Controlled redox and hemin utilization essential for Porphyromonas gingivalis persistence

Abstract

OBJECTIVES: Antibiotic-tolerant bacterial persisters critically account for common long-lasting complex infections and inflammation. Our recent in vitro study shows for the first time the presence of metronidazole-tolerant P. gingivalis persisters and hemin as an important modifier of these noxious persisters. This study further investigated the underlying survival mechanisms of P. gingivalis persisters by shotgun proteomics. METHODS: P. gingivalis ATCC 33277 was cultured anaerobically in broth containing 10μg/ml of hemin, and treated with 100μg/ml of metronidazole for 6 h. Subsequently, cellular proteins were extracted from the P. gingivalis persister fractions and controls followed by in solution digestion with trypsin. The resulting peptides were loaded on an LTQ-Orbitrap system and analyzed by LC-MS/MS. The proteomic data were subjected to statistical and bioinformatical analysis. RESULTS: Over 300 proteins were identified, and their expression was to different extent altered in the metronidazole-tolerant P. gingivalis persisters, with reference to the controls. Notably, the persisters exhibited significantly decreased protein expression of pyruvate:ferredoxin oxidoreductase (PFOR, e.g. PGN_1530 and PGN_1753) that is crucial for the activation of metronidazole. Additional proteins responsible for redox regulation (e.g. PGN_0033 and PGN_0302) were differentially expressed in the persisters. Interestingly, the hemin/iron uptake-associated proteins such as PGN_0659 and PGN_0728 were markedly downregulated, whereas a key iron-storage protein (PGN_0604) was significantly upregulated, which could result in reduced biosynthesis of redox-active proteins and impaired activation of metronidazole. CONCLUSIONS: This pioneering study demonstrates that regulation of cellular redox state may be essential for the tolerance of P. gingivalis persisters to metronidazole, and repression of hemin/iron utilization could contribute to the survival of these persisters.