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Comprehensive characterization of Mycobacterium tuberculosis strains after acquisition of isoniazid resistance




Nieto Ramirez, Luisa Maria, author
Dobos, Karen M., advisor
Lenaerts, Anne, committee member
Prenni, Jessica, committee member
Slayden, Richard, committee member

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Despite the global efforts to reduce tuberculosis (TB) rates, the emergence of drug resistant TB has not allowed effective control of this disease. In the last decade, there were roughly 10 million new TB cases per year and isoniazid resistant (INHr) TB accounted for 9.5% of these cases around the world. In 2012, United States had an interruption in the supply of isoniazid (INH), which increased the likelihood of INH resistance rates. Although INH resistance in Mycobacterium tuberculosis (Mtb) is multigenic, mutations in the catalase-peroxidase (katG) gene predominate amongst INHr Mtb strains. The characterization of the Mtb proteome before and after acquiring INH resistance remains understudied. Additionally, the effect of these drug-resistance-conferring mutations on Mtb fitness and virulence is variable. The purpose of this work is to describe a complete biochemical and immunological characterization of the INHr acquisition in Mtb. In this way, a global exploration of the protein and mycolic acids differences in Mtb cultures, as well as differences in the immune response and bacterial virulence in the mouse model comparing clonal susceptible and INHr pairs of Mtb were evaluated. After this, common trends were analyzed and the findings were interpreted in the context of bacterial metabolism and host-interaction. For this work, two clonal clinical Mtb strains and one laboratory clonal pair of the H37Rv strain with different susceptibility profiles to INH were studied. The H37Rv INHr strain was isolated from a mouse that was exposed to INH in the lab and developed the same katG mutation that one of the clinical INHr strain has (V1A). In all cases, the first strain was susceptible to all tested drugs (mostly known as the INHs strain in this dissertation) while the second strain was resistant only to INH (named INHr throughout this work). The clinical pairs were confirmed as clonal pairs of the Beijing and T genotype respectively by spoligotyping and restriction fragment polymorphism analysis that uses the patterns given by the distribution of the insertion sequence (IS)-6110. Previous whole genome sequencing analysis of the clinical clonal pairs showed a katG mutation and the presence of some additional non-synonymous polymorphisms in the INHr strains. After the proteomic analysis, a katG PCR sequencing confirmed two mutations in katG for the T INHr pair (V1A and E3V) while the L101R mutation previously identified for the Beijing INHr was not confirmed. This mutation was highly unstable and the Beijing INHr might have reversed its phenotype after the absence of INH during in vitro growth. Therefore, the analysis with the Beijing clonal pair is only presented in chapter II. Protein comparison of secreted and cellular fractions (membrane, cytosol and cell wall) between clinical and lab clonal pairs of Mtb before and after acquisition of INH resistance revealed at least 25 commonly altered proteins looking at the same cellular fractions. These proteins were involved in ATP synthase machinery, lipid metabolism, regulatory events, virulence, detoxification and adaptation processes. Western blot analysis supported some of our findings, particularly the lower level of bacterial enzyme KatG in the INHr strains. Mycolic acid (MA) analysis in these clonal pairs did not reveal a common trend in these molecules for INHr strains but generated supporting information about an alternative fatty acid biosynthetic pathway in the clinical INHr strain. These analyses are further described in chapter III. Additionally, differences in bacterial growth, immune response and pathology induced by Mtb strains harboring mutations at the N-terminus of KatG were evaluated in the C57BL/6 mouse model. The results in the mouse study support the idea of the individual effect of specific located mutations in the katG gene together with the associated changes in the bacterial proteome induce differences in the Mtb virulence and pathogenicity. In addition, the in vivo results also suggest the contribution of innate immune response via TLR-2 in the clearance of the INHr-attenuated Mtb strains. Further details of this work are described in chapter IV. This work provides a better understanding of new compensatory mechanisms in Mtb after INH resistance acquisition providing novel information that could be used to address alternative combined therapies as well as the identification of new drug targets in INHr strains. The results presented here also contribute to the generation of new hypothesis regarding RNA decay in Mtb and the need to evaluate if the observed biochemical differences are also associated with the bacterial exposure to the first line drug therapy that occurred in the patient. After the results obtained in this study, a subsequent biochemical analysis of Mtb strains obtained from patients before and after drug treatment is proposed to improve the description of the evolution of the acquired drug resistant phenomena observed in TB cases that limit the global disease control and hence its eradication (chapter V).


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