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.

由于其流行率、持久性、多药耐药性、致病性和治疗的不断增加 挑战，非结核分枝杆菌(NTM)脓肿分枝杆菌(Mab)已成为 严重威胁患有诸如支气管扩张症和囊性纤维化等慢性肺部疾病的人类，以及 免疫功能受损的病人。然而，与其他致病因子相比，单抗的研究还不够深入。 分枝杆菌。此外，目前的动物模型存在一个局限性，这对 Smooth(S)和Rough(R)两种单抗形态在肺组织中的作用 免疫病理学、持久性和宿主免疫反应。单抗在免疫活性和免疫缺陷中的清除 即使是免疫受损的小鼠也阻止了慢性感染的研究，而对于斑马鱼来说，胚胎是 它们是研究单抗感染的工具，不能用来模拟肺部感染和T细胞 参与其中。因此，有必要建立互补的动物模型。由于与斑马鱼胚胎一样，莱维氏X.laevis 蝌蚪是透明的，可以看到病原体的传播，但除此之外，它们确实有 肺功能正常，并表现出包括T细胞在内的免疫系统与人类非常相似，我们建议 开发一种模仿人类肺部疾病的单抗感染的莱维氏X.laevis蝌蚪模型。依赖于一个 建立比较生物学方法研究蝌蚪和我们最近发现的分枝杆菌免疫 证明S和R型单抗都容易感染蝌蚪，在蝌蚪肺和 持续50天，探索性研究提案的目标是在体内进行超过40天的研究 天数单抗持续期和免疫发病机制。具体地说，为了解决持续R 在华支睾吸虫蝌蚪体内，单抗比S单抗具有更活跃的增殖和免疫致病作用，我们提出： ，通过比较 R和S单抗在肺等组织中的活体免疫应答和持久性 (1)持续感染MAb对蝌蚪肺免疫发病机制的影响 显微镜、流式细胞术和转录组学以及荧光标记的单抗和转基因蝌蚪 有荧光的巨噬细胞。 (2)探讨体内R型转换与S发病及持久性的相关性。 ，由 用启动子系统控制S感染后的R开关及开关的效果检测 持久性、致病性和宿主免疫反应。 (3)探讨单抗免疫致病的遗传决定因素 ，使用选定的单抗删除 单抗特异性基因的等位基因交换产生的突变体以及Gram-Gram共享的基因同源物 肺部病原体阴性。 我们期待与人类相关的新奇见解将被收集起来。我们的协作团队独一无二 由于我们的背景和互补的专业知识，我们有能力进行这些研究。