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.

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Revised 30 April 2008