Evasion of complement-mediated killing by pathogenic rickettsial species

描述(申请人提供)：革兰氏阴性-蛋白立克次体细菌小(0.3-0.5×0.8-1.0米)，专性胞内生物体。斑点热组(SFG)和斑疹伤寒组(TG)可以通过抗原性和基于细胞内肌动蛋白的运动性来区分。该属的成员对人类的严重疾病负责，包括康氏杆菌和立克次体在内的几个物种已被美国国家过敏和传染病研究所(NIAID)列为B类和C类优先病原体，并被疾病控制和预防中心(CDC)列为选择性制剂(R.rickettsii)，因为它们可能被用作生物恐怖主义的工具。 我们最近确定，在缺乏中和抗体的情况下，地中海斑点热(MSF)的病原体--地中海斑点热(MSF)的模式立克次体--康氏杆菌能够逃避补体介导的人和小鼠血清中的杀伤。这些结果表明，康氏立克次体和可能的其他立克次体物种可能进化出了活性机制，以逃避补体沉积的杀菌作用，因为病原体在血液中传播到靶器官和组织。我们已经确定了两个高度保守的立克次体抗原rOmpB/SCA5和RC1281/Adr1，它们足以在体外人血清立克次体菌血症模型中介导存活。我们已经证明了孔氏杆菌与人因子H(Fh)相互作用，人因子H(Fh)是替代补体途径的液体相调节器，rOmpB与人Fh共免疫沉淀，证明了它作为真正的Fh结合蛋白(FHBP)的能力。我们还证明，当康氏立克次体在缺乏FH的人血清中孵育时，存活会受到影响，这表明与血清成分的特异性立克次体相互作用是存活的关键。ROmpB和RC1281的同源物存在于所有致病立克次体中，这表明在血液中主动干扰补体介导的杀伤的能力是这类病原体的一种新的毒力属性。本提案中概述的实验将涉及以下研究兴趣： I.立克次体FH结合蛋白对血清抵抗力的贡献将在VITR中使用在大肠杆菌中的替代表达系统进行分析。我们将初步确定rOmpB和RC1281在体外对血清抵抗的意义，并将确定补体介导的杀伤逃逸的机制。 二、使用康诺里和立克次杆菌感染的体外和体内模型，将分析在RompB/SCA5和rc1281中产生的靶向插入突变体的贡献。

DESCRIPTION (provided by applicant): The Gram-negative ¿-proteobacteria of the genus Rickettsia are small (0.3-0.5 x 0.8-1.0 ?m), obligate intracellular organisms. They are categorized into two major groups, the Spotted Fever Group (SFG) and Typhus Group (TG), which can be distinguished by antigenicity and intracellular actin-based motility. Members of this genus are responsible for severe human diseases and several species including R. conorii and R. rickettsii, have been classified as Category B and C Priority Pathogens by the National Institute of Allergy and Infectious Diseases (NIAID) and as the Select Agents (R. rickettsii) by the Centers for Disease Control and Prevention (CDC) for their potential use as tools for biological terrorism. We have recently determined that in the absence of neutralizing antibodies, a model SFG rickettsial species, R. conorii the etiologic agent of Mediterranean spotted fever (MSF), is able to evade complement-mediated killing in human and murine serum. These results suggested that R. conorii and possibly other rickettsial species likely have evolved active mechanisms to evade the bactericidal effects of complement deposition as the pathogen disseminates in the bloodstream to target organs and tissues. We have identified two highly conserved rickettsial antigens, rOmpB/Sca5 and RC1281/Adr1, which are sufficient to mediate survival in ex-vivo human serum models of rickettsia bacteremia. We have demonstrated that R. conorii interact with human Factor H (fH), a fluid phase regulator of the alternative complement pathway and that rOmpB co-immunopreciptates with human fH, demonstrating its ability to serve as a bona fide fH binding protein (fHbp). We have also demonstrated that R. conorii survival is compromised when incubated in human serum depleted of fH suggesting that specific rickettsial interactions with serum components are critical to survival. Homologues to rOmpB and RC1281 exist in all pathogenic rickettsiae suggesting that the ability to actively perturb complement-mediated killing in the blood is a novel virulence attribute for this class of pathogens. The experiments outlined in this proposal will address the following research interests: i. The contribution of rickettsial fH binding proteins to serum resistance will be analyzed in vitr using surrogate expression systems in E. coli. We will initially determine the significance of rOmpB and RC1281 to serum resistance in vitro and will also determine the mechanisms underlying the evasion of complement-mediated killing. ii. The contribution of generated targeted insertion mutants in rompB/sca5 and rc1281 will be analyzed using in vitro and in vivo models of R. conorii and R. rickettsii infection.