Elucidation of Gene Essentiality and Genetic Determinants of Intrinsic p-Aminosalicylic Acid Resistance in Mycobacterium Kansasii

Author

Keith J. Levendosky, The Graduate Center, City University of New YorkFollow

Date of Degree

9-2022

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Luis E.N. Quadri

Committee Members

Shaneen Singh

Anjana Saxena

Weigang Qiu

Dean Crick

Subject Categories

Bacteriology | Molecular Genetics | Pathogenic Microbiology

Keywords

mycobacteria, antibiotic resistance, gene essentiality, forward genetics

Abstract

Mycobacterium kansasii (Mk) is an opportunistic pathogen capable of causing tuberculosis-like pulmonary disease in immunocompromised individuals and those with other risk factors including chronic obstructive pulmonary disease or malignancy. Mk is frequently isolated from man-made water sources where it forms resilient biofilms, posing a health risk to these susceptible individuals. Despite its medical relevance as an environmental pathogen, and a close phylogenetic relationship with the obligate pathogen Mycobacterium tuberculosis (Mtb), few studies to date have probed the molecular biology and genetics of Mk. Here, we sought to apply a transposon (Tn) mutagenesis tool to dissect gene function in Mk using both deep sequencing and forward genetics approaches. Through deep sequencing of 2.5 million Mk Tn mutants, we defined the in vitro essential gene set of Mk and then compared these results to essentiality data available for several other species of mycobacteria. Importantly, this analysis shows that several orthologs of essential Mtb genes encoding drug target candidates were identified as nonessential in Mk, suggesting they may not be viable drug targets in Mk. Next, we carried out a forward genetic screen of 10,000 individually arrayed Mk Tn mutants and identified those displaying increased susceptibility to the antitubercular agent p-aminosalicylic acid (PAS), to which Mk is naturally resistant. Interestingly, most of the PAS-susceptible Mk Tn mutants analyzed carried insertions in genes sharing orthologs in Mtb. This finding suggests that the natural resistance of Mk to PAS may not be due to the presence of dedicated Mk-specific PAS resistance mechanisms, but rather it is a result of fundamental differences in physiology between Mk and Mtb. Finally, we performed preliminary characterization and complementation analysis of select PAS-susceptible Mk Tn mutants to further explore these genotype-phenotype associations. Altogether, our findings from both approaches represent valuable resources to assist in the process of identifying and prioritizing potential Mk drug target candidates and to guide future studies on Mk biology.

Recommended Citation

Levendosky, Keith J., "Elucidation of Gene Essentiality and Genetic Determinants of Intrinsic p-Aminosalicylic Acid Resistance in Mycobacterium Kansasii" (2022). CUNY Academic Works.
https://academicworks.cuny.edu/gc_etds/5028