Regulation of the bacterial second messenger cyclic-di-GMP in Salmonella Typhimurium allows it to delicately alter phenotypes to optimize invasion and survive intracellularly in epithelial cells and macrophages to become virulent and cause infection. The concentration of cyclic-di-GMP is regulated by the presence of external stimuli, sensor CMEs (diguanylate cyclases, DGCs, and phosphodiesterases, PDEs), and cyclic-di-GMP binding effectors. Previous studies established that maintenance of low cyclic-di-GMP concentrations is required for survival in macrophages and that the deletion of 3 active PDEs reduces this survival. This study aimed to further investigate the regulation of cyclic-di-GMP for survival in macrophages and epithelial cells. Salmonella Typhimurium mutants were generated and used for an infection assay with RAW 264.7 macrophage and HeLa epithelial cell lines to determine active CMEs via intracellular survival. Intracellular survival was quantified by plate counting of cell lysates at 1-, 4-, and 24-hours post-infection. Our result showed that the previously identified 3 PDEs also influenced the infection of epithelial cells. We re-established the decreased survival in the RAW 264.7 macrophage cell line and determined that the cyclic-di-GMP-binding cellulose synthase BcsA was responsible for decreased survival in macrophages. Finally, we identified an active DGC whose deletion within the 3xKO PDEs restores survival levels, suggesting that this enzyme is responsible for the synthesis of cyclic-di-GMP during macrophage infection. Further studies on how cyclic-di-GMP regulates Salmonella Typhimurium intracellular survival could lead to identifying a potential alternative drug target for treating its infections.
| Identifer | oai:union.ndltd.org:ETSU/oai:dc.etsu.edu:asrf-2191 |
| Date | 25 April 2023 |
| Creators | Musa, Abdulafiz, Petersen, Erik |
| Publisher | Digital Commons @ East Tennessee State University |
| Source Sets | East Tennessee State University |
| Detected Language | English |
| Type | text |
| Source | Appalachian Student Research Forum |
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