The Rv2623-Rv1747 interaction: regulation of the in vivo fate of M. tuberculosis

Rv2623-Rv1747 相互作用：结核分枝杆菌体内命运的调节

• 批准号: 10529446
• 负责人: John R. Chan
• 金额: $ 88.5万
• 依托单位: RBHS-NEW JERSEY MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-16 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10529446
• 关键词: ATP-Binding Cassette Transporters; Acute; Affect; Alanine; Amino Acids; Animal Model; Attenuated; Bacillus; Bacteria; Binding; Biochemical Genetics; Biogenesis; Bioinformatics; Biological Process; C3H/HeJ Mouse; Cavia; Cell Wall; Cells; Chronic; Chronic Phase; Complex; Cues; Data; Data Analyses; Development; Disease; Exhibits; FHA Domain; Generations; Genes; Genus Mycobacterium; Glycolipids; Goals; Growth; Heat shock proteins; Homologous Protein; Hypoxia; Immunologics; In Vitro; Individual; Infection; Inositol; Kinetics; Knock-out; Knowledge; Life Cycle Stages; Light; Mannosides; Mediating; Membrane; Modeling; Mus; Mutation; Mycobacterium smegmatis; Mycobacterium tuberculosis; Nitric Oxide; Oranges; Pathogenesis; Pathway interactions; Persons; Phase; Phenotype; Phosphorylation; Phosphothreonine; Physiological Processes; Play; Population; Positioning Attribute; Property; Proteins; Regulation; Regulon; Research; Role; Signal Transduction; Signaling Protein; Starvation; Stress; System; Testing; Threonine; Tissues; Tuberculosis; cell envelope; chronic infection; design; differential expression; disease transmission; immunopathology; immunoregulation; in vivo; insight; latent infection; lipooligosaccharide; mutant; mycobacterial; nitrosative stress; novel; overexpression; pathogen; pathogenic bacteria; reactivation from latency

Abstract The life cycle of Mycobacterium tuberculosis (Mtb) is complex, encompassing an acute phase, during which the pathogen replicates exponentially; a chronic phase, when bacterial burden is stably maintained, and a latent paucibacillary state that can reactivate. Chronic tuberculosis (TB) is associated with the development of tissue-damaging immunopathology and can promote disease transmission. It has been estimated that approximately 1/4 of the world's population are infected with Mtb, and a significant proportion of these individuals harbor latent bacilli that can reactivate to cause diseases. Unraveling the mechanisms that regulate Mtb growth in an infected host in the different phases of infection is paramount to understanding TB pathogenesis. It is generally thought that certain host environmental conditions (e.g., hypoxia, nitrosative stress, starvation) can promote the establishment of a latent infection. However, the precise mechanisms that regulate TB latency are incompletely defined. Mtb Rv2623, which is among the most upregulated genes in the dormancy regulon, encodes a universal stress protein (USP) that can regulate bacillary growth both in vivo and in vitro. A deletion mutant ΔRv2623 is hypervirulent in susceptible mice and Guinea pigs, and in the latter, it is defective in establishing a chronic persistent infection. In vitro, overexpression of Rv2623 in mycobacteria retards growth in recipient cells; and Mtb ΔRv2623 exits from the non-replicative phase of the hypoxia-induced Wayne latency model more expeditiously than wild-type (WT) Mtb upon transfer into O2-sufficient media. These results provide evidence that Rv2623 regulates Mtb growth, including possibly during the latent/reactivation phase of infection. We showed that Rv2623 interacts with the FHA domain-containing Mtb Rv1747, a putative exporter of lipooligosaccharides. The FHA domain is a signaling protein module that mediates a wide variety of biological processes via phosphorylation-dependent mechanisms. We further showed that the Rv2623-Rv1747 interaction is mediated through binding of the FHAI domain of Rv1747 with a phosphothreonine (at position 237)-containing motif of Rv2623, and that the T237 residue is essential for mediating the growth-regulatory attribute of Rv2623. In contrast to the hypervirulent ΔRv2623, ΔRv1747 is attenuated for growth in vivo. And while the hypervirulent ΔRv2623 expresses enhanced levels of the immunoregulatory phosphatidyl-myo- inositol mannosides (PIMs) relative to WT Mtb, the hypovirulent ΔRv1747 is a hypo-producer of PIMs. In addition, we showed that Rv1747-overexpressing strains hyperproduce PIMs. The correlation of Rv1747's expression levels and Mtb cell wall PIMs amounts suggests that Rv1747 may function as an exporter of Mtb cell wall biogenesis intermediates. This, together with the opposing PIMs phenotype and in vivo growth phenotype of ΔRv2623 and ΔRv1747, has led us to hypothesize that Rv2623 negatively regulates the functional activity of Rv1747 to modulate the levels of Mtb cell wall PIMs, which immunoregulatory properties can alter Mtb-host interactions, thereby influencing the in vivo fate of the tubercle bacillus. We will use biochemical, genetics, and immunological approaches, in conjunction with animal modeling and integrative bioinformatics and computational data analysis, to rigorously test this hypothesis. Finally, accumulating knowledge derived from functional and structural analysis of Rv1747, and the discovery of the relationship between Rv1747 expression and PIM levels, will enable the generation of a set of isogenic Mtb mutants expressing graded levels of PIMs, which can be used to stringently probe the significance of these immunoregulatory glycoplids in influencing the in vivo fate of Mtb. The proposed studies should illuminate how the Rv2623-Rv1747-PIM pathway regulates in vivo Mtb growth. The data generated may help gain insight into the function of Rv1747 in modulating the cell wall PIM levels, the roles of PIMs in impacting the fate of the tubercle bacillus in an infected host, Mtb cell wall biogenesis, and potentially the mechanisms that regulate TB latency and reactivation.

抽象的 结核分枝杆菌（MTB）的生命周期很复杂，包括急性期 病原体复制品指数;慢性相，当细菌燃烧稳定时，并且 可以重新激活的潜在paucibaCillarry状态。慢性结核病（TB）与 损害组织免疫病理学并可以促进疾病传播。据估计 世界上约有1/4人口感染了MTB，其中很大一部分 个体藏有潜在的细菌，可以重新激活以引起疾病。阐明调节的机制 在不同感染阶段，受感染宿主的MTB生长对于理解结核病至关重要 发病。人们普遍认为某些宿主环境条件（例如缺氧，氮 压力，饥饿）可以促进潜在感染的建立。但是，确切的机制 调节结核病延迟未完全定义。 MTB RV2623，它是该基因最新的基因之一 休眠状态调节，编码可以调节体内低音生长的通用应激蛋白（USP） 体外。缺失突变体ΔRV2623在易感的小鼠和豚鼠中是高度毒性的，在后者中，它是 在建立慢性持续感染方面有缺陷。分枝杆菌中RV2623的体外，过表达 抑制受体细胞的生长； MTBΔRV2623从缺氧诱导的非复制阶段退出 韦恩潜伏期模型比野生型（WT）MTB更快地转移到O2充足的媒体后。 这些结果提供了RV2623调节MTB增长的证据，包括在 感染的潜在/重新激活阶段。 我们表明RV2623与含FHA域的MTB RV1747相互作用，这是一种推定的出口商 Lipoligosacharides。 FHA结构域是一种信号蛋白模块，可介导各种生物学 通过磷酸化依赖机制的过程。我们进一步表明RV2623-RV1747 通过RV1747的FHAI结构域与磷酸硫氨酸的结合（在位置）介导相互作用 237） - 含RV2623的主题，T237居住对于介导生长调节至关重要 RV2623的属性。与高毒性的ΔRV2623相反，ΔRV1747被衰减以在体内生长。和 而高持高度的ΔRV2623表示免疫调节磷脂酰myo-的水平增强 肌醇甘露糖苷（PIMS）相对于WT MTB，下病毒ΔRV1747是PIMS的低产生剂。在 此外，我们表明了RV1747过表达的菌株超生产PIM。 RV1747的相关性 表达水平和MTB细胞壁PIMS量表明RV1747可能起到MTB的出口商 细胞壁生物发生中间体。这与相反的PIMS表型和体内生长 ΔRV2623和ΔRV1747的表型使我们假设RV2623负调节 RV1747的功能活性调节MTB细胞壁PIM的水平，该水平是免疫调节特性的 可以改变MTB-host相互作用，从而影响结节芽孢杆菌的体内命运。我们将使用 生化，遗传学和免疫学方法与动物建模和综合性结合 生物信息学和计算数据分析，以严格检验该假设。最后，积累 从RV1747的功能和结构分析得出的知识，并发现了关系 在RV1747表达和PIM水平之间，将能够产生一组等源的MTB突变体 表达分级的PIM级别，可用于严格探测这些级别的重要性 影响MTB体内命运的免疫调节性糖脂。拟议的研究应阐明如何 RV2623-RV1747-PIM途径调节体内MTB生长。生成的数据可能有助于了解 RV1747在调节细胞壁PIM水平的功能，PIM在影响命运中的作用 感染宿主，MTB细胞壁生物发生中的结节芽孢杆菌，可能是调节结核病的机制 潜伏期和重新激活。