AIM To elucidate the genetic basis for the pronounced resistance that the oral pathogen, Porphyromonas gingivalis (P. gingivalis), exhibits towards the cationic antimicrobial peptide, polymyxin B. METHODOLOGY A genetic screen of P. gingivalis clones generated by a Tn4400'-based random insertion mutagenesis strategy was performed to identify bacteria harboring novel genetic mutations that render P. gingivalis susceptible to killing by the cationic antimicrobial peptide, polymyxin B (PMB, 50 microg x mL(-1)). RESULTS P. gingivalis (ATCC 33277) is unusually resistant to the cationic antimicrobial peptide, PMB at relatively high concentrations (200 microg x mL(-1)). Approximately 2,700 independent Tn4400'-derived mutants of P. gingivalis were examined for increased sensitivity to PMB killing at a relatively low dose (50 microg x mL(-1)). A single PMB-sensitive mutant was obtained in this phenotypic screen. We determined that the Tn4400' transposon was integrated into the gene encoding the lipid A 4'-phosphatase, PGN_0524, demonstrating that this insertion event was responsible for its increased susceptibility of this clone to PMB-dependent killing. The resulting mutant strain, designated 0524-Tn4400', was highly sensitive to PMB killing relative to wild-type P. gingivalis, and exhibited the same sensitivity as the previously characterized strain, 0524KO, which bears a genetically engineered deletion in the PGN_0524 locus. Positive ion mass spectrometric structural (MALDI-TOF MS) analyses revealed that lipid A isolates from 0524-Tn4400' and 0524KO strains displayed strikingly similar MALDI-TOF MS spectra that were substantially different from the wildtype P. gingivalis lipid A spectrum. Finally, intact 0524-Tn4400' and 0524KO mutant bacteria, as well as their corresponding LPS isolates, were significantly more potent in stimulating Toll-like receptor 4 (TLR4)-dependent E-selectin expression in human endothelial cells relative to intact wild-type P. gingivalis or its corresponding LPS isolate. CONCLUSION The combined molecular evidence provided in this report suggests that PGN_0524, a lipid A 4'-phosphatase, is the sole genetic element conferring the ability of the periodontopathogen, P. gingivalis, to evade the killing activity of cationic antimicrobial peptides, such as PMB. These data strongly implicate PGN_0524 as a critical virulence factor for the ability of P. gingivalis to evade front-line host innate defenses that are dependent upon cationic antimicrobial peptide activity and TLR 4 sensing.

中文翻译：

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牙龈卟啉单胞菌对多粘菌素 B 的抗性由脂质 A 4'-磷酸酶 PGN_0524 决定。

目的阐明口腔病原体牙龈卟啉单胞菌（P. gingivalis）对阳离子抗菌肽多粘菌素 B 表现出显着抗性的遗传基础。执行插入诱变策略以识别含有新基因突变的细菌，这些突变使 P. gingivalis 容易被阳离子抗菌肽、多粘菌素 B（PMB，50 microg x mL(-1)）杀死。结果 牙龈卟啉单胞菌 (ATCC 33277) 对阳离子抗菌肽、PMB 的浓度相对较高 (200 microg x mL(-1)) 具有异常抗性。大约 2,700 独立 Tn4400' 派生的 P.gingivalis 突变体进行了检查，以提高对 PMB 杀死在相对低剂量 (50 microg x mL(-1)) 的敏感性。在该表型筛选中获得了单个 PMB 敏感突变体。我们确定 Tn4400' 转座子被整合到编码脂质 A 4'-磷酸酶 PGN_0524 的基因中，表明该插入事件是该克隆对 PMB 依赖性杀伤的敏感性增加的原因。由此产生的突变菌株，命名为 0524-Tn4400'，相对于野生型牙龈卟啉单胞菌对 PMB 杀伤高度敏感，并表现出与先前表征的菌株 0524KO 相同的敏感性，0524KO 在 PGN_0524 基因座中带有基因工程缺失。正离子质谱结构 (MALDI-TOF MS) 分析显示，从 0524-Tn4400' 和 0524KO 菌株中分离出的脂质 A 显示出惊人相似的 MALDI-TOF MS 谱，与野生型 P. 牙龈脂质A谱。最后，与完整的野生型相比，完整的 0524-Tn4400' 和 0524KO 突变细菌及其相应的 LPS 分离株在刺激人内皮细胞中 Toll 样受体 4 (TLR4) 依赖性 E-选择素表达方面显着更有效P. gingivalis 或其相应的 LPS 分离株。结论 本报告中提供的综合分子证据表明，PGN_0524（一种脂质 A 4'-磷酸酶）是赋予牙周病原菌 P. gingivalis 逃避阳离子抗菌肽（如 PMB）杀伤活性能力的唯一遗传元件. 这些数据强烈暗示 PGN_0524 是 P. 能力的关键毒力因子。