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ItemDivalent metal cations increase the activity of the antimicrobial peptide kappacinDashper, SG ; O'Brien-Simpson, NM ; Cross, KJ ; Paolini, RA ; Hoffmann, B ; Catmull, DV ; Malkoski, M ; Reynolds, EC (AMER SOC MICROBIOLOGY, 2005-06-01)Kappacin, nonglycosylated kappa-casein(106-169), is a novel antimicrobial peptide produced from kappa-casein found in bovine milk. There are two major genetic forms of kappacin, A and B, and using synthetic peptides corresponding to the active region, kappa-casein(138-158), of these forms, we have shown that the Asp148 to Ala148 substitution is responsible for the lesser antibacterial activity of kappa-casein-B(106-169). Kappacin was shown to have membranolytic action at concentrations above 30 microM at acidic pH when tested against artificial liposomes. There was little membranolytic activity at neutral pH, which is consistent with the lack of antibacterial activity of kappacin against Streptococcus mutans at this pH. Kappacin specifically bound two zinc or calcium ions per mol, and this binding enhanced antibacterial activity at neutral pH. Nuclear magnetic resonance analysis indicated that a kappa-casein-A(138-158) synthetic peptide undergoes a conformational change in the presence of the membrane solvent trifluoroethanol and excess divalent metal ions. This change in conformation is presumably responsible for the increase in antibacterial activity of kappacin detected in the presence of excess zinc or calcium ions at neutral pH. When tested against the oral bacterial pathogen S. mutans cultured as a biofilm in a constant-depth film fermentor, a preparation of 10 g/liter kappacin and 20 mM ZnCl2 reduced bacterial viability by 3 log10 and suppressed recovery of viability. In contrast 20 mM ZnCl2 alone reduced bacterial viability by approximately 1 log10 followed by rapid recovery. In conclusion, kappacin has a membranolytic, antibacterial effect that is enhanced by the presence of divalent cations.
ItemA novel Porphyromonas gingivalis FeoB plays a role in manganese accumulationDashper, SG ; Butler, CA ; Lissel, JP ; Paolini, RA ; Hoffmann, B ; Veith, PD ; O'Brien-Simpson, NM ; Snelgrove, SL ; Tsiros, JT ; Reynolds, EC (AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2005-07-29)FeoB is an atypical transporter that has been shown to exclusively mediate ferrous ion transport in some bacteria. Unusually the genome of the periodontal pathogen Porphyromonas gingivalis has two genes (feoB1 and feoB2) encoding FeoB homologs, both of which are expressed in bicistronic operons. Kinetic analysis of ferrous ion transport by P. gingivalis W50 revealed the presence of a single, high affinity system with a K(t) of 0.31 microM. FeoB1 was found to be solely responsible for this transport as energized cells of the isogenic FeoB1 mutant (W50FB1) did not transport radiolabeled iron, while the isogenic FeoB2 mutant (W50FB2) transported radiolabeled iron at a rate similar to wild type. This was reflected in the iron content of W50FB1 grown in iron excess conditions which was approximately half that of the wild type and W50FB2. The W50FB1 mutant had increased sensitivity to both oxygen and hydrogen peroxide and was avirulent in an animal model of infection whereas W50FB2 exhibited the same virulence as the wild type. Analysis of manganous ion uptake using inductively coupled plasma-mass spectrometry revealed a greater than 3-fold decrease in intracellular manganese accumulation in W50FB2 which was also unable to grow in manganese-limited media. The protein co-expressed with FeoB2 appears to be a novel FeoA-MntR fusion protein that exhibits homology to a manganese-responsive, DNA-binding metalloregulatory protein. These results indicate that FeoB2 is not involved in iron transport but plays a novel role in manganese transport.