Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
Vanderbilt Institute for Infection Immunology and Inflammation, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
J Bacteriol. 2023 Apr 25;205(4):e0005223. doi: 10.1128/jb.00052-23. Epub 2023 Mar 27.
The localization of lipoprotein (Lol) system is used by Gram-negative bacteria to export lipoproteins to the outer membrane. Lol proteins and models of how Lol transfers lipoproteins from the inner to the outer membrane have been extensively characterized in the model organism Escherichia coli, but in numerous bacterial species, lipoprotein synthesis and export pathways deviate from the E. coli paradigm. For example, in the human gastric bacterium Helicobacter pylori, a homolog of the E. coli outer membrane component LolB is not found, E. coli LolC and LolE correspond to a single inner membrane component (LolF), and a homolog of the E. coli cytoplasmic ATPase LolD has not been identified. In the present study, we sought to identify a LolD-like protein in H. pylori. We used affinity-purification mass spectrometry to identify interaction partners of the H. pylori ATP-binding cassette (ABC) family permease LolF and identified the ABC family ATP-binding protein HP0179 as its interaction partner. We engineered H. pylori to conditionally express HP0179 and showed that HP0179 and its conserved ATP binding and ATP hydrolysis motifs are essential for H. pylori growth. We then performed affinity purification-mass spectrometry using HP0179 as the bait and identified LolF as its interaction partner. These results indicate that H. pylori HP0179 is a LolD-like protein and provide a more complete understanding of lipoprotein localization processes in H. pylori, a bacterium in which the Lol system deviates from the E. coli paradigm. Lipoproteins are critical in Gram-negative-bacteria for cell surface assembly of LPS, insertion of outer membrane proteins, and sensing envelope stress. Lipoproteins also contribute to bacterial pathogenesis. For many of these functions, lipoproteins must localize to the Gram-negative outer membrane. Transporting lipoproteins to the outer membrane involves the Lol sorting pathway. Detailed analyses of the Lol pathway have been performed in the model organism Escherichia coli, but many bacteria utilize altered components or are missing essential components of the E. coli Lol pathway. Identifying a LolD-like protein in Helicobacter pylori is important to better understand the Lol pathway in diverse bacterial classes. This becomes particularly relevant as lipoprotein localization is targeted for antimicrobial development.
脂蛋白（Lol）系统的定位被革兰氏阴性细菌用于将脂蛋白输出到外膜。Lol 蛋白及其将脂蛋白从内膜转移到外膜的模型已在模式生物大肠杆菌中得到广泛描述，但在许多细菌物种中，脂蛋白的合成和外排途径偏离了大肠杆菌的模式。例如，在人类胃细菌幽门螺杆菌中，没有发现大肠杆菌外膜成分 LolB 的同源物，大肠杆菌 LolC 和 LolE 对应于单个内膜成分（LolF），并且尚未鉴定出大肠杆菌细胞质 ATP 酶 LolD 的同源物。在本研究中，我们试图在幽门螺杆菌中鉴定出一种 LolD 样蛋白。我们使用亲和纯化质谱法鉴定了幽门螺杆菌 ABC 家族渗透酶 LolF 的相互作用伙伴，并鉴定出 ABC 家族 ATP 结合蛋白 HP0179 是其相互作用伙伴。我们构建了条件表达 HP0179 的幽门螺杆菌，并表明 HP0179 及其保守的 ATP 结合和 ATP 水解基序对幽门螺杆菌的生长是必不可少的。然后，我们使用 HP0179 作为诱饵进行亲和纯化质谱分析，并鉴定出 LolF 是其相互作用伙伴。这些结果表明，幽门螺杆菌 HP0179 是一种 LolD 样蛋白，并为脂蛋白在幽门螺杆菌中的定位过程提供了更完整的理解，幽门螺杆菌的 Lol 系统偏离了大肠杆菌的模式。脂蛋白对于革兰氏阴性菌的细胞表面 LPS 组装、外膜蛋白的插入和 envelope stress 的感应至关重要。脂蛋白也有助于细菌发病机制。对于许多这些功能，脂蛋白必须定位于革兰氏阴性的外膜。将脂蛋白运送到外膜涉及 Lol 分拣途径。Lol 途径的详细分析已在模式生物大肠杆菌中进行，但许多细菌利用改变的成分或缺失大肠杆菌 Lol 途径的必需成分。在幽门螺杆菌中鉴定出一种 LolD 样蛋白对于更好地理解不同细菌类别的 Lol 途径很重要。当脂蛋白定位成为抗菌药物开发的目标时，这一点尤其重要。
