CAREER: Convergent molecular mechanisms regulating dormancy in Vibrios

在诸如饥饿或抗生素暴露等不利条件下，许多微生物进入休眠状态以促进其生存。休眠的细菌细胞与抗生素治疗失败、与食物有关的疾病暴发和环境疾病传播有关。尽管休眠状态的普遍性和生物学意义，这些关键现象的几个方面仍然不清楚。在本研究项目中，将研究控制细菌休眠的调节网络和驱动这种现象的环境条件。在这个研究项目中，霍乱的病原体霍乱弧菌将被用作模型系统来研究转录因子ToxR在休眠中的作用。然而，实验方法和潜在的发现将适用于形成这些休眠细胞类型的许多微生物。这一建议的结果可能对公共卫生和经济产生重大影响，因为这些细胞通常对抗生素具有耐药性，并且是食物和水污染的主要来源。一项主要的综合教育倡议，协同学者计划，将通过创建一个围绕学者指导的支持性社区，促进中佛罗里达大学生物医学科学专业代表性不足的学生的保留。中佛罗里达大学是一所为西班牙裔服务的大型机构。为来自波多黎各美洲大学（Aguadilla）的学生提供一个桥梁项目，让他们在UCF就本提案所涵盖的领域进行研究，并在UCF开设一门关于“细菌应激反应”的最新课程，该课程将包括本提案中的主题，如细菌休眠。本研究项目的重点是控制细菌休眠的趋同调节网络，以及不同的环境条件如何驱动这一现象。除产孢外，休眠细菌细胞有两种主要状态，即持久菌和可活但不可培养（VBNC）。持久性细胞通常是在抗生素暴露后选择的，而VBNC细胞是在自然环境中由于暴露于生物和非生物因素而产生的。尽管存在这些差异，但这两个州之间仍有一些主要的重叠之处。在本研究项目中，将使用霍乱弧菌作为模型系统和主调节剂ToxR对VBNC进入和持久性细胞形成的假设进行测试，该假设是由不同的刺激启动的，但会聚到共同的调节途径中。主要假设将在三个独立但互补的目标中进行测试：目标1将解决常见的水生非生物和生物驱动因素如何通过严格的反应以依赖toxr的方式协同调节VBNC；目的2将确定ToxR和细胞包膜应激反应在持久性细胞形成调控中的作用；并且，目标3将检查toxr调节的小RNA在这些休眠表型趋同中的作用。总的来说，在一个可处理的模型系统中使用遗传工具，并整合有关调节网络和环境条件的数据，将提供对休眠细胞动力学的深入了解。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Under unfavorable conditions such as starvation or antibiotic exposure, numerous microorganisms enter dormant states that facilitate their survival. Dormant bacterial cells have been linked to the failure of antibiotic therapy, food-related outbreaks, and environmental disease transmission. Despite the prevalence and biological significance of dormant states, several aspects of these critical phenomenon remain obscure. In this research project, the regulatory networks controlling bacterial dormancy and environmental conditions driving this phenomenon will be investigated. In this research project, Vibrio cholerae, the causative agent of cholera, will be used as a model system to study the role of a transcription factor, ToxR, in dormancy. However, the experimental approaches and potential findings will be applicable to numerous microorganisms that form these types of dormant cells. Results from this proposal may have major public health and economic implications as these cells are typically resistant to antibiotics and are a major source of food and water contamination. A major integrated education initiative, the Synergy Scholars Program, will foster the retention of underrepresented students in Biomedical Sciences at University of Central Florida, a large Hispanic-Serving Institution by creating a supportive community that revolves around the mentoring of scholars. A bridge program for students from the Universidad Interamericana de Puerto Rico (Aguadilla) to perform research at UCF in areas covered in this proposal and an updated course on “Bacterial Stress Responses” at UCF that will include topics from this proposal such as bacterial dormancy will be implemented.This research project focuses on the convergent regulatory networks controlling bacterial dormancy and how diverse environmental conditions drive this phenomenon. Aside from sporulation, there are two major states of dormant bacterial cells, persisters and viable but non-culturable (VBNC). Persister cells are typically selected after antibiotic exposure, whereas VBNC cells arise in natural environments due to exposure to biotic and abiotic factors. In spite of these differences, there are major overlaps between the two states. In this research project, the hypothesis that VBNC entry and persister cell formation are initiated by distinct stimuli but converge into common regulatory pathways will be tested using Vibrio cholerae as a model system and the master regulator ToxR. The main hypothesis will be tested in three independent but complementary aims: aim 1 will address how common aquatic abiotic and biotic drivers synergistically regulate VBNC in a ToxR-dependent manner via the stringent response; aim 2 will define the roles of ToxR and the cell envelope stress response in the regulation of persister cell formation; and, aim 3 will examine the role of a ToxR-regulated small RNA in the convergence of these dormant phenotypes. Overall, the use of genetic tools in a tractable model system and the integration of data on the regulatory networks and environmental conditions will provide insight into the dynamics of dormant cells.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.