EXPRESSION OF YERSINIA PESTIS CAPSULE ATTENUATES WILD-TYPE SALMONELLA

这个子项目是利用资源的许多研究子项目之一 由NIH/NCRR资助的中心拨款提供。次级项目的主要支助 子项目的主要研究者可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 定点诱变通常用于鉴定野生型（wt）革兰氏阴性细菌。 然而，毒力基因往往没有被确定，从而使减毒株难以构建。我们假设通过强制表达各种附属物（例如，菌毛、针或胶囊）代表了灭活WT细菌的替代方法，从而允许这些突变体用作活疫苗，同时仍然刺激强免疫应答。为了验证这一假设，选择鼠疫耶尔森氏菌荚膜抗原F1（F1-Ag）。 F1-Ag的分泌依赖于由caf操纵子编码的分泌装置的形成，所述caf操纵子包括在细菌外膜（OM）中形成通道的引导蛋白，从而允许F1-Ag的分泌。 我们评估了这种蛋白分泌装置在OM中的过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。 我们假设，过度表达的usher Caf 1A蛋白，当组装成通道，将减毒沙门氏菌，这种无毒突变体将提供保护，对野生型沙门氏菌的挑战。为了研究这种可能性，我们研究了在鼠伤寒沙门氏菌中过表达整个caf操纵子是否会减弱沙门氏菌，并发现过表达Caf 1胶囊可以显著减弱沙门氏菌。鼠伤寒沙门氏菌在体外和体内。 通过一系列的基因缺失的caf操纵子，我们证明了所观察到的胶囊介导的沙门氏菌减毒可以引起的任何caf操纵子基因，包括caf 1A，caf 1 M，和caf 1。 目前，我们正在研究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型减毒策略的潜在机制。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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. We hypothesized that attenuation of 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 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 that includes an usher protein that forms into channels in the bacterial outer membrane (OM), allowing secretion of F1-Ag. We evaluated whether overexpression of this protein secretion apparatus in the OM would adversely affect the bacterium, thereby, influencing channel-mediated attenuation. We hypothesize that overexpression of the 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 overexpression of the entire caf operon in wt Salmonella enterica serovar Typhimurium would attenuate Salmonella and found that overexpression 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 can be caused by any of the caf operon genes, including caf1A, caf1M, and caf1. Currently, we are investigating whether the attenuated Salmonella will be 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.