Year of Award

2020

Document Type

Dissertation

Degree Type

Doctor of Philosophy (PhD)

Degree Name

Cellular, Molecular and Microbial Biology

Other Degree Name/Area of Focus

Microbiology

Department or School/College

Division of Biological Sciences

Committee Chair

Michael Minnick

Commitee Members

Scott Samuels, Steve Lodmell, Dan Drecktrah, Kent Sugden

Keywords

Bartonella bacilliformis, Coxiella burnetii, Selfish Genetic Elements, Small RNAs

Abstract

Coxiella burnetii is a Gram-negative gammaproteobacterium and zoonotic agent of Q fever in humans. Previous work in our lab has demonstrated that C. burnetii codes for several small RNAs (sRNAs) that are differentially expressed between in vivo and in vitro growth conditions. sRNAs serve as post-transcriptional regulatory effectors involved in the control of nearly all biological processes. We demonstrated that several of the identified sRNAs, namely Coxiella burnetii small RNA 3 (CbsR3), Cbsr13, and CbsR16, represent members of two novel families of miniature inverted-repeat transposable elements (MITEs), termed QMITE1 and QMITE2. Furthermore, we have characterized a highly expressed, infection-specific sRNA, CbsR12, and have determined that it is necessary for expansion of the C. burnetii intracellular niche in a human monocyte-derived alveolar macrophage cell line. We have determined that CbsR12 may participate in broad gene regulation by acting as an "RNA sponge" for the global regulatory RNA-binding protein CsrA. Additionally, CbsR12 is a trans-acting sRNA that targets transcripts of the carA, metK, and cvpD genes in vitro and in vivo.

Bartonella bacilliformis is a Gram-negative alphaproteobacterium and the etiological agent of Carrión's disease in humans. B. bacilliformis is spread between humans through the bite of female phlebotomine sand flies. As a result, the pathogen encounters significant environmental shifts during its life cycle, including changes in pH and temperature. Bacterial sRNAs can serve as a means of rapid regulation under shifting environmental conditions. We therefore performed total RNA-sequencing analyses on B. bacilliformis grown in vitro then shifted to one of ten distinct conditions that simulate various environments encountered by the pathogen during its life cycle. From this, we identified 160 sRNAs significantly expressed under at least one of the conditions tested. Northern blot analysis was used to confirm the expression of eight novel sRNAs. We also characterized a Bartonella bacilliformis group I intron (BbgpI) that disrupts an un-annotated tRNACCUArg gene and determined that the intron splices in vivo and self-splices in vitro. Furthermore, we verified the predicted molecular targeting of a sand fly-specific sRNA, Bartonella bacilliformis small RNA 9 (BbsR9), to transcripts of the ftsH, nuoF, and gcvT genes, in vitro.

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© Copyright 2020 Shaun Gregory Wachter