Gill Cameron P, Phan Christopher, Platt Vivien, Worrell Danielle, Andl Thomas, Roy Hervé
Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, Florida, USA.
Microbiol Spectr. 2023 Sep 28;11(5):e0142923. doi: 10.1128/spectrum.01429-23.
Lysyl-diacylglycerol (Lys-DAG) was identified three decades ago in , but the biosynthetic pathway and function of this aminoacylated lipid have since remained uncharacterized. Combining genetic methods, mass spectrometry, and biochemical approaches, we show that the multiple peptide resistance factor (MprF) homolog LysX from and two mycobacterial species is responsible for Lys-DAG synthesis. LysX is conserved in most Actinobacteria and was previously implicated in the synthesis of another modified lipid, lysyl-phosphatidylglycerol (Lys-PG), in . Although we detected low levels of Lys-PG in the membrane of , our data suggest that Lys-PG is not directly synthesized by LysX and may require an additional downstream pathway, which is as yet undefined. Our results show that LysX in is a major factor of resistance against a variety of positively charged antibacterial agents, including cationic antimicrobial peptides (e.g., human peptide LL-37 and polymyxin B) and aminoglycosides (e.g., gentamycin and apramycin). Deletion of caused an increase in cellular membrane permeability without dissipation of the membrane potential, suggesting that loss of the protein does not result in mechanical damage to the cell membrane. Furthermore, -deficient cells exhibited an attenuated virulence phenotype in a infection model, supporting a role for LysX during infection. Altogether, Lys-DAG represents a novel molecular determinant for antimicrobial resistance and virulence that may be widespread in Actinobacteria and points to a richer landscape than previously realized of lipid components contributing to overall membrane physiology in this important bacterial phylum. IMPORTANCE In the past two decades, tRNA-dependent modification of membrane phosphatidylglycerol has been implicated in altering the biochemical properties of the cell surface, thereby enhancing the antimicrobial resistance and virulence of various bacterial pathogens. Here, we show that in several Actinobacteria, the multifunctional protein LysX attaches lysine to diacylglycerol instead of phosphatidylglycerol. We found that lysyl-diacylglycerol (Lys-DAG) confers high levels of resistance against various cationic antimicrobial peptides and aminoglycosides and also enhances virulence. Our data show that Lys-DAG is a lipid commonly found in important actinobacterial pathogens, including and species.
赖氨酰二酰甘油（Lys-DAG）在三十年前就已被发现，但此后这种氨酰化脂质的生物合成途径和功能一直未得到明确。通过结合遗传方法、质谱分析和生化方法，我们发现来自[具体物种1]和两种分枝杆菌的多肽抗性因子（MprF）同源物LysX负责Lys-DAG的合成。LysX在大多数放线菌中保守，此前在[具体物种2]中被认为参与另一种修饰脂质赖氨酰磷脂酰甘油（Lys-PG）的合成。尽管我们在[具体物种3]的膜中检测到低水平的Lys-PG，但我们的数据表明Lys-PG不是由LysX直接合成的，可能需要一条额外的下游途径，目前尚不清楚。我们的结果表明，[具体物种3]中的LysX是抵抗多种带正电荷抗菌剂的主要因素，包括阳离子抗菌肽（如人肽LL-37和多粘菌素B）和氨基糖苷类（如庆大霉素和阿普拉霉素）。[具体物种3]的缺失导致细胞膜通透性增加，而膜电位没有消散，这表明该蛋白的缺失不会导致细胞膜的机械损伤。此外，[具体物种3]缺陷型细胞在[具体感染模型]中表现出毒力减弱的表型，支持LysX在感染过程中的作用。总之，Lys-DAG代表了一种新的抗微生物抗性和毒力分子决定因素，可能在放线菌中广泛存在，并且表明在这个重要细菌门中，对整体膜生理学有贡献的脂质成分比以前认识的更为丰富。重要性在过去二十年中，膜磷脂酰甘油的tRNA依赖性修饰被认为与改变细胞表面的生化特性有关，从而增强了各种细菌病原体的抗微生物抗性和毒力。在这里，我们表明在几种放线菌中，多功能蛋白LysX将赖氨酸连接到二酰甘油而不是磷脂酰甘油上。我们发现赖氨酰二酰甘油（Lys-DAG）赋予对各种阳离子抗菌肽和氨基糖苷类的高水平抗性，并且还增强毒力。我们的数据表明Lys-DAG是在重要的放线菌病原体中常见的脂质，包括[具体物种1]和[具体物种2]。
