Exploring the function of the putative fratricins of Streptococcus mutans and Streptococcus vestibularis
Master thesis
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https://hdl.handle.net/11250/3147940Utgivelsesdato
2024Metadata
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- Master's theses (KBM) [927]
Sammendrag
Antibiotic resistance is a growing problem in modern society and is largely caused by bacteria being able to exchange genetic material with each other through horizontal gene transfer. Streptococci have been shown to be competent for natural transformation, a process in which bacteria can acquire new genetic material from the environment. The enzyme responsible for making DNA available is called fratricin, and this enzyme has been extensively studied in Streptococcus pneumoniae. However, there is limited knowledge about fratricins from other streptococcal species. Therefore, the purpose of this thesis was to investigate the putative fratricins LytF and CbpD from Streptococcus mutans and Streptococcus vestibularis. The aim was to investigate if the fratricins are competence induced and to identify their function in the transformation process.
Using a luciferase reporter assay it was shown that transcription from PlytF and PcbpD increased upon competence induction. Although the signal strengths were relatively weak, the results indicate that the genes are competence induced. By knocking out lytF in S. mutans, it was also discovered that LytF has an important role in the DNA-uptake process, as the transformability was severely reduced. CbpD from S. vestibularis, on the other hand, had limited impact on this process, as knockout of the fratricin reduced the transformation efficiency minimally. It was therefore speculated that CbpD in S. vestibularis only functions as a fratricin, while LytF on the other hand may have an additional role in the DNA-uptake process in competent S. mutans cells. Since the transformation pilus is an important part of the uptake mechanism, it has been suggested that the fratricin-like proteins may help the pilus to penetrate the peptidoglycan layer of the cell wall, allowing the pilus to exit the cell. However, immunofluorescence microscopy suggested that the pilus protein ComGC was present on the cell surface of ΔlytF cells, although it was present in slightly smaller amounts in the mutant compared to the wildtype. These findings may indicate that certain pores within the cell wall of S. mutans are wide enough for unassisted pilus penetration. However, since the activity of LytF increases the transformability of the bacterium, it is possible that the fratricin enlarges the pores to ensure a more efficient pilus penetration through the cell wall. Further experiments are needed to confirm this finding, as there is a possibility that the results are a consequence of another function performed by LytF. Lastly, it was also investigated whether LytF functions as a cell wall hydrolase, but zymography revealed no muralytic activity for this protein.