MOLECULAR MECHANISMS OF YERSINIA PESTIS PERSISTENCE IN THE FLEA VECTOR

描述（由申请人提供）：鼠疫是一种跳蚤传播的人畜共患疾病，其特征是野生啮齿动物种群及其相关跳蚤的森林循环。这些森林循环由动物流行病阶段和地方性动物流行病/静止阶段组成，前者增加了传播和人类疾病的风险，后者致病细菌鼠疫耶尔森氏菌持续存在并维持宿主。虽然动物流行病可以通过生物膜介导的肠道阻塞传播机制得到支持，但地方性/静止阶段的基础尚不清楚。我们认为这种静止是由一种“休眠”形式的细菌介导的，这种细菌在跳蚤体内持续存在，但在有利的条件下可以恢复为传染性感染。了解鼠疫杆菌在动物流行间期持续存在的分子基础将解决知识上的重大空白，并可能导致确定预防蚤媒传播和动物流行的新靶点。我们最近对鼠疫耶尔森菌的比较转录谱分析？phoP突变株与野生型株的对比支持在？phoP在跳蚤肠道内突变。在描述休眠的其他支持现象中，许多毒素：抗毒素（TA）模块通过下调基本细胞过程负责休眠持久细胞的形成，在？phoP突变。这导致了一种假设，即鼠疫杆菌进入休眠的自我保护生存状态，使其能够在跳蚤宿主中持续存在，并且这种表型是可逆的，由TA模块表达驱动。R21探索性研究的目的是确定休眠是否是持久性的功能，以及TA模块的表达是否负责驱动鼠疫菌生物生物膜肠道阻塞传播感染的休眠和再激活。在实验上，这将通过评估休眠鼠疫杆菌的长期生存能力和对抗生素的高耐受性，分别确定休眠是否是表型和机制上的持久性功能来实现。跳蚤肠道中的phoP突变体。接下来，通过创建一个有条件诱导的phoP菌株，我们将测试休眠细胞是否可以重新激活以引起传染性感染。最后，我们将通过制造条件诱导毒素表达菌株并测定它们的长期存活、抗生素耐受性和生物膜肠道阻塞，具体测试TA模块在驱动休眠和重新激活的鼠疫菌肠道阻塞传递表型中的作用。这项研究符合R21机制的探索性/发育性（如PA-10-069所述），因为它探索了驱动鼠疫杆菌在跳蚤媒介中持续存在的未表征的分子机制，这可能导致鉴定新的靶点，以防止跳蚤传播和动物流行病。

DESCRIPTION (provided by applicant): Plague is a flea-borne zoonotic disease characterized by sylvatic cycles in wild rodent populations and their associated fleas. These sylvatic cycles are composed of an epizootic phase, which increases the risk of transmission and human disease, and an enzootic/quiescent stage in which the causative bacterium, Yersinia pestis, persists and maintains the reservoir. While epizootics can be supported by a biofilm-mediated gut blockage transmission mechanism, the basis of the enzootic/quiescent stage is unknown. We propose that this quiescence is mediated by a 'dormant' form of the bacterium which sustains persistence within the flea but can then revert to a transmissible infection upon favorable conditions. Understanding the molecular underpinnings of Y. pestis persistence during inter-epizootic periods would resolve a significant gap in knowledge and may lead to identification of new targets to prevent flea-borne transmission and epizootics. Our recent comparative transcriptional profiling of a Yersinia pestis ?phoP mutant versus a wildtype strain supports the presence of an alternate dormant survival state in the ?phoP mutant within the flea gut. Among other supporting phenomena descriptive of dormancy, numerous toxin:antitoxin (TA) modules that are responsible for dormant persister cell formation by downregulating essential cellular processes have exclusively elevated expression in the ?phoP mutant. This lead to the hypothesis that Y. pestis transitions into the self-protective survival state of dormancy which allows it to persist in the flea host, and that this phenotype is reversible and driven by TA module expression. The goal of the R21 exploratory research is to determine whether dormancy is a function of persistence and whether expression of TA modules are responsible for driving dormancy and reactivation of the biological biofilm gut blockage transmissible infection in Y. pestis. Experimentally this will be achieved by establishing whether dormancy is phenotypically and mechanistically a function of persistence by assessing the prolonged survivability and high tolerance to antibiotics, respectively of the dormant Y. pestis ?phoP mutant in the flea gut. Next, by creating a conditionally inducible phoP strain we will test whether dormant cells can be reactivated to cause a transmissible infection. Finally we will specifically test the role of TA modules in driving the dormant and reactivated gut blocked transmitting phenotypes of Y. pestis by creating conditionally inducible toxin expressing strains and assaying them for long term survival, antibiotic tolerance and biofilm gut blockage. This research is consistent with the exploratory/developmental nature of the R21 mechanism (as described in PA-10-069) because it explores uncharacterized molecular mechanisms that drive persistence of Y. pestis in the flea vector, which may lead to identification of new targets to prevent flea-borne transmission and epizootics. PUBLIC HEALTH RELEVANCE: Bubonic plague has re-emerged as a public health problem and is a bioweapons threat, in both events due to transmission by infected fleas. The objective of the research is to characterize whether dormancy drives persistence of Yersinia pestis in the flea vector and to define the molecular mechanisms responsible for this persistence during inter-epizootic periods. This may lead to identification of new targets to prevent flea-borne transmission and epizootics/epidemics.