Unveiling Proteomic Insights: Histatin-5 Degradation by Oral Bacteria

Objectives: Salivary proteins called histatins are broken down by oral proteolytic enzymes produced by host and microbial cells. Histatin-5 degrades differently in healthy people compared to those who have periodontitis, suggesting that proteases produced by periodontopathogenic microbes may be essential to this process. The aim of this study was to identify whether oral microorganisms Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum and Streptococcus mutans degrade histatin-5 and to determine the protein fragments (peptides) produced during the degradation process.
Methods: Synthetic histatin-5 was individually incubated with each microorganism in Phosphate-buffered saline (PBS) at a final concentration of 0.5μg/μL for 0, 2, and 6h at 37°C. At each time point, aliquots samples were boiled for 5-min to stop any proteolytic activity and then processed for cationic polyacrylamide gel electrophoresis (PAGE). The experiment was done in triplicate. Proteolytic fragments of histatin-5 were extracted intact from the gel and together with supernatant were desalted before Mass Spectrometry analysis.
Results: The results showed that histatin-5 was degraded only by P. gingivalis, with histatin-5 degradation fragments were evident at 2 and 6h. On the contrary, the other microorganisms did not degrade histatin-5 throughout the duration of the experiment. Moreover, 19 fragments of histatin-5 were identified; including two unique peptides at 2h and seven unique peptides at 6h. The study identified key proteases in the supernatant of P. gingivalis, particularly trypsin-like enzymes.
Conclusions: Since P. gingivalis’ mainly produces trypsin-like enzymes as a virulence factor, and this is consistent with the results obtained in the supernatant of our samples with histatin-5, these findings highlight the key role of P. gingivalis in the degradation of histatin-5. Our study may have revealed a new, crucial, and promising biomarker of periodontal disease that has the potential for developing diagnostic tools and improving dental public health in many locales.