Mechanisms of Incorporation for D-Amino Acid Probes That Target Peptidoglycan Biosynthesis
Mechanisms of Incorporation for D-Amino Acid Probes That Target Peptidoglycan Biosynthesis
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DOI：
10.1021/acschembio.9b00664
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发表时间：
2019-12-01
影响因子：
4
通讯作者：
VanNieuwenhze, Michael S.
中科院分区：
文献类型：
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作者：
Kuru, Erkin;Radkov, Atanas;VanNieuwenhze, Michael S.
细菌通过合成和修饰其必需肽聚糖（PG）细胞壁，呈现出无数不同的形态。我们发现了一种基于荧光d -氨基酸（FDAA）的PG标记方法，为观察这些形态变化的发生提供了一种强有力的方法。鉴于PG是细菌细胞所特有的，也是抗生素的共同靶点，了解(F) daa探针结合的精确机制对于理解PG合成在细菌细胞生物学中的作用至关重要，并且可以为开发新的抗微生物药物以治疗耐药抗菌感染提供有价值的工具。本研究利用大肠杆菌（革兰氏阴性）和枯草芽孢杆菌（革兰氏阳性）两种模式生物，系统探讨了FDAA探针与PG结合的机制。我们的体外和体内数据明确表明，这些细菌通过两种胞质外途径结合FDAAs：通过它们的D、D转肽酶的活性，如果存在的话，通过它们的L、D转肽酶，而不是通过细胞质结合到D- ala -D- ala二肽前体。我们的数据还揭示了前所未有的发现，除了已知的对d -丙氨酸消旋酶和D-Ala-D-Ala连接酶的抑制活性外，daa药物d -环丝氨酸还可以被这些转肽酶结合到肽干中。这些机制的发现促进了一种新的、基于fda的体外标记方法的发展，该方法可以报告多肽的亚细胞分布，这是研究生长模式不明确的细菌的一个特别重要的属性。在解释高分辨率实验结果和强调合成DAAs的抗菌潜力时，提高对DAAs探针所利用的掺入机制的理解是必不可少的。
Bacteria exhibit a myriad of different morphologies, through the synthesis and modification of their essential peptidoglycan (PG) cell wall. Our discovery of a fluorescent D-amino acid (FDAA)-based PG labeling approach provided a powerful method for observing how these morphological changes occur. Given that PG is unique to bacterial cells and a common target for antibiotics, understanding the precise mechanism(s) for incorporation of (F)DAA-based probes is a crucial determinant in understanding the role of PG synthesis in bacterial cell biology and could provide a valuable tool in the development of new antimicrobials to treat drug-resistant antibacterial infections. Here, we systematically investigate the mechanisms of FDAA probe incorporation into PG using two model organisms Escherichia coli (Gram-negative) and Bacillus subtilis (Gram-positive). Our in vitro and in vivo data unequivocally demonstrate that these bacteria incorporate FDAAs using two extracytoplasmic pathways: through activity of their D,D-transpeptidases, and, if present, by their L,D-transpeptidases and not via cytoplasmic incorporation into a D-Ala-D-Ala dipeptide precursor. Our data also revealed the unprecedented finding that the DAA-drug, D-cycloserine, can be incorporated into peptide stems by each of these transpeptidases, in addition to its known inhibitory activity against D-alanine racemase and D-Ala-D-Ala ligase. These mechanistic findings enabled development of a new, FDAA-based, in vitro labeling approach that reports on subcellular distribution of muropeptides, an especially important attribute to enable the study of bacteria with poorly defined growth modes. An improved understanding of the incorporation mechanisms utilized by DAA-based probes is essential when interpreting results from high resolution experiments and highlights the antimicrobial potential of synthetic DAAs.