Controlled cellular redox, repressive hemin utilization and adaptive stress responses are crucial to metronidazole tolerance of Porphyromonas gingivalis persisters

Abstract

Antimicrobial-tolerant microbial persisters critically account for various infections and inflammation. This study identified the characteristics of Porphyromonas gingivalis persisters, and explored their underlying survival mechanisms through proteomic profiling. Porphyromonas gingivalis cultured with different concentrations of hemin was treated with 100μg/ml of metronidazole (MTZ). The viability of P.gingivalis persisters was determined by colony-forming unit assay and LIVE/DEAD staining. The proteomic signature of P.gingivalis persister fractions was examined using LC-MS/MS and bioinformatic analysis. A small fraction of P.gingivalis persisters survived from lethal MTZ treatment without heritability. At late exponential phase, the frequency of these persisters significantly increased when incubated with 1μg/ml of hemin compared to 10μg/ml. Higher levels of P.gingivalis persisters formed at stationary phase than the late exponential phase. High-throughput proteomic analysis showed that the persisters markedly downregulated multiple proteins involved in electron transfer and heme/iron utilization essential for redox regulation and MTZ activation. Moreover, the persisters enabled to shut down major cellular activities (e.g. translation) and overexpress stress proteins. The presence and survival of metronidazole-tolerant P.gingivalis persisters may be dominated by regulation of cellular redox state and enhanced via repression of heme/iron utilization, dormancy and stress responses.