Analysis of the substrate specificity of the Staphylococcus aureus sortase transpeptidase SrtA
Analysis of the substrate specificity of the Staphylococcus aureus sortase transpeptidase SrtA
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DOI：
10.1021/bi035920j
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发表时间：
2004-02-17
期刊：
影响因子：
2.9
通讯作者：
McCafferty, DG
中科院分区：
文献类型：
--
作者：
Kruger, RG;Otvos, B;McCafferty, DG
金黄色葡萄球菌分选酶转肽酶SrtA同种型负责毒力和定殖相关蛋白与细菌肽聚糖的共价连接。SrtA利用两种底物，十一烯醇-焦磷酸-MurNAc（GlcNAc）-Ala-D-isoGlu-Lys（ε-GIY（5））-D-Ala-D-Ala（支链脂质II）和含有高度保守的C-末端LPXTG序列的分泌蛋白。SrtA同时切割含LPXTG蛋白的Thr-Gly键，并与分支脂质II的Gly（5）部分的亲核氨基形成新的酰胺键，将蛋白锚定到该关键中间体，随后聚合成肽聚糖。在这里，我们描述了一个通用的体外方法阐明分选酶的底物特异性的发展。此外，利用免疫荧光、细胞粘附试验和透射电子显微镜，我们建立了S.金黄色葡萄球菌分选酶同种型。这些研究的结果为SrtA亚型在S.金黄色葡萄球菌粘附和宿主定殖，说明SrtA和SrtB同种型之间缺乏特异性串扰，并强调SrtA作为开发针对革兰氏阳性细菌病原体的抗病力化疗药物的靶标的潜力。
The Staphylococcus aureus sortase transpeptidase SrtA isoform is responsible for the covalent attachment of virulence and colonization-associated proteins to the bacterial peptidoglycan. SrtA utilizes two substrates, undecaprenol-pyrophosphoryl-MurNAc(GlcNAc)-Ala-D-isoGlu-Lys(epsilon-GIY(5))-D-Ala-D-Ala (branched Lipid II) and secreted proteins containing a highly conserved C-terminal LPXTG sequence. SrtA simultaneously cleaves the Thr-Gly bond of the LPXTG-containing protein and forms a new amide bond with the nucleophilic amino group of the Gly(5) portion of branched Lipid II, anchoring the protein to this key intermediate that is subsequently polymerized into peptidoglycan. Here we describe the development of a general in vitro method for elucidating the substrate specificity of sortase enzymes. In addition, using immunofluorescence, cell adhesion assays, and transmission electron microscopy, we establish links between in vitro substrate specificity and in vivo function of the S. aureus sortase isoforms. Results from these studies provide strong supporting evidence of a primary role of the SrtA isoform in S. aureus adhesion and host colonization, illustrate a lack of specificity cross talk between SrtA and SrtB isoforms, and highlight the potential of SrtA as a target for the development of antivirulence chemotherapeutics against Gram-positive bacterial pathogens.