Date of Award

5-2008

Document Type

Thesis

Degree Name

Master of Dental Science (MDS)

Program

Periodontology

Research Advisor

Edwin L. Thomas, Ph.D.

Committee

Paul Bland, D.D.S. Jegdish P. Babu, Ph. D. David A. Tipton, D.D.S., Ph.D.

Abstract

Human neutrophils (PMN cells) contain four peptides known as alpha (α)-defensins or HNP 1-4. The peptides have broad-spectrum antibiotic activity. HNP 1-3 are major neutrophil components. They differ by only one amino acid residue. HNP-4 is found in small amounts, and has a different sequence. HNP-4 purified from neutrophils was reported to be 100-times more active than HNP-1 against Escherichia coli, suggesting that HNP-4 plays a special role in helping neutrophils kill gram-negative bacteria.

Activity of HNP-4 against gram-negative bacteria is of special interest because Aggregatibacter (Actinobacillus) actinomycetemcomitans (A.a.) and other periodontal pathogens are gram-negative. These bacteria are resistant to HNP 1-3. Defensin-resistance may contribute to the ability of these bacteria to cause disease. There have been no studies of HNP-4 activity against oral bacteria, because HNP-4 is found in small amounts and is difficult to purify. Recently, HNP-4 was chemically synthesized, making it possible to study its antibacterial activity. However, synthetic HNP-4 was reported to be no more active than HNP-1 against E. coli.

Lipopolysaccharide (LPS) covers the surface of gram-negative bacteria. Each LPS molecule has three parts: a long carbohydrate chain (oligosaccharide) that is exposed on the cell surface, a short carbohydrate core, and lipid A that anchors LPS in the outer membrane. LPS on the bacterial cell surface may be the receptor for HNP-4 and other peptides that kill gram-negative bacteria.

Our aims were to: (1) Compare the purity and physical properties of purified and synthetic HNP-4. (2) Compare activity of synthetic and purified HNP-4 against E. coli. (3) Determine whether synthetic HNP-4 binds to purified E. coli LPS. (4) Locate the HNP-4 binding site for synthetic HNP-4 on E. coli LPS molecules. (5) Measure activity of synthetic HNP-4 against A.a. (6) Compare binding of synthetic HNP-4 to LPS purified from E. coli and A.a.

Results: (1) Purified and synthetic HNP-4 were of high purity. They had the same molecular mass, which was equal to the mass calculated from the amino acid sequence. (2) Activity of purified and synthetic HNP-4 against E. coli was the same, and similar to the activity of HNP-1. (3) LPS purified from E. coli absorbed synthetic HNP-4. (4) Experiments with LPS fragments showed that synthetic HNP-4 was absorbed to the LPS inner core. (5) Synthetic HNP-4 partially inhibited growth of A.a. but did not kill these bacteria. (6) There was no difference in the ability of E. coli or A.a. LPS to absorb synthetic HNP-4.

The results suggest that HNP-4 is not more active than other neutrophil α-defensins against gram-negative bacteria. The results also indicate that LPS on the surface of gram-negative bacteria could be the receptor for HNP-4. Because HNP-4 binds to the LPS inner core, HNP-4 may help neutrophils kill gram-negative bacteria that lack oligosaccharide and the LPS outer core.

The periodontal pathogen A.a. is resistant to all of the human leukocyte α-defensins including HNP-4. Therefore, synthetic HNP-4 is not likely to be used in dental practice to prevent or treat oral disease. Resistance of A.a. to HNP-4 is not the result of a difference in LPS structure that interferes with HNP-4 binding. Further studies are needed to determine the mechanism of defensin resistance.

DOI

10.21007/etd.cghs.2008.0137

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