Keywords

CRISPR Interference; Mycobacterium abscessus; antibiotic resistance; L,D-transpeptidase; infectious disease; molecular bioogy

Abstract

Mycobacterium tuberculosis (Mtb), a bacterium that causes Tuberculosis, results in over 1.5 million deaths annually and is typically treated with a cocktail of four drugs over 4-6 months. Mycobacterium abscessus (Mab), which affects patients with pre-existing conditions such as Cystic Fibrosis, is a rapidly growing and drug-resistant pathogen. Mab, which is a non-tuberculous mycobacterium (NTM), grows more rapidly and is more virulent than other NTMs. Bacteria in the genus Mycobacteria are especially difficult to treat due to their highly impermeable cell wall. To identify the most critical targets for β-lactam antibiotics, which can inhibit multiple peptidoglycan synthesis enzymes, I generated single CRISPRi knockdowns of L,D-transpeptidases (Ldts), enzymes involved in cell wall cross-linking. Mtb and Mab encode five Ldt homologs, each with distinct functions. Using CRISPRi technology and Golden GATEway multiplex cloning, I simultaneously silenced multiple genes and tested Mab Ldt combinations for synergistic interactions. Growth and viability were monitored by spot plating to estimate CFU following anhydrotetracycline induction to identify priority Ldts for antibiotic targeting. With the goal of validating and prioritizing a class of cell wall cross-linking enzymes as drug targets in mycobacteria, I determined which Ldt knockdown(s) had the greatest inhibitory effect by itself and which combination(s) of Ldts exhibited the greatest synergy to define the optimal target profile for β-lactams.

Thesis Completion Year

2026

Thesis Completion Semester

Spring

Thesis Chair

Rohde, Kyle

College

College of Medicine

Department

Burnett School of Biomedical Sciences

Thesis Discipline

Biomedical Sciences

Language

English

Access Status

Campus Access

Length of Campus Access

5 years

Campus Location

Orlando (Main) Campus

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Restricted to the UCF community until 5-15-2031; it will then be open access.

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In Copyright