ABSTRACT
Aggregatibacter (Actinobacillus) actinomycetemcomitans P(7-20) strain isolated from a periodontally diseased patient has produced a bacteriocin (named as actinomycetemcomitin) that is active against Peptostreptococcus anaerobius ATCC 27337. Actinomycetemcomitin was produced during exponential and stationary growth phases, and its amount decreased until it disappeared during the decline growth phase. It was purified by ammonium sulphate precipitation (30-60% saturation), and further by FPLC (mono-Q ionic exchange and Phenyl Superose hydrophobic interaction) and HPLC (C-18 reversed-phase). This bacteriocin loses its activity after incubation at a pH below 7.0 or above 8.0, following heating for 30 min at 45 degrees C, and after treatment with proteolytic enzymes such as trypsin, alpha-chymotrypsin, and papain. Actinomycetemcomitin has a molecular mass of 20.3 KDa and it represents a new bacteriocin from A. actinomycetemcomitans.
Subject(s)
Aggregatibacter actinomycetemcomitans/metabolism , Anti-Bacterial Agents/biosynthesis , Bacteriocins/biosynthesis , Aggregatibacter actinomycetemcomitans/isolation & purification , Amino Acid Sequence , Ammonium Sulfate , Anti-Bacterial Agents/pharmacology , Bacteriocins/chemistry , Bacteriocins/isolation & purification , Bacteriocins/pharmacology , Chromatography, High Pressure Liquid , Chromatography, Liquid , Fractional Precipitation , Humans , Hydrogen-Ion Concentration , Molecular Sequence Data , Molecular Weight , Peptide Hydrolases/metabolism , Peptostreptococcus/drug effects , Periodontitis/microbiology , Sequence Alignment , TemperatureABSTRACT
We evaluated the influence of abiotic factors on antagonistic activity of Actinobacillus actinomycetemcomitans strains isolated from periodontal pockets and two reference strains (ATCC 29523 and FDC Y4). Antagonistic assays were performed by the overlayer method on tryptic soy agar (TSA), brain heart infusion agar, thioglycollate agar and brucella agar, added with yeast extract and supplemented (S) or not with L-cystine and sodium bicarbonate. Iso-, auto-, and heteroantagonism against a wide range of indicator strains were assayed. The influence of incubation atmosphere (anaerobic chamber, anaerobic and candle jars) and pH (5.0 to 11.0) was also evaluated. Autoantagonism was not observed. TSA-S was shown to be the most adequate medium for antagonistic activity expression. The widest spectrum of heteroantagonistic activity was also observed on TSA-S. The incubation atmosphere affected only the isoantagonistic activity expression. Only at pH 8.0 did A. actinomycetemcomitans express bacteriocinogenic activity. The lack of standardized methodology to detect antagonistic activity can lead to discrepant results and can make data difficult to be compared. This study provides information on abiotic factors that influence bacteriocinogenic activity expression and suggests adequate culture conditions for testing A. actinomycetemcomitans bacteriocin production, contributing to the establishment of a reproducible and reliable methodology.
Subject(s)
Actinobacillus Infections/microbiology , Aggregatibacter actinomycetemcomitans/drug effects , Aggregatibacter actinomycetemcomitans/growth & development , Antibiosis , Bacteriocins/pharmacology , Periodontal Pocket/microbiology , Aggregatibacter actinomycetemcomitans/metabolism , Anaerobiosis , Bacteriocins/biosynthesis , Bacteriological Techniques/standards , Culture Media/chemistry , Humans , Hydrogen-Ion Concentration , Microbial Sensitivity TestsABSTRACT
Actinobacillus actinomycetemcomitans is a clinically relevant periodontopathogenic Gram-negative coccobacillus that produces a leukotoxin of the RTX cytolysin family. In this study, we evaluated the leukotoxic activity of A. actinomycetemcomitans strains isolated from human and marmosets by Trypan blue exclusion and by the chemiluminescence assays. Among eight A. actinomycetemcomitans human strains studied, two (P2.17 and P8.12) were classified as high leukotoxin producers and among eight marmoset strains, one (M22.11) showed high leukotoxin production, as determined by Trypan blue exclusion assay. The reference strains ATCC 29523 and FDC Y4 respectively behaved like moderate and low producers. The chemiluminescence assay was used to evaluate the leukotoxic activity of M22.11 and P2.17 strains submitted to different growth conditions. Leukotoxic activity was detected on cells at the logarithmic phase and was similar under anaerobic and microaerophilic growth conditions. It was greatly reduced when cells were grown at glucose concentrations lower or higher than 0.75per cent (0.25per cent and 1.5per cent) in thioglycolate medium. Leukotoxin production mainly by the M22.11 strain was low in BHI broth, whereas production in TSB medium showed a similar level as in thioglycolate broth medium. Sodium bicarbonate at 10 mM did not affect leukotoxin production.
