EXPRESSION OF YERSINIA PESTIS CAPSULE ATTENUATES WILD-TYPE SALMONELLA

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 定点突变通常用于灭活野生型(Wt)革兰氏阴性细菌。然而，毒力基因往往没有被鉴定，因此很难构建减毒株。通过迫使各种附件，如菌毛、针或胶囊的表达来减弱毒力细菌，是灭活wt细菌的另一种方法，从而允许这些突变体用作活疫苗，同时仍然刺激强大的免疫反应。为了验证这一假说，我们选择了鼠疫耶尔森氏菌的囊膜抗原F1(F1-Ag)。F1-Ag的分泌依赖于由Caf操纵子编码的分泌机构的形成，该机构包括一种引座蛋白，该蛋白形成细菌外膜(OM)中的通道，从而允许F1-Ag的分泌。我们质疑这种蛋白质分泌装置在OM中的过度表达是否会对细菌产生不利影响，从而影响通道介导的衰减。我们推测，过度表达的迎宾Caf1a蛋白，当组装成通道时，会使沙门氏菌减毒，而这个无毒的突变体将对wt沙门氏菌的挑战提供保护。为了研究这种可能性，我们研究了在鼠伤寒沙门氏菌(鼠伤寒沙门氏菌)中过表达完整的caf操纵子是否会抑制沙门氏菌，并发现在体外和体内过表达CAF1胶囊都能显著抑制鼠伤寒沙门氏菌。通过CAF操纵子中的一系列基因缺失，我们证明了观察到的被膜介导的沙门氏菌减毒是由于Caf1A的过度表达所致。在未来，我们将研究Caf1a减毒沙门氏菌是否能够作为沙门氏菌病的活疫苗。我们将通过微阵列技术进一步研究这种新的衰减策略所涉及的潜在机制。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Site-directed mutagenesis is typically used to inactivate wild-type (wt) Gram-negative bacteria. However, virulence genes have often not been identified, thus making attenuated strains difficult to construct. Attenuating virulent bacteria by forcing the expression of various appendages, e.g., fimbriae, needles, or capsules, represents an alternative approach to inactivating wt bacteria, thereby allowing these mutants to be used as live vaccines while still stimulating a robust immune response. To test this hypothesis, the Yersinia pestis (Y. pestis) capsule antigen F1 (F1-Ag) was selected. Secretion of F1-Ag is dependent upon the formation of a secretion apparatus encoded by the caf operon, which includes an usher protein that forms into channels in the bacterial outer membrane (OM), allowing secretion of F1-Ag. We questioned whether over expression of this protein secretion apparatus in the OM would adversely affect the bacterium, thereby, influencing channel-mediated attenuation. We hypothesize that over expressed usher Caf1A protein, when assembled into channels, will attenuate Salmonella, and this avirulent mutant will render protection against wt Salmonella challenge. To study this possibility, we investigated whether over expression of the entire caf operon in wt Salmonella enterica serovar Typhimurium (S. typhimurium) would attenuate Salmonella and found that over expression of the Caf1 capsule can significantly attenuate S. typhimurium both in vitro and in vivo. Through a series of gene deletions in the caf operon, we demonstrated that the observed capsule-mediated Salmonella attenuation resulted from over expression of Caf1A. In the future, we will investigate whether the Caf1A-attenuated Salmonella are able to serve as a live vaccine for salmonellosis. We will further investigate the underlying mechanism involved in this novel attenuation strategy via microarray technology.