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

描述(申请人提供)：霍乱弧菌(VC)引起霍乱。它作为病原体的成功依赖于在不同环境中适当的转录调节，如人类肠道或水生环境，在这些环境中，生长条件是次优的。基因调控导致操纵子的表达，操纵子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，感染性皱纹VC变异体的特征是菌落呈波纹状，能够形成广泛的生物膜，对渗透胁迫和氧化应激的抵抗力增强。与光滑的VC相比，皱纹VC生物膜提高了在胆汁存在下的存活率，这可能解释了为什么最近脱落的VC生物膜聚集体具有生物膜特性，对人类更具传染性。粗糙度和生物膜结构调节剂基因簇(RBMA-F)已经被鉴定，它与其他几种分泌蛋白一起，是形成El Tor VC皱纹生物膜所必需的。针对RBM蛋白的抗体(Abs)的保护作用尚未被研究。RBM蛋白是由胆汁诱导的，因此作为感染接种的一部分和在定植阶段表达；RBMA、RbmC和Bap-1是野生型定植所必需的。我们假设，RBMA、RbmC和Bap-1(RbmC的同源物)分泌的蛋白质，已知调节生物膜基质，是保护性抗原，能够诱导抗体(Abs)调节VC生物膜，从而调节发病机制。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非常重要。这项拟议的研究汇集了韦德博士和耶尔迪兹博士两位科学家的专业知识，他们都在霍乱研究的不同方面拥有丰富的研究经验。W.Wade博士是一位分子免疫学家，他率先开发了合成VC-LPS抗原与载体蛋白结合的疫苗接种方案。韦德博士是一位久负盛名的免疫学家，在制备和鉴定针对VC抗原(TCPA、LPS)的保护性抗体方面有着广泛的记录。耶尔迪兹博士是一位公认的分子生物学家，他分离和鉴定了RBM蛋白(在申请中也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。 与公共卫生相关：霍乱仍然是一种每年导致数百万人患病和数千人死亡的疾病。我们建议根据霍乱弧菌生物膜形成所需蛋白质中保护性表位的鉴定，研制一种新的亚单位霍乱疫苗。韦德博士的实验室之前的工作表明，抗TCPA抗体可以改变生物膜的生长。耶尔迪兹博士团队的其他工作已经确定了生物膜形成所需的蛋白质，这些蛋白质将作为免疫原来诱导抗体。

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.