Date of Degree

9-2022

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Luis E.N. Quadri

Committee Members

Shaneen Singh

Peter Lipke

Haiping Cheng

Thomas Dick

Subject Categories

Bacteria | Biology | Diseases | Molecular Biology

Keywords

mycobacterium, tuberculosis, iron, siderophores, model system, TB

Abstract

Mycobacterium tuberculosis (Mtb) is the pathogenic bacterium causative of tuberculosis. According to the 2021 World Health Organization (WHO) Global Tuberculosis (TB) Report, there were approximately 5.8 million TB cases in 2020, and 1.3 million TB deaths among HIV negative people in that year. The rise of multi-drug resistant Mtb strains has increased the need for more potent microbials against this pathogen. Biosynthesis of iron scavenging molecules, known as siderophores (e.g., mycobactin and carboxymycobactin), has been identified as a potential drug target candidate since iron acquisition has been directly linked to the survival and virulence of Mtb. Salicyl-AMS (Sal-AMS) is an antibiotic that targets the enzyme MbtA, responsible for initiating mycobactin/carboxymycobactin scaffold biosynthesis. This antibiotic has been found to inhibit the growth of Mtb in iron-limiting conditions by inhibiting MbtA’s ability to catalyze the first reaction in the biosynthetic pathway of mycobactin. Using a M. smegmatis (Msm) model system, we aimed to further investigate the mycobacterial susceptibility to Sal-AMS and analogues in iron-limiting conditions. Considering Msm produces an additional siderophore type (exochelin), our Msm model were engineered to lack the ability to produce exochelin, depending solely on the mycobactins/carboxymycobactins for iron acquisition under iron-limiting conditions, mirroring Mtb. Further generation of a siderophore deficient strain of Msm allowed for examination of mycobactin/carboxymycobactin dependent growth in iron-limiting medium and susceptibility to MbtA inhibitors. It also served as a host strain for expression of the potentially clinically relevant target, MbtA from Mtb (MbtAtb) and the generation of strains expressing MbtAtb with mutations hypothesized to result in resistance to specific Sal-AMS analogues. Lastly, our Msm model strain allowed for further exploration of the relationship between production of salicylic acid (SA) (which is the acyl substrate of MbtA) and Sal-AMS susceptibility via the generation of Msm mbtIsm deletion strains deficient in SA production necessary to produce mycobactin/carboxymycobactin. Overall, due to its fast-doubling time in liquid culture and non-pathogenicity, our findings demonstrate the usefulness of a Msm model system for exploring inhibition of not only MbtA by Sal-AMS and analogues, but for the examination of other mycobactin/carboxymycobactin inhibitors such as MbtI inhibitors.

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