Zebrafish as a natural host model for Vibrio cholerae

描述(申请人提供)：霍乱弧菌是一种水生细菌，当人类在受污染的水中摄入时，会导致严重的腹泻疾病霍乱。据估计，每年有500万人感染霍乱，造成超过10万人死亡。目前霍乱弧菌的主要动物模型是幼鼠和兔。然而，这些模型昂贵、困难、令人不快，而且不能再现真实的疾病状态，因为它们不是霍乱弧菌的自然宿主。霍乱弧菌自然生活在几个水生生态位中，包括与贝类、昆虫卵块和浮游生物有关的生态位。最近，霍乱弧菌也被发现定植于脊椎鱼的肠道。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，这将使新的致病机理和环境定植研究成为可能。在过去的20年里，斑马鱼被广泛地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，当霍乱弧菌通过灌胃直接进入消化道或简单地添加到孵化斑马鱼的水中时，霍乱弧菌在斑马鱼肠道的定殖率非常高。后者是一种比任何现有动物模型更自然、更具代表性的殖民模型，我们可能会通过利用斑马鱼作为动物模型来了解关于殖民过程的重要新信息。霍乱弧菌除了在斑马鱼的肠道中定植外，还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发病过程的一个很好的模型。这项建议的目的是建立斑马鱼作为霍乱弧菌的功能性自然动物模型。我们将确定霍乱弧菌在斑马鱼上的定植地点。我们将建立一个殖民时间表，这样未来的实验就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上定植所需的感染量，这对未来实验的设计非常重要，并将阐明霍乱弧菌和鱼类在自然环境中自由游泳的动态。最后，我们将确定霍乱弧菌的分泌因子如何导致鱼类死亡。由于导致斑马鱼死亡的主要毒力调节基因是必需的，因此霍乱弧菌的毒力基因必须在斑马鱼存在的情况下被诱导。我们的长期目标是识别在人类感染期间诱导毒力的信号，以便毒力可以在治疗上被破坏。斑马鱼模型为识别这些信号提供了一个全新的机会。斑马鱼作为一种天然动物宿主模型的建立，对于霍乱弧菌研究人员不仅研究霍乱弧菌的定殖和疾病过程，而且研究霍乱弧菌的环境生活方式，都是非常有用的，这对于了解霍乱弧菌能引起霍乱流行或大流行的水库是非常重要的。因此，这一新模型的建立应该会对全世界的霍乱弧菌研究产生广泛的影响。 与公共卫生有关：霍乱是许多发展中国家和遭受战争或自然灾害影响的地区的一个主要公共卫生问题，估计每年新增病例500万例，死亡10万人。这项提议的目标是建立斑马鱼作为霍乱弧菌的新的自然宿主模型。这一新模型将有助于更好地了解霍乱弧菌如何在环境中生活，以及它们如何成为人类病原体。

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.