Virulence of the opportunistic pathogen Rhodococcus equi

机会性病原体马红球菌的毒力

• 批准号: 7174191
• 负责人: MARY HONDALUS
• 金额: $ 32.16万
• 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/7174191
• 关键词: Actinobacteria class; Actinomyces; Address; Affect; Attenuated; Bacteria; Binding; Binding Sites; Biological Assay; Biology; Breathing; Cellular biology; Clinical; Complex; Data; Defect; Development; Disease; Disruption; Epidemic; Event; Gene Family; Genes; Genetic; Goals; Granulomatous; Growth; HIV; High temperature of physical object; Homologous Gene; Host Defense; Immunity; In Vitro; Individual; Infection; Inflammation; Investigation; Ions; Knowledge; Life; Light; Lipoproteins; Location; Lysosomes; Macrophage Cell Biology; Malignant Neoplasms; Methods; Molecular; Molecular Mechanisms of Action; Monitor; Mus; Mutagenesis; Mycobacterium tuberculosis; Nature; Obstruction; Oxidative Stress; Pathogenesis; Pathway interactions; Persons; Phagocytosis; Phagosomes; Phenotype; Physiology; Plasmids; Pneumonia; Positioning Attribute; Process; Production; Proteins; Recombinants; Regulation; Regulatory Pathway; Regulon; Reporter; Research; Research Personnel; Resources; Rhodococcus equi; Role; Signal Transduction; Soil; Stimulus; Sum; Surface; System; Temperature; Testing; Time; Virulence; Work; attenuation; base; chemotherapy; expectation; genetic analysis; genetic manipulation; in vivo; innovation; insight; intracellular parasitism; killings; macrophage; member; mortality; mouse model; mutant; novel; pathogen; programs; promoter; resistance factors; response; tool; trafficking

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(申请人提供)：随着艾滋病毒疫情的到来和接受各种癌症化疗的人数增加，未被研究的人畜共患机会性病原体马红球菌已成为免疫力受损人群中危及生命的肺炎的原因。流行性肺炎以脓性肉芽肿性炎伴空洞为特征，易误诊为结核分枝杆菌感染。马放线菌是一种常见的土传兼性放线菌，当被吸入时，它可以抵抗先天的杀伤机制，并很容易在易感宿主的巨噬细胞中繁殖，这可能是由于内体转运的扰动。人们对马立克杆菌致病的分子基础知之甚少，在我们最近的工作之前，几乎没有工具可以对这种细菌进行彻底的遗传分析。最近，我们发展了遗传方法来构建马立克氏杆菌的明确突变体，并建立了一个高效的转座子诱变系统。使用这些新的工具，我们证明了VapA(毒力相关蛋白A)是细胞内生长和完全毒力所必需的，它是马立克氏杆菌毒力质粒上存在的一个基因。VapA的缺失会削弱细菌，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马立克氏杆菌毒力的遗传基础。我们建议首先扩大我们对VapA的理解，它是迄今为止唯一已知的R等毒力决定因素。这项工作的第一个目的是确定VapA的作用机制。为此，我们将定义野生型马R和VapA缺失突变体吞噬后的细胞内事件。具体地说，我们将描述野生型马立克杆菌感染的巨噬细胞吞噬小体成熟的过程，并测试VapA对吞噬小体酸化和与溶酶体融合的影响。我们将确定VapA相互作用的宿主蛋白伙伴。第二个目的是通过对VIPA表达调控因子VirR和YAP调节因子VARA的研究，来研究等毒力调节子的特性。我们将识别由这些调节器转导的特定信号，并识别由它们控制的其他基因。我们将建立病毒和VARA缺失突变体对马立克氏杆菌毒力的影响。最后，我们将确定调节子与VapA启动子的结合位点。这些针对马放线菌致病的分子机制的研究将进一步加深我们对巨噬细胞生物学的理解，并将从总体上深入了解放线菌宿主与病原体之间的生物学关系。