Human neutrophils contain alpha-defensin peptides with broad-spectrum antibiotic activity. Gram-negative periodontal pathogens are resistant to human alpha-defensins. Defensin-resistance could contribute to the ability of these bacteria to cause disease. To investigate resistance, we studied binding of purified human alpha-defensin HNP-1 to LPS (lipopolysaccharide), which makes up the outer layer of the outer membrane of gram-negative bacteria. Objectives: To determine whether HNP-1 binds to LPS from E. coli., which is killed by HNP-1, and to LPS from resistant bacteria including A.a., P.g., P.d., and F.n. (Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella denticola, and Fusobacterium nucleatum). Methods: E. coli ML-35 bacteria (106 CFU/ml) were incubated 4 h at 37°C with 0 or 5 µM HNP-1. Purified LPS from E. coli, A.a., P.g., P.d., or F.n. was added to determine whether purified LPS would absorb HNP-1 and block killing of E. coli. Viability was measured by plating serial dilutions and counting colonies. Results: LPS had no effect on growth or viability of E. coli. HNP-1 killed 99.7 ± 0.1% of the bacteria. Killing was blocked by adding 50 µg/mL of purified E. coli or A.a. LPS. Assuming an average molecular weight of 10,000 for LPS, about one molecule of LPS per molecule of HNP-1 was needed to block killing. About twice as much P.d. LPS or up to eight times as much P.g. or F.n. LPS was needed to block. Conclusions: HNP-1 binds equally well to purified LPS from E. coli or A.a. indicating that A.a. resistance is not a result of weak binding of HNP-1 to LPS on the surface of A.a. On the other hand, weak HNP-1 binding to LPS of P.g., P.d., and F.n. could contribute to resistance of these pathogens to human alpha-defensins.
Supported by the UTHSC Dental Alumni Endowment Fund for Research