Negative regulation of virulence in Pseudomonas aeruginosa

铜绿假单胞菌毒力的负调控

• 批准号: 8600237
• 负责人: Albert Siryaporn
• 金额: $ 5.89万
• 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/8600237
• 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，例如养分，温度和pH值的可用性。尚未详细表征的毒力表达的一种潜在刺激剂是固体表面的存在。我在铜绿假单胞菌中进行的初步实验表明，在附着在非生物表面的细胞中，毒力基因的转录被上调，表面连接的细胞也对宿主的distyostelium discoideum也更具毒性。我将检验以下假设：毒力受到表面附着的调节。特别是，我将使用荧光蛋白转录报告器以及使用微阵列分析和细胞毒性测定法表征铜绿假单胞菌对体内表面附着的反应。我会验证 表面附着的细胞表现出对小鼠巨噬细胞和人肺上皮细胞的毒力增加。在对浮游细胞和表面附着的细胞的微阵列分析的指导下，我还将荧光蛋白转录报告基因向受表面附着调节的基因开发。我将使用精细调整的微流体设备跟踪这些基因的表达动力学，因为细胞从浮游生长到表面跟踪过渡。我还将检验以下假设：细胞具有检测表面存在的分子传感器。这将通过在荧光蛋白转录报告基因菌株中拆除候选传感器和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，针头蛋白PCRV是表面诱导的毒力激活所必需的。虽然III型分泌针在将毒素传递到宿主细胞中具有明显的作用，但我将测试以下假设：III型分泌针也是检测表面存在的传感器。我将测试其相互作用伴侣PCRG的PCRV突变体和突变体是否对表面的存在敏感。以前，我对浮游生物生长过程中过度刺激性的突变体进行了屏幕。这些细胞表现出对柱状细胞，小鼠巨噬细胞和人肺上皮细胞的毒力显着增加。我将检验以下假设：这些菌株使用上面的荧光蛋白转录报告基因，微阵列分析和细胞毒性测定法检测表面有缺陷。特别是，我将最初的努力集中在表征一个突变体上，该突变体含有破坏I的ROEA，该突变体编码了从浮游生物生长到表面上的无柄生长的过渡涉及的二烷基酸环化酶。如果时间允许，我将通过淘汰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中高率的机制。这项研究的发现将阐明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。

项目成果

Familial aggregation of cerebral malaria and severe malarial anemia.

脑型疟疾和严重疟疾贫血有家族聚集性。

• DOI: 10.1086/427238
• 发表时间: 2005
• 期刊: The Journal of infectious diseases
• 作者: Ranque,Stephane;Safeukui,Innocent;Poudiougou,Belco;Traore,Abdoulaye;Keita,Modibo;Traore,Diamori;Diakite,Mahamadou;Cisse,MahamadouB;Keita,MaroufM;Doumbo,OgobaraK;Dessein,AlainJ
• 通讯作者: Dessein,AlainJ

Space-time clustering of childhood malaria at the household level: a dynamic cohort in a Mali village.

• DOI: 10.1186/1471-2458-6-286
• 发表时间: 2006-11-21
• 期刊: BMC public health
• 影响因子: 4.5
• 作者: Gaudart J;Poudiougou B;Dicko A;Ranque S;Toure O;Sagara I;Diallo M;Diawara S;Ouattara A;Diakite M;Doumbo OK
• 通讯作者: Doumbo OK