For the next funding period of AI-26289, our overall goal is to understanding the biology of five pathogenic microorganisms (Vibrio cholerae, Pseudomonas aeruginosa, Helicobacter pylori, Haemophilus influenzae, and Escherichia coli) in order to discover anti-infective strategies based on the use of small molecule inhibitors. These studies will be horizontally integrated by use of common technologies and genetic approaches. For example, we will use differential fluorescence induction (DFI) to define and study genes expressed in vivo or associated with virulence regulons of biological interest. Genomic microarrays will be used to measure gene expression at the global transcriptional level under various growth conditions, in various defined mutants, and under a series of imposed stresses. These stress challenges will include exposure to host milieu during infection, exposure to host cells, withdrawal of essential gene products through conditional expression, and exposure to toxic compounds, toxic protein aptamers, and inhibitory antibiotics. We will use a variety of methods including the new DNA-chip based """"""""TraSH method"""""""" to identify genes required for bacterial growth and viability in vitro and in vivo. We will attempt to identify the function of new essential proteins by characterizing complexes they form with other proteins through micro liquid chromatography tandem mass spectrometry (microLCMS) analysis. Other proteomic projects will include the use of microLCMS to define proteins expressed on the surface of bacteria grown in vitro and in vivo. We will also explore the use of """"""""protein chips"""""""" as a means of doing protein-protein interaction analysis and as a new tool for studying host immune responses. Computational methods will be used to mine expression and genomic databases for interesting potential virulence or essential gene products, which will then be analyzed genetically for attenuation in experimental animals. We will use the information we gain on essential processes in pathogenic bacteria to devise sensitive bioscreens that can detect small molecule inhibitors of these processes. Finally, we will screen diverse combinatorial compound libraries in high-throughput formats for """"""""hits"""""""" that pharmacologically interfere with essential and virulence related functions. ? ?
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