Biotype-specific evolution

项目概要 目前霍乱是由 O1 血清群、El Tor 生物型霍乱弧菌引起，该血清群于 1961 年出现，引发了霍乱 正在进行的第七次大流行。之前的 6 次霍乱大流行都是由 O1 血清群经典生物型 V 引起的。 霍乱。过去60年来，经典生物型基本上从水生环境中消失 并作为霍乱的原因。然而，其原因尚不清楚。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 V 斑马鱼模型。 霍乱弧菌，可以研究霍乱弧菌和覆盖整个海洋的天然水生宿主之间的相互作用 传染周期。之前的研究发现斑马鱼在斑马鱼殖民时间上存在显着差异 和 El Tor 生物型，经典型在 72 小时内被清除，El Tor 能够在具有以下特征的情况下殖民长达 14 天： 高水平的细菌复制。我们假设这种长期的鱼类定殖和复制提供了 El Tor 具有强大的选择优势，使其能够在环境领域取代经典。霍乱弧菌 Tor 生物型有 2 个致病岛，称为 VSP-1 和 VSP-2，这是经典生物型所缺乏的。初步数据 实验表明删除 VSP-1 的 El Tor 菌株具有正常的鱼类定植表型，而 VSP-2 缺失菌株在长时间定植方面存在缺陷。因此，VSP-2 内的基因很可能 对于长期殖民来说至关重要。该提案的目标 1 将使用渐进式删除策略来识别 并表征 VSP-2 内对长期定植很重要的基因，并评估这些基因是否 基因足以延长经典殖民化。在宿主方面，先天性和适应性免疫提供 防止病原体入侵。鱼类具有与哺乳动物非常相似的先天免疫反应， 在生命的前 4-6 周内形成的适应性免疫反应。我们假设经典生物型 可以通过严格的先天反应快速清除，而 El Tor 只能通过适应性免疫清除 回应。该提案的目标 2-1 将使用斑马鱼幼虫来测试这些假设，斑马鱼具有完全功能 天生的反应，但尚未发展的适应性反应。目标 2-2 将进一步检验 El Tor 的假设 清除需要使用适应性免疫缺陷的斑马鱼突变体进行适应性免疫。 使用斑马鱼作为环境霍乱弧菌宿主模型的拟议工作的完成将显着 增进我们对天然储存库中霍乱弧菌进化和选择压力的理解。长期来看 这项工作的目标是更好地了解霍乱弧菌的生命周期，它如何影响人类的发病机制， 并确定对抗霍乱弧菌疾病和传播的新策略。 。

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. .