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: 9973940
• 负责人: John R. Chan
• 金额: $ 92.6万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 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 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9973940
• 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; Structure; 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.

摘要 结核分枝杆菌（Mtb）的生命周期是复杂的，包括急性期，在此期间， 病原体以指数方式复制;慢性期，此时细菌负荷稳定维持， 潜伏性少菌状态可以重新激活。慢性结核病（TB）与以下疾病的发生有关 组织损伤免疫病理学，并可促进疾病传播。据估计 世界上大约四分之一的人口感染了结核分枝杆菌，其中很大一部分人感染了结核分枝杆菌。 个体体内潜伏着可以重新激活而引起疾病的杆菌。揭示了调节 在感染的不同阶段，结核分枝杆菌在受感染宿主中的生长对于了解结核病至关重要 发病机制通常认为某些宿主环境条件（例如，亚硝化缺氧 压力，饥饿）可以促进潜伏感染的建立。然而， 规范TB延迟的定义不完全。Mtb Rv 2623，它是在大肠杆菌中上调最多的基因之一。 休眠调节子，编码一种通用应激蛋白（USP），可以调节体内和体外的细菌生长， 体外缺失突变体Δ Rv 2623在易感小鼠和豚鼠中是高毒力的，并且在后者中， 在建立慢性持续性感染方面有缺陷。在体外，Rv 2623在分枝杆菌中的过表达 Mtb Δ Rv 2623从缺氧诱导的非复制期退出， 韦恩潜伏期模型比野生型（WT）Mtb转移到氧气充足的培养基更迅速。 这些结果提供了Rv 2623调节结核分枝杆菌生长的证据，包括可能在结核分枝杆菌生长期间。 感染的潜伏/再激活阶段。 我们发现Rv 2623与含有FHA结构域的Mtb Rv 1747相互作用，Rv 1747是Mtb的推定输出者。 脂寡糖FHA结构域是一种信号蛋白模块，其介导多种生物学功能。 通过磷酸化依赖机制的过程。我们进一步表明，Rv 2623-Rv 1747 相互作用是通过Rv 1747的FHAI结构域与磷酸苏氨酸（在位置 237)-含有Rv 2623的基序，并且T237残基对于介导生长调节是必需的。 Rv 2623的属性。与高毒力Δ Rv 2623相反，Δ Rv 1747在体内生长减弱。和 而高毒力Δ Rv 2623表达增强水平的免疫调节性磷脂酰-肌- 相对于WT Mtb，低毒性Δ Rv 1747是低生产的肌醇甘露糖苷（PIM）。在 此外，我们还发现Rv 1747过表达的菌株能过度产生PIM。Rv 1747的相关性 表达水平和Mtb细胞壁PIM的量表明Rv 1747可能作为Mtb的输出者起作用， 细胞壁生物合成中间体。这与相反的PIM表型和体内生长一起 Δ Rv 2623和Δ Rv 1747的表型，使我们假设Rv 2623负调节 Rv 1747调节Mtb细胞壁PIM水平的功能活性， 可以改变结核杆菌-宿主相互作用，从而影响结核杆菌的体内命运。我们将使用 生物化学，遗传学和免疫学方法，结合动物建模和综合 生物信息学和计算数据分析，以严格测试这一假设。最后，积累 从Rv 1747的功能和结构分析中获得的知识，以及关系的发现 Rv 1747表达和PIM水平之间的差异，将能够产生一组同基因Mtb突变体 表达PIM的分级水平，其可用于严格地探测这些PIM的重要性。 免疫调节糖体影响Mtb的体内命运。拟议的研究应阐明如何 Rv 2623-Rv 1747-PIM途径调节体内Mtb生长。生成的数据可能有助于深入了解 Rv 1747在调节细胞壁PIM水平中的功能，PIM在影响细胞壁PIM的命运中的作用， 结核杆菌在受感染宿主中的作用、结核分枝杆菌细胞壁的生物发生，以及可能调节结核病的机制 潜伏期和再激活。