Department of Immunology and Microbiology, Wayne State University School of Medicine, Detroit, MI 48201, USA.
J Bacteriol. 2011 Jan;193(2):411-20. doi: 10.1128/JB.01098-10. Epub 2010 Nov 19.
Many streptococcal pathogens require a polysaccharide capsule for survival in the host during systemic infection. The highly conserved CpsA protein is proposed to be a transcriptional regulator of capsule production in streptococci, although the regulatory mechanism is unknown. Hydropathy plots of CpsA predict an integral membrane protein with 3 transmembrane domains and only 27 cytoplasmic residues, whereas other members of the LytR_cpsA_psr protein family are predicted to have a single transmembrane domain. This unique topology, with the short cytoplasmic domain, membrane localization, and large extracellular domain, suggests a novel mechanism of transcriptional regulation. Therefore, to determine the actual membrane topology of CpsA, specific protein domains were fused to beta-galactosidase or alkaline phosphatase. Enzymatic assays confirmed that the predicted membrane topology for CpsA is correct. To investigate how this integral membrane protein may be functioning in regulation of capsule transcription, purified full-length and truncated forms of CpsA were used in electrophoretic mobility shift assays to characterize the ability to bind the capsule operon promoter. Assays revealed that full-length, purified CpsA protein binds specifically to DNA containing the capsule promoter region. Furthermore, the large extracellular domain is not required for DNA binding, but all cytoplasmic regions of CpsA are necessary and sufficient for specific binding to the capsule operon promoter. This is the first demonstration of a member of this protein family interacting with its target DNA. Taken together, CpsA, as well as other members of the LytR_cpsA_psr protein family, appears to utilize a unique mechanism of transcriptional regulation.
许多链球菌病原体需要多糖荚膜才能在全身感染期间在宿主体内存活。CpsA 蛋白高度保守，据推测它是链球菌荚膜产生的转录调节剂，尽管其调节机制尚不清楚。CpsA 的疏水性图预测它是一种具有 3 个跨膜结构域和仅 27 个细胞质残基的完整膜蛋白，而 LytR_cpsA_psr 蛋白家族的其他成员则预测具有单个跨膜结构域。这种独特的拓扑结构，具有短的细胞质结构域、膜定位和大的细胞外结构域，表明存在一种新的转录调节机制。因此，为了确定 CpsA 的实际膜拓扑结构，将特定的蛋白质结构域融合到β-半乳糖苷酶或碱性磷酸酶中。酶测定证实了 CpsA 的预测膜拓扑结构是正确的。为了研究这种完整的膜蛋白如何在调节荚膜转录中发挥作用，使用纯化的全长和截断形式的 CpsA 进行电泳迁移率变动分析，以表征结合荚膜操纵子启动子的能力。分析表明，全长、纯化的 CpsA 蛋白特异性结合含有荚膜启动子区域的 DNA。此外，不需要大的细胞外结构域进行 DNA 结合，但 CpsA 的所有细胞质区域对于与荚膜操纵子启动子的特异性结合都是必需和充分的。这是首次证明该蛋白质家族的成员与目标 DNA 相互作用。总之，CpsA 以及 LytR_cpsA_psr 蛋白家族的其他成员似乎利用一种独特的转录调节机制。
