在革兰氏阳性细菌包膜内的菌毛组装过程中,膜结合分选酶通过细胞壁分选信号(CWSS)顺序交联特定的菌毛蛋白单体,从指定的小费Pilin开始,接着是另一个pilin做的轴,最终将纤维基菌毛固定在细胞壁上。迄今为止,控制菌毛尖端组装和潜在机制的分子决定因素仍然未知。这里,我们在放线菌模型中解决了这个问题。这种口腔微生物聚集了一个致病重要的菌毛(称为2型菌毛),用Fimapilins做的,显示两个交替的尖端菌毛-FimB或共聚集因子CafA之一,它们具有明显相似的CWSS。我们证明,将CafA的CWSS与FimB的CWSS交换会产生功能杂种,它位于菌毛尖端并介导多微生物共聚集,而CWSS中保守FLIAG基序的丙氨酸取代阻碍了这些过程。值得注意的是,将正常细胞壁锚定的糖蛋白GspA的CWSS与CafA的CWSS交换可促进杂合GspA在FimA菌毛尖端的组装。最后,将白喉棒杆菌轴菌毛SpaA的CWSS与CafA的CWSS交换导致异源菌毛蛋白SpaA在A.oris的FimA菌毛尖端的FLIAG基序依赖性定位。显然,CafA的CWSS和FLIAG基序对于其目的地到由生物体中指定的分选酶特异性组装的同源菌毛尖端都是必要和充分的。
目标:革兰氏阳性菌毛,其前体具有分选酶介导的菌毛组装所需的细胞壁分选信号(CWSS),通常包括菌毛轴和尖端粘附素。菌毛是如何变成菌毛的,然而,仍然不确定。WedemonstratehereinActencesoristhattheCWSSofthetippilinCafAisnecessaryandsufficienttopromotepilustipassembly,此功能组件涉及CWSS内的保守FLIAG基序。这可以通过以下事实来证明:A.oris细胞壁锚定的糖蛋白,GspA,或来自白喉棒杆菌的异源轴菌丝,SpaA,设计了CafA的CWSS来代替他们的CWSS,在需要FLIAG图案的过程中定位在菌毛尖端。我们的发现为分选酶催化的菌毛尖端组装提供了分子基础,其他革兰氏阳性细菌和潜在的生物工程应用很可能在受控的表面距离上显示抗原。
During pilus assembly within the Gram-positive bacterial envelope, membrane-bound sortase enzymes sequentially crosslink specific pilus protein monomers through their cell wall sorting signals (CWSS), starting with a designated tip pilin, followed by the shaft made of another pilin, ultimately anchoring the fiber base pilin to the cell wall. To date, the molecular determinants that govern pilus tip assembly and the underlying mechanism remain unknown. Here, we addressed this in the model organism Actinomyces oris. This oral microbe assembles a pathogenically important pilus (known as type 2 fimbria) whose shafts, made of FimA pilins, display one of two alternate tip pilins-FimB or the coaggregation factor CafA-that share a markedly similar CWSS. We demonstrate that swapping the CWSS of CafA with that of FimB produces a functional hybrid, which localizes at the pilus tip and mediates polymicrobial coaggregation, whereas alanine-substitution of the conserved FLIAG motif within the CWSS hampers these processes. Remarkably, swapping the CWSS of the normal cell wall-anchored glycoprotein GspA with that of CafA promotes the assembly of hybrid GspA at the FimA pilus tip. Finally, exchanging the CWSS of the Corynebacterium diphtheriae shaft pilin SpaA with that of CafA leads to the FLIAG motif-dependent localization of the heterologous pilus protein SpaA at the FimA pilus tip in A. oris. Evidently, the CWSS and the FLIAG motif of CafA are both necessary and sufficient for its destination to the cognate pilus tip specifically assembled by a designated sortase in the organism.
OBJECTIVE: Gram-positive pili, whose precursors harbor a cell wall sorting signal (CWSS) needed for sortase-mediated pilus assembly, typically comprise a pilus shaft and a tip adhesin. How a pilin becomes a pilus tip, nevertheless, remains undetermined. We demonstrate here in Actinomyces oris that the CWSS of the tip pilin CafA is necessary and sufficient to promote pilus tip assembly, and this functional assembly involves a conserved FLIAG motif within the CWSS. This is evidenced by the fact that an A. oris cell-wall anchored glycoprotein, GspA, or a heterologous shaft pilin from Corynebacterium diphtheriae, SpaA, engineered to have the CWSS of CafA in place of their CWSS, localizes at the pilus tip in a process that requires the FLIAG motif. Our findings provide the molecular basis for sortase-catalyzed pilus tip assembly that is very likely employed by other Gram-positive bacteria and potential bioengineering applications to display antigens at controlled surface distance.