Master of Science
Haniford, David B.
Salmonella Typhimurium is a leading contributor to non-typhoid diseases with complex regulatory networks that are key to understanding its pathogenicity and virulence. I explore the role of the sRNA IS200, where its deletion in Salmonellaleads to premature induction of the Cys regulon. The premature induction in ∆IS200 was validated through qRT-PCR and GFP transcriptional fusions and occurs at late exponential to early stationary phase. In addition, ∆IS200 leads to increased sensitivity to oxidative stress. The focus of this work has been to understand how ∆IS200 leads to premature Cys regulon induction, and I present three models: the futile import/export, the metabolic burden, and LrhA deprivation models. I conclude that the third model, which proposes a novel function for the global regulator LrhA, provides a promising direction for future research. Understanding this problem will help uncover how IS200 has integrated itself into the metabolic machinery of a medically important pathogen.
Summary for Lay Audience
Pathogenesis and virulence are what allow bacteria to infect, persists and spread within populations. Proteins are functioning units in the cell to carry out jobs, and these proteins are regulated at multiple steps throughout their production. For example, transcription is the conversion of reading a DNA template or gene and copy it to messenger RNA (mRNA), and translation is turning that mRNA into a protein. Small RNAs (sRNA) will regulate post-transcriptionally, and the sRNA of interest in this project is the insertion sequence 200 (IS200).
Salmonella Typhimurium is a pathogenetic bacterium and a significant contributor to non-typhoid gastrointestinal diseases. Our lab has previously discovered that IS200 can regulate genes in its host Salmonella. When IS200 is deleted from the Salmonella strain, it causes the expression of the pathways responsible for pathogenesis to increase. Another consequence of deleting IS200 is that a pathway for creating cysteine, an important protein building block, is turned on earlier than expected. In this project, I validate and characterize this premature turn-on of the cysteine pathway in an IS200 deletion strain. When IS200 is removed, I show that the Salmonella strain is at a disadvantage when exposed to oxidative stress, which comes from harmful reactive oxygen molecules.
In addition to validating and characterizing the turning on of the cysteine pathway in Salmonella with IS200 deleted, I developed and tested three models to explain how IS200 might be causing this premature turn-on. Through a series of experiments, I show that the most promising of the three models I present involved a protein called LrhA. This protein regulates pathways involved in pathogenesis by preventing their expression, and I provide evidence that LrhA could be involved in regulating the cysteine pathway. My results imply that this is a novel function of LrhA and in Salmonella with IS200 deleted, LrhA could influence the cysteine pathway via direct and indirect regulation.
Scherba, Naomi-Jean Q., "Disruption of Insertion Sequence 200 (IS200) leads to a premature induction of the Cysteine regulon in Salmonella Typhimurium" (2021). Electronic Thesis and Dissertation Repository. 8158.
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Available for download on Thursday, August 18, 2022