Department of Science, University Roma Tre, Rome, Italy.
Department of Pharmacological and Biomolecular Sciences, University of Milano, Milan, Italy.
mSphere. 2024 Feb 28;9(2):e0067723. doi: 10.1128/msphere.00677-23. Epub 2024 Feb 2.
The outer membrane (OM) is an essential structure of Gram-negative bacteria that provides mechanical strength and protection from large and/or hydrophobic toxic molecules, including many antibiotics. The OM is composed of glycerophospholipids (GPLs) and lipopolysaccharide (LPS) in the inner and outer leaflets, respectively, and hosts integral β-barrel proteins and lipoproteins. While the systems responsible for translocation and insertion of LPS and OM proteins have been elucidated, the mechanism(s) mediating transport of GPLs from the inner membrane to the OM has remained elusive for decades. Very recently, studies performed in proposed a role in this process for AsmA-like proteins that are predicted to share structural features with eukaryotic lipid transporters. In this study, we provide the first systematic investigation of AsmA-like proteins in a bacterium other than , the opportunistic human pathogen . Bioinformatic analyses revealed that possesses seven AsmA-like proteins. Deletion of -like genes in many different combinations, coupled with conditional mutagenesis, revealed that four AsmA-like proteins are redundantly essential for growth and OM integrity in , including a novel AsmA-like protein (PA4735) that is not present in . Cells depleted of AsmA-like proteins showed severe defects in the OM permeability barrier that were partially rescued by lowering the synthesis or transport of LPS. Since fine balancing of GPL and LPS levels is crucial for OM integrity, this evidence supports the role of AsmA-like proteins in GPL transport toward the OM.
Given the importance of the outer membrane (OM) for viability and antibiotic resistance in Gram-negative bacteria, in the last decades, several studies have focused on the characterization of the systems involved in OM biogenesis, which have also been explored as targets for antibacterial drug development. However, the mechanism mediating translocation of glycerophospholipids (GPLs) to the OM remained unknown until recent studies provided evidence that AsmA-like proteins could be responsible for this process. Here, we demonstrate for the first time that AsmA-like proteins are essential and redundant for growth and OM integrity in a Gram-negative bacterium other than the model organism and demonstrate that the human pathogen has an additional essential AsmA-like protein that is not present in , thus expanding the range of AsmA-like proteins that play key functions in Gram-negative bacteria.
外膜（OM）是革兰氏阴性细菌的重要结构，为其提供机械强度和保护，使其免受大的和/或疏水性有毒分子的侵害，包括许多抗生素。OM 由内、外叶的甘油磷脂（GPLs）和脂多糖（LPS）组成，并含有整合β-桶蛋白和脂蛋白。虽然已经阐明了负责 LPS 和 OM 蛋白易位和插入的系统，但几十年来，介导 GPL 从内膜向 OM 转运的机制仍然难以捉摸。直到最近，在[细菌名称]中进行的研究提出了 AsmA 样蛋白在这个过程中的作用，这些蛋白被预测与真核脂质转运蛋白具有结构特征。在这项研究中，我们首次对除机会性病原体[细菌名称]之外的细菌中的 AsmA 样蛋白进行了系统研究。生物信息学分析表明，[细菌名称]含有七种 AsmA 样蛋白。在许多不同组合中删除 AsmA 样基因，并结合条件突变，表明四种 AsmA 样蛋白在[细菌名称]中对生长和 OM 完整性是冗余必需的，包括一种新型的 AsmA 样蛋白（PA4735），它不存在于[细菌名称]中。缺乏 AsmA 样蛋白的细胞在外膜通透性屏障方面表现出严重缺陷，这部分通过降低 LPS 的合成或运输得到挽救。由于 GPL 和 LPS 水平的精细平衡对 OM 完整性至关重要，因此这一证据支持 AsmA 样蛋白在 GPL 向 OM 转运中的作用。
鉴于外膜（OM）对革兰氏阴性细菌的生存能力和抗生素耐药性的重要性，在过去几十年中，已经有几项研究集中在参与 OM 生物发生的系统的表征上，这些系统也被探索为抗菌药物开发的靶点。然而，直到最近的研究提供了证据表明 AsmA 样蛋白可能负责这个过程，介导甘油磷脂（GPLs）向 OM 易位的机制仍然未知。在这里，我们首次证明了 AsmA 样蛋白在除模式生物[细菌名称]以外的革兰氏阴性细菌中对生长和 OM 完整性是必需和冗余的，并证明了人类病原体[细菌名称]有一种额外的必需的 AsmA 样蛋白，它不存在于[细菌名称]中，从而扩大了在革兰氏阴性细菌中发挥关键功能的 AsmA 样蛋白的范围。
