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: 10685658
• 负责人: John R. Chan
• 金额: $ 13.54万
• 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/10685658
• 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.

摘要 结核分枝杆菌(Mtb)的生命周期是复杂的，包括一个急性期，在此期间 病原体呈指数级复制；慢性期，细菌负荷稳定维持； 潜伏的少杆菌状态，可以重新激活。慢性结核病(TB)与 破坏组织的免疫病理学，并可促进疾病传播。据估计， 大约四分之一的世界人口感染了结核分枝杆菌，其中相当大一部分人 个体体内有潜伏的杆菌，它们可以重新激活而导致疾病。解开调控的机制 结核杆菌在感染的不同阶段在受感染宿主中的生长对了解结核病是至关重要的 发病机制。一般认为，某些宿主环境条件(如低氧、亚硝酸盐 压力、饥饿)可促进潜伏感染的形成。然而，准确的机制是 调整结核病潜伏期的定义并不完整。结核分枝杆菌Rv2623，它是在 休眠调节子，编码一种通用应激蛋白(USP)，可调节细菌在体内和 在试管中。缺失突变体ΔRv2623在易感小鼠和豚鼠中超强毒力，在后者中，它是 在确定慢性持续性感染方面存在缺陷。Rv2623基因在分枝杆菌中的体外过表达 抑制受体细胞的生长；MTBΔRv2623退出低氧诱导的非复制阶段 在转移到氧气充足的介质中时，韦恩潜伏期模型比野生型(WT)Mtb更快。 这些结果提供了Rv2623调控结核分枝杆菌生长的证据，可能包括在 感染潜伏期/再活跃期。 我们发现Rv2623与含有FHA结构域的Mtb Rv1747相互作用，Mtb Rv1747是 脂寡糖。FHA结构域是一个信号蛋白模块，它介导了多种生物 通过磷酸化依赖的机制进行加工。我们进一步表明，Rv2623-Rv1747 相互作用是通过Rv1747的FHAI结构域与磷酸苏氨酸(在 237)-含有Rv2623基序，并且T237残基是介导生长调节所必需的 Rv2623的属性。与超强毒力ΔRv2623不同，ΔRv1747在体内生长较弱。和 而超强毒力ΔRv2623则表达免疫调节磷脂酰肌醇的增强水平。 肌醇甘露糖苷(PIMs)相对于WT Mtb，低毒力的ΔRv1747是PIMs的低产生菌。在……里面 此外，我们还发现Rv1747过表达菌株可以高水平产生PIMs。Rv1747‘S的相关性 Mtb的表达水平和细胞壁PIMs的数量表明Rv1747可能是Mtb的输出子 细胞壁生物发生的中间体。这与相反的PIMs表型和体内生长有关 ΔRv2623和ΔRv1747的表型使我们假设Rv2623负性调节 Rv1747调节Mtb细胞壁PIMs水平的功能活性 可以改变结核分枝杆菌与宿主的相互作用，从而影响结核杆菌在体内的命运。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，以严格检验这一假说。最后，积累 从Rv1747的功能和结构分析中获得的知识，以及发现它们之间的关系 在Rv1747表达和PIM水平之间，将能够产生一组等基因Mtb突变体 表达PIM的分级水平，可以用来严格探索这些指标的意义 免疫调节糖链对结核分枝杆菌体内命运的影响。拟议的研究应该说明如何 Rv2623-Rv1747-PIM通路在体内调节Mtb的生长。生成的数据可能有助于深入了解 Rv1747在调节细胞壁PIM水平中的作用，PIM在影响细胞命运中的作用 感染宿主中的结核杆菌，结核分枝杆菌细胞壁的生物发生，以及潜在的调节结核的机制 延迟和重新激活。