EXPRESSION OF YERSINIA PESTIS CAPSULE ATTENUATES WILD-TYPE SALMONELLA

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 中心，不一定是研究者的机构。 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。 通过迫使各种附属物的表达来减弱有毒细菌，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，鼠疫耶尔森氏菌（Y。鼠疫荚膜抗原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，其包括在细菌外膜（OM）中形成通道的引导蛋白，允许F1-Ag的分泌。 我们质疑这种蛋白分泌装置在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。 我们假设过度表达的usher Caf 1A蛋白，当组装成通道时，将减弱沙门氏菌，并且这种无毒突变体将提供针对野生型沙门氏菌挑战的保护。 为了研究这种可能性，我们研究了是否在鼠伤寒沙门氏菌中过表达整个caf操纵子。鼠伤寒沙门氏菌（Salmonella typhimurium）会使沙门氏菌减毒，并且发现过表达Caf 1荚膜可以显著地使沙门氏菌减毒。鼠伤寒沙门氏菌在体外和体内。 通过在caf操纵子中的一系列基因缺失，我们证明了所观察到的胶囊介导的沙门氏菌减毒是由Caf 1A的过度表达引起的。 在未来，我们将研究是否Caf 1A减毒沙门氏菌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. 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.