Regulatory Oxygenases in Vasculopathic Rickettsioses

描述（由申请人提供）：致病性立克次体是人类已知的一些最严重的细菌性疾病的病原。其中分别由立克次体致病菌引起的落基山斑疹热和普拉兹克氏体致病菌引起的流行性斑疹伤寒。立克次体是专性细胞内寄生虫，其特点是对哺乳动物宿主的血管内皮有趋向性。越来越多的证据表明活性氧（ROS）在内皮功能障碍和立克次体发病中起重要作用。作为生理稳态的维护者，内皮维持血管张力和血管通透性，并调节对过多有害刺激的炎症反应。我们发表的和初步的数据进一步表明：1。内皮细胞（EC）感染诱导血红素加氧酶(HO)-1的表达，这种酶负责将促氧化血红素降解为具有多种细胞保护功能的生物活性分子（胆红素、一氧化碳和铁蛋白）；2. 斑点热组（SFG）立克次体感染时环氧化酶(Cox)-2表达升高导致前列腺素（PG）分泌增加；和3。SF和斑疹伤寒组（TG）生物触发ROS生成和激活抗氧化防御机制的能力存在显著差异，这可能是由于它们的胞浆内行为存在明显差异。这些观察结果导致了这样的假设：HO和Cox酶系统的调节以及HO和Cox信号传导机制之间的相互作用是宿主细胞存活、炎症的发生/程度和血管通透性变化的关键决定因素，所有这些都是立克次体病病理表现的关键决定因素。拟议的研究集中在监管机制阐明未知控制氧化还原内稳态,急性炎症,血管渗透性使用人工培养的EC(体外)感染和传播内皮易感小鼠感染菌株(体内)SFG (r . rickettsii r . conorii和r .南极光)和TG (r . prowazekii r .伤寒)作为独立的模型系统。采用不同毒力程度的立克次体菌株、细胞成像、同工酶特异性抑制剂/激活剂或显性阴性突变体、基于sirna的敲除、组织靶向过表达和敲除小鼠，我们将解决以下主题和机制相关的特定目标：目的1将(i)定义使用Sheila Smith（高毒力）、HLP（相对致病性较低）和Iowa（无毒）立克次体的立克次体菌株感染诱导HO-1的信号机制和功能后果的相似性和/或潜在差异，以及（ii）研究Cox-2的调节及其通过产生pg和立克次体复制控制血管通透性的作用。目的2将描述SFG与TG立克次体的差异效应，以及个体物种中具有不同毒力程度的代表性菌株对内皮HO-1和Cox-2的影响，并确定调节宿主细胞反应的特定立克次体机制。目的3将确定HO-1在宿主保护性适应中的作用，以及Cox-2在体内SFG和TG立克次体病的病理生理中的作用。总之，这些研究将剖析HO-1和Cox-2活性的血管活性产物在SFG和TG立克次体病的发病和毒力机制中的生理意义和潜在差异，并为确定特定物种特异性干预或血管致病性立克次体病的新治疗靶点提供有用的见解。

DESCRIPTION (provided by applicant): Pathogenic Rickettsia species are etiologic agents of some of the most severe bacterial diseases known to mankind worldwide. Among them are Rocky Mountain spotted fever and epidemic typhus caused respectively by R. rickettsii and R. prowazekii. Rickettsiae are obligate intracellular parasites characterized by tropism for vascular endothelium of their mammalian hosts. Accruing evidence indicates an important role for reactive oxygen species (ROS) in endothelial dysfunction and rickettsial pathogenesis. As an upholder of physiological homeostasis, endothelium maintains vessel tone and vascular permeability and regulates inflammatory responses to a plethora of noxious stimuli. Our published and preliminary data further suggest that: 1. Infection of endothelial cells (EC) induces the expression of heme oxygenase (HO)-1, an enzyme responsible for the degradation of pro-oxidant heme into biologically active molecules (bilirubin, carbon monoxide, and ferritin) with diverse cytoprotective functions; 2. Increased expression of cyclooxygenase (Cox)-2 during infection with spotted fever group (SFG) rickettsiae results in enhanced prostaglandin (PG) secretion; and 3. There are significant differences in the abilities of SF and typhus group (TG) organisms to trigger ROS generation and activate anti-oxidant defense mechanisms, likely due to distinct differences in their intracytoplasmic behavior. These observations have led to the hypothesis that regulation of HO and Cox enzyme systems and interplay between HO and Cox signaling mechanisms in the vasculature are critical determinants of host cell survival, onset/degree of inflammation, and changes in vascular permeability, all of which are critical determinants of pathologic manifestations of rickettsioses. The proposed studies are focused on elucidating heretofore unknown regulatory mechanisms controlling redox homeostasis, acute inflammation, and vascular permeability using infection of cultured human EC (in vitro) and disseminated endothelial infection of susceptible mice strains (in vivo) with SFG (R. rickettsii, R. conorii and R. australis) and TG (R. prowazekii, R. typhi) as independent model systems. Employing Rickettsia strains with varying degree of virulence, cell imaging, isozyme-specific inhibitors/activators or dominant- negative mutants, siRNA-based knockdown, tissue-targeted over-expression, and knockout mice, we will address the following thematically and mechanistically interrelated specific aims: Aim 1 will (i). define similarities and/or potential differences in signaling mechanisms and functional consequences of infection-induced HO-1 using Sheila Smith (highly virulent), HLP (comparatively less pathogenic), and Iowa (avirulent) strains of R rickettsii, and (ii). investigate the regulation of Cox-2 and its role in the control of vascular permeability via production of PGs and rickettsial replication. Aim 2 will characterize the differential effects of SFG versus TG rickettsiae and representative strains with varying degree of virulence within individual species on endothelial HO-1 and Cox-2 and identify specific rickettsial mechanisms that regulate host cell responses. Aim 3 will determine the role of HO-1 in the host-protective adaptations and Cox-2 in the pathophysiology of in vivo SFG and TG rickettsioses. Together, these studies will dissect the physiological significance and potential differences in the contributions of vasoactive products of HO-1 and Cox-2 activities in the pathogenetic and virulence mechanisms of SFG and TG rickettsioses and provide useful insight to identify novel therapeutic targets for species-specific interventions in particular or vasculopathic rickettsial diseases in general.