Coordinate Regulation of Bacterial Virulence Factors

描述（由申请人提供）：对于AI-26289的下一个资助期，我们的总体目标是了解五种致病微生物（霍乱弧菌，铜绿假单胞菌，幽门螺杆菌，流感嗜血杆菌和大肠杆菌）的生物学，以便发现基于使用小分子抑制剂的抗感染策略。这些研究将通过使用共同技术和遗传方法进行横向整合。例如，我们将使用差分荧光诱导（DFI）来定义和研究体内表达的基因或与生物学感兴趣的毒力调控相关的基因。基因组微阵列将用于在各种生长条件下，在各种定义的突变体中，以及在一系列施加的压力下，在全球转录水平上测量基因表达。这些应激挑战将包括感染期间暴露于宿主环境、暴露于宿主细胞、通过条件表达退出必需基因产物、暴露于有毒化合物、有毒蛋白质适体和抑制性抗生素。我们将使用多种方法，包括新的基于dna芯片的“TraSH方法”，在体外和体内鉴定细菌生长和生存所需的基因。我们将尝试通过微液相色谱串联质谱（microLCMS）分析来表征它们与其他蛋白质形成的复合物，从而确定新的必需蛋白的功能。其他蛋白质组学项目将包括使用微lcms来定义体外和体内生长的细菌表面表达的蛋白质。我们还将探索使用“蛋白质芯片”作为进行蛋白质-蛋白质相互作用分析的手段和作为研究宿主免疫反应的新工具。计算方法将用于挖掘表达和基因组数据库，以获取有趣的潜在毒力或必要的基因产物，然后对其进行遗传分析，以便在实验动物中进行衰减。我们将利用我们在致病细菌的基本过程中获得的信息来设计敏感的生物筛选，可以检测这些过程的小分子抑制剂。最后，我们将以高通量格式筛选各种组合化合物文库，以寻找在药理学上干扰基本和毒力相关功能的“命中”。

DESCRIPTION (provided by applicant): 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.