Negative regulation of virulence in Pseudomonas aeruginosa

铜绿假单胞菌毒力的负调控

• 批准号: 8313355
• 负责人: Albert Siryaporn
• 金额: $ 5.39万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-01 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8313355
• 关键词: A549; Amoeba genus; Antibiotics; Attenuated; Bacteria; Biological Assay; Cell Death; Cell Line; Cell surface; Cell-Matrix Junction; Cells; Chemotaxis; Complex; Cystic Fibrosis; Data; Defect; Detection; Development; Dictyostelium; Dictyostelium discoideum; Elastases; Employee Strikes; Environment; Epithelial; Epithelial Cells; Event; Exhibits; Flagella; Foundations; Future; Gene Expression; Genes; Genetic Transcription; Genome; Goals; Gram-Negative Bacteria; Growth; Hospitals; Human; Infection; Investigation; Knock-out; Libraries; Light; Lipase; Lung; Mammalian Cell; Measures; Mechanics; Membrane; Microarray Analysis; Microbial Biofilms; Microfluidic Microchips; Molecular; Motor; Mus; Needles; Nutrient; Pathogenesis; Patients; Phenotype; Pilum; Pneumonia; Production; Proteins; Pseudomonas aeruginosa; Regulation; Reporter; Role; Screening procedure; Sepsis; Signal Pathway; Signal Transduction; Solid; Staging; Stimulus; Surface; System; Temperature; Testing; Therapeutic; Time; Toxin; Transcriptional Regulation; Urinary tract infection; Virulence; Virulence Factors; Virulent; Work; Wound Infection; cell motility; cytotoxicity; diguanylate cyclase; extracellular; improved; in vivo; macrophage; mutant; novel; pathogen; porin; protein-histidine kinase; quorum sensing; research study; response; sensor

A number of environmental signals regulate the expression of virulence factors, such as the availability of nutrients, temperature and pH. One potential stimulator of virulence expression that has not been characterized in detail is the presence of solid surfaces. My preliminary experiments in Pseudomonas aeruginosa suggest that transcription of virulence genes is upregulated in cells that are attached to abiotic surfaces Surface-attached cells are also more virulent towards the host Dictyostelium discoideum. I will test the hypothesis that virulence is regulated by surface attachment. In particular, I will characterize the P. aeruginosa response to surface attachment in vivo using fluorescent protein transcriptional reporters and using microarray analysis and cell cytotoxicity assays. I will verify that surface-attached cells exhibit increased virulence towards mouse macrophage and human lung epithelial cells. Guided by microarray analysis of planktonic and surface-attached cells, I have also developed fluorescent protein transcriptional reporters to genes that are regulated by surface attachment. I will track the expression dynamics of these genes using finely-tuned microfluidic devices as cells transition from planktonic growth to surface-attachment. I will also test the hypothesis that cells possess a molecular sensor that detects the presence of surfaces. This will be achieved by knocking out candidate sensors in the fluorescent protein transcriptional reporter strains and testing strains for transcriptionally insensitivity to the presence of surfaces. In particular, my preliminary results indicate that the needle tip protein PcrV is required for surface-induced activation of virulence. While the type III secretion needle has a clear role in delivery of toxins to host cells, I will test the hypothesis that the type III secretion needle is also a sensor that detects the presence of surfaces. I will test whether pcrV mutants and mutants of its interacting partner PcrG are transcriptionally sensitive to the presence of surfaces. Previously, I performed a screen for mutants that are hyper-virulent during planktonic growth. These cells exhibit a striking increase in virulence towards Dictyostelium cells, mouse macrophage cells and human lung epithelial cells. I will test the hypothesis that these strains are defective in the detection of surfaces using the fluorescent protein transcriptional reporters above, microarray analysis and cytotoxicity assays. In particular, I will focus my initial efforts on characterizing a mutant that contains a disruption i roeA, which encodes a diguanylate cyclase that is involved in the transition from planktonic growth to sessile growth on surfaces. If time permits, I will identify the mechanisms of hyper-virulence in these mutants by knocking out known virulence factors and screening for mutants with attenuated virulence. The findings from this study will shed light on how bacterial cells detect surfaces and how virulence is regulated. Additionally, it may also provide further details about the initial stages of biofilm formation.

许多环境信号调节毒力因子的表达，例如营养物质的可用性、温度和 pH 值。尚未详细表征的一种潜在的毒力表达刺激因素是固体表面的存在。我在铜绿假单胞菌中的初步实验表明，附着在非生物表面的细胞中毒力基因的转录上调。表面附着的细胞对宿主盘基网柄菌也更具毒性。 我将检验毒力受表面附着调节的假设。特别是，我将使用荧光蛋白转录报告基因并使用微阵列分析和细胞毒性测定来表征铜绿假单胞菌对体内表面附着的反应。我将验证表面附着的细胞对小鼠巨噬细胞和人肺上皮细胞表现出更强的毒力。在浮游细胞和表面附着细胞的微阵列分析的指导下，我还开发了针对受表面附着调节的基因的荧光蛋白转录报告基因。当细胞从浮游生长过渡到表面附着时，我将使用微调的微流体装置跟踪这些基因的表达动态。 我还将检验细胞拥有检测表面存在的分子传感器的假设。这将通过敲除荧光蛋白转录报告菌株中的候选传感器并测试菌株对表面存在的转录不敏感性来实现。特别是，我的初步结果表明，针尖蛋白 PcrV 是表面诱导毒力激活所必需的。虽然 III 型分泌针在将毒素输送到宿主细胞方面具有明显的作用，但我将检验以下假设：III 型分泌针也是检测表面存在的传感器。我将测试 pcrV 突变体及其相互作用伙伴 PcrG 的突变体是否对表面的存在转录敏感。 此前，我对浮游生长过程中的高毒力突变体进行了筛选。这些细胞对盘基网柄菌细胞、小鼠巨噬细胞和人肺上皮细胞的毒力显着增加。我将测试以下假设：这些菌株在使用上述荧光蛋白转录报告基因、微阵列分析和细胞毒性测定进行表面检测方面存在缺陷。特别是，我最初的努力将集中于表征包含 roeA 破坏的突变体，该突变体编码一种二鸟苷酸环化酶，参与从浮游生长到表面无柄生长的转变。如果时间允许，我将通过敲除已知的毒力因子并筛选毒力减弱的突变体来确定这些突变体的高毒力机制。这项研究的结果将揭示细菌细胞如何检测表面以及如何调节毒力。此外，它还可以提供有关生物膜形成初始阶段的更多详细信息。