Interaction of adrenomedullin and calcitonin gene-related peptide with the periodontal pathogen Porphyromonas gingivalis.

The nature of the interaction between Porphyromonas gingivalis and the multifunctional peptides adrenomedullin and calcitonin gene-related peptide (CGRP) was investigated. Growth of P. gingivalis was not inhibited in the presence of either of these peptides [minimal inhibitory concentration (MIC)>250 microg mL(-1)]. The ability of the arginine- and lysine-specific proteases from P. gingivalis to breakdown these peptides was investigated. Adrenomedullin and CGRP were incubated with culture supernatants from wild-type and protease gene knockout strains. No significant effect on antimicrobial activity against the indicator organism Escherichia coli BUE55 was found (MIC=6.25 microg mL(-1) in all cases). The role of anionic components on the surface of P. gingivalis, which may alter binding of these cationic peptides, was also investigated in relation to adrenomedullin. Growth of gene knockout strains lacking surface polysaccharide and capsule components was not inhibited (MIC>250 microg mL(-1)). It is suggested that a lack of sensitivity to adrenomedullin and CGRP may enable P. gingivalis to persist in the oral cavity and cause disease.

Antimicrobial activity does not predict cytokine response to adrenomedullin or its shortened derivatives.

The aim of this study was to investigate cytokine release from oral keratinocytes and fibroblasts in response to AM and shortened derivatives previously characterised in terms of their antimicrobial activities. Cells were incubated with AM or its fragments (residues 1-12, 1-21, 13-52, 16-21, 16-52, 22-52, 26-52, and 34-52), and culture supernatants collected after 1, 2, 4, 8, and 24 hours. A time-dependant increase in production of interleukin1-alpha and interleukin 1-beta from keratinocytes in response to all peptides was demonstrated. However, exposure to fragments compared to whole AM resulted in reduced production of these cytokines (60% mean reduction at 24 hours, P<.001). No consistent differences were shown between the cytokine response elicited by antimicrobial and nonantimicrobial fragments. The production of interleukin-6 and interleukin-8 did not change significantly with time or peptide used. Fibroblast cells were relatively unresponsive to all treatments. This study demonstrates that antimicrobial activity does not predict cytokine response to adrenomedullin or its shortened derivatives.

Mechanisms of adrenomedullin antimicrobial action.

The mechanism of antimicrobial action of the multifunctional peptide adrenomedullin (AM) against Escherichia coli and Staphylococcus aureus was investigated. AM (52 residues) and AM fragments (1-12, 1-21, 13-52, 16-21, 16-52, 22-52, 26-52 and 34-52 residues) were tested for activity. Carboxy-terminal fragments were shown to be up to 250-fold more active than the parent molecule. Minimum inhibitory concentration values of the most active fragments (13-52 and 16-52) and the parent molecule were 4.9 x 10(-2) and 12.5 microg/ml, respectively, with E. coli. Ultrastructural analyses of AM treated cells demonstrated marked cell wall disruption with E. coli within 0.5 h. Abnormal septum formation with no apparent peripheral cell wall disruption was observed with S. aureus after 2 h. Outer membrane permeabilisation assays with E. coli confirmed that the C-terminal fragments were significantly (P < 0.05) more active. It is suggested that postsecretory processing may generate multiple AM congeners that have enhanced antimicrobial activities against a range of potential targets.