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"Role of Human Salivary Cystatin SA in Aggregatibacter actinomycetemcomitans Infections"

by
Krishnaraj Ganeshnarayan
Oral Biology
B.D.S. 1999, VS Dental College, Bangalore
MDS, 2004, SDM, Dharwad


Thesis Advisor: Daniel H. Fine, D.M.D.
Professor and Chair
Department of Oral Biology

Tuesday, February 7, 2012
10:00 A.M., MSB Room C-600


Abstract

Healthy subjects who do not have Aggregatibacter actinomycetemcomitans (Aa) in their oral cavity may possess factors in saliva that might demonstrate antibacterial activity against the bacterium. Previous studies have shown that healthy subjects who do not possess this bacterium in their oral cavity have the ability to kill the bacteria. In this thesis, we have identified and purified a protein from the saliva of subjects who do not have Aa in the oral cavity, which demonstrates significant antimicrobial effect against Aa. We have also investigated its mechanism of action of and finally correlated the levels of this protein in health and disease.
Saliva was collected from subjects who demonstrated antimicrobial activity against Aa. After clarifying the saliva, it was subjected to an affinity chromatography column with Aa. The proteins bound to Aa were eluted and identified using mass spectrometry. Based on the proteins that bound Aa in the column, we hypothesized that proteins in saliva that bound to Aa, especially Cystatin SA might demonstrate antibacterial activity against Aa. By using several clinical isolates of Aa, we demonstrate that Cystatin SA demonstrates statistically significant antimicrobial effect against different clinical isolates of Aa. The effect of Cystatin SA decreased with lower concentration (0.01 mg/ml) showing no antibacterial effect. The addition of Cystatin SA antibodies to the purified sample completely negated the antimicrobial effect. The bacteria were also treated with a proteinase inhibitor, leupeptin, to clarify if the antimicrobial effect was related to tis protease inhibitory function. We demonstrate that the antimicrobial effect of cystatin SA is not related to its protease inhibitory function. By identifying the proteins on the surface of Aa to which Cystatin SA binds to, we identified two major potential targets of Cystatin SA, namely omp34 and omp39. By using molecular genetics and cloning, we show that Cystatin SA demonstrates antibacterial activity against E. coli cells expressing omp34 on its surface while cells expressing omp39 showed no effect. By using mutagenesis experiments, we demonstrate that cells expressing mutant omp34 become resistant to killing by Cystatin SA.
Another important aspect of the role of Cystatin SA in the pathogenesis of periodontal disease is the inhibition of colonization of Aa in the oral cavity. Pretreatment of Aa with Cystatin SA reduced the ability of the bacteria to bind to buccal epithelial cells, the initial site for colonization of Aa in the oral cavity. Additionally, E. coli expressing omp34 but not omp39 demonstrated significant binding to Buccal epithelial cells (BEC) and this was reduced to baseline levels when they were treated with Cystatin SA. Finally, we also demonstrate that the levels of salivary Cystatin SA in patients suffering from Localized Aggressive Periodontitis (LAP) were significantly lower than those observed in healthy patients.


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