Monitoring the uptake of protein-derived peptides by Porphyromonas gingivalis with fluorophore-labeled substrates.
The aim of this study was to develop a simple method to quantify peptide uptake by the periodontopathogenic bacterium Porphyromonas gingivalis. After incubation of bacterial cells with self-quenched fluorescent bovine serum albumin (DQ Green BSA), the fluorescence measured in the supernatant of the assay mixture indicated the degree of protein degradation, whereas the fluorescence associated with the lysate of washed cells indicated the amount of BSA-derived fragments incorporated by the bacteria. The optimal conditions for uptake of fluorophore-labeled albumin fragments were found to be mid-log grown cells, 150 m M NaCl in phosphate buffer, pH 7, 37 degrees C, and anaerobiosis. Among the protease inhibitors tested, 4-(2-aminoethyl)-benzene sulfonyl-fluoride hydrochloride (AEBSF) and cathepsin B inhibitor II caused a significant inhibition of the uptake of BSA-derived peptides. This assay was applicable for other commercially available fluorescent substrates. This simple method may be useful to investigate protein processing in proteolytic bacteria and for studying the effects of environmental parameters or cell treatments on the peptide uptake.
Yoshioka
Masami
M
Groupe de Recherche en Ecologie Buccale, Faculté de Médecine Dentaire, Université Laval, Québec, Canada, G1K 7P4.
Grenier
Daniel
D
Mayrand
Denis
D
D003470:Culture Media; D005453:Fluorescence; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D009994:Osmolar Concentration; D010455:Peptides / Q000378:metabolism*; D016966:Porphyromonas gingivalis / Q000254:growth & development / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D012710:Serum Albumin, Bovine / Q000378:metabolism*
D003470:Culture Media; D005456:Fluorescent Dyes; D010455:Peptides; D011480:Protease Inhibitors; D012710:Serum Albumin, Bovine