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Degradation of Antimicrobial Histatin-variant Peptides in Staphylococcus aureus and Streptococcus mutans

Department of Dental Basic Sciences, Section of Oral Biochemistry, Academic Centre for Dentistry Amsterdam (ACTA), Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands;

View larger version (78K): [in a new window]   Figure 1. Peptide degradation by bacterial proteinases. (A) S. aureus cells (6.4 x 108 cells/mL PBS) were incubated with the dhvar peptides for 4 hrs at 37°C. Samples were analyzed on high-density SDS-PAGE gels in combination with Coomassie Brilliant Blue staining. Lane 1, dhvar1 (200 µg/mL) not incubated with bacteria; lanes 2–4, samples taken at 15, 120, and 240 min, respectively. A peptide band with lower molecular weight gradually appeared over time, indicating progressive degradation. Incubation of S. mutans with dhvar1 and dhvar2 showed similar patterns (not shown). (B) Lane 1 contained the native peptide dhvar1 (200 µg/mL). Lane 2: Dhvar1 incubated with P. gingivalis cells (1.6 x 106 cells/mL PBS) for 60 min at 37°C. Lane 3: Dhvar1 incubated (4:1) with supernatant of 6 days’ cultured P. gingivalis (five-minute incubation). Incubations with P. aeruginosa showed similar results (not shown).

View larger version (26K): [in a new window]   Figure 2. Degradation of dhvar1 by S. mutans analyzed by MALDI-TOF. A suspension of S. mutans (6.4 x 108 cells/mL PBS) was incubated with 200 µg/mL dhvar1 for 5 hrs at 37°C. Subsequently, cells were removed by centrifugation, proteinases were inactivated by being heated for 5 min at 100°C, and samples were analyzed by MALDI-TOF. The original peptide (molecular mass 1855) was degraded into two fragments with molecular masses of 821 and 1054, corresponding to the fragments Lys1-Glu6 and Leu7-Tyr14, respectively. Potassium and sodium adducts contribute to minor adjacent peaks.