Department of Diagnostic and Biological Sciences, University of Minnesota School of Dentistry, Minneapolis, Minnesota, United States of America.
University of Minnesota Informatics Institute, University of Minnesota, Minneapolis, Minnesota, United States of America.
PLoS One. 2024 Oct 24;19(10):e0312200. doi: 10.1371/journal.pone.0312200. eCollection 2024.
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 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 enzyme-linked immunosorbent assay 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 bacteria 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.
牙龈卟啉单胞菌是牙周病的关键病原体。该细菌呈黑色色素沉着，生长需要血红素。牙龈卟啉单胞菌对许多抗菌肽具有抗性，这有助于它们在口腔中成功生存。牙龈卟啉单胞菌 W50 对抗菌肽 LGL13K 具有抗性，但对全 D-氨基酸立体异构体 DGL13K 敏感。经过长时间暴露于 DGL13K，分离出一种新型非色素沉着突变体。暴露于 L-异构体 LGL13K 不会产生非色素沉着突变体。本研究的目的是描述在 DGL13K 处理后导致非色素沉着表型的基因组和细胞变化。非色素沉着突变体显示出较低的最小抑菌浓度和两倍延长的最小杀菌时间，表明对该肽具有耐受性。DGL13K 耐受细菌在 hagA 基因中显示同义突变。这些突变并未阻止 mRNA 表达，但预计会改变 mRNA 结构。非色素沉着细菌在血凝和血红蛋白结合方面存在缺陷，表明 HagA 蛋白未表达。这得到了全细胞酶联免疫吸附试验和牙龈蛋白酶活性试验的支持，这些试验表明 HagA 缺失，但两种密切相关的牙龈蛋白酶未缺失。在 Galleria mellonella 模型中，野生型和非色素沉着细菌的体内毒力相似。结果表明，与 LGL13K 不同，DGL13K 可以克服多种细菌耐药机制，但细菌可以通过 hagA 基因突变获得对 DGL13K 的耐受性。
