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

NRSA 支持 Meg Nas：发现嗜麦芽寡养单胞菌的新型 IV 型分泌系统。

• 批准号: 9761206
• 负责人: Megan Yasemin Nas
• 金额: $ 4.02万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9761206
• 关键词: A/J Mouse; Address; Antibiotic Resistance; Apoptosis; Apoptotic; Assimilations; Bacteria; Bartonella; Bioinformatics; Biological Assay; Blood; Blood Circulation; Caspase; Cell Death; Cell Line; Cell surface; Cells; Cessation of life; Clinical; Coculture Techniques; Colony-forming units; Complex; Computer software; Coupling; Cystic Fibrosis; Cytoplasm; DNA delivery; Data; Drug Targeting; Drug resistance; Epithelial Cells; Escherichia coli; Eye; Fellowship; Femur; Genes; Genome; Genomics; Growth; Health; Hour; Human; Immune system; In Vitro; Individual National Research Service Award; Infection; Knowledge; Lead; Lung; Measures; Mediating; Microbe; Multiple Bacterial Drug Resistance; Mus; Mutate; Mutation; National Research Service Awards; Nutrient; PAWR protein; Pathogenesis; Pathway interactions; Phenotype; Pneumonia; Prevalence; Primary Infection; Process; Protein Secretion; Proteins; Pseudomonas aeruginosa; Pulmonary Fibrosis; Research; Risk Factors; Scientist; Serine Protease; Siderophores; Skin; Staurosporine; Stenotrophomonas maltophilia; Stream; System; Testing; Two-Hybrid System Techniques; Type IV Secretion System Pathway; Urinary tract infection; Virulence; Virulence Factors; Work; Xanthomonas; antimicrobial; bactericide; cell type; cystic fibrosis infection; cystic fibrosis patients; drug development; extracellular; in vivo; macrophage; monolayer; mortality; multi-drug resistant pathogen; mutant; novel; pathogen; pre-doctoral; prevent; programs; soft tissue

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.

摘要 嗜麦芽窄食单胞菌（Stenotrophomonasmaltophilia，Sm）是一种革兰氏阴性、多重耐药菌，近年来在临床上日益受到重视 被认为是一种重要的机会致病菌和院内致病菌。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、软组织和泌尿道 在囊性纤维化（CF）患者中，肺感染是肺恶化的危险因素。尽管如此， 对Sm毒力的了解最少。我的实验室已经证明Sm会导致肺上皮细胞 分离和死亡，并已表明Sm编码II型蛋白分泌系统， 肺上皮细胞凋亡。在完成关于另一种Sm分泌因子的工作后， 我确定这是一种新的儿茶酚，我开始描述IV型分泌系统（T4 SS）编码 在临床分离株K279 a的基因组中。我证明了Sm T4 SS核心部分的突变 复合物（virB 10）导致肺上皮细胞脱落和死亡增加，表明SmT 4SS 抑制细胞死亡。我发现Sm T4 SS抑制星形孢菌素诱导的细胞死亡以及半胱天冬酶， 激活，从而确定Sm T4 SS对人上皮细胞具有抗凋亡作用。我能够 以证实Sm感染原代人支气管/气管上皮细胞后的这种表型。我测试了 Sm T4 SS对人巨噬细胞系（U937）和从人巨噬细胞系（U937）中获得的巨噬细胞的作用 A/J小鼠股骨，并确定Sm T4 SS阐述了对巨噬细胞的促凋亡作用。两 表型需要细菌与宿主细胞接触，并且在其他四种Sm临床分离株中是明显的。 我还确定了两种凋亡表型都不是由野生型（WT）与virB 10的差异引起的 突变体Sm附着于宿主细胞。此外，通过鼻内感染WT A/J小鼠， virB 10突变体Sm在小鼠肺中的生长受到T4 SS的促进。我还确定， T4 SS与E.大肠杆菌和铜绿假单胞菌（按菌落测定） 共培养24小时后的CFU。这是一个表型，以前只归因于 VI型分泌系统直到最近才被归于黄单胞菌属和巴尔通体属T4 SS。在 使用多种软件程序进行生物信息学分析，我开发了一个18个假定的Sm T4 SS的列表， 效应器通过进行细菌双杂交试验，我确定了这18种蛋白质确实与 Sm T4 SS装置通过其偶联蛋白VirD 4。总之，这些数据表明Sm具有独特的 T4 SS具有三种不同的细胞依赖性表型。因此，我建议i）确定Sm T4 SS效应子 调节宿主细胞凋亡（目的1）和ii）评估Sm T4 SS依赖性生长优势对P。 铜绿假单胞菌和共存于CF肺的其他细菌（目的2）。除了提供批判性的理解 这项工作将对其他细菌基因组中存在的许多未定义的T4 SS产生影响 并帮助我们评估T4 SS作为新抗微生物药物的靶点。