Biotype-specific evolution

项目总结 霍乱目前由O1血清群El Tor生物型霍乱弧菌引起，该菌于1961年出现，引发了霍乱 正在进行的第七次大流行。前6次霍乱流行是由O1血清群经典生物型V型引起的。 霍乱弧菌。在过去的60年里，经典的生物型基本上已经从水环境中消失了 也是霍乱的原因之一。然而，造成这种情况的原因尚不清楚。在环境中发现霍乱弧菌 与许多脊椎动物鱼类有关。拟议的工作将使用斑马鱼模型来研究V. 霍乱弧菌可以研究霍乱弧菌与覆盖整个 感染性循环。先前的研究发现，在斑马鱼定居的时间线上，经典的 和El Tor生物型，经典的在72小时内被清除，El Tor能够用 高水平的细菌复制。我们推测，这种长期的鱼类定居和繁殖提供了 对El Tor有很强的选择性优势，使其能够在环境利基领域取代经典产品。霍乱弧菌EL Tor生物型有两个致病岛，分别称为VSP-1和VSP-2，这是经典菌株所缺乏的。初步数据 实验表明，缺失VSP-1的El Tor菌株具有正常的鱼类定植表型，而 缺失VSP-2的菌株在长期定殖中存在缺陷。因此，很可能是VSP-2中的基因(S) 对于长期殖民来说是必不可少的。该提案的目标1将使用渐进式删除策略来确定 并确定VSP-2中对长期定植至关重要的基因(S)的特征，并评估这种 基因足以延长经典的殖民。在宿主方面，先天免疫和获得性免疫提供 防止病原体入侵。鱼具有与哺乳动物非常相似的先天免疫反应，以及 在生命的前4-6周内产生的适应性免疫反应。我们假设经典生物型 通过严格的先天反应迅速清除，而El Tor只能通过适应性免疫才能清除 回应。该提案的目标2-1将使用斑马鱼幼体来验证这些假设，斑马鱼幼体具有完全功能 与生俱来的反应，但尚未形成的适应性反应。Aim 2-2将进一步检验El Tor的假设 清除需要通过使用在适应性免疫方面有缺陷的斑马鱼突变体进行适应性免疫。 利用斑马鱼作为霍乱弧菌环境宿主模型的拟议工作的完成，将大大 促进我们对霍乱弧菌在天然水库中的进化和选择压力的理解。从长远来看 这项工作的目标是更好地了解霍乱弧菌的生命周期，它如何在人类发病中起作用， 并确定抗击霍乱弧菌疾病和传播的新战略。 。

PROJECT SUMMARY Cholera is presently caused by O1 serogroup, El Tor biotype V. cholerae, which emerged in 1961 to initiate the ongoing seventh pandemic. The prior 6 cholera pandemics were caused by O1 serogroup classical biotype V. cholerae. Over the past 60 years, classical biotype has essentially disappeared from the aquatic environment and as a cause of cholera. However, the reasons for this are unknown. V. cholerae in the environment is found in association with numerous vertebrate fish species. The proposed work will use a zebrafish model for V. cholerae that can investigate interactions between V. cholerae and natural aquatic hosts covering the entire infectious cycle. Previous work found dramatic differences in the timeline of zebrafish colonization by classical and El Tor biotypes, with classical being cleared within 72 h and El Tor able to colonize for up to 14 days with high levels of bacterial replication. We hypothesize that this prolonged fish colonization and replication provided a strong selective advantage to El Tor, allowing it to replace classical in environmental niches. V. cholerae El Tor biotype has 2 pathogenicity islands termed VSP-1 and VSP-2 that classical lacks. Data from preliminary experiments indicate an El Tor strain deleted for VSP-1 has a normal fish colonization phenotype, whereas a strain deleted for VSP-2 is defective in prolonged colonization. Therefore, it is likely that gene(s) within VSP-2 are essential for prolonged colonization. Aim 1 of this proposal will use a progressive deletion strategy to identify and characterize gene(s) within VSP-2 that are important for prolonged colonization and assess whether such genes are sufficient to prolong classical colonization. On the host side, innate and adaptive immunity provide protection from invading pathogens. Fish have innate immune responses very similar to mammals, as well as adaptive immune responses that develop over the first 4-6 weeks of life. We hypothesize that classical biotype is rapidly cleared by a strictly innate response, whereas El Tor can only be cleared by an adaptive immune response. Aim 2-1 of this proposal will test these hypotheses using larval zebrafish, which have a fully functioning innate response but an undeveloped adaptive response. Aim 2-2 will further test the hypothesis that El Tor clearance requires adaptive immunity by using zebrafish mutants that are defective in adaptive immunity. Completion of the proposed work, using zebrafish as an environmental V. cholerae host model, will significantly advance our understanding of V. cholerae evolution and selective pressures in a natural reservoir. The long term goal of this work is to better understand the V. cholerae life cycle, how it contributes to pathogenesis in humans, and identify new strategies to combat V. cholerae disease and transmission. .