V. cholerae proteins are required for biofilm growth-vaccine targets

描述（由申请人提供）：霍乱弧菌（Vc）引起霍乱。它作为病原体的成功取决于在不同环境中适当的转录调控，如人类肠道或水生环境，那里的生长条件不是最佳的。基因调控导致操纵子的表达，这对生物膜的产生至关重要。生物膜是由细菌细胞、蛋白质和多糖组成的复杂结构，使细菌能够在恶劣的环境中生存。与光滑Vc相比，传染性rugose Vc变体的特点是具有波纹状菌落，能够形成广泛的生物膜，并增强了对渗透和氧化应激的抵抗力。与光滑Vc相比，褶皱Vc生物膜在胆汁存在下的存活率更高，这可以解释为什么最近脱落的具有生物膜特征的Vc生物膜聚集体对人类更具传染性。皱褶性和生物膜结构调节基因簇（rbmA-F）已被确定，它与其他几种分泌蛋白一起，是El Tor Vc皱褶生物膜形成所必需的。抗体（Abs）对Rbm蛋白的保护作用尚未被研究。Rbm蛋白由胆汁诱导，因此作为感染接种物的一部分和在定植阶段表达；野生型定植需要RbmA、RbmC和Bap-1。我们假设RbmA、RbmC和Bap-1 （RbmC的同源物）分泌的蛋白是调节生物膜基质的保护性抗原，能够诱导调节Vc生物膜和发病机制的抗体（Abs）。这些研究对于确定生物膜抗原是否是免疫系统和霍乱疫苗的相关靶标具有重要意义。这项拟议中的研究汇集了两位科学家的专业知识。Wade和Yildiz在霍乱研究的不同方面都有丰富的研究经验。W. Wade博士是一名分子免疫学家，他开创了合成Vc LPS抗原结合载体蛋白的疫苗接种方案的发展。Wade博士是一位成熟的免疫学家，在产生和表征Vc抗原（TcpA， LPS）的保护性抗体方面有着广泛的记录。F. Yildiz博士是公认的分子生物学家，他分离和表征了Rbm蛋白（在应用中也被称为生物膜基质或基质蛋白）及其对Vc生物膜生物学的要求。

DESCRIPTION (provided by applicant): Vibrio cholerae (Vc) causes cholera. Its success as a pathogen depends on appropriate transcriptional regulation in different surroundings, such as the human intestinal tract or the aquatic environment, where growth conditions are sub-optimal. Gene regulation results in expression of operons that are critical for generation of a biofilm. Biofilms are complex structures composed of bacterial cells, proteins, and polysaccharides which allow the bacteria to exist in hostile environments. Compared to smooth Vc, the infectious rugose Vc variant is characterized by corrugated colonies, the capacity to develop extensive biofilms, and increased resistance to osmotic and oxidative stresses. Compared to smooth Vc, rugose Vc biofilms have enhanced survival in the presence of bile which may explain why recently shed Vc biofilm aggregates, that have biofilm characteristics, are more infectious for human. The rugosity and biofilm structure modulator gene cluster (rbmA-F) has been identified, which along with several other secreted proteins, are required for the formation of El Tor Vc rugose biofilms. The protective role of antibodies (Abs) to the Rbm proteins has not been studied. Rbm proteins are induced by bile and thus are expressed as part of the infecting inoculum and during the stages of colonization; RbmA, RbmC, and Bap-1 are required for wild type colonization. We hypothesize that the RbmA, RbmC, and Bap-1 (a homolog to RbmC) secreted proteins, known to regulate the biofilm matrix, are protective antigens able to induce antibodies (Abs) that modulate Vc biofilms and thus pathogenesis. These studies are important to determine if biofilm antigens are relevant targets for the immune system and thus cholera vaccines. The proposed study brings together the expertise of two scientists, Drs. Wade and Yildiz who both have extensive research experience in different aspects of cholera research. Dr. W. Wade is a molecular immunologist who pioneered the development of the vaccination protocols for synthetic Vc LPS antigens conjugated to carrier proteins. Dr. Wade is an established immunologist with an extensive track record in generating and characterizing protective Abs to Vc antigens (TcpA, LPS). Dr. F. Yildiz is a recognized molecular biologist who isolated and characterized the Rbm proteins (also referred to in the application as biofilm matrix or matrix proteins) and their requirement for Vc biofilm biology. PUBLIC HEALTH RELEVANCE: Cholera is still a disease that sickens millions and kills thousands yearly. We propose to formulate a new subunit cholera vaccine based on identification of protective epitopes in proteins known to be required for V. cholerae biofilm generation. Previous work from Dr. Wade's laboratories has shown that anti-TcpA antibodies can modify biofilm growth. Other work by Dr. Yildiz's group has identified proteins required for biofilm development that will serve as immunogens to induce antibodies.