Salmonella-Host Cell Interactions

鼠伤寒沙门氏菌（Salmonella Typhimurium）是人类小肠结肠炎最常见的病因之一。这种兼性细胞内病原体的发病机制依赖于侵入非吞噬细胞的能力，例如在肠上皮中发现的那些。入侵依赖于III型分泌系统（T3 SS 1），其用于将一组细菌效应蛋白易位到宿主细胞中。在内化之后，细胞内沙门氏菌存活并在修饰的吞噬体（含沙门氏菌的空泡（SCV））内复制。第二种III型系统（T3 SS 2）在细胞内诱导，并且与SCV的细胞内存活/复制和生物发生相关。为了了解沙门氏菌的发病机制，我们必须剖析T3 SS 1和T3 SS 2效应蛋白的作用，以及控制它们在宿主细胞内的表达和活性的机制。由于这些毒力因子的表达和功能非常依赖于细胞内环境，因此我们专注于开发适当的体外系统以在分子水平上研究其活性。我们目前正在使用原代人巨噬细胞模型系统和极化上皮细胞。我们还与德克萨斯A&M大学的Garry亚当斯合作，研究了沙门氏菌与宿主细胞在体内牛肠上皮细胞中的相互作用。 该项目的一个独立部分是开发用于研究效应器功能的新工具。我们采取的一种方法是通过向小鼠注射合并的效应蛋白来产生单克隆抗体。大约一半的单克隆抗体，我们的特点是特异性的肌动蛋白结合效应SiPA。肽扫描方法已被用于鉴定这些抗体识别的表位。表位位于SipA的所有三个特征结构域内，包括肌动蛋白结合结构域。我们还通过免疫荧光显微镜、ELISA和免疫沉淀法筛选了抗SipA抗体。 这些筛选表明，在这19种抗体中，有一些抗体将用于特定的技术。例如，三种抗体特异性地检测沙门氏菌感染的HeLa细胞的胞质溶胶中的易位SipA。大多数其他抗体识别已知的效应蛋白，但一些识别尚未被鉴定为T3 SS 1效应子的蛋白。我们还在开发新的分子生物学工具，例如使用可调合成启动子或诱导型启动子来研究单个毒力基因的功能。

Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) is one of the most common causes of enterocolitis in humans. Pathogenesis of this facultative intracellular pathogen is dependent on the ability to invade non-phagocytic cells, such as those found in the intestinal epithelium. Invasion is dependent on a type III secretion system (T3SS1), which is used to translocate a set of bacterial effector proteins into the host cell. Following internalization, intracellular Salmonella survive and replicate within a modified phagosome, the Salmonella-containing vacuole (SCV). A second type III system (T3SS2) is induced intracellularly and is associated with intracellular survival/replication and biogenesis of the SCV. To understand Salmonella pathogenesis we must dissect the roles of the individual T3SS1 and T3SS2 effector proteins as well as the mechanisms that control their expression and activity inside host cells. Since the expression and function of these virulence factors is exquisitely dependent on the intracellular environment, we are focusing on developing appropriate in vitro systems to study their activities at the molecular level. We are currently using a both primary human macrophage model system and polarized epithelial cells. We have also collaborated with Garry Adams at Texas A&M to look at the involvement of Salmonella-host cell interactions in the bovine intestinal epithelium in vivo. A separate part of this project is to develop novel tools for studying effector function. One approach we have taken is to raise monoclonal antibodies by injecting mice with pooled effector proteins. Approximately half of the monoclonals that we have characterized are specific for the actin-binding effector SipA. A peptide scanning method has been used to identify the epitopes recognized by these antibodies. The epitopes are located within all three characterized domains of SipA, including the actin binding domain. We have also screened the anti-SipA antibodies for by immunofluorescence microscopy, ELISA and immunoprecipitation. And these screens indicate that within this group of 19 antibodies there are ones that will be useful for specific techniques. For example, three of the antibodies specifically detect translocated SipA in the cytosol of Salmonella-infected HeLa cells. Most of the other antibodies recognize known effector proteins but some recognize proteins that have not been identified as T3SS1 effectors. We are also developing new molecular biology tools, for example using tunable synthetic promoters or inducible promotors to study the functions of individual virulence genes.