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HIV流行的到来和因各种癌症接受化疗的个体的增加，未充分研究的人畜共患病机会性病原体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。红球菌性肺炎的特征是脓肉芽肿性炎症伴空洞，可误诊为结核分枝杆菌感染。R.马是一种常见的土传兼性放线菌，当被吸入时，其抵抗先天性杀伤机制，并容易在易感宿主的巨噬细胞中增殖，这可能是通过内体运输的扰动。关于R的分子基础知之甚少。马病原体，并且在我们最近的工作之前，很少有工具存在，以允许这种细菌的彻底的遗传分析。最近，我们发展了遗传学方法来构建确定的R. Equi，并且还创建了有效的转座子诱变系统。利用这些新的工具，我们证明了vapA（毒力相关蛋白A），一个存在于R。马，是细胞内生长和完全毒力所必需的。vapA的缺失使细菌变弱，使其不能在体内生长并且不能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R的遗传基础。等毒力。我们建议首先扩展我们对vapA的理解，这是迄今为止唯一已知的R。等毒力决定因子这项工作的第一个目的是确定vapA的作用机制。为此，我们将定义两种野生型R. equi和vapA缺失突变体。具体来说，我们将描述的过程中吞噬体成熟的野生型R。马感染的巨噬细胞，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主机蛋白伴侣的VapA。第二个目的是对R.通过检测vapA、VirR（yjrulence regulator）和VarA（雅普regulator）的表达调节子，对马毒力调节子进行了初步的研究。我们将鉴定这些调节因子转导的特定信号，并鉴定由它们控制的其他基因。我们将建立virR和varA缺失突变体对R.等毒力。最后，我们将确定的vapA启动子的监管机构的结合位点。这些研究阐明了R.马的发病机制将进一步我们的巨噬细胞生物学的理解，并将产生洞察生物学放线菌宿主-病原体的关系一般。