Negative regulation of virulence in Pseudomonas aeruginosa

许多环境信号调节毒力因子的表达，如营养物质的可获得性、温度和pH。一个潜在的毒力表达刺激物尚未被详细描述，那就是固体表面的存在。我在铜绿假单胞菌中的初步实验表明，在附着在非生物表面的细胞中，毒力基因的转录上调。表面附着的细胞对宿主盘基网柄菌的毒力也更强。 我将测试毒力受表面附着调控的假设。特别是，我将在体内使用荧光蛋白转录报告以及微阵列分析和细胞毒性分析来表征铜绿假单胞菌对表面附着的反应。我将验证附着在表面的细胞对小鼠巨噬细胞和人肺上皮细胞的毒力增强。在对浮游细胞和表面附着细胞的微阵列分析的指导下，我还开发了受表面附着调控的基因的荧光蛋白转录报告。当细胞从浮游生长过渡到表面附着时，我将使用微流控设备跟踪这些基因的表达动态。 我还将测试这样一种假设，即细胞拥有检测表面存在的分子传感器。这将通过敲除荧光蛋白转录报告菌株中的候选传感器和测试菌株对表面存在的转录不敏感来实现。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pcrV突变体和其相互作用伙伴pcrG的突变体是否在转录上对表面的存在敏感。 之前，我对浮游生长过程中超强毒力的突变体进行了筛选。这些细胞对网盘基细胞、小鼠巨噬细胞和人肺上皮细胞的毒力显著增加。我将使用上面的荧光蛋白转录报告、微阵列分析和细胞毒性分析来检验这些菌株在检测表面方面存在缺陷的假设。特别是，我将把我最初的努力集中在鉴定一种含有中断I roeA的突变体，它编码一种参与表面从浮游生长到固着生长的转换的二鸟苷环化酶。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鉴定这些突变体的超强毒力机制。这项研究的发现将阐明细菌细胞如何检测表面，以及毒力是如何受到调控的。此外，它还可以提供有关生物膜形成的初始阶段的更多细节。

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.