Nobbs Angela H, Vajna Reka M, Johnson Jeremy R, Zhang Yongshu, Erlandsen Stanley L, Oli Monika W, Kreth Jens, Brady L Jeannine, Herzberg Mark C
Department of Diagnostic and Biological Sciences, School of Dentistry, Medical School, University of Minnesota, Minneapolis, MN 55455, USA.
Department of Genetics, Cell Biology and Development, Medical School, University of Minnesota, Minneapolis, MN 55455, USA.
Microbiology (Reading). 2007 Dec;153(Pt 12):4088-4097. doi: 10.1099/mic.0.2007/007252-0.
Sortase A (SrtA) is required for cell-wall anchoring of LPXTG-containing Gram-positive surface proteins. It was hypothesized, therefore, that disruption of the srtA gene would alter surface anchoring and functions of target LPXTG motif-bearing SspA and SspB proteins of Streptococcus gordonii. Mutant strains in srtA (V288srtA(-), DL1srtA(-)) were constructed in S. gordonii V288 (wtV288) and DL1 (wtDL1). When compared to wtV288, the V288srtA(-) mutant showed decreased biofilm formation on polystyrene, and reduced binding to immobilized purified salivary agglutinin (BIAcore analysis). The wtV288 and V288srtA(-) strains were similar in ultrastructure, but immunogold-labelled SspA/SspB surface expression was reduced on the V288srtA(-) mutant. DL1srtA(-) was also complemented to obtain DL1srtA(+). From the wild-type strains (wtV288, wtDL1), srtA(-) mutants (V288srtA(-), DL1srtA(-)), and the complemented mutant (DL1srtA(+)), cytoplasmic, cell-wall and released extracellular protein fractions were isolated. Each fraction was analysed by SDS-PAGE and immunoblotting with anti-P1. Spent medium from srtA(-) mutant cells contained over-represented proteins, including SspA/SspB (P1 antigen). Mutants showed less P1 on the cell surface than wild-types, as estimated using whole-cell ELISA, and no P1 appeared in the cytoplasmic fractions. Expression of several adhesin genes (sspA/B, cshA/B, fbpA) was generally upregulated in the mutants (V288srtA(-), DL1srtA(-)), but restored to wild-type levels in DL1srtA(+). These data therefore imply that in addition to its role in processing LPXTG-containing adhesins, sortase A has the novel function of contributing to transcriptional regulation of adhesin gene expression.
分选酶A（SrtA）是含LPXTG的革兰氏阳性表面蛋白细胞壁锚定所必需的。因此，有人推测，srtA基因的破坏会改变戈登链球菌中目标含LPXTG基序的SspA和SspB蛋白的表面锚定及功能。在戈登链球菌V288（野生型V288）和DL1（野生型DL1）中构建了srtA的突变菌株（V288srtA(-)、DL1srtA(-)）。与野生型V288相比，V288srtA(-)突变体在聚苯乙烯上的生物膜形成减少，与固定化纯化唾液凝集素的结合减少（BIAcore分析）。野生型V288和V288srtA(-)菌株在超微结构上相似，但V288srtA(-)突变体上免疫金标记的SspA/SspB表面表达减少。DL1srtA(-)也进行了互补，得到DL1srtA(+)。从野生型菌株（野生型V288、野生型DL1）、srtA(-)突变体（V288srtA(-)、DL1srtA(-)）和互补突变体（DL1srtA(+)）中分离出细胞质、细胞壁和释放的细胞外蛋白组分。每个组分通过SDS-PAGE分析并用抗P1进行免疫印迹。srtA(-)突变体细胞的用过培养基中含有过量表达的蛋白质，包括SspA/SspB（P1抗原）。使用全细胞ELISA估计，突变体在细胞表面的P1比野生型少，并且在细胞质组分中没有出现P1。几种粘附素基因（sspA/B、cshA/B、fbpA）的表达在突变体（V288srtA(-)、DL1srtA(-)）中通常上调，但在DL1srtA(+)中恢复到野生型水平。因此，这些数据表明，分选酶A除了在处理含LPXTG的粘附素中起作用外，还具有促进粘附素基因表达转录调控的新功能。
