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<p>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.</p><p>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.</p><p>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.
</p><p>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.</p><p>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.</p>
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