Negative regulation of virulence in Pseudomonas aeruginosa

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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.