Regulatory Oxygenases in Vasculopathic Rickettsioses

描述(申请人提供)：致病性立克次体是一些人类已知的最严重的细菌性疾病的病原体。其中落基山斑热病和流行性斑疹伤寒分别由立克次体和普罗瓦泽克立克次体引起。立克次体是一种专性细胞内寄生虫，其特征是嗜食宿主的血管内皮细胞。越来越多的证据表明，活性氧簇(ROS)在内皮功能障碍和立克次体发病机制中起着重要作用。作为生理动态平衡的维持者，内皮细胞维持血管张力和血管通透性，并调节对过多伤害性刺激的炎症反应。我们已发表的和初步的数据进一步表明：1.感染内皮细胞(EC)诱导血红素加氧酶(HO)-1的表达，这是一种负责将促氧化剂血红素降解为具有不同细胞保护功能的生物活性分子(胆红素、一氧化碳和铁蛋白)的酶；2.斑点热立克次体感染时环氧合酶(COX)-2的表达增加导致前列腺素(PG)分泌增加；以及3.斑点热立克次体(SF)和斑疹伤寒(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，这可能是由于它们在胞浆内的行为不同。这些观察结果导致假设，血管中HO和COX酶系统的调节以及HO和COX信号机制之间的相互作用是宿主细胞存活、炎症开始/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理表现的关键决定因素。本研究以SFG(里氏立克次体、康氏立克次体和澳洲立克次体)和TG(普氏立克次体和伤寒杆菌)作为独立的模型系统，以体外培养的人EC和体内易感小鼠的播散性内皮细胞感染为研究对象，研究氧化还原动态平衡、急性炎症和血管通透性的调控机制。使用具有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/激活剂或显性阴性突变体、基于siRNA的敲除、组织靶向过度表达和敲除小鼠，我们将解决以下主题和机械上相互关联的特定目标：目标1将(I)。使用希拉·史密斯(高毒力)、HLP(相对较低致病性)和爱荷华州(无毒)立克次体菌株，确定感染诱导的HO-1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PGs的产生和立克次体复制在控制血管通透性中的作用。目的研究SFG和TG立克次体以及不同物种内毒力不同的代表性菌株对内皮细胞HO-1和COX-2的不同作用，并确定调节宿主细胞反应的特定立克次体机制。目的3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立克次体的病理生理学中的作用。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立克次体致病和致病机制中的生理意义和潜在差异，并为为特定物种或一般血管致病性立克次体疾病确定新的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.