Date of Award

8-31-2019

Document Type

Doctoral Dissertation - Open Access

Degree Name

Doctor of Philosophy

Department

Microbiology and Immunology

First Advisor

Chandra Shekhar Bakshi, DVM, Ph.D.

Abstract

The Tier 1 Select Agent, Francisella tularensis causes an acute and fatal disease known as tularemia. Many studies have devoted enormous efforts to understand how F. tularensis avoids host defense mechanisms, replicates within an extremely secure immune system, and eventually causes the deadly disease tularemia. The extremely high virulence of Francisella depends on its ability to manipulate gene expression according to the surrounding environment. This process requires the involvement of unique transcriptional regulators. Francisella possesses very few transcriptional regulators, and a majority of them characterized to-date have been shown to regulate genes involved in virulence and cellular functions. The role of a transcriptional regulator of F. tularensis belonging to the AraC/XylS in gene regulation and virulence remains uncharacterized to-date. This study characterized the role of AraC in gene regulation, intramacrophage survival, and virulence of F. tularensis.

In specific aim 1, we generated a deletion mutant (DaraC) of FTL_0689 gene encoding AraC of F. tularensis Live Vaccine Strain (LVS), and its transcomplemented strain (DaraC+paraC). Characterization of the DaraC mutant demonstrated that AraC does not regulate genes involved in arabinose utilization. Genomic organization of the araC gene suggested that it may have a role in the regulation of a unique multidrug efflux pump (MEP) located downstream of it. Our results revealed that the phenotype of the DaraC mutant mirrors that of the emrA1 and the silC mutants, the components of the MEP. However, these phenotypic similarities are not due to the direct regulation of MEP genes by AraC. Further characterization revealed that AraC is involved in providing resistance against oxidative stress.

In specific aim 2, we investigated the role of AraC as a global transcriptional regulator in F. tularensis LVS strain. We studied gene expression profiles of the wild type F. tularensis LVS and the DaraC mutant under normal and oxidative stress conditions. The results revealed that AraC serves as a transcriptional regulator only when the bacteria are exposed to oxidative stress conditions. AraC is involved in the regulation of virulence genes encoded on Francisella Pathogenicity Island, stress response genes, energy production, genes encoding enzymes in the tricarboxylic acid (TCA) cycle, metabolism and regulatory proteins indicating its role as a global regulator. The differential expression of these genes also impaired the ability of the DaraC mutant to survive in macrophages and attenuated its virulence in mice.

Collectively, this study identified and characterized a novel transcription factor, AraC, required for adaptation of Francisella to oxidative stress conditions encountered outside or within a host.

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