Calprotectin is a complex of two anionic proteins found in abundance in the cytosol of neutrophils, certain macrophages, and oral epithelial keratinocytes. Bacteria of the genus Capnocytophaga are pathogens of periodontal origin which can cause systemic infection in neutropenic subjects. Recently, it has been observed that Capnocytophaga may be internalized by neutrophils within the cytosol rather than within a membrane-delimited phagosome. The purpose of this study was to test the in vitro antibacterial effect of the cytosolic complex, calprotectin, against Capnocytophaga sputigena. Calprotectin was purified from the cytosol of human neutrophils by gel filtration and anion exchange FPLC, and it exerted potent in vitro antimicrobial effects against C. sputigena. Net bacteriostatic activity was exerted up to 18 h, after which bactericidal effects were observed. Both net bacteriostatic and bactericidal activity occurred at concentrations above 20 micrograms/mL and exhibited identical dose-response characteristics. Particle counts increased in the presence of calprotectin, despite net bacteriostasis as assessed by changes in colony-forming units (CFU). Dose-response characteristics and direct particle counts suggested that net bacteriostatic effects were the result of balanced cell division and death, rather than suspension of cell division. We conclude that calprotectin can be a significant contributor to host defense against infection by Capnocytophaga.

Neutrophils play a major role in defending the periodontium against infection by oral, gram-negative, facultative bacteria, such as Actinobacillus actinomycetemcomitans, Eikenella corrodens, and Capnocytophaga spp. We examined the sensitivity of these bacteria to a mixture of low-molecular-weight peptides and highly purified individual defensin peptides (HNP-1, HNP-2, and HNP-3) isolated from human neutrophils. Whereas the Capnocytophaga spp. strains were killed significantly by the mixed human neutrophil peptides, the A. actinomycetemcomitans and E. corrodens strains were resistant. Killing was attributable to the defensins. The bactericidal activities of purified defensins HNP-1 and HNP-2 were equal, and both of these activities were greater than HNP-3 activity against strains of Capnocytophaga sputigena and Capnocytophaga gingivalis. The strain of Capnocytophaga ochracea was more sensitive to defensin-mediated bactericidal activity than either C. sputigena or C. gingivalis was. The three human defensins were equipotent in killing C. ochracea. C. ochracea was killed under aerobic and anaerobic conditions and over a broad pH range. Killing was most effective under hypotonic conditions but also occurred at physiologic salt concentrations. We concluded that Capnocytophaga spp. are sensitive to oxygen-independent killing by human defensins. Additional studies will be required to identify other components that may equip human neutrophils to kill A. actinomycetemcomitans, E. corrodens, and other oral gram-negative bacteria.

The Capnocytophaga are inhabitants of the hypoxic human gingival crevice that are normally prevented by neutrophils from causing periodontal and systemic infection. To identify potential nonoxidative bactericidal mechanisms against Capnocytophaga within human neutrophils, gel filtration chromatography was used to fractionate neutrophil granule extracts.Seven granule fractions, designated A through G, were obtained. The Capnocytophaga were most sensitive to killing by fraction D. Fraction D exhibited substantial bactericidal activity under aerobic and anaerobic conditions. The bactericidal activity associated with ion-exchange subfractions D8-D11, which contained primarily cathepsin G as assessed by enzymatic activity, amino acid composition, and NH2-terminal sequence. Heat-inactivation, diisopropylfluorophosphate, PMSF, and N-benzyloxycarbonylglycylleucylphenylalanylchloromethyl ketone inhibited bactericidal activity against Capnocytophaga sputigena but not Escherichia coli. We conclude that (a) human neutrophil cathepsin G is an important antimicrobial system against the Capnocytophaga, (b) the bactericidal activity of cathepsin G against Capnocytophaga is oxygen independent, and (c) an intact enzyme active site is involved in the killing of C. sputigena but not E. coli. We suggest that human neutrophil cathepsin G is an important antimicrobial system against certain oral bacteria and that cathepsin G kills bacteria by two distinct mechanisms. (J. Clin. Invest.

Oral, gram-negative, facultative bacteria, including Actinobacillus actinomycetemcomitans, Eikenella corrodens, and Capnocytophaga spp. have been associated with destructive periodontal infection. Neutrophils play a critical role in defending the periodontium against destructive infection. Defensins are antimicrobial peptides that have been isolated in human, rabbit, guinea pig, and rat leukocytes that may constitute an important nonoxidative mechanism of killing. The purpose of this study was to examine the sensitivity of a battery of oral, gram-negative, facultative bacteria to the bactericidal effects of the isolated rabbit peptide NP-1. All species tested were killed by NP-1; however, there was strain-to-strain variation in sensitivity. The bactericidal effect was not dependent on net bacterial growth, although metabolic activity was evident as assessed by bacterial oxygen consumption. We conclude that bacteria are sensitive to the cidal mechanism involved in defensin-mediated bacterial killing and that the conditions of this assay system support the killing of bacteria by the defensin peptides.

The purpose of this study was to determine whether granule fractions of human neutrophils differentially kill Actinobacillus actinomycetemcomitans and Capnocytophaga spp. Granule extracts were subjected to gel filtration, and seven fractions (designated A through G) were obtained. Under aerobic conditions at pH 7.0, representative strains of A. actinomycetemcomitans were killed by fraction D and variably by fraction B. In contrast, the Capnocytophaga spp. were killed by fractions C, D, F, and G. Fractions A (containing lactoferrin and myeloperoxidase) and E (containing lysozyme) exerted little bactericidal activity under these conditions. Anaerobiosis had little effect on the bactericidal activity of fractions D and F but inhibited that of fractions B and C. Electrophoresis, zymography, determination of amino acid composition, and N-terminal sequence analysis revealed that fraction C contained elastase, proteinase 3, and azurocidin. Fraction D contained lysozyme, elastase, and cathepsin G. Subfractions of C and D containing elastase (subfraction C4), a mixture of elastase and azurocidin (subfraction C5), and cathepsin G (subfraction D9) were found to be bactericidal. The bactericidal effects of fraction D and subfraction D9 against A. actinomycetemcomitans was not inhibited by heat inactivation, phenylmethylsulfonyl fluoride, or N-benzyloxycarbonylglycylleucylphenylalanylchloromethyl ketone. We conclude that (i) A. actinomycetemcomitans and Capnocytophaga spp. were sensitive to the bactericidal effects of different neutrophil granule components, (ii) both were sensitive to the bactericidal effects of neutral serine proteases, and (iii) the killing ofA. actinomycetemcomitans by cathepsin G-containing fractions was independent of oxygen and neutral serine protease activity.

The purpose of this study was to compare the killing of Actinobacillus actinomycetemcomitans with that of Capnocytophaga spp. by purified cathepsin G and elastase in vitro. Both were sensitive to killing by purified cathepsin G, but only the Capnocytophaga spp. were killed by elastase. Killing by cathepsin G exhibited logarithmic kinetics, was enhanced slightly by alkaline pH, and was enhanced greatly under hypotonic conditions. Treatment of cathepsin G with diisopropyl fluorophosphate significantly reduced its bactericidal activity against Capnocytophaga spp. but not against Escherichia coli or A. actinomycetemcomitans. The bactericidal effects of cathepsin G against Capnocytophaga sputigena and A. actinomycetemcomitans were inhibited by a-1-antichymotrypsin, aL-1-antitrypsin, and a-2-macroglobulin but not by bovine serum albumin. We conclude that (i) cathepsin G kills Capnocytophaga spp. and A. actinomycetemcomitans, (ii) elastase kills Capnocytophaga spp., (iii) the bactericidal activity of cathepsin G is enzyme dependent against Capnocytophaga spp. and enzyme independent against A. actinomycetemcomitans, and (iv) natural plasma antiproteases may control both enzyme-dependent and enzyme-independent bactericidal activities of cathepsin G.

Azurocidin was purified in the presence of phenylmethylsulfonyl fluoride. Electrophoresis revealed at least seven species which exhibited N-terminal sequences consistent with azurocidin. Azurocidin exhibited no bactericidal activity against Capnocytophaga sputigena or other oral bacteria but synergized the bactericidal activity of enzymatically active elastase. Azurocidin also interacted synergistically with cathepsin G.

ActinobaciUus actinomycetemcomitans and Capnocytophaga spp. are gram-negative bacteria implicated in the etiology of periodontal disease (particularly in individuals with neutrophil defects) and life-threatening systemic infections. They are resistant to many antibiotics of microbial origin but are sensitive to the nonoxidative microbicidal action of neutrophils. These organisms are susceptible to the microbicidal effect of cathepsin G but are killed by two distinct mechanisms. The purpose of this study was to assess their sensitivity to the antibiotic effects of IIGGR and HPQYNQR, antimicrobial peptides derived from human neutrophil cathepsin G. The efficacies of the synthetic peptides IIGGR and HPQYNQR were tested by single-dose screening, dose-response, and kinetic assays against three representative strains (each) ofA. actinomycetemcomitans and Capnocytophaga spp. and one strain of Eikenella corrodens. Strains ofA. actinomycetemcomitans were sensitive to IIGGR and HPQYNQR at equal concentrations (wt/vol), whereas strains of Capnocytophaga and E. corrodens were more sensitive to IIGGR than to HPQYNQR. These differential antibiotic effects occurred over both time and dose ranges too narrow to be of therapeutic significance but are consistent with the premise that cathepsin G kills these oral bacteria by two distinct mechanisms.