COORDINATE REGULATION OF BACTERIAL VIRULENCE FACTORS

在接下来的支持期间，我们将继续研究 三种不同菌株毒力基因表达的协同调控 细菌、鼠伤寒沙门氏菌、霍乱弧菌和波氏杆菌 百日咳。为了便于毒力基因的鉴定， 我们设计了一种遗传策略，允许直接选择 在动物组织中表达的基因。我们称这种方法论为“In” 活体表达技术(IVET)，现在提出在 三种目标生物中的每一种都表现出一种调节模式 与毒力基因一致(即在生长过程中表达较差 在实验室介质中，但在动物组织中高度表达)。基因 显示这一调节的融合将被克隆和核苷酸 确定了融合点的序列。野生型靶基因 也将被克隆并用于构建相应的空突变 目标生物体。这些突变体将接受毒力缺陷测试。 在适当的动物模型中。监管部门的特殊反应 选定的融合将在体外进行研究，以努力确定 控制毒力基因表达的寄主环境信号。这个 介导宿主特异性调控反应的调控基因将是 通过遗传方法进行鉴定(例如，分离构成突变 这在体外解除了融合的抑制)。我们还将分离新的基因 由同一监管系统协调监管的融合。 我们进一步建议构造一系列改进的依维特向量之一 这将允许识别编码分泌物或膜的基因 在宿主组织中选择性表达的蛋白质，而另一种 这将提供对组织特异性基因的灵敏测量 利用定点重组作为报告基因进行表达。正在进行中 还将继续进行的研究将包括在S。 鼠伤寒沙门氏菌将解决O-抗原合成的调节 体内诱导反义RNA。在百日咳杆菌中，我们将研究 通过细胞内生长诱导Vir抑制基因。最后，在V。 我们将研究霍乱期间基因表达的时间阶段性 感染周期关注既被激活又被激活的基因 受ToxR调节系统和热休克反应的抑制。 这些实验应该在以下方面提供一些实际应用 疫苗和抗菌药物的开发。

During the next period of support we, will continue our studies on the coordinate regulation of virulence gene expression in three different bacterial species, Salmonella typhimurium, Vibrio cholerae and Bordetella pertussis. In order to facilitate the identification of virulence genes, we have devised a genetic strategy that allows the direct selection of genes that are expressed in animal tissues. We call this methodology "in vivo expression technology (IVET)" and now propose to identify genes in each of the three target organisms that show a pattern of regulation that is consistent with a virulence gene (i.e., expressed poorly during grow on laboratory media but highly expressed in animal tissues). The gene fusions displaying this regulation will be cloned and the nucleotide sequence of the fusion junction determined. The wild-type target gene will also be cloned and used to construct corresponding null mutations in the target organism. These mutants will be tested for virulence defects in appropriate animal models. The particular regulatory response of selected fusions will be studied in vitro in an effort to ascertain the host environmental signals controlling virulence gene expression. The regulatory genes mediating the host specific regulatory responses will be identified by genetic methods (e.g., isolation of constitutive mutations that derepress the fusion in vitro). We will also isolate new gene fusions that are coordinately regulated by the same regulatory system. We further propose to construct a series of improved IVET vectors one of which will allow identification of genes encoding secreted or membrane proteins that are expressed selectively in host tissues, while another that will provide a sensitive measurement of tissue-specific gene expression by using site-specific recombination as a reporter. Ongoing studies that will also be continued will include studies in S. typhimurium that will address the regulation of O-antigen synthesis by an in vivo induced anti-sense RNA. In B. pertussis, we will study the induction of Vir-repressed genes by intracellular growth. Finally, in V. cholerae we will study the temporal phasing of gene expression during the infection cycle focusing on genes that are both activated as well as repressed by the ToxR regulatory system and the heat shock response. These experiments should provide several practical applications in vaccine and antimicrobial drug development.