In vivo persistence and immuno-pathogenesis of Mycobacterium abscessus in a new Xenopus tadpole model

脓肿分枝杆菌在新爪蟾蝌蚪模型中的体内持久性和免疫发病机制

• 批准号: 10608077
• 负责人: Martin S. Pavelka
• 金额: $ 19.25万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-11 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10608077
• 关键词: Acids; Address; Alleles; Amphibia; Animal Model; Antibiotic Resistance; Antibiotic Therapy; B-Lymphocytes; Bacteria; Biological Assay; Breathing; Bronchiectasis; Burkholderia cepacia; Chronic; Chronic lung disease; Comparative Biology; Cystic Fibrosis; Development; Disease; Embryo; Embryo Research; Exhibits; Fertilization; Flow Cytometry; Gene Mutation; Genes; Genetic Determinism; Genus Mycobacterium; Histology; Homologous Gene; Human; Immune; Immune response; Immune system; Immunity; Immunocompetent; Immunocompromised Host; Infection; Interferons; Investigation; Knock-out; Knockout Mice; Label; Laboratory Organism; Leukocytes; Liver; Lower respiratory tract structure; Lung; Lung diseases; Lung infections; Macrophage; Mammals; Microbial Biofilms; Microscopy; Modeling; Morphology; Multi-Drug Resistance; Mus; Mycobacterium abscessus; Mycobacterium marinum; Myeloid Cells; Pathogenesis; Pathogenicity; Pathology; Patients; Phenotype; Positioning Attribute; Prevalence; Production; Proliferating; Pseudomonas aeruginosa; Refractory; Research Proposals; Resistance; Role; Skin; Soil; System; T-Lymphocyte; Tadpoles; Tissues; Transgenic Organisms; Virulence Factors; Visualization; Water; Xenopus; Xenopus laevis; Zebrafish; adaptive immunity; arm; cell envelope; chronic infection; emerging pathogen; genome annotation; host colonization; human disease; human tissue; immunopathology; in vivo; insight; intravital microscopy; lung pathogen; mutant; non-tuberculosis mycobacteria; novel; pathogen; prevent; promoter; respiratory pathogen; response; transcriptomics

Owing to its increasing prevalence, persistence, multidrug resistance, pathogenicity and treatment challenges, the non-tuberculosis mycobacterium (NTM) Mycobacterium abscessus (Mab) has become a serious threat to humans with chronic lung diseases such as bronchiectasis and cystic fibrosis, as well as immunocompromised patients. However, Mab is still understudied compared to other pathogenic mycobacteria. In addition, there is a limitation in current animal models that poses a real challenge for investigating the respective roles of two Mab morphotypes, smooth (S) and rough (R), in lung immunopathology, persistence and host immune response. The clearance of Mab in immunocompetent and even immunocompromised mice prevent the study of chronic infection, and while for zebrafish embryos are instrumental for investigating Mab infection, they cannot be used to model a pulmonary infection and T cell involvement. Thus, there is a need for complementary animal models. Since like zebrafish embryos, X. laevis tadpoles are transparent, allowing visualization of pathogen dissemination, but that in addition they do have functional lungs and exhibit an immune system including T cells remarkably similar to humans, we propose to develop a X. laevis tadpole model of Mab infection that mimics human pulmonary disease. Relying on an established comparative biology approach to study immunity to mycobacteria in tadpoles and our recent demonstration that both S and R Mab morphotypes readily infect tadpoles, disseminate in tadpole lungs and persist up to 50 days, the objective of the exploratory research proposal is to investigate in vivo for over 40 days Mab persistence, and immuno-pathogenesis. Specifically, to address the hypothesis that persistent R Mab is more actively proliferating and immunopathogenic than S Mab in X. laevis tadpoles we propose: , by comparing the host immune response and persistence of R and S Mab in the lung and other tissues using intravital (1) Characterize immune-pathogenesis in tadpole lungs during persisting Mab infection microscopy flow cytometry and transcriptomics as well as fluorescently labeled Mab and transgenic tadpoles with fluorescent macrophages. (2) Investigate the relevance of S to R morphotype switch in vivo for pathogenesis and persistence , by using a promoter system to control the S to R switch following infection and examining effect of switches on persistence, pathogenicity and host immune response. (3) Investigate genetic determinants of Mab immune-pathogenesis , using selected Mab deletion mutants generated by allelic exchange of Mab-specific genes as well as gene homologs shared by Gram- negative lung pathogens. We anticipate that novel insights relevant to human will be gathered. Our collaborative team is uniquely positioned to carry these studies due to our background and complementary expertise.

由于其日益流行、持久性、多重耐药性、致病性和治疗 挑战中，非结核分枝杆菌 (NTM) 脓肿分枝杆菌 (Mab) 已成为 对患有支气管扩张和囊性纤维化等慢性肺部疾病以及 免疫功能低下的患者。然而，与其他致病菌相比，Mab 的研究仍然不足。 分枝杆菌。此外，当前的动物模型存在局限性，这对 研究两种 Mab 形态型（光滑 (S) 和粗糙 (R)）在肺中各自的作用 免疫病理学、持久性和宿主免疫反应。免疫功能健全者中 Mab 的清除 即使是免疫功能低下的小鼠也无法进行慢性感染的研究，而对于斑马鱼胚胎来说 有助于研究 Mab 感染，但不能用于模拟肺部感染和 T 细胞 参与。因此，需要补充动物模型。因为像斑马鱼胚胎一样，X. laevis 蝌蚪是透明的，可以观察到病原体的传播，但除此之外，它们还具有 具有功能性的肺部并表现出与人类非常相似的包括 T 细胞在内的免疫系统，我们建议 开发模拟人类肺部疾病的 Mab 感染的 X. laevis 蝌蚪模型。依靠一个 建立了比较生物学方法来研究蝌蚪对分枝杆菌的免疫力，以及我们最近的研究 证明 S 和 R Mab 形态型都很容易感染蝌蚪，在蝌蚪肺中传播， 持续长达 50 天，探索性研究提案的目标是在体内研究 40 多年 天 Mab 持久性和免疫发病机制。具体来说，为了解决持久 R 的假设 在非洲虎蝌蚪中，Mab 比 S Mab 具有更活跃的增殖和免疫致病性，我们认为： ，通过比较 宿主免疫反应以及 R 和 S Mab 在肺和其他组织中的持久性 (1) 表征持续 Mab 感染期间蝌蚪肺的免疫发病机制 显微镜流式细胞术和转录组学以及荧光标记的单克隆抗体和转基因蝌蚪 与荧光巨噬细胞。 (2) 研究体内S到R形态类型转换与发病机制和持久性的相关性 ， 经过 使用启动子系统控制感染后的S到R开关并检查开关的效果 持久性、致病性和宿主免疫反应。 (3) 研究Mab免疫发病机制的遗传决定因素 ，使用选定的 Mab 删除 通过 Mab 特异性基因以及 Gram- 共享的基因同源物的等位基因交换产生的突变体 肺部病原体呈阴性。 我们预计将收集与人类相关的新颖见解。我们的协作团队是独一无二的 由于我们的背景和互补的专业知识，我们有能力开展这些研究。