Understanding and preventing Mycobacterium avium infections
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Mycobacterium avium (Mav) complex (MAC) is a group of slow growing, environmental opportunist pathogens, that have been associated with animal and human infections. Mav is one of the species belonging to MAC, which can be a human pathogen. As a human pathogen, Mav can cause pulmonary infections, lymphadenitis, and disseminated infection. Since Mav species are present in a diverse milieu, they are observed to have high genetic diversity. These genetic variations may affect virulence and pathogenesis of Mav within the host. Inside the human host, the control of mycobacterial pathogenesis is largely dependent on cell-mediated immunity. The adaptive immune response ranges from the activation of antigen presenting cells to the containment of the infection. In Mav infections, the CD4+ T cells play a major role in containing the spread of infection albeit a minimal role is played by CD8+T cells. Cytokines like IFNγ and TNFα produced by effector T cells have been observed to be crucial in curtailing the spread of Mav infection; however, IL-17 producing effector T cells have only a small role in controlling Mav infection. It is therefore important to understand not only the pathogenesis of Mav but also to determine the correlates of protection against it. In the current study, the first aim was to understand the pathogenesis of Mav isolated from patients. It was hypothesized that host adapted Mav undergo genetic modifications to cope with the host environment. In addition, the genetic polymorphisms within the bacteria could lead to a varied host response. To achieve this, pulsed-field gel electrophoresis (PFGE) and whole genome sequencing (WGS) was performed on, 40 MAC isolates sequentially isolated from 15 patients. The WGS data revealed the accumulation of genetic mutations within the Mav serial isolates. The immunological response to these serial isolates was tested in mouse macrophages and mice. It was observed that the serial isolates exhibited a propensity towards increased persistence by downregulating important proinflammatory cytokines over time. In the end, high rates of genetic polymorphisms and subsequent alteration of virulence properties were revealed in host-adapted Mav from chronically infected patients. To understand the function of genes required for bacterial survival, a positively regulated expression system was constructed, to control gene expression within mycobacteria. The XylS/Pm expression system was modified from Pseudomonas putida, to give robust time and dose-dependent expression within M. smegmatis and M. tuberculosis. The modified XylS/Pm expression system was able to achieve low basal expression and robust time and dose dependent expression of genes in mycobacteria. Lastly, the aim of this study was also to investigate the attributes of protection against Mav, by employing a vaccine mouse model. For this, M. smegmatis ΔespG3 overexpressing a Mav antigen was employed as a vaccine strain. It was observed that Mav104-infection of mice vaccinated with M. smegmatis ΔespG3::mpt64 exhibited reduced bacterial loads and increased frequency of CD8+IL-17 (Tc17) cells compared to sham-vaccinated mice. The role of Tc17 in mycobacterial protection is unknown, so to decipher their role adoptive transfer of Tc17 into naïve and pre-infected mice was performed. It was observed that Tc17 cells were not protective and rather required an inflammatory milieu within the host to aid in the clearing of Mav infection. Ultimately, M. smegmatis ΔespG3::mpt64 provided comparable protection to BCG, during Mav infection. In addition, a therapeutic role for Tc17 cells in mycobacterial protection was elicited. In the end, this study provides important insights into Mav host adaptation, tools to study gene function and correlates involved in limiting Mav infection.