Date of Award

5-2011

Document Type

Thesis

Degree Name

Master of Dental Science (MDS)

Program

Periodontology

Research Advisor

Edwin L. Thomas, Ph.D.

Committee

Pradeep C. Adatrow, D.D.S., M.P.H., M.D.S. Anastasios Karydis, D.D.S., Ph.D. Swati Y. Rawal, B.D.S., M.D.S. Sidney H. Stein, D.D.S., Ph.D.

Keywords

Defensin, Epithelium, Lipopolysaccharide

Abstract

Defensins are cationic (positive-charged) peptides with broad-spectrum antibiotic activity. In humans, there are two types of defensins, alpha (α) and beta (β). Human neutrophils contain four α-defensins known as Human Neutrophil Peptide (HNP) 1-4. Epithelial cells produce four β-defensins known as Human Beta Defensin (HBD) 1-4. Gram-negative anaerobic bacteria that are associated with periodontal disease are resistant to human α-defensins, but are killed by β-defensins.

HBD-3 is the most active β-defensin. HBD-3 is a longer peptide than HNP 1-4. HBD-3 has additional amino acid residues with hydrophobic side chains near the N-terminus and residues with cationic side chains at the C-terminus.

Objectives: (1) Confirm that the periodontal pathogen A.a. (Aggregatibacter actinomycetemcomitans) is resistant to HNP-1 but killed by HBD-3; (2) Determine if the N-terminal or C-terminal portion of HBD-3 can account for activity against A.a.; (3) Determine whether HBD-3 binds to lipopolysaccharide (LPS), which covers the surface of gram-negative bacteria; (4) Determine whether binding of the hydrophobic N-terminus of HBD-3 to the hydrophobic lipid A portion of LPS accounts for activity of HBD-3 against A.a.

Methods: Non-pathogenic Escherichia coli and pathogenic A.a. Y4 bacteria were incubated with recombinant HBD-3 or HNP-1 purified from human neutrophils. Bacteria were also incubated with synthetic peptides CHRG07 and CHRG01. These peptides have sequences derived from the HBD-3 N-terminus and C-terminus, respectively. The number of viable bacteria was determined by diluting, plating on solid growth medium, and counting colonies.

Bacteria were also incubated with HBD-3 and purified LPS from E. coli or A.a. to determine whether purified LPS absorbs HBD-3 and blocks killing. Similar experiments used purified lipid A or deacylated-LPS, which lacks the hydrophobic fatty acids of the lipid Aportion of LPS.

Results: HBD-3 had strong bactericidal activity against A.a. under the usual assay conditions for α-defensins (in dilute culture medium) and the usual assay conditions for β-defensins (in buffer without nutrients). HBD-3 at 5 µM gave 90 to 99% killing of A.a.within 2 to 4 h. In contrast, HNP-1 had no activity against A.a. regardless of assay conditions, confirming that A.a. is resistant to HNP-1 but killed by HBD-3.

Both CHRG07 and CHRG01 killed A.a., but CHRG07 was much more active. The activity of CHRG07 was equal to that of HBD-3, indicating that the mixture of hydrophobic and cationic amino acid residues at the N-terminus can account for HBD-3 activity againstA.a.

Purified LPS from E. coli or A.a. blocked the activity of HBD-3 at a 1:1 ratio of LPS to HBD-3, indicating that one molecule of HBD-3 binds to each molecule of LPS. Deacylated-LPS also blocked HBD-3 at a 1:1 ratio, but purified lipid A did not block. Although HBD-3 binds to LPS, and hydrophobic residues near the N-terminus of HBD-3 appear to be important for killing of A.a., the hydrophobic lipid A portion of LPS was not the binding site for HBD-3. Binding of HBD-3 to other hydrophobic substances such as membrane proteins or phospholipids may be important to HBD-3 activity against A.a.

Conclusions: Resistance of A.a. to leukocyte α-defensins is probably important to the ability of A.a. to cause disease. On the other hand, the epithelial cell β-defensins probably help to protect healthy individuals against oral disease. Small synthetic peptides such as CHRG07 that contain the portion of HBD-3 active against the periodontal pathogen A.a. may be useful to prevent or treat gingivitis and periodontitis.

DOI

10.21007/etd.cghs.2011.0093

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