The Mla system of the diderm Firmicute Veillonella parvula reveals an ancestral envelope-spanning core for phospholipid trafficking

Despite extensive characterisation of envelope biogenesis systems in diderm (Gram-negative) bacteria, glycerophospholipid (GPL) trafficking remains poorly understood, and has only been studied in a handful of model species. In classical model diderms (Proteobacteria), the Mla system has been proposed to facilitate retrograde GPL trafficking and is composed of six proteins, MlaA-F. GPLs are extracted from the outer leaflet of the outer membrane (OM) by the lipoprotein MlaA which associates with porin trimers, then shipped through the periplasmic space by the periplasmic chaperone MlaC, which delivers GPLs to the inner membrane (IM) complex formed by MlaBDEF. Here, we investigate GPL trafficking in Veillonella parvula, a diderm member of the Firmicutes which encodes an Mla system devoid of MlaA and MlaC. V. parvula ∆mla mutants display phenotypes characteristic of disrupted lipid asymmetry such as hypervesiculation and detergent hypersensitivity. Lipid content analysis from outer membrane vesicles (OMVs) reveals an enrichment for the major lipid component phosphatidylethanolamine (PE). Interestingly, suppressor analysis identifies mutations in tamB that rescue detergent hypersensitivity of ∆mla strains, supporting the involvement of these two systems in antagonistic GPL trafficking functions across diverse bacterial lineages. A combination of structural modeling and subcellular localisation assays shows that MlaDVp is longer than in classical diderm models and forms a transenvelope bridge, encoding both an IM-localised MCE domain and an OM ß-barrel. These results strongly suggest that V. parvula possesses a minimal Mla system for GPL trafficking replacing the need for chaperones and OM lipoproteins by directly connecting the two membranes. Finally, phylogenomic analysis indicates that this MlaEFD self-contained architecture is widely distributed in diderm bacteria and most likely represents the ancestral functional core of the Mla system, which subsequently increased in complexity in Proteobacteria and closely related phyla following the emergence of MlaABC. Our work broadens the diversity of current models of GPL trafficking in diderm bacteria, challenging the paradigm set by classical models and shedding light on the evolution of a crucial system in the biogenesis and maintenance of the bacterial outer membrane.

尽管对双膜（diderm，即革兰氏阴性）细菌的包膜生物发生系统已有广泛表征，但甘油磷脂（glycerophospholipid, GPL）的运输过程仍知之甚少，且仅在少数模式物种中得到研究。在经典双膜模式生物——变形菌门（Proteobacteria）中，Mla系统被提出可介导逆行甘油磷脂运输，该系统由MlaA至F共六种蛋白组成。甘油磷脂由与孔蛋白三聚体结合的脂蛋白MlaA从外膜（outer membrane, OM）的外小叶中提取，随后通过周质伴侣蛋白MlaC在周质空间中转运，并将甘油磷脂递交给由MlaBDEF组成的内膜（inner membrane, IM）复合物。本研究对韦荣氏球菌（Veillonella parvula）的甘油磷脂运输过程展开探究，该菌属于厚壁菌门（Firmicutes）的双膜细菌，其编码的Mla系统缺失MlaA与MlaC。V. parvula的Δmla突变体表现出脂质不对称破坏的典型表型，包括过度囊泡化与去污剂超敏性。对外膜囊泡（outer membrane vesicles, OMVs）的脂质含量分析显示，其主要脂质组分磷脂酰乙醇胺（phosphatidylethanolamine, PE）出现富集。值得注意的是，抑制子分析发现tamB基因的突变可挽救Δmla菌株的去污剂超敏性，这支持两类系统在不同细菌类群中发挥拮抗的甘油磷脂运输功能。结合结构建模与亚细胞定位实验的结果显示，V. parvula来源的MlaD（MlaDVp）比经典双膜模型中的同源蛋白更长，可形成跨包膜桥，同时包含内膜定位的MCE结构域与外膜β桶结构域。上述结果强烈表明，V. parvula拥有一套简化版的Mla系统用于甘油磷脂运输，无需依赖伴侣蛋白与外膜脂蛋白，而是直接连接两层细胞膜。最后，系统发育基因组学分析显示，这种由MlaEFD组成的自主架构广泛分布于双膜细菌中，极有可能代表了Mla系统的祖先功能核心；在MlaABC元件出现后，变形菌门及相关类群的Mla系统复杂度随之提升。本研究拓宽了当前双膜细菌甘油磷脂运输模型的多样性，挑战了经典模型确立的研究范式，并为细菌外膜生物发生与维持这一关键系统的演化提供了新的认识。