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DEVELOPMENT OF LUMINESCENT TOOLS FOR USE IN THE STUDY OF MYCOBACTERIUM TUBERCULOSIS
Title
DEVELOPMENT OF LUMINESCENT TOOLS FOR USE IN THE STUDY OF MYCOBACTERIUM TUBERCULOSIS.
Creator
Moore, Krista A, Rohde, Kyle, University of Central Florida
Abstract / Description
Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is a growing problem worldwide due to the emergence of multi-drug resistant and extensively-drug resistant strains of the bacteria. A key to combatting the spread of these strains lies in the understanding of gene expression occurring in Mtb. This study focuses on the development and optimization of a luciferase-based bioluminescent transcriptional reporter that can be used to monitor gene expression in Mtb. The...
Show moreMycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is a growing problem worldwide due to the emergence of multi-drug resistant and extensively-drug resistant strains of the bacteria. A key to combatting the spread of these strains lies in the understanding of gene expression occurring in Mtb. This study focuses on the development and optimization of a luciferase-based bioluminescent transcriptional reporter that can be used to monitor gene expression in Mtb. The luminescent signal emitted from the reporter can be measured and correlated with the level of transcription of certain genes. This study focuses specifically on a gene called whiB7 which encodes a transcription factor known to contribute to the drug resistance of Mtb. The drug-inducible whiB7 promoter was cloned into various locations in the luciferase plasmid in order to determine the ideal configuration of the reporter for maximum luminescence. The optimized luciferase reporter was then compared with a fluorescent transcriptional reporter, mCherry, also under control of the whiB7 promoter. Fluorescent reporters present some disadvantages including delayed kinetics and inability to accurately reflect gene downregulation due to long half-life of reporter proteins. It was hypothesized that the luciferase reporter would solve these problems by offering a more sensitive and dynamic tool to monitor gene expression. Quantitative real-time PCR was used to measure whiB7 mRNA present in cultures containing either the luciferase or mCherry reporters. The luminescent and fluorescent signal given from these reporters was then compared to actual mRNA expression. It was observed that the signal from the luciferase reporter more closely matched mRNA expression at each timepoint, indicating that the luciferase reporter is a better gauge of actual gene expression levels than the mCherry reporter.
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Date Issued
2019
Identifier
CFH2000478, ucf:45912
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFH2000478
Characterization of Novel Borrelia burgdorferi Transcripts Expressed during Tick and Mammalian Infection
Title
Characterization of Novel Borrelia burgdorferi Transcripts Expressed during Tick and Mammalian Infection.
Creator
Adams, Philip, Jewett, Mollie, Rohde, Kyle, Moore, Sean, Fernandez-Valle, Cristina, University of Central Florida
Abstract / Description
The purpose of this dissertation is to characterize the transcriptome of Borrelia (Borreliella) burgdorferi to discover novel transcripts, important for pathogenesis. As a spirochete and the etiological agent of Lyme disease, the foremost vector-borne bacterial infection in the world, B. burgdorferi fulfills a distinctive niche among bacterial pathogens. Persisting in the disparate environments of a tick vector and mammalian reservoirs, it is absolutely dependent on its hosts for transmission...
Show moreThe purpose of this dissertation is to characterize the transcriptome of Borrelia (Borreliella) burgdorferi to discover novel transcripts, important for pathogenesis. As a spirochete and the etiological agent of Lyme disease, the foremost vector-borne bacterial infection in the world, B. burgdorferi fulfills a distinctive niche among bacterial pathogens. Persisting in the disparate environments of a tick vector and mammalian reservoirs, it is absolutely dependent on its hosts for transmission and nutrient acquisition. B. burgdorferi harbors a complex fragmented genome which is largely linear, unlike that of most prokaryotes, lacks an array of classically described metabolic genes, and contains an unusually large percentage of unique genomic sequences specific to Borrelia (Borreliella) species. To date, few regulatory mechanisms have been identified which contribute to the ability of the spirochete to sense and respond to its environment. Efforts to use global transcript analysis to elucidate the molecular mechanisms of B. burgdorferi host adaptation have proven challenging due to the low numbers of the pathogen present during infection. Previously, our laboratory successfully developed an in vivo expression technology based approach for B. burgdorferi (BbIVET) to identify spirochete promoter sequences that are active during a murine infection. This screen identified 233 unique putative promoters which mapped to locations across the entire genome. These putative infection-active B. burgdorferi promoters were not only located at the 5' end of annotated open reading frames (ORFs), but also mapped to unannotated locations antisense, intergenic, and intragenic to ORFs. Given the limited characterization of the B. burgdorferi transcriptome, this dissertation applies an RNA sequencing approach (5'RNA-seq) to globally annotate the transcriptional start sites (TSSs) and 5' processed ends of the spirochete's RNA during in vitro cultivation. This resulted in the discovery of numerous novel internal, intergenic, and antisense transcripts. Synergistic analysis combining Northern blotting techniques, alignments of these transcripts to BbIVET proposed promoters, and interrogation of promoter activity via in vivo live imaging of mice, confirmed the expression of a variety of RNAs during laboratory culture and mammalian infection. Further, as a means to improve quantitation of the expression of these transcripts, a new methodology was developed and applied to measure B. burgdorferi promoter activity during tick-pathogen interactions, in a strand specific manner. Finally, because the Lyme disease spirochete harbors many unclassified and unique genomic sequences, the mammalian infection-expressed gene bb0562, identified through BbIVET and 5'RNA-seq, was selected for targeted deletion and evaluation throughout B. burgdorferi's infectious cycle. This demonstrated that gene bb0562 encodes a membrane associated protein, whose presence is critical for establishing murine infection through the bite of an infected tick. In sum, this work contributes significant insight into the transcriptome of B. burgdorferi, provides an innovative approach for the analysis of RNA transcripts at the tick-pathogen interface, and identifies a novel gene critical for Lyme disease pathogenesis.
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Date Issued
2017
Identifier
CFE0006707, ucf:51915
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0006707