title: Human salivary cystatin SA exhibits antimicrobial effect against Aggregatibacter actinomycetemcomitans.
abstract: OBJECTIVE
  Healthy subjects who do not have Aggregatibacter actinomycetemcomitans in their oral cavity may possess factors in saliva that might demonstrate antibacterial activity against the bacterium. The aim of this study was to identify and purify proteins from saliva of healthy subjects that might demonstrate antibacterial activity against A. actinomycetemcomitans and test the same against the bacteria.
  
  
  METHODS
  Saliva from 10 healthy volunteers was tested individually for its anti-A. actinomycetemcomitans activity. Among the 10 subjects, eight demonstrated anti-A. actinomycetemcomitans activity. Saliva was collected from one healthy volunteer who demonstrated the highest antimicrobial activity against A. actinomycetemcomitans. After clarifying the saliva, it was subjected to an affinity chromatography column with A. actinomycetemcomitans. The proteins bound to A. actinomycetemcomitans were eluted from the column and identified using mass spectrometry (MALDI-TOF/TOF MS). Among other proteins that bound to A. actinomycetemcomitans, which included lactoferrin, immunoglobulin A and kallikrein, cystatin SA was observed in significantly higher concentrations, and this was purified from the eluate. The purified cystatin SA was tested at different concentrations for its ability to kill A. actinomycetemcomitans in a 2 h cell killing assay. The bacteria were also treated with a proteinase inhibitor, leupeptin, to clarify whether the antimicrobial effect of cystatin SA was related to its protease inhibitory function. Cystatin SA was also tested for its ability to prevent binding of A. actinomycetemcomitans to buccal epithelial cells (BECs) in an A. actinomycetemcomitans-BEC binding assay.
  
  
  RESULTS
  Cystatin SA (0.1 mg/mL) demonstrated a statistically significant antimicrobial activity against A. actinomycetemcomitans. The effect of cystatin SA decreased with lower concentrations, with 0.01 mg/mL showing no effect. The addition of monoclonal cystatin SA antibodies to the purified sample completely negated the antimicrobial effect. Treatment of A. actinomycetemcomitans with leupeptin resulted in no antimicrobial effect, suggesting that the antimicrobial activity of cystatin SA is independent of its protease inhibitory function. A. actinomycetemcomitans pretreated with cystatin SA showed reduced binding to BECs, suggesting a potential role for cystatin SA in decreasing the colonization of A. actinomycetemcomitans.
  
  
  CONCLUSIONS
  The present study shows that cystatin SA demonstrates antimicrobial activity against the periodontopathogen A. actinomycetemcomitans, and future studies determining the mechanism of action are necessary. The study also shows the ability of cystatin SA to reduce significantly the binding of A. actinomycetemcomitans to BECs.
authors.affiliation: Department of Oral Biology, UMDNJ, Newark, NJ, USA.
authors.forename: K
authors.initials: K
authors.lastname: Ganeshnarayan
authors.forename: K
authors.initials: K
authors.lastname: Velliyagounder
authors.forename: D
authors.initials: D
authors.lastname: Furgang
authors.forename: D H
authors.initials: DH
authors.lastname: Fine
mesh_terms: D016976:Aggregatibacter actinomycetemcomitans / Q000187:drug effects*; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001422:Bacterial Adhesion / Q000187:drug effects; D002403:Cathepsins / Q000037:antagonists & inhibitors; D002846:Chromatography, Affinity; D015853:Cysteine Proteinase Inhibitors / Q000302:isolation & purification / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K / Q000494:pharmacology; D004847:Epithelial Cells / Q000382:microbiology; D016967:Fusobacterium nucleatum / Q000187:drug effects; D006801:Humans; D007071:Immunoglobulin A, Secretory / Q000302:isolation & purification; D007610:Kallikreins / Q000302:isolation & purification; D007781:Lactoferrin / Q000302:isolation & purification; D007976:Leupeptins / Q000494:pharmacology; D018613:Microscopy, Confocal; D009061:Mouth Mucosa / Q000166:cytology / Q000382:microbiology; D016966:Porphyromonas gingivalis / Q000187:drug effects; D012463:Saliva / Q000187:drug effects; D055333:Salivary Cystatins / Q000302:isolation & purification / Q000494:pharmacology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors
chemical_list: D000900:Anti-Bacterial Agents; C525172:CST2 protein, human; D015853:Cysteine Proteinase Inhibitors; D007071:Immunoglobulin A, Secretory; D007976:Leupeptins; D055333:Salivary Cystatins; D002403:Cathepsins; D007610:Kallikreins; D007781:Lactoferrin; D019286:Endopeptidase K; C032854:leupeptin