Generation of <i>Mycobacterium smegmatis</i> Model Systems for Testing Inhibitors of the Mycobactin Siderophore of <i>Mycobacterium tuberculosis</i>

<p><em>Mycobacterium tuberculosis (Mtb)</em> 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 <em>Mtb </em>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 <em>Mtb</em>. 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 <em>Mtb</em> in iron-limiting conditions by inhibiting MbtA’s ability to catalyze the first reaction in the biosynthetic pathway of mycobactin. Using a <em>M. smegmatis (Msm)</em> model system, we aimed to further investigate the mycobacterial susceptibility to Sal-AMS and analogues in iron-limiting conditions. Considering <em>Msm </em>produces an additional siderophore type (exochelin), our <em>Msm</em> model were engineered to lack the ability to produce exochelin, depending solely on the mycobactins/carboxymycobactins for iron acquisition under iron-limiting conditions, mirroring <em>Mtb</em>. Further generation of a siderophore deficient strain of <em>Msm</em> 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 <em>Mtb</em> (MbtA_{tb)<em> </em>}and the generation of strains expressing MbtA_{tb<em> </em>}with mutations hypothesized to result in resistance to specific Sal-AMS analogues. Lastly, our <em>Msm</em> 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 <em>Msm</em> <em>mbtI_sm</em> 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 <em>Msm</em> 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.</p>

Name thesis:degree_name

Doctor of Philosophy

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Biology

Grantor

The Graduate School and University Center of The City University of New York

Year dc:date.available

2022

Author dc:creator

• Germain, Gabrielle A

Advisor dc:contributor.advisor

• Luis E.N. Quadri

Committee members dc:contributor.committeemember

• Shaneen Singh
• Peter Lipke
• Haiping Cheng
• Thomas Dick

• Bacteria
• Biology
• Diseases
• Molecular Biology
• mycobacterium
• tuberculosis
• iron
• siderophores
• model system
• TB

Repository record dc:identifier

https://academicworks.cuny.edu/gc_etds/5042

OAI identifier oai:identifier

oai:academicworks.cuny.edu:gc_etds-6106