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.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 定点诱变通常用于灭活野生型（WT）革兰氏阴性细菌。 然而，毒力基因通常尚未被鉴定，因此使菌株难以构造。我们假设通过强迫各种附属物的表达（例如膜片，针或胶囊）的表达来衰减毒细菌，这代表了一种灭活WT细菌的替代方法，从而使这些突变体可以用作活疫苗，同时仍可以刺激强大的免疫免疫反应。为了检验该假设，选择了耶尔森氏菌囊囊抗原F1（F1-AG）。 F1-AG的分泌取决于由CAF操纵子编码的分泌设备的形成，该仪式包括一个迎接蛋白质的蛋白质，该蛋白会形成细菌外膜（OM）中的通道，从而允许F1-AG分泌。 我们评估了OM中这种蛋白质分泌设备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usher Caf1a蛋白的过表达在组装成通道时会减弱沙门氏菌，而这种无毒的突变体将对WT沙门氏菌挑战提供保护。为了研究这种可能性，我们调查了WT沙门氏菌肠球形鼠伤寒的整个CAF操纵子的过表达是否会减弱沙门氏菌，发现Caf1胶囊的过表达可以显着减轻体外和体内的Typhimurium S. typhimurium。 通过CAF操纵子中的一系列基因缺失，我们证明了观察到的胶囊介导的沙门氏菌衰减可能是由CAF1A，CAF1M和CAF1在内的任何CAF操纵子基因引起的。 目前，我们正在研究减毒的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衰减策略所涉及的潜在机制。