Examining Mycobacterial Interactions with Host Cellular Pathways
Tuberculosis is a devastating disease that has been plaguing humankind for millennia. Co-evolution of humans with Mycobacterium tuberculosis, the causative agent of the disease, has allowed for the pathogen to possess an abundance of survival mechanisms. The outcome of this is the ability of the bacterium to create an intracellular niche lifestyle inside host cells where it can successfully evade the host immune system. While there is a vaccine available, named the BCG vaccine, it confers little protection to adults in the pulmonary form of the disease. The lack of an effective vaccine and the rise of Multidrug-Resistant (MDR) and Extensively Drug-Resistant (XDR) tuberculosis highlight the need for more research into combating Mycobacterium tuberculosis. The purpose of this work is to enhance the field of knowledge of how mycobacterial virulence factors affect host cellular pathways so that the interactions can be exploited for novel therapeutics and vaccine development.
One of the hypotheses for the poor efficacy of the BCG vaccine is that it fails to elicit a strong CD8+ T cell response during infection. Studies have found that vaccinating mice with apoptotic bodies containing mycobacterial antigens were able to protect mice to a greater degree than BCG and that this is dependent on CD8+ T cell activation. Thus, we hypothesized that a pro-apoptotic mutant of M. tuberculosis could be utilized as a novel vaccine candidate. Through screening a library of M. tuberculosis transposon mutants, we identified an Enhanced Cell Death mutant (ECD19) that functions through caspase 3 mediated apoptosis. Sequencing revealed that the mutant has a transposon insertion in Rv2456c, a probable integral membrane transport protein. Immunogenicity testing via Enzyme-Linked ImmunoSpot (ELISPOT) and Intracellular Cytokine Staining (ICS) assays demonstrated that ECD19 induced an altered immune response when compared to the parental strain M. tuberculosis H37Rv. Additionally, ECD19 has reduced survival in an in vitro THP-1 cell model and in an in vivo mouse model. Taken together, our data suggest that Rv2456c is important to the survival of H37Rv in host cells and that deletion of the gene may enhance the immunogenicity of the bacterium.
Inappropriate dosing and poor adherence to antibiotics in the treatment of tuberculosis has led to MDR and XDR, the highest incidences of which can be found in the KwaZulu-Natal (KZN) province of South Africa. Little is known about the virulence of these strains, but it is hypothesized that the drug resistance mechanisms come at a cost to the bacteria. In an in vitro assay, we have found that clinical isolates from the KZN region induce higher levels of necrosis than virulent laboratory strains of M. tuberculosis. Additionally, our in vivo studies show that the drug-resistant isolates do not disseminate as well as susceptible strains, and in both immunocompetent and immunocompromised mouse models, mice infected with the drug-resistant strains are able to live longer than mice infected with drug-sensitive strains. As all strains are highly related on a genetic level, we can say that the drug-resistant mechanisms acquired by the strains come at a cost of reduced virulence. Thus, it is likely that higher prevalence of the MDR and XDR in the KZN province is due to the high rate of HIV+, immunocompromised individuals living in the region.
Lastly, we are interested in building on the knowledge that avirulent mycobacteria are able to induce autophagy in a murine macrophage cell line. Through the use of Mammalian Target of Rapamycin (mTOR) inhibitors and autophagy-deficient macrophages, we were able to show that Mycobacterium smegmatis is able to induce both mTOR and autophagy during infection. Additionally, we found that mycobacterial killing occurs in the absence of autophagy when mTOR is inhibited. This effect is not due to a bactericidal effect of the mTOR inhibitors. From these data, we show that there is an underappreciated role in the induction of mTOR after mycobacterial infection. By studying the interplay of mTOR and autophagy, therapies targeted to favoring host defenses could be developed.
In summary, the insights from this work enhance the knowledge of how mycobacteria are able to be successful pathogens. This data may be useful in the creation of novel vaccine candidates or the identification of potential drug targets to bolster the therapeutic options in treating those afflicted with tuberculosis.
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