Dissertation
UNDERSTANDING THE ROLE OF DIMETHYL ADENOSINE TRANSFERASE (KSGA) IN SALMONELLA ENTERITIDIS CELL-ENVELOPE FITNESS
Doctor of Philosophy (PhD), Washington State University
01/2018
Handle:
https://hdl.handle.net/2376/117104
Abstract
We previously reported that inactivation of a universally conserved dimethyl adenosine transferase (KsgA) attenuates virulence and increases sensitivity to oxidative and osmotic stress in Salmonella Enteritidis. Here, we show a role of KsgA in cell-envelope fitness as a potential mechanism underlying these phenotypes in Salmonella. We assessed structural integrity of the cell-envelope by transmission electron microscopy, permeability barrier function by determining intracellular accumulation of ethidium bromide and electrophysical properties by dielectrophoresis in wild-type and ksgA knock-out mutants of S. Enteritidis. Deletion of ksgA resulted in disruption of the structural integrity, permeability barrier and distorted electrophysical properties of the cell-envelope. The cell-envelope fitness defects were alleviated by expression of wild-type KsgA (WT-ksgA) but not by its catalytically inactive form (ksgAE66A), suggesting that the dimethyl transferase activity of KsgA is important for cell-envelope fitness in S. Enteritidis. Upon expression of WT-ksgA and ksgAE66A in inherently permeable E. coli cells, the former strengthened and the latter weakened the permeability barrier, suggesting that KsgA also contributes to the cell-envelope fitness in E. coli. Lastly, expression of ksgAE66A exacerbated the cell-envelope fitness defects, resulting in impaired S. Enteritidis interactions with human intestinal epithelial cells, and human and avian phagocytes. KsgA modifies the structure of the small ribosomal subunit (30S) region that recruits bacterial transcripts and facilitates their translation. To elucidate mechanisms behind KsgA activities in Salmonella, we identified highly expressed transcripts whose expected protein abundance may be impaired in KsgA-deficiency due to faulty interaction with 30S. Using in silico prediction of gene expression and RNA-seq technology, we identified a core of 39 non-ribosomal highly expressed genes that have optimal binding interactions with 30S to ensure abundant protein levels and are thus sensitive to disturbances in these interactions. These genes are relevant for metabolism, stress response and cell-envelope biogenesis in S. Enteritidis, providing a potential mechanistic basis for the phenotypes observed in KsgA-deficiency. This study shows that KsgA contributes to cell-envelope fitness and opens new avenues to modulate cell-envelopes via use of KsgA-antagonists.
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Details
- Title
- UNDERSTANDING THE ROLE OF DIMETHYL ADENOSINE TRANSFERASE (KSGA) IN SALMONELLA ENTERITIDIS CELL-ENVELOPE FITNESS
- Creators
- Kim Lam Chiok Casimiro
- Contributors
- Devendra H Shah (Advisor)Troy Bankhead (Committee Member)Santanu Bose (Committee Member)Brian Clowers (Committee Member)Jean Guard (Committee Member)
- Awarding Institution
- Washington State University
- Academic Unit
- College of Veterinary Medicine
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Number of pages
- 105
- Identifiers
- 99900581420401842
- Language
- English
- Resource Type
- Dissertation