The O-Antigen Flippase Wzk Can Substitute for MurJ in Peptidoglycan Synthesis in <i>Helicobacter pylori</i> and <i>Escherichia coli</i>

The peptidoglycan (PG) cell wall is an essential component of the cell envelope of most bacteria. Biogenesis of PG involves a lipid-linked disaccharide-pentapeptide intermediate called lipid II, which must be translocated across the cytoplasmic membrane after it is synthesized in the inner leaflet of this bilayer. Accordingly, it has been demonstrated that MurJ, the proposed lipid II flippase in Escherichia coli, is required for PG biogenesis, and thereby viability. In contrast, MurJ is not essential in Bacillus subtilis because this bacterium produces AmJ, an unrelated protein that is functionally redundant with MurJ. In this study, we investigated why MurJ is not essential in the prominent gastric pathogen, Helicobacter pylori. We found that in this bacterium, Wzk, the ABC (ATP-binding cassette) transporter that flips the lipid-linked O- or Lewis- antigen precursors across the inner membrane, is redundant with MurJ for cell viability. Heterologous expression of wzk in E. coli also suppresses the lethality caused by the loss of murJ. Furthermore, we show that this cross-species complementation is abolished when Wzk is inactivated by mutations that target a domain predicted to be required for ATPase activity. Our results suggest that Wzk can flip lipid II, implying that Wzk is the flippase with the most relaxed specificity for lipid-linked saccharides ever identified.肽聚糖（peptidoglycan, PG）细胞壁是绝大多数细菌细胞包膜的核心必需组分。肽聚糖的生物合成依赖于一种名为脂质II（lipid II）的脂质连接型二糖-五肽中间体，该中间体在细胞质膜双分子层的内膜小叶内合成后，必须跨膜转运至膜外侧。已有研究证实，大肠杆菌（Escherichia coli）中被认定为脂质II翻转酶的MurJ蛋白，是肽聚糖生物合成以及细菌存活所不可或缺的。与之形成对比的是，MurJ在枯草芽孢杆菌（Bacillus subtilis）中并非必需基因，这是因为该菌可表达AmJ蛋白——一种与MurJ功能冗余的非同源蛋白。本研究针对幽门螺杆菌（Helicobacter pylori，一种重要的胃部致病原）中MurJ非必需的原因展开探究。我们发现，在该致病菌中，负责将脂质连接的O型或路易斯（Lewis）抗原前体跨内膜翻转的ATP结合盒（ATP-binding cassette, ABC）转运蛋白Wzk，在维持细胞生存方面与MurJ存在功能冗余。在大肠杆菌中异源表达wzk基因，同样能够抑制murJ基因缺失所引发的致死效应。进一步实验证实，当Wzk蛋白被针对其预测的ATP酶活性必需结构域的突变灭活后，这种跨物种互补现象便会完全消失。本研究结果表明，Wzk具备翻转脂质II的能力，这意味着Wzk是目前已发现的对脂质连接糖类底物特异性最为宽松的翻转酶。