Investigation for regulatory mechanisms of GcrR protein for S.mutans gbp Cexpression

作者研究了gcrR基因作为双组分调控系统对gbpC基因表达的反应调控机制。反应调节因子的信号转导是通过它们的对应物，传感器-传递蛋白的磷酸化转移来完成的。GcrR蛋白的第53个氨基酸天冬氨酸通过位点定向诱变被改变为丙氨酸，并将该突变引入S.mutans染色体。尽管该突变体中改变的GcrR蛋白不应被磷酸化，但该突变体表现出与野生型菌株相同的葡聚糖依赖聚集表型。因此，作者得出结论，GcrR蛋白不是作为反应调节因子，而是作为抑制因子。GbpC蛋白是附着于肽聚糖层的壁锚蛋白成员。作者通过Western blot分析证实了GbpC在细胞壁处的表达，并且使用BiaCore系统表明s.a mutans细胞能够更多地结合由GbpC蛋白介导的固定化葡聚糖。蛋白质的细胞壁锚定是由分选酶介导的。S.mutans的分选酶阴性突变体没有表现出预期的葡聚糖依赖的聚集表型。最初在菌株GS-5中发现了gbpC基因的无义突变，这导致在变形链球菌菌株中检测到gbpC基因的单核苷酸多态性。gbpC基因的保守区和多态区将有助于估计gbpC蛋白的功能域和鉴定s.a mutans菌株。利用该保守的gbpC蛋白信息设计引物，用PCR方法成功鉴定了一种来自macacae链球菌的gbpC基因同源物。作者发现了另一种聚集表型，称为冷凝集。利用Himarl转座子体外转位的随机诱变方法，从一些s.a mutans菌株中鉴定出胶原结合黏附素基因。这是首次在人类口腔原生菌群中发现来自绿绿链球菌的胶原结合粘连蛋白。少

The auther has investigated the regulatory mechanisms of the gcrR gene as a response regulator of two-component regulatory systems for gbpC gene expression. Signal transduction of response regulators is performed by their phosphoryl transfer from their counterparts, sensor-transmitter proteins. The 53rd amino acid aspartate of GcrR protein that is expected as the signal receptor site was changed to aranine by site-directed mutagenesis, and this mutation was introduced into S.mutans chromosome. Albeit this altered GcrR protein in this mutant should not be phosphorylated, this mutant exhibited the same dextran-dependent aggregation phenotype as the wild type strain. Therefore, the authers concluded that GcrR protein does not act as a response regulator but act as a represser. GbpC protein is a member of wall-anchored proteins tethered to peptidoglycan layer. The authers confirmed GbpC expression at cell wall by the Western blot analysis, and the S.mutans cells were indicated to be able t … More o bind to immobilized glucan mediated by the GbpC protein using the BiaCore system. Cell wall anchoring of the protein is mediated by the sortase enzyme. Sortase negative mutant of S.mutans did not exhibit dextran-dependent aggregation phenotype as expected. A nonsense mutation of the gbpC gene was initially found in strain GS-5 and this lead to detection of single nucleotide polymorphisms in the gbpC gene among S.mutans strains. Both conserved and polymorphic regions of the gbpC gene will be useful for an estimation of functional domains of GbpC protein and identification of S.mutans strains. A gbpC gene homologue from Streptococcus macacae was successfully identified by PCR method with primers designed from this conserved GbpC protein information. The authers found another aggregation phenotype designated as cold agglutination. The collagen-binding adhesin gene was identified from some strains of S.mutans by random mutagenesis method using in vitro transposition of Himarl transposon. This is the first report that demonstrates a collagen-binding adhesin from viridans streptococci in human oral indigenous flora. Less