title: Microbe sample from Porphyromonas gingivalis
organism: Porphyromonas gingivalis
description: Porphyromonas gingivalis is a keystone pathogen for periodontal disease. The bacteria are black-pigmented and require heme for growth. P. gingivalis exhibit resistance to many antimicrobial peptides, which contributes to their success in the oral cavity. P. gingivalis W50 was resistant to the antimicrobial peptide LGL13K but susceptible to the all-D-amino acid stereoisomer, DGL13K. Upon prolonged exposure to DGL13K, a novel non-pigmented mutant was isolated. Exposure to the L-isomer, LGL13K, did not produce a non-pigmented mutant. The goal of this study was to characterize the genomic and cellular changes that led to the non-pigmented phenotype upon treatment with DGL13K. The non-pigmented mutant (hagA23/167) showed a low minimum inhibitory concentration and two-fold extended minimum duration for killing by DGL13K, consistent with tolerance to this peptide. The DGL13K tolerant bacteria exhibited synonymous mutations in the hagA gene. The mutations did not prevent mRNA expression but were predicted to alter mRNA structure. The non-pigmented bacteria were deficient in hemagglutination and hemoglobin binding, suggesting that the HagA protein was not expressed. This was supported by whole cell ELISA and gingipain activity assays, which suggested the absence of HagA but not of two closely related gingipains. In vivo virulence was similar for wild-type and non-pigmented hagA23/167 in the Galleria mellonella model. The results suggest that, unlike LGL13K, DGL13K can defeat multiple bacterial resistance mechanisms but bacteria can gain tolerance to DGL13K through mutations in the hagA gene.