Infectious Diseases Translational Research Programme and Department of Microbiology and Immunology, National University of Singaporegrid.4280.e, Singapore.
Department of Pathology and Laboratory Medicine, KK Women's and Children's Hospital Singapore.
mBio. 2021 Dec 21;12(6):e0261521. doi: 10.1128/mBio.02615-21. Epub 2021 Nov 2.
MOP (Multidrug/Oligosaccharidyl-lipid/Polysaccharide) family transporters are found in almost all life forms. They are responsible for transporting lipid-linked precursors across the cell membrane to support the synthesis of various glycoconjugates. While significant progress has been made in elucidating their transport mechanism, how these transporters select their substrates remains unclear. Here, we systematically tested the MOP transporters in the Streptococcus pneumoniae capsule pathway for their ability to translocate noncognate capsule precursors. Sequence similarity cannot predict whether these transporters are interchangeable. We showed that subtle changes in the central aqueous cavity of the transporter are sufficient to accommodate a different cargo. These changes can occur naturally, suggesting a potential mechanism of expanding substrate selectivity. A directed evolution experiment was performed to identify gain-of-function variants that translocate a noncognate cargo. Coupled with a high-throughput mutagenesis and sequencing (Mut-seq) experiment, residues that are functionally important for the capsule transporter were revealed. Lastly, we showed that the expression of a flippase that can transport unfinished precursors resulted in an increased susceptibility to bacitracin and mild cell shape defects, which may be a driving force to maintain transporter specificity. All licensed pneumococcal vaccines target the capsular polysaccharide (CPS). This layer is highly variable and is important for virulence in many bacterial pathogens. Most of the CPSs are produced by the Wzx/Wzy mechanism. In this pathway, CPS repeating units are synthesized in the cytoplasm, which must be flipped across the cytoplasmic membrane before polymerization. This step is mediated by the widely conserved MOP (Multidrug/Oligosaccharidyl-lipid/Polysaccharide) family transporters. Here, we systematically evaluated the interchangeability of these transporters and identified the residues important for substrate specificity and function. Understanding how CPS is synthesized will inform glycoengineering, vaccine development, and antimicrobial discovery.
MOP（多药/寡糖脂/多糖）家族转运蛋白几乎存在于所有生命形式中。它们负责将脂质连接的前体跨细胞膜运输，以支持各种糖缀合物的合成。尽管在阐明其运输机制方面取得了重大进展，但这些转运蛋白如何选择其底物仍不清楚。在这里，我们系统地测试了肺炎链球菌荚膜途径中的 MOP 转运蛋白，以研究它们转运非同源荚膜前体的能力。序列相似性不能预测这些转运蛋白是否可以互换。我们表明，转运蛋白中心水腔中的细微变化足以容纳不同的货物。这些变化可能自然发生，表明了扩大底物选择性的潜在机制。进行了定向进化实验以鉴定可转运非同源货物的功能获得变体。结合高通量诱变和测序（Mut-seq）实验，揭示了对荚膜转运蛋白功能重要的残基。最后，我们表明，表达可以转运未完成前体的翻转酶会导致对杆菌肽的敏感性增加和轻微的细胞形状缺陷，这可能是维持转运蛋白特异性的驱动力。所有许可的肺炎球菌疫苗都针对荚膜多糖（CPS）。这一层高度可变，对许多细菌病原体的毒力很重要。大多数 CPS 都是通过 Wzx/Wzy 机制产生的。在这个途径中，CPS 重复单元在细胞质中合成，在聚合之前必须翻转穿过细胞质膜。这一步由广泛保守的 MOP（多药/寡糖脂/多糖）家族转运蛋白介导。在这里，我们系统地评估了这些转运蛋白的可互换性，并确定了对底物特异性和功能重要的残基。了解 CPS 是如何合成的将为糖工程、疫苗开发和抗菌药物发现提供信息。
