EXPRESSION OF YERSINIA PESTIS CAPSULE ATTENUATES WILD-TYPE SALMONELLA

鼠疫耶尔森氏菌胶囊的表达可减弱野生型沙门氏菌

• 批准号: 8360166
• 负责人: Xinghong Yang
• 金额: $ 10.47万
• 依托单位: MONTANA STATE UNIVERSITY - BOZEMAN
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8360166
• 关键词: Affect; Antigens; Attenuated; Attenuated Vaccines; Bacteria; Emerging Communicable Diseases; Funding; Gene Deletion; Genes; Gram-Negative Bacteria; Grant; Immune response; In Vitro; Mediating; Membrane; Microarray Analysis; National Center for Research Resources; Needles; Operon; Principal Investigator; Protein Secretion; Proteins; Research; Research Infrastructure; Resources; Salmonella; Salmonella enterica; Salmonella infections; Salmonella typhimurium; Series; Site-Directed Mutagenesis; Source; Testing; United States National Institutes of Health; Virulence; Virulent; Yersinia pestis; appendage; attenuation; capsule; cost; fimbria; in vivo; mutant; novel; overexpression

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.

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。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 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。我们假设，通过迫使各种附件(例如，菌毛、针或胶囊)的表达来衰减毒力细菌是灭活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菌囊膜抗原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。我们评估了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。我们推测，过表达的Usher Caf1A蛋白，当组装到通道中时，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中过表达完整的caf操纵子是否会减弱沙门氏菌，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衰减可以由任何CAF操纵子基因引起，包括ca1a、af1M和CAF1。目前，我们正在研究减毒沙门氏菌能否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。