Kruger Ryan G, Otvos Balint, Frankel Brenda A, Bentley Matthew, Dostal Patrick, McCafferty Dewey G
Johnson Research Foundation and Department of Biochemistry and Biophysics, The University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6059, USA.
Biochemistry. 2004 Feb 17;43(6):1541-51. doi: 10.1021/bi035920j.
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-Gly(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.
金黄色葡萄球菌分选酶转肽酶SrtA亚型负责将与毒力和定植相关的蛋白质共价连接到细菌肽聚糖上。SrtA利用两种底物，十一异戊烯焦磷酸化的MurNAc（GlcNAc）-Ala-D-异谷氨酰胺-Lys（ε-Gly(5)）-D-丙氨酸-D-丙氨酸（分支脂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II的Gly(5)部分的亲核氨基形成新的酰胺键，将蛋白质锚定到这个关键中间体上，该中间体随后聚合成肽聚糖。在此，我们描述了一种用于阐明分选酶底物特异性的通用体外方法的开发。此外，通过免疫荧光、细胞黏附试验和透射电子显微镜，我们建立了金黄色葡萄球菌分选酶亚型的体外底物特异性与体内功能之间的联系。这些研究结果为SrtA亚型在金黄色葡萄球菌黏附和宿主定植中的主要作用提供了有力的支持证据，说明了SrtA和SrtB亚型之间缺乏特异性串扰，并突出了SrtA作为开发针对革兰氏阳性细菌病原体的抗毒力化学疗法靶点的潜力。
