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

基本信息

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
项目类别：
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

项目摘要

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，包括一个急性期，在此期间病原体呈指数复制；慢性阶段，细菌负荷稳定维持，并且可重新激活的潜伏性少杆菌状态与慢性结核病 (TB) 的发展有关。据估计，组织损伤性免疫病理学可以促进疾病传播。世界上大约 1/4 的人口感染了 Mtb，其中很大一部分个体体内潜藏的细菌可以重新激活从而引发疾病。受感染宿主在不同感染阶段的结核分枝杆菌生长对于了解结核病至关重要一般认为与某些宿主环境条件（例如缺氧、亚硝化）有关。压力、饥饿）可以促进潜伏感染的建立，然而，其确切机制并不明确。 Mtb Rv2623 是结核病潜伏期中调节程度最高的基因之一。休眠调节子，编码通用应激蛋白（USP），可以调节体内和体内的细菌生长缺失突变体 ΔRv2623 在易感小鼠和豚鼠中具有高毒力，而在后者中，它是高毒力的。体外，分枝杆菌中 Rv2623 过度表达。延缓受体细胞的生长；并且 Mtb ΔRv2623 退出缺氧诱导的非复制期在转移至 O2 充足的培养基中时，Wayne 潜伏期模型比野生型 (WT) Mtb 更迅速。这些结果提供了 Rv2623 调节 Mtb 生长的证据，包括可能在感染的潜伏/再激活阶段。我们发现 Rv2623 与包含 FHA 结构域的 Mtb Rv1747 相互作用，Mtb Rv1747 是假定的 FHA 结构域是介导多种生物的信号蛋白模块。我们进一步证明了 Rv2623-Rv1747 通过磷酸化依赖性机制进行的过程。相互作用是通过 Rv1747 的 FHAI 结构域与磷酸苏氨酸（位于位置 237) 包含 Rv2623 的基序，并且 T237 残基对于介导生长调节至关重要与高毒力 ΔRv2623 相比，ΔRv1747 的体内生长减弱。而高毒力 ΔRv2623 表达的免疫调节磷脂酰肌醇水平增强。相对于 WT Mtb，低毒力的 ΔRv1747 是 PIM 的低生产者。此外，我们还发现 Rv1747 过表达菌株过度产生 PIM。 Rv1747 的相关性。表达水平和 Mtb 细胞壁 PIM 量表明 Rv1747 可能充当 Mtb 的输出者这与相反的 PIM 表型和体内生长有关。 ΔRv2623 和 ΔRv1747 的表型，使我们解决了 Rv2623 负调节 Rv1747 调节 Mtb 细胞壁 PIM 水平的功能活性，其免疫调节特性可以改变结核分枝杆菌与宿主的相互作用，从而影响结核杆菌的体内命运。生物化学、遗传学和免疫学方法，结合动物建模和综合最后，通过生物信息学和计算数据分析来严格检验这一假设。来自 Rv1747 功能和结构分析的知识，以及发现的关系 Rv1747 表达和 PIM 水平之间的差异，将能够产生一组等基因 Mtb 突变体表达 PIM 的分级水平，可用于严格探究这些的重要性免疫调节糖苷酸对 Mtb 体内命运的影响拟议的研究应阐明如何影响 Mtb 的体内命运。 Rv2623-Rv1747-PIM 通路调节体内 Mtb 生长。生成的数据可能有助于深入了解 Mtb 的生长。 Rv1747 在调节细胞壁 PIM 水平中的功能、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生物发生以及调节结核病的潜在机制潜伏期和重新激活。