Zebrafish as a natural host model for Vibrio cholerae

描述（由申请人提供）：霍乱弧菌是一种水生细菌，当人类摄入受污染的水时，会引起严重的腹泻病霍乱。估计每年有500万人感染霍乱，造成10万多人死亡。目前霍乱弧菌的主要动物模型是幼鼠和家兔。然而，这些模型昂贵、困难、令人不快，并且不能再现真实的疾病状态，因为它们不是霍乱弧菌的天然宿主。霍乱弧菌自然栖息于几种水生生态位，包括与贝类、虫卵群和浮游生物有关的生态位。最近还发现霍乱弧菌在脊椎动物鱼类的肠道中定植。我们的中心假设是斑马鱼，Danio rerio，是霍乱弧菌理想的自然动物宿主模型，这将使新的发病机制和环境定植研究成为可能。在过去的20年里，斑马鱼被广泛用作许多不同生物过程的模式生物，包括传染病模型。初步数据表明，霍乱弧菌在斑马鱼肠道内的定殖水平非常高，无论是通过灌胃直接进入消化道，还是简单地添加到斑马鱼孵育的水中。后者是一个比任何现有动物模型更自然、更有代表性的定植模型，我们很可能通过利用斑马鱼作为动物模型来了解关于定植过程的重要新信息。除了定植斑马鱼肠道外，霍乱弧菌还具有功能性毒力调节系统，通过一种未知的机制杀死斑马鱼。因此，斑马鱼也可能是研究霍乱弧菌疾病过程的良好模型。本提案的目的是建立斑马鱼作为霍乱弧菌的功能天然动物模型。我们将确定斑马鱼霍乱弧菌定植的地点。我们将建立一个殖民的时间表，以便未来的实验可以利用最相关的时间点来收集数据。我们将确定霍乱弧菌在斑马鱼中定植所需的感染剂量，这对未来实验的设计具有重要意义，并将阐明自然环境中霍乱弧菌和鱼类自由游动的动力学。最后，我们将确定霍乱弧菌分泌因子是如何导致鱼类死亡的。由于主要的毒力调控是导致斑马鱼死亡所必需的，因此霍乱弧菌毒力基因必须在斑马鱼存在的情况下被诱导。我们的长期目标是确定在人类感染期间诱导毒力的信号，以便在治疗上破坏毒力。斑马鱼模型为识别这些信号提供了一个全新的机会。建立斑马鱼作为天然动物宿主模型对霍乱弧菌的研究人员非常有用，不仅可以研究霍乱弧菌的定植和疾病过程，还可以研究霍乱弧菌的环境生活方式，这对于了解霍乱弧菌的宿主可以产生流行病或大流行霍乱疫情非常重要。因此，这一新模型的建立将对全球霍乱弧菌研究产生广泛的影响。

DESCRIPTION (provided by applicant): Vibrio cholerae are aquatic bacteria that cause the severe diarrheal disease cholera when ingested by humans in contaminated water. Cholera afflicts an estimated 5 million people annually, causing over 100,000 deaths. Currently the predominant animal models for V. cholerae are infant mice and rabbits. However, these models are expensive, difficult, unpleasant, and do not reproduce true disease states, as they are not natural hosts for V. cholerae. V. cholerae naturally inhabit several aquatic niches, including associations with shellfish, insect egg masses, and plankton. Recently V. cholerae were also found to colonize the intestinal tracts of vertebrate fish. Our central hypothesis is that the zebrafish, Danio rerio, is an ideal natural animal host model for V. cholerae that will make new studies of both pathogenesis and environmental colonization possible. Zebrafish have been utilized extensively in the past 20 years as model organisms for many different biological processes, including infectious disease models. Preliminary data indicate that V. cholerae colonize the zebrafish intestine to very high levels when administered either directly into the digestive tract via gavage or when simply added to water in which zebrafish are incubated. The latter is a much more natural, representative model for colonization than any existing animal model, and we are likely to learn important new information about the colonization process by utilizing zebrafish as an animal model. In addition to colonizing the zebrafish intestine, V. cholerae that have a functional virulence regulatory system also kill zebrafish through an unknown mechanism. Therefore, zebrafish may also be a good model for studying V. cholerae disease processes. The objective of this proposal is to establish zebrafish as a functional natural animal model for V. cholerae. We will determine the sites of V. cholerae colonization of zebrafish. We will establish a timeline for colonization, such that future experiments can utilize the most relevant time points for collecting data. We will determine the infectious dose required for V. cholerae colonization of zebrafish, which is important for the design of future experiments and will illuminate the dynamics of free swimming V. cholerae and fish in the natural environment. Finally, we will determine how secreted factors from V. cholerae cause fish death. Because the major virulence regulon is required for causing zebrafish death, V. cholerae virulence genes must be induced in the presence of zebrafish. Our long term goal is to identify the signals that induce virulence during human infection such that virulence can be disrupted therapeutically. The zebrafish model presents an entirely new opportunity for identifying these signals. Establishment of zebrafish as a natural animal host model should prove extremely useful to V. cholerae researchers studying not only colonization and disease processes but also the environmental lifestyle of V. cholerae, which is very important for understanding the V. cholerae reservoirs that can produce epidemic or pandemic cholera outbreaks. Therefore establishment of this new model should have a broad impact on V. cholerae research worldwide. PUBLIC HEALTH RELEVANCE: Cholera is a major public health problem in many developing countries and areas suffering from the effects of war or natural disaster, with an estimated 5,000,000 new cases and 100,000 deaths each year. The goal of this proposal is to establish zebrafish as a new natural host model for V. cholerae, the cause of cholera. This new model will facilitate better understanding of both how V. cholerae live in the environment and how they become human pathogens.