From the Department of Microbiology, Harvard Medical School, Boston, Massachusetts 02115.
From the Department of Microbiology, Harvard Medical School, Boston, Massachusetts 02115.
J Biol Chem. 2018 Nov 16;293(46):17985-17996. doi: 10.1074/jbc.RA118.004561. Epub 2018 Sep 20.
Modifications to the Gram-positive bacterial cell wall play important roles in antibiotic resistance and pathogenesis, but the pathway for the d-alanylation of teichoic acids (DLT pathway), a ubiquitous modification, is poorly understood. The d-alanylation machinery includes two membrane proteins of unclear function, DltB and DltD, which are somehow involved in transfer of d-alanine from a carrier protein inside the cell to teichoic acids on the cell surface. Here, we probed the role of DltD in the human pathogen using both cell-based and biochemical assays. We first exploited a known synthetic lethal interaction to establish the essentiality of each gene in the DLT pathway for d-alanylation of lipoteichoic acid (LTA) and confirmed this by directly detecting radiolabeled d-Ala-LTA both in cells and in vesicles prepared from mutant strains of We developed a partial reconstitution of the pathway by using cell-derived vesicles containing DltB, but no other components of the d-alanylation pathway, and showed that d-alanylation of previously formed lipoteichoic acid in the DltB vesicles requires the presence of purified and reconstituted DltA, DltC, and DltD, but not of the LTA synthase LtaS. Finally, based on the activity of DltD mutants in cells and in our reconstituted system, we determined that Ser-70 and His-361 are essential for d-alanylation activity, and we propose that DltD uses a catalytic dyad to transfer d-alanine to LTA. In summary, we have developed a suite of assays for investigating the bacterial DLT pathway and uncovered a role for DltD in LTA d-alanylation.
革兰氏阳性菌细胞壁的修饰在抗生素耐药性和发病机制中起着重要作用，但对于普遍存在的修饰物——肽聚糖的 d-丙氨酸化途径（DLT 途径），其途径仍知之甚少。d-丙氨酸化机制包括两种功能尚不清楚的膜蛋白，DltB 和 DltD，它们在某种程度上参与了将胞内的 d-丙氨酸从载体蛋白转移到细胞表面的肽聚糖上。在这里，我们使用基于细胞和生化的测定方法来研究 DltD 在人类病原体中的作用。我们首先利用已知的合成致死相互作用，确定了 DLT 途径中的每个基因对于脂磷壁酸（LTA）的 d-丙氨酸化的必要性，并通过直接检测来自突变株的细胞和囊泡中的放射性标记的 d-Ala-LTA 来证实这一点。我们通过使用含有 DltB 但没有其他 d-丙氨酸化途径成分的细胞衍生囊泡来部分重建途径，并表明 DltB 囊泡中先前形成的脂磷壁酸的 d-丙氨酸化需要纯化和重建的 DltA、DltC 和 DltD 的存在，但不需要 LTA 合酶 LtaS。最后，基于 DltD 突变体在细胞和我们重建的系统中的活性，我们确定 Ser-70 和 His-361 对于 d-丙氨酸化活性是必需的，并且我们提出 DltD 使用催化偶联将 d-丙氨酸转移到 LTA 上。总之，我们开发了一系列用于研究细菌 DLT 途径的测定方法，并揭示了 DltD 在 LTA d-丙氨酸化中的作用。
