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以及各种癌症接受化疗的个体的增加，研究的人畜共患病原体病原体rohodococcus equi已成为受损免疫力损害的人的肺炎。犀牛肺炎的特征是秋水神经瘤炎症伴气炎，可以误诊为结核分枝杆菌感染。 R. Equi是一种常见的土壤培养基放线菌，当吸入时，它可以抵抗先天的杀戮机制，并且很容易在易感宿主的巨噬细胞中乘以繁殖，这可能是由于内体贩运的扰动。关于Equi发病机理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该细菌进行彻底的遗传分析。最近，我们开发了构建岩石河的定义突变体的遗传方法，并创建了一个有效的转座子诱变系统。使用这些新工具，我们证明了vapa（毒力相关的蛋白A），一种存在于R. equi的毒力质粒上的基因，是细胞内生长和充分毒力所必需的。 VAPA的缺失减弱了细菌，使其无法体内生长，并且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肠杆菌的遗传基础。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R. Equi毒力决定因素。这项工作的第一个目的是确定VAPA的作用机理。为此，我们将定义野生型R. Equi和Vapa缺失突变体的吞噬作用后的细胞内事件。具体而言，我们将表征野生型吞噬体成熟的过程。Equi感染了巨噬细胞，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VAPA的相互作用的宿主蛋白伴侣。第二个目的是通过检查VAPA表达，VIRR（YJROULESENCULTATER）和VARA（YAP调节剂）的调节剂来表征R. equi毒力调节。我们将确定这些调节器传输的特定信号，并确定由其控制的其他基因。我们将建立Virr和Vara缺失突变体对股E. Equi毒力的影响。最后，我们将确定调节器与VAPA启动子的结合位点。这些针对R. equi发病机理的分子机制的研究将进一步了解我们对巨噬细胞生物学的理解，并将深入了解放线菌的宿主 - 病原体关系的生物学。