Proteomics Reveals MUC7-12-mer Induces Cysteine Synthetase Downregulation in S. mutans

Objective: The cationic antimicrobial peptide, MUC7-12-mer, exhibits strong antimicrobial activity against Streptococcus mutans. The objective of this study was to confirm the preliminary results that determined the proteins whose expression was altered in MUC7-12-mer- treated versus untreated S. mutans cells, and to investigate their function.

Methods: Protein isolation: MUC7-12-mer (RKSYKCLHKRCR, aa 40-51 of the parent MUC7) was added to cells grown in ¼ BHI medium (OD 0.4) at 15 uM, and incubated for two hours. Proteins were isolated from cells by Fast Prep. DIGE analysis: Three 50 ug samples of each: treated, untreated and an internal standard (each labeled with one of three cyanide dyes) were run on 3 gels, with 2 subsequent gels run for spot-picking (500 ug of untreated sample for each). Isoelectric focusing with a nonlinear pH gradient (3-10) was followed by SDS-PAGE. Gels were scanned and data analysis was performed using DeCyder software. Identification of proteins: spots were excised from the gel and digested with trypsin. Peptides were cleaned with ZipTip and analysed on MALDI-TOF mass spectrometer. Mass spectra were acquired and peak lists were searched in NCBI database using Mascot software.

Results: Several differentially expressed proteins were found in the treated cells. The downregulation of cysteine synthetase was found to be the most statistically significant, suggesting that MUC7-12-mer may interact with this enzyme. Although the connection between this enzyme and action of antimicrobial peptides is presently unknown, the literature indicates that cysteine synthetase functions in the regulation of pyruvate formate lyase (PFL), the first enzyme in a pathway to convert pyruvate to acid fermentation products besides lactate.

Conclusions: The study confirmed the preliminary results that cysteine synthetase is the most downregulated protein in MUC7-12-mer-treated cells.

Supported by AADR Student Research Fellowship, and NIH/NIDCR grants DE007034 and DE009820.