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

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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.