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，如营养物质的可用性、温度和ph。一个尚未被详细描述的毒力表达的潜在刺激因素是固体表面的存在。我对铜绿假单胞菌的初步实验表明，在附着于非生物表面的细胞中，毒力基因的转录是上调的。附着于非生物表面的细胞对宿主盘状盘基钢柱的毒力也更强。