Biofilm accounts for 65%-80% of microbial infections in humans. Considerable evidence links biofilm formation to oral disease and consequently systemic infections. Eradication of biofilm-associated infections is important. Streptococcus sanguinis, a Gram-positive bacterium, is one of the most abundant species in oral biofilm. It contributes to biofilm development in oral cavities and is one of the recognized causes of infective endocarditis. To study and identify biofilm genes in S. sanguinis, biofilm formation of 51 mutants was compared with the wild type SK36 strain using crystal violet (CV) staining in a microtiter plate. Confocal laser scanning microscopy (CLSM) and image analysis was done to compare biofilm formation by the mutant to the wild type SK36 strain. A biofilm mutant XG2_0351, encoding a type I signal peptidase (SPase I), was further investigated. SPase I cleaves proteins that are transported through secretory machinery and is necessary for the release of translocated preproteins from a cytoplasmic site of synthesis to extracytoplasmic/membrane destinations. S. sanguinis, like many Gram-positive bacteria, has multiple SPases I. The objective of this project is to investigate the distinctive role that SPase I plays in biofilm formation in S. sanguinis. Using a plate reader, the growth curves of the wild type strain SK36 and XG2_0351 were compared. The scanning electron microscope (SEM) was utilized to compare the cell surface morphologies. Coomassie staining was done to narrow the list of potential substrates of XG2_0351. CV staining and CLSM images indicated phenotypic differences between the SPase I mutant and SK36. The growth curves of XG2_0351 and SK36 showed no significant difference although SEM illustrated a difference in the cell surface morphologies. Coomassie staining illustrated a number of substrates that were present in SK36 but not XG2_0351. In addition bioinformatics was used to understand the gene function. In conclusion, XG2_0351 reduces biofilm formation in S. sanguinis but further research is necessary to elucidate the specific proteins that are involved. Clarifying the vii role that SPase I plays in reduced biofilm formation in S. sanguinis will give a better understanding of the biofilm formation mechanism.
| Identifer | oai:union.ndltd.org:vcu.edu/oai:scholarscompass.vcu.edu:etd-5463 |
| Date | 01 January 2016 |
| Creators | Aynapudi, Jessica |
| Publisher | VCU Scholars Compass |
| Source Sets | Virginia Commonwealth University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | © The Author |
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