Negative regulation of virulence in Pseudomonas aeruginosa

铜绿假单胞菌毒力的负调控

• 批准号: 8423822
• 负责人: Albert Siryaporn
• 金额: $ 5.57万
• 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/8423822
• 关键词: Antibiotics; Attenuated; Bacteria; Biological Assay; Cell surface; Cell-Matrix Junction; Cells; Chemotaxis; Complex; Cystic Fibrosis; Data; Detection; Development; Dictyostelium; Dictyostelium discoideum; Elastases; Employee Strikes; Environment; Epithelial Cells; Event; Exhibits; Flagella; Gene Expression; Genes; Genetic Transcription; Goals; Gram-Negative Bacteria; Growth; Hospitals; Human; Infection; Investigation; Knock-out; Light; Lipase; Lung; Measures; Membrane; Microarray Analysis; Microbial Biofilms; Microfluidic Microchips; Molecular; Motor; Mus; Needles; Nutrient; Patients; Pilum; Pneumonia; Production; Proteins; Pseudomonas aeruginosa; Regulation; Reporter; Role; Sepsis; Signal Transduction; Solid; Staging; Surface; System; Temperature; Testing; Therapeutic; Time; Toxin; Transcriptional Regulation; Urinary tract infection; Virulence; Virulence Factors; Virulent; Wound Infection; cytotoxicity; diguanylate cyclase; extracellular; improved; in vivo; macrophage; mutant; novel; pathogen; porin; protein-histidine kinase; quorum sensing; research study; response; screening; sensor

DESCRIPTION (provided by applicant): 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.

描述（由申请人提供）：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。一种尚未详细表征的毒力表达的潜在刺激物是固体表面的存在。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，毒力基因的转录上调。表面附着的细胞对宿主盘基网柄藻也更具毒性。我将检验毒力受表面附着调节的假设。特别是，我将表征铜绿假单胞菌在体内表面附着的反应，使用荧光蛋白转录报告，并使用微阵列分析和细胞毒性试验。我会核实 表面附着细胞对小鼠巨噬细胞和人肺上皮细胞表现出增强的毒力。通过微阵列分析的荧光和表面附着细胞的指导下，我还开发了荧光蛋白转录报告基因的表面附着调节。我将使用微调的微流控装置跟踪这些基因的表达动态，因为细胞从非稳态生长过渡到表面附着。我也将测试假设，即细胞拥有一个分子传感器，检测表面的存在。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是所需的表面诱导激活的毒力。虽然III型分泌针在将毒素递送到宿主细胞中具有明确的作用，但我将测试III型分泌针也是检测表面存在的传感器的假设。我将测试是否pcrV突变体和突变体的相互作用的合作伙伴PcrG是转录敏感的表面的存在。以前，我进行了一个屏幕的突变体是超毒性的，在无菌生长。这些细胞表现出对网骨藻细胞、小鼠巨噬细胞和人肺上皮细胞的毒力显著增加。我将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来检验这些菌株在表面检测中存在缺陷的假设。特别是，我将集中我的初步努力，表征突变体，其中包含一个中断i roeA，它编码的二鸟苷酸环化酶，参与从无菌生长到固着生长的过渡表面。如果时间允许，我将通过敲除已知的毒力因子和筛选毒力减弱的突变体来确定这些突变体的超强毒力机制。这项研究的发现将揭示细菌细胞如何检测表面以及毒力如何调节。此外，它还可以提供关于生物膜形成的初始阶段的进一步细节。