Activity of Epithelial Defensin HBD-3 Against a Periodontal Pathogen

<p>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. </p> <p> 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.</p> <p> Objectives: (1) Confirm that the periodontal pathogen <em>A.a. </em>(<em>Aggregatibacter actinomycetemcomitans</em>) 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 <em>A.a.</em>; (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 <em>A.a.</em></p> <p> Methods: Non-pathogenic <em>Escherichia coli</em> and pathogenic <em>A.a.</em> 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.</p> <p> Bacteria were also incubated with HBD-3 and purified LPS from <em>E. coli</em> or <em>A.a.</em> 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.</p> <p> Results: HBD-3 had strong bactericidal activity against <em>A.a.</em> 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 <em>A.a.</em>within 2 to 4 h. In contrast, HNP-1 had no activity against <em>A.a.</em> regardless of assay conditions, confirming that <em>A.a.</em> is resistant to HNP-1 but killed by HBD-3.</p> <p> Both CHRG07 and CHRG01 killed <em>A.a.</em>, 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 against<em>A.a.</em></p> <p> Purified LPS from <em>E. coli</em> or <em>A.a.</em> 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 <em>A.a.</em>, 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 <em>A.a.</em></p> <p> Conclusions: Resistance of <em>A.a.</em> to leukocyte α-defensins is probably important to the ability of <em>A.a.</em> 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 <em>A.a.</em> may be useful to prevent or treat gingivitis and periodontitis.</p>

Name thesis:degree_name

Master of Dental Science (MDS)

Level thesis:degree_level

Thesis

Discipline thesis:degree_discipline

Periodontology

Year dc:date.available

2011

Author dc:creator

• Fine, Norman B.

Contributors dc:contributor

• Edwin L. Thomas, Ph.D.

• Defensin
• Epithelium
• Lipopolysaccharide
• Dentistry
• Medicine and Health Sciences
• Periodontics and Periodontology

Repository record dc:identifier

https://dc.uthsc.edu/dissertations/81

OAI identifier oai:identifier

oai:dc.uthsc.edu:dissertations-1082