NYMC Student Theses and Dissertations

Date of Award

11-21-2025

Document Type

Doctoral Dissertation - Open Access

Degree Name

Doctor of Philosophy

Department

Basic Medical Sciences

First Advisor

Chandra Shekhar Bakshi

Abstract

Francisella tularensis (F. tularensis) is a highly infectious, gram-negative intracellular bacterium that causes the severe zoonotic disease tularemia. Because of its low infectious dose, ease of aerosolization, high virulence, and the absence of an effective vaccine, F. tularensis is designated by the CDC as a Tier 1 Category A Select Agent due to its potential use as a bioterror agent. Development of novel genetic manipulation techniques is crucial for advancing F. tularensis research. CRISPR technology has revolutionized genomic studies in eukaryotic systems, but its application in prokaryotes has lagged. CRISPR interference is a modified CRISPR system that employs a catalytically inactive Cas9 protein to repress gene expression transcriptionally. In this work, we developed a plasmid-encoded CRISPR interference expression system for F. tularensis that is easily modifiable and enables efficient targeting of genes of interest. This system achieved transcriptional repression sufficient to generate phenotypes in knockdown mutants comparable to full loss-of-function mutants for certain gene targets. A genome-wide investigation of F. tularensis enables large-scale discovery of virulence-associated genes. Our laboratory generated an F. tularensis Tn5 transposon mutant library comprising 756 individual mutants to enable genome-wide screening. Through this work, we identified several previously undescribed genes involved in bacterial stress responses to acidic, oxidative, and host macrophage environments. Use of lipid-rich complete media further revealed potential links between F. tularensis lipid metabolism, stress responses, and overall bacterial fitness. Targeted characterization of one previously uncharacterized gene identified through the transposon screen (F. tularensis Live Vaccine Strain gene locus FTL_0690, encoding a putative acyl-CoA synthetase) uncovered several key functions of the acyl-CoA synthetase enzyme in the bacterium’s xv biology. We discovered that loss of acyl-CoA synthetase gene function results in a significantly altered metabolic profile, hypersensitivity to oxidizing conditions, and decreased growth capability within host macrophages compared to F. tularensis LVS wild-type bacteria. Furthermore, membrane-targeted sensitivity assays revealed a plausible link between membrane integrity and oxidative stress resistance in F. tularensis. Taken together, our work demonstrated a unique approach that complements previously established methods for novel gene-function discovery to advance the field of F. tularensis research, while simultaneously uncovering essential roles for several Francisella-encoded factors, especially acyl-CoA synthetase.

Keywords

Francisella tularensis, Tularemia, Transposon, Mutagenesis, CRISPR, Oxidative Stress, Metabolomics

Disciplines

Bacteriology | Medicine and Health Sciences

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