NRSA in Support of Meg Nas: Discovering the Novel Type IV Secretion System of Stenotrophomonas Maltophilia.

摘要 嗜麦芽窄食单胞菌(Sm)是一种革兰氏阴性、多药耐药的细菌，其感染日益严重。 被认为是一种重要的机会性和院内病原体。SM感染通常表现为 肺炎和血流感染，但也可引起中枢神经系统、眼睛、皮肤、软组织和尿路 感染并作为囊性纤维化(CF)患者肺部恶化的危险因素。尽管如此，还是有 对Sm毒力的最低了解。我的实验室已经证明了Sm能引起肺上皮细胞 脱离和死亡，还表明Sm编码一种II型蛋白分泌系统，触发 肺上皮细胞的凋亡。在完成关于另一个Sm分泌因子的工作后，一个铁载体 在我确定是一种新的儿茶酚后，我开始鉴定一种编码的IV型分泌系统(T4SS) 在临床分离株K279a的基因组中。我证明了Sm T4SS核心成分的突变 复合体(VirB10)可导致肺上皮细胞脱离和死亡，提示Sm T4SS 抑制细胞死亡。我发现Sm T4SS可以抑制星状孢子素诱导的细胞死亡和caspase 因此确定Sm T4SS对人上皮细胞具有抗凋亡作用。我有能力 确认Sm感染原代人支气管/气管上皮细胞的表型。我测试了 Sm T4SS对人巨噬细胞系(U937)及其体外培养巨噬细胞的影响 A/J小鼠股骨，确定Sm T4SS对巨噬细胞有促凋亡作用。两者都有 表型需要细菌与宿主细胞接触，在其他四株Sm临床分离株中也有明显表现。 我还确定，这两种细胞凋亡表型都不是野生型(Wt)与virB10的差异所致 突变的Sm附着在宿主细胞上。此外，我还通过鼻腔感染WT和WT感染了A/J小鼠 VirB10突变体Sm的T4SS促进了Sm在小鼠肺内的生长。我还确定了Sm 通过菌落测定，T4SS与大肠杆菌和铜绿假单胞菌共培养时具有生长优势。 24小时共培养后形成单位(CFU)。这是一种表型，以前只被归因于 VI型分泌系统直到最近才被归类为Xanthomonas和Bartonella T4SS。在……里面 通过使用多个软件程序进行生物信息学分析，我列出了18个可能的Sm T4s 效应器。通过进行细菌双杂交试验，我确定这18种蛋白质确实与 SM T4SS装置通过其偶联蛋白VirD4。综上所述，这些数据表明，Sm具有独特的 T4SS具有三种不同的细胞依赖表型。因此，我建议i)确定Sm T4SS效应器 调节宿主细胞凋亡(目标1)和II)评估Sm T4SS依赖的生长优势。 铜绿假单胞菌和其他共生于肺的细菌(目标2)。除了提供批判性的理解 对于Sm，这项工作将对存在于其他细菌基因组中的许多未定义的T4s产生影响 并帮助我们评估T4SS作为新的抗微生物药物的靶标。

Abstract Stenotrophomonas maltophilia (Sm), a gram negative, multi-drug resistant bacterium, is increasingly recognized as an important opportunistic and nosocomial pathogen. Sm infection commonly manifests as pneumonia and blood stream infections but can also cause CNS, eye, skin, soft tissue, and urinary tract infections and act as a risk factor for lung exacerbations in Cystic Fibrosis (CF) patients. Despite this, there is minimal understanding of Sm virulence. My lab has demonstrated that Sm causes lung epithelial cell detachment and death and has also shown that Sm encodes a type II protein secretion system which triggers apoptosis in lung epithelial cells. After completing work regarding another Sm secreted factor, a siderophore that I determined to be a novel catecholate, I began characterizing a type IV secretion system (T4SS) encoded in the genome of clinical isolate K279a. I demonstrated that a mutation in a component of the Sm T4SS core complex (virB10) results in enhanced lung epithelial cell detachment and death indicating that the Sm T4SS inhibits cell death. I showed that the Sm T4SS inhibits staurosporine induced cell death as well as caspase activation and thus determined the Sm T4SS has an anti-apoptotic effect on human epithelial cells. I was able to confirm this phenotype upon Sm infection of primary human bronchial/tracheal epithelial cells. I tested the effect of the Sm T4SS on a human macrophage cell line (U937) and explanted macrophages obtained from A/J mouse femurs and determined that the Sm T4SS elaborates a pro-apoptotic effect on macrophages. Both phenotypes necessitated bacterial contact with the host cell and were evident in four other Sm clinical isolates. I also determined that neither apoptosis phenotype was attributed to a difference in wildtype (WT) vs virB10 mutant Sm attachment to the host cells. Moreover, I showed by intranasally infecting A/J mice with WT and virB10 mutant Sm that the T4SS enhanced the growth of Sm in mouse lungs. I also determined that the Sm T4SS confers a growth advantage when co-cultured with E. coli and P. aeruginosa as measured by colony forming units (CFUs) after a 24 hour co-culture. This is a phenotype that was previously only attributed to the type VI secretion system until recently when it was attributed to the Xanthomonas and Bartonella T4SS. In performing bioinformatic analysis using multiple software programs, I developed a list of 18 putative Sm T4SS effectors. By performing a bacterial two-hybrid assay, I determined that the 18 proteins indeed interact with the Sm T4SS apparatus via its coupling protein VirD4. Taken together, these data indicate Sm possesses a unique T4SS with three different cell-dependent phenotypes. Thus, I propose to i) determine the Sm T4SS effectors that modulate host cell apoptosis (Aim 1) and ii) assess Sm T4SS dependent growth advantages against P. aeruginosa and other bacteria that co-habitate the CF lung (Aim 2). Aside from providing critical understanding of Sm, this work will have implications for the many undefined T4SS that exist in the genomes of other bacteria and aid in our assessment of T4SS as a target for new anti-microbials.