ORCID Identifier(s)

0000-0002-5911-2563

Graduation Semester and Year

Spring 2024

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Quantitative Biology

Department

Biology

First Advisor

Cara C. Boutte

Second Advisor

Allan C. Clark

Third Advisor

Alison Ravenscraft

Fourth Advisor

Joseph Boll

Fifth Advisor

Shawn Christensen

Abstract

The disease Tuberculosis (TB) is caused by the pathogen Mycobacterium tuberculosis and was the cause of over one million deaths worldwide in 2023 (World Health Organization, 2023). TB treatment is difficult due to antibiotic tolerance and emerging resistance in Mtb strains. Mycobacterial cells have different regulatory mechanisms that allow them to adjust to myriad environmental stressors: these responses often lead to antibiotic tolerance. Therefore, studying responses to stress will lead to insights about antibiotic tolerance and treatment failure. Here, we specifically study two essential mycobacterial proteins, Wag31 and MmpL3, involved in cell wall metabolism. We show that Wag31 has distinct roles in the cell cycle, where it promotes polar elongation at the poles while inhibiting the initiation of elongation at the new pole, thus promoting asymmetric growth. Also, we find that Wag31 has a negative regulatory role in septation. Our data suggests that wag31 and mmpl3 are genetically linked and work together to regulate the elongation of the old pole. MmpL3 is a trehalose monomycolate transporter that is required for mycolic acid synthesis. We find that Wag31 and PlrA, another essential polar growth factor, recruit MmpL3 to the poles, and these three proteins may be required for the new pole to transition into the old pole. We also propose a novel role for the C-terminus of MmpL3 in regulating polar cell wall metabolism broadly in growth and stress, in promoting antibiotic resistance. In addition, we show that peptidoglycan and mycolic acid synthesis are tightly connected, which means that inhibition of MmpL3 reduces peptidoglycan metabolism and vice versa. Our work ultimately describes the molecular function of Wag31 and proposes a new model for cell wall metabolism in mycobacterial cells.

Keywords

Mycobacteria, Polar growth, elongation, deptation, Wag31, DivIVA, Mmpl3, peptidoglycan, Mycolic acid, trehalose mycolates

Disciplines

Bacteriology | Biochemistry | Cell Biology | Microbial Physiology | Molecular Biology | Structural Biology

Available for download on Wednesday, May 14, 2025

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