Virulence Mechanisms of the Emerging Pathogen Stenotrophomonas maltophilia

 描述（由申请人提供）：嗜麦芽窄食单胞菌（Sm）是一种环境革兰氏阴性细菌，最近已成为重要的机会致病菌和医院病原体，特别是呼吸道和血流。链球菌感染嗜麦芽窄食单胞菌通常很难治疗，因为S.嗜麦芽窄食单胞菌菌株对多种类型的抗生素具有敏感性。尽管Sm在肺炎、血流感染和各种其他疾病（包括尿路、CNS、眼、皮肤和软组织感染）中的作用，但Sm的发病机制一直未得到充分研究。在一个小NIH R 03补助金的帮助下，我们以前开始了旨在开发研究Sm的方法的研究。在建立了Sm肺炎的小鼠模型并开发了用于诱变测序的Sm菌株K279 a的方法之后，我们研究了Sm中II型蛋白分泌（T2 S）的功能。我们实验室和其他人以前的工作表明，T2 S系统是各种病原体毒力的主要促进因素，包括肺炎和血流感染的其他病原体。通过T2 S分泌的蛋白质通常包括多种降解性和组织破坏性酶。我们记录了Xps T2 S系统在菌株K279 a中是功能性的，分泌至少七种蛋白质。重要的是，我们确定Xps介导两种丝氨酸蛋白酶（StmPr 1和StmPr 2）的输出，这两种丝氨酸蛋白酶引起人肺上皮细胞系（即，I型A549细胞）经历变圆、肌动蛋白重排、脱离和死亡。因此，我们假设Xps T2 S及其底物，包括StmPr 1和StmPr 2，在S.嗜麦芽窄食症基因组数据进一步表明，Sm具有第二个T2 S系统（Gsp）以及IV型（T4 S）和I型（T1 S）蛋白分泌系统。我们推测，这些其他系统分泌的蛋白质也代表Sm的毒力因子。为了解决这些假设，我们提出i）在两种不同的疾病鼠模型（公开的肺炎模型和新的菌血症模型）中测试Xps突变体（Aim 1），ii）在肺炎和菌血症模型中测试stmPr 1 stmPr 2突变体，并且随着StmPr 1和/或StmPr 2的重要性的确认，开始确定蛋白酶如何介导宿主细胞死亡（Aim 2），以及iii）测试Gsp，T4 S和T1 S突变体。从这项探索性R21资助的结果来看，我们将为新生的Sm发病机制领域建立一个工作基础，以便未来的努力能够集中于表征最重要的分泌系统和分泌蛋白。除了提供急需的洞察Sm发病机制，获得的数据有可能发现新类型的毒力决定因素，并导致新形式的疾病诊断，治疗或预防。.

 DESCRIPTION (provided by applicant): Stenotrophomonas maltophilia (Sm) is an environmental, Gram-negative bacterium that has recently emerged as an important opportunistic and nosocomial pathogen, particularly of the respiratory tract and bloodstream. Infection with S. maltophilia is often very difficult to treat because of the resistance that S. maltophilia strains have toward multiple types of antibiotics. Despite the role of Sm in pneumonia, bloodstream infections, and various other diseases, including urinary tract, CNS, eye, skin, and soft tissue infections, the pathogenesis of Sm has been grossly understudied. With the aid of a small NIH R03 grant, we previously embarked upon research aimed at developing methods to study Sm. After having established a murine model for Sm pneumonia and developed means for mutagenizing the sequenced Sm strain K279a, we investigated the functioning of type II protein secretion (T2S) in Sm. Previous work from our lab and others had shown that T2S systems are major facilitators of virulence in various pathogens, including other agents of pneumonia and bloodstream infection. Proteins secreted via T2S typically include a wide variety of degradative and tissue- destructive enzymes. We documented that the Xps T2S system is functional in strain K279a, secreting at least seven protein species. Importantly, we determined that Xps mediates the export of two serine proteases (StmPr1 and StmPr2) that cause a human lung epithelial cell line (i.e., type I A549 cells) to undergo rounding, actin-rearrangement, detachment, and death. Thus, we hypothesize that Xps T2S and its substrates, including StmPr1 and StmPr2, are critical in the pathogenesis of S. maltophilia. Genomic data further indicate that Sm possesses a second T2S system (Gsp) as well as type IV (T4S) and type I (T1S) protein secretion systems. We hypothesize that proteins secreted by these other systems also represent virulence factors of Sm. To address these hypotheses, we propose to i) test the Xps mutant in two different murine models of disease, the published pneumonia model and a new bacteremia model (Aim 1), ii) test a stmPr1 stmPr2 mutant in the pneumonia and bacteremia models, and with confirmation of the importance of StmPr1 and/or StmPr2 begin to determine how the proteases mediate host cell death (Aim 2), and iii) test Gsp, T4S, and T1S mutants in the two disease models. From the results of this exploratory R21 grant, we will establish a working foundation for the nascent Sm pathogenesis field such that future efforts will be able to focus on characterizing the secretion system(s) and secreted proteins that are most important. In addition to providing much-needed insight into Sm pathogenesis, the data obtained have the potential to both uncover new types of virulence determinants and lead to new forms of disease diagnosis, treatment, or prevention. .