Virulence of the Opportunistic Pathogen Rhodococcis Equi

机会性病原体马红球菌的毒力

• 批准号: 7064408
• 负责人: MARY HONDALUS
• 金额: $ 33.12万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-02-01 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7064408
• 关键词: genes; intracellular; macrophage; mutant; proteins; virulence

DESCRIPTION (provided by applicant): With the arrival of the HIV epidemic and the increase in individuals undergoing chemotherapy for various cancers, the understudied zoonotic opportunistic pathogen Rhodococcus equi has emerged as cause of life-threatening pneumonia in persons of compromised immunity. Rhodococcal pneumonia is characterized by pyogranulomatous inflammation with cavitation, which can be misdiagnosed as Mycobacterium tuberculosis infection. R. equi is a common soil-borne facultative actinomycete which, when inhaled, resists innate killing mechanisms and readily multiplies in macrophages of susceptible hosts, likely by perturbation of endosomal trafficking. Little is known about the molecular basis for R. equi pathogenesis, and prior to our recent work, few tools existed to allow a thorough genetic analysis of this bacterium. Recently, we have developed the genetic methods to construct defined mutants of R. equi and have also created an efficient transposon mutagenesis system. Using these new tools, we demonstrated that vapA (virulence associated protein A), a gene present on the virulence plasmid of R. equi, is required for intracellular growth and full virulence. Deletion of vapA attenuates the bacterium, rendering it incapable of growth in vivo and unable to replicate in macrophages cultured in vitro. Our long-term goal is to completely dissect the genetic basis for R. equi virulence. We propose to do so by first expanding our understanding of vapA, which is to date the only known R. equi virulence determinant. The first AIM of this work is to identify the mechanism of action of vapA. To do so, we will define the intracellular events post phagocytosis of both wild type R. equi and the vapA deletion mutant. Specifically we will characterize the process of phagosomal maturation in wild type R. equi infected macrophages and test the influence of VapA on phagosome acidification and fusion with lysosomes. We will identify interacting host protein partners of VapA. The second AIM is to characterize the R. equi virulence regulon through the examination of regulators of vapA expression, VirR (yjrulence regulator) and VarA (yap regulator). We will identify the specific signals transduced by these regulators and identify additional genes controlled by them. We will establish the effects of deletion mutants of virR and varA on R. equi virulence. Finally, we will identify the binding sites of the regulators to the vapA promoter. These studies addressing the molecular mechanisms of R. equi pathogenesis will further our understanding of macrophage cell biology and will yield insight into the biology of actinomycete host-pathogen relationships in general.

描述（由申请人提供）：随着艾滋病毒流行的到来和接受各种癌症化疗的个体的增加，尚未得到充分研究的人畜共患机会致病菌马红球菌已成为免疫力低下人群中危及生命的肺炎的原因。红球菌性肺炎的特点是脓肉芽肿性炎症伴空化，可误诊为结核分枝杆菌感染。马氏放线菌是一种常见的土壤传播的兼性放线菌，当吸入时，它抵抗先天杀伤机制，并容易在易感宿主的巨噬细胞中繁殖，可能是通过内体运输的扰动。我们对马链球菌发病机制的分子基础知之甚少，在我们最近的工作之前，很少有工具可以对这种细菌进行彻底的遗传分析。近年来，我们已经建立了一套高效的转座子诱变系统。利用这些新工具，我们证明了存在于马鼠毒力质粒上的基因vapA（毒力相关蛋白A）是细胞内生长和完全毒力所必需的。删除vapA会削弱细菌，使其无法在体内生长，也无法在体外培养的巨噬细胞中复制。我们的长期目标是彻底剖析平毛犀毒力的遗传基础。我们建议通过首先扩展我们对vapA的理解来做到这一点，这是迄今为止唯一已知的麻黄毒力决定因素。本工作的第一个目的是确定vapA的作用机制。为此，我们将定义野生型马鼠和vapA缺失突变体吞噬后的细胞内事件。具体来说，我们将描述野生型马鼠感染巨噬细胞吞噬体成熟的过程，并测试VapA对吞噬体酸化和与溶酶体融合的影响。我们将确定相互作用的VapA宿主蛋白伴侣。第二个目的是通过对vapA表达调控因子、VirR（毒力调控因子）和VarA（毒力调控因子）的研究来表征猪链球菌的毒力调控。我们将识别由这些调节因子转导的特定信号，并识别由它们控制的其他基因。我们将确定virR和varA缺失突变体对相等鼠毒力的影响。最后，我们将确定调节剂与vapA启动子的结合位点。这些研究将进一步加深我们对巨噬细胞生物学的理解，并将深入了解放线菌宿主-病原体的生物学关系。