MOLECULAR MECHANISMS OF YERSINIA PESTIS PERSISTENCE IN THE FLEA VECTOR

描述(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周期变化。这些生物循环由两个阶段组成：一个是流行病阶段，它增加了传播和人类疾病的风险；另一个是地方性/静止期，在这个阶段，引起鼠疫耶尔森氏菌的细菌持续存在并维持着宿主。虽然家禽流行病可以由生物膜介导的肠道阻塞传播机制来支持，但流行病/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可传播的感染。了解鼠疫耶尔森氏菌在动物间流行期间持续存在的分子基础将解决知识上的重大差距，并可能导致确定新的目标，以防止跳蚤传播和流行性疾病。我们最近对鼠疫耶尔森氏菌Phop突变体与野生型菌株的比较转录图谱支持在跳蚤肠道内？Phop突变体中存在交替的休眠生存状态。在描述休眠的其他支持现象中，许多毒素：抗毒素(TA)模块通过下调必要的细胞过程来负责休眠持久细胞的形成，在？Phop突变体中唯一地上调了表达。这导致了假设，鼠疫杆菌过渡到自我保护的休眠生存状态，使其能够在跳蚤宿主中持续存在，并且这种表型是可逆的，并受TA模块表达的驱动。R21探索性研究的目的是确定休眠是否是持久性的函数，以及TA模块的表达是否负责驱动鼠疫杆菌生物膜肠道阻塞可传播感染的休眠和重新激活。在实验上，这将通过评估休眠的鼠疫杆菌突变体在跳蚤肠道中的延长存活率和对抗生素的高耐受性来确定休眠是否在表型和机械上是持久性的函数。接下来，通过创造一种条件可诱导的Phop菌株，我们将测试休眠细胞是否可以被重新激活以导致可传播的感染。最后，我们将通过建立有条件诱导的毒素表达菌株，并检测它们的长期存活、抗生素耐受性和生物膜肠道堵塞，来具体测试TA模块在驱动休眠和重新激活的肠道阻断鼠疫杆菌传播表型方面的作用。这项研究与R21机制的探索性/发育性(如PA-10-069中所述)是一致的，因为它探索了驱动鼠疫杆菌在跳蚤媒介中持续存在的未知分子机制，这可能导致识别新的靶点，以防止跳蚤传播和家禽流行性疾病。 与公共卫生相关：在这两起事件中，由于受感染的跳蚤传播，淋巴腺鼠疫再次成为一个公共卫生问题，并对生物武器构成威胁。这项研究的目的是确定休眠是否驱动鼠疫耶尔森氏菌在跳蚤媒介中的持续存在，并确定在动物流行间期导致这种持续存在的分子机制。这可能导致确定新的目标，以防止跳蚤传播和家畜/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.