Mechanisms for Vibrio cholerae colonization and pathogenesis in zebrafish

项目总结 水生细菌霍乱弧菌会引起人类腹泻疾病，从轻微到致命不等。 大流行的O1/O139菌株会导致霍乱，这是一种潜在的致命疾病，而非O1/O139菌株也 霍乱弧菌被称为“环境”霍乱弧菌，可引起从轻微到严重的一系列腹泻疾病。动物模型 在确定霍乱弧菌主要毒力因子方面很有用，但兔子和小鼠不是天然的霍乱弧菌。 霍乱宿主，需要缺乏或损坏的微生物群才能定居。脊椎鱼是天然的V。 霍乱宿主和最近的研究已经建立了斑马鱼作为概括整个传染性霍乱的模型 在存在完整的肠道微生物区系的情况下进行。暴露在霍乱弧菌中的鱼类迅速定居 水，发展腹泻，排出大量霍乱弧菌，并将感染传播给幼稚的鱼。 因为这是一个自然宿主的模型，它为霍乱弧菌的研究提供了新的机会，而不是 在哺乳动物模型上是可能的。然而，在鱼类发病中起作用的霍乱弧菌因子尚不清楚。 O1和非O1/O1319菌株。这项建议将检查O1和O1使用的感染过程 和非O1/O139霍乱弧菌，并检验以下假设：1)霍乱弧菌产生特异性定植 鱼中的因素在人类感染中也很重要。这些因素在所有霍乱弧菌中都有 菌株。将使用TN-Seq来验证这一假设，以确定对鱼类定居至关重要的基因。2)V。 霍乱弧菌产生的因子(S)直接导致腹泻。这一假设将通过剔除已知数据来检验。 每个菌株中的毒素和测量腹泻。3)霍乱弧菌扰乱肠道的特定成分 微生物区系，为殖民创造了一个利基。这一假说将通过评估微生物组来验证 在感染之后。4)第六型分泌系统(T6ss)在与肠道的竞争中起重要作用。 微生物区系。这一假设将通过敲除T6SS并进一步识别T6SS效应器来验证 对这场比赛很重要。5)鱼排出的霍乱弧菌具有高度传染性，就像它们在人类霍乱中一样 粪便，这可能是霍乱弧菌暴发的来源。这一假设将通过比较 从体外培养的和通过鱼传播的霍乱弧菌定植所需的感染量。6)霍乱弧菌 在宿主逃逸之前改变其基因表达程序，以实现传播并有助于 高度传染性。这一假说将使用RNA-Seq进行验证，以确定基因表达的全球变化 在体外培养、活跃定植和排泄霍乱弧菌之间。完成拟议的工作，使用 斑马鱼作为霍乱弧菌的天然宿主模型，将极大地促进我们对霍乱弧菌 霍乱弧菌在天然水库中的环境生活方式，霍乱弧菌成为 病原体，并应发现治疗、环境补救和疾病的新靶点 预防。该项目的长期目标是利用斑马鱼模型更好地了解霍乱弧菌。 并确定抗击霍乱弧菌疾病和传播的新策略。 。

PROJECT SUMMARY The aquatic bacterium Vibrio cholerae causes human diarrheal disease that can range from mild to deadly. Pandemic O1/O139 strains cause cholera, a potentially deadly disease, whereas non-O1/O139 strains, also known as “environmental” V. cholerae, cause a range of diarrheal diseases from mild to severe. Animal models have been useful in characterizing major V. cholerae virulence factors, but rabbits and mice are not natural V. cholerae hosts and require absent or damaged microbiota to enable colonization. Vertebrate fish are natural V. cholerae hosts and recent work has established zebrafish as a model that recapitulates the entire infectious process in the presence of intact intestinal microbiota. Fish are rapidly colonized by V. cholerae exposure in water, develop diarrhea, excrete large numbers of V. cholerae, and transmit the infection to naïve fish. Because this is a model in a natural host, it presents new opportunities for V. cholerae study that are not possible with mammalian models. However, the V. cholerae factors that act in fish pathogenesis are unknown for both O1 and non-O1/O1319 strains. This proposal will examine the infectious processes used by both O1 and non-O1/O139 V. cholerae and test the following hypotheses: 1) V. cholerae produce specific colonization factors in fish that are also important in human infections. These factors are shared among all V. cholerae strains. This hypothesis will be tested using Tn-Seq to identify genes important for fish colonization. 2) V. cholerae produces factor(s) that directly cause diarrhea. This hypothesis will be tested by knocking out known toxins in each strain and measuring diarrhea. 3) V. cholerae perturbs specific components of the intestinal microbiota, creating a niche for colonization. This hypothesis will be tested by assessing microbiome before and after infection. 4) The type six secretion system (T6SS) is important for competition with intestinal microbiota. This hypothesis will be tested by knocking out T6SS and further identifying T6SS effectors that are important for this competition. 5) V. cholerae excreted by fish are hyperinfectious, as they are in human cholera stool, and this is a likely source of V. cholerae outbreaks. This hypothesis will be tested by comparing infectious doses required for colonization from in vitro grown and fish-passaged V. cholerae. 6) V. cholerae shifts its gene expression program prior to host escape to enable transmission and contribute to hyperinfectivity. This hypothesis will be tested using RNA-Seq to determine global changes in gene expression between in vitro grown, actively colonizing, and excreted V. cholerae. Completion of the proposed work, using zebrafish as a natural V. cholerae host model, will significantly advance our understanding of the environmental lifestyle of V. cholerae in a natural reservoir, the requirements for V. cholerae to become a pathogen, and should uncover new targets for therapeutics, environmental remediation, and disease prevention. The long term goal of this project is to use the zebrafish model to better understand V. cholerae pathogenesis in humans and identify new strategies to combat V. cholerae disease and transmission. .