Interspecific Competition Between Rickettsiae in Ticks

描述（由申请方提供）：尽管蜱传斑点热群立克次体具有多样性和广泛分布，但田间和实验室研究均表明，尽管蜱能够获得多种立克次体感染，但大多数蜱调查显示，在受感染的蜱中仅存在单一立克次体种属。在之前的资助期间，我们已经证明，稳定的蒙大拿立克次体感染阻断了蜱卵巢的维持和第二个新引入的立克次体的传播。随后的研究提供了经验证据，细胞进入可能是一个限制步骤，以多种立克次体定殖蜱卵巢系统。在本申请中，我们专注于通过跨卵传播调节立克次体维持的潜在机制，特别是促成跨卵干扰现象的蜱基因产物。特别是，我们将测试的假设，即斑点热群立克次体在宿主细胞进入或通过竞争的蜱卵巢生态位相互竞争。本研究的目的是：（1）确定细胞进入或生长是否是阻止立克次体双重感染的限制性步骤;孔雀感染的安氏革蜱对卵巢作为小生境的竞争将通过分子或通过目视检查感染有GFP或mCherry表达立克次体的细胞来测量。在这个目标下，我们还将研究立克次体致病性的程度是否与蜱卵巢维持强毒立克次体的作用降低有关。立克次体D. andersoni;和（2）研究在具有共生R. peacockii或R.因此，在卵巢中抑制立克次体的重复感染。我们将检验这一假设，即免疫反应持续在一个高度的水平在蜱组成性感染立克次体，这有助于干扰现象表型。使用qRT-PCR，我们将比较未感染和R. peacockii或R.蒙大拿州固有感染蜱虫。然后，我们将进行一个相互的挑战与组成型立克次体感染的蜱，并比较防御基因的表达水平，以未挑战的组成型感染的蜱。 公共卫生相关性：在美国有九种不同的蜱传斑点热群立克次体，包括高致病性立克次体，它是落基山斑点热（RMSF）的病原体。RMSF的特点是头痛，发烧，肌痛，恶心和呕吐，如果不治疗，它可能会发展为多器官衰竭和死亡。虽然人类RMSF病例自2000年以来增加了一倍多，但美国立克次体/蜱调查表明，R。立克次体在媒介蜱中不太普遍，并且在大多数情况下，阳性蜱携带非致病性立克次体，例如立克次体。peacockii，R. montanensis和R.裂头兽R.立克次体在阳性蜱中的感染产生了有趣的问题，从而引导我们调查蜱中立克次体多重感染的后果和流行病学意义。特别是，我们的研究集中在调节蜱类立克次体维持的潜在机制上。

DESCRIPTION (provided by applicant): Despite the diversity and wide distribution of tick-borne spotted fever group rickettsiae both field and laboratory studies have demonstrated that while ticks are capable of acquiring multiple rickettsial infections most often tick surveys revealed the presence of only a single rickettsial species in infected tick. During previous funding period, we have shown that stable Rickettsia montanensis infection blocks the tick ovarian maintenance and transmission of a second newly introduced rickettsiae. Studies following ours have offered empirical evidence that cell entry may be a limiting step to multiple rickettsial colonization of tick ovarial system. In this application we are focusing on underlying mechanisms that regulate rickettsial maintenance via transovarial transmission and in particular the tick gene products contributing to the transovarial interference phenomenon. In particular, we will test the hypothesis that spotted fever group rickettsiae outcompete each other for the tick ovarian niche during host cell entry or through competition. The aims of the proposed research are: (1) To determine if cell entry or growth is the limiting step preventing dual rickettsial infection in R. peacockii - infected Dermacentor andersoni ticks. Competition for the ovary as a niche will be measured molecularly or via visual inspection of cells infected with GFP- or mCherry- expressing rickettsiae. Under this aim we will also investigate whether the degree of rickettsial pathogenicity correlates with reduced role for tick ovarian maintenance of virulent R. rickettsii in D. andersoni; and (2) To investigate if immune activity persists at a heightened level in ticks with a symbiotic R. peacockii- or R. montanensis-infected ticks thereby prohibiting rickettsial superinfection in the ovaries. We will test the hypothesis that the immune response persists at a heightened level in ticks constitutively infected with rickettsiae, which contributes to the interference phenomenon phenotype. Using qRT-PCR, we will compare defensin, lysozyme and a kunitz-type protease inhibitor (DvKPI) expression levels in uninfected and R. peacockii or R. montanensis-constitutively infected ticks. We will then perform a reciprocal challenge of ticks with a constitutive rickettsia infection and compare the expression level of defense genes to unchallenged constitutively infected ticks. PUBLIC HEALTH RELEVANCE: Nine different tick-transmitted spotted fever group rickettsiae are recognized in the United States including the highly pathogenic Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF). RMSF is characterized by headache, fever, myalgia, nausea and vomiting early in the illness and if untreated it could progress to multi-organ failure and death. While the human RMSF cases more than doubled since 2000 overall the U.S. Rickettsia/tick surveys indicate that R. rickettsii is less prevalent in vector ticks and in most cases the positive ticks harbor nonpathogenic rickettsiae such as R. peacockii, R. montanensis and R. rhipicephali. The low prevalence of R. rickettsii in positive ticks has generated intriguing questions, thus leading us to investigate the consequences and epidemiological significance of multiple rickettsial infections in ticks. In particular, our research focused on underlying mechanisms that regulate rickettsial maintenance in ticks.