Structural determinants of Actinomyces sortase SrtC2 required for membrane localization and assembly of type 2 fimbriae for interbacterial coaggregation and oral biofilm formation.
Structural determinants of Actinomyces sortase SrtC2 required for membrane localization and assembly of type 2 fimbriae for interbacterial coaggregation and oral biofilm formation.
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放线菌分选酶 SrtC2 的结构决定因素是膜定位和 2 型菌毛组装所需的，以实现细菌间共聚集和口腔生物膜形成。
DOI：
10.1128/jb.00093-12
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发表时间：
2012
影响因子：
3.2
通讯作者：
Ton-That,Hung
中科院分区：
文献类型：
--
作者：
Wu,Chenggang;Mishra,Arunima;Reardon,MelissaE;Huang,I-Hsiu;Counts,SarahC;Das,Asis;Ton-That,Hung
作为口腔的先驱定植菌，口放线菌表达蛋白质菌毛（也称为菌毛），以介导生物膜形成中的以下两个关键事件：牙釉质上唾液沉积物的粘附和细菌间关联。直接促进口腔链球菌和放线菌生物膜发育的 2 型菌毛的组装需要 C 类分选酶 SrtC2。尽管与分选酶相关的一般机制已被阐明，但 C 类分选酶特有的几个结构属性还需要进行功能研究。这里报告的突变研究表明，预计含有信号肽序列的 SrtC2 N 端跨膜 (TM) 区域从成熟蛋白质中被切除，并且这种加工对于酶在细胞质膜上的正确整合至关重要，这是由包含 TM 结构域和细胞质尾部的扩展疏水性 C 末端介导的。删除这个假定的 TM 或整个细胞质结构域就消除了酶的定位和功能。保守催化 Cys-His 二联体的丙氨酸取代消除了 SrtC2 酶活性。相比之下，旨在改变覆盖 C 类分选酶催化口袋的“盖子”结构域的突变对酶活性没有影响。最后，影响SrtC2活性或膜定位的每一个有害突变也消除了放线菌物种生物膜的形成以及细菌与链球菌的共聚集。我们得出的结论是，SrtC2 的 N 末端包含信号序列，是正确的蛋白质易位和成熟所必需的，而延伸的 C 末端疏水区域则作为适当酶功能的稳定膜锚定。
As a pioneer colonizer of the oral cavity,Actinomyces orisexpresses proteinaceous pili (also called fimbriae) to mediate the following two key events in biofilm formation: adherence to saliva deposits on enamel and interbacterial associations. Assembly of type 2 fimbriae that directly facilitate coaggregation with oral streptococci andActinomycesbiofilm development requires the class C sortase SrtC2. Although the general sortase-associated mechanisms have been elucidated, several structural attributes unique to the class C sortases require functional investigation. Mutational studies reported here suggest that the N-terminal transmembrane (TM) region of SrtC2, predicted to contain a signal peptide sequence, is cleaved off the mature protein and that this processing is critical for the proper integration of the enzyme at the cytoplasmic membrane, which is mediated by the extended hydrophobic C terminus containing a TM domain and a cytoplasmic tail. Deletion of this putative TM or the entire cytoplasmic domain abolished the enzyme localization and functionality. Alanine substitution of the conserved catalytic Cys-His dyad abrogated the SrtC2 enzymatic activity. In contrast, mutations designed to alter a “lid” domain that covers the catalytic pocket of a class C sortase showed no effect on enzyme activity. Finally, each of the deleterious mutations that affected SrtC2 activity or membrane localization also eliminatedActinomycesspecies biofilm development and bacterial coaggregation with streptococci. We conclude that the N terminus of SrtC2, which contains the signal sequence, is required for proper protein translocation and maturation, while the extended C-terminal hydrophobic region serves as a stable membrane anchor for proper enzyme functionality.