenzyme U (ExoU) by P. aeruginosa is associated with a cytotoxic effect on epithelial cells in vitro and virulence in animal models. Insertional mutations in the exoU gene confer a noncytotoxic phenotype on mutant strains and decrease virulence for animals. We used the model of acute pneumonia to determine whether introduction of the exoU gene into noncytotoxic strains of P. aeruginosa lacking this gene affected virulence. Seven phenotypically noncytotoxic P. aeruginosa strains were transformed with pUCP19exoUspcU which carries the exoU gene and its associated chaperone, Three of these strains became cytotoxic to cultured epithelial cells in vitro. These strains all secreted ExoU, as confirmed by detection of the ExoU protein with specific antisera, The 50% lethal dose of exoU-expressing strains was significantly lower for all three P. aeruginosa isolates carrying plasmid pUCP19exoUspcU than for the isogenic exoU-negative strains. mRNA specific for ExoU was readily detected in the lungs of animals infected with the transformed P. aeruginosa strains. Introduction of the exoU gene confers a cytotoxic phenotype on some, but not all, otherwise-noncytotoxic P. aeruginosa strains and, for recombinant strains that could express ExoU, there was markedly increased virulence in a murine model of acute pneumonia and systemic spread.