Substitutions in the N-terminal alpha helical spine of Neisseria gonorrhoeae pilin affect type IV pilus assembly, dynamics and associated functions
Substitutions in the N-terminal alpha helical spine of Neisseria gonorrhoeae pilin affect type IV pilus assembly, dynamics and associated functions
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DOI：
10.1111/j.1365-2958.2006.05482.x
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发表时间：
2007-01-01
影响因子：
3.6
通讯作者：
Koomey, Michael
中科院分区：
文献类型：
--
作者：
Aas, Finn Erik;Winther-Larsen, Hanne C.;Koomey, Michael
IV型菌毛(TFP)是一种多功能表面附属物，由许多具有医疗、环境和工业重要性的革兰氏阴性菌表达。这些细胞器中最保守的结构特征是N-末端定位的，也就是所谓的α-螺旋结构。流行的菌毛组装和结构模型总是暗示它对膜运输、细胞器结构和相关功能的重要性。然而，相对较少的研究考察了这一领域内的错义替换的影响。以淋病奈瑟菌为模型系统，我们构建了定位于菌毛蛋白亚单位堆这一区域的单一和多个氨基酸替换的突变体，并对它们的菌毛蛋白稳定性、细胞器表达和相关表型进行了表征。同时检测了同时表达突变型和野生型堆型的后果。这一发现首次在明确的遗传背景下证明了菌毛蛋白的分子间互补现象，即组装缺陷的菌毛蛋白可以通过野生型桩的共表达而被拯救成可纯化的TFP。结果进一步证明，菌毛蛋白亚基的组成可以通过监控亚单位-亚单位相互作用的效率的过程，影响由PILT收缩蛋白介导的细胞器动力学。除了证实和扩大了桩多聚体作为自然遗传转化能力的重要组成部分的证据外，这项工作还为详细研究TFP亚单位-亚单位之间的相互作用铺平了道路，包括膜内的自我识别和菌毛聚合物内的堆积。
Type IV pili (Tfp) are multifunctional surface appendages expressed by many Gram negative species of medical, environmental and industrial importance. The N-terminally localized, so called alpha-helical spine is the most conserved structural feature of pilin subunits in these organelles. Prevailing models of pilus assembly and structure invariably implicate its importance to membrane trafficking, organelle structure and related functions. Nonetheless, relatively few studies have examined the effects of missense substitutions within this domain. Using Neisseria gonorrhoeae as a model system, we constructed mutants with single and multiple amino acid substitutions localized to this region of the pilin subunit PilE and characterized them with regard to pilin stability, organelle expression and associated phenotypes. The consequences of simultaneous expression of the mutant and wild-type PilE forms were also examined. The findings document for the first time in a defined genetic background the phenomenon of pilin intermolecular complementation in which assembly defective pilin can be rescued into purifiable Tfp by coexpression of wild-type PilE. The results further demonstrate that pilin subunit composition can impact on organelle dynamics mediated by the PilT retraction protein via a process that appears to monitor the efficacy of subunit-subunit interactions. In addition to confirming and extending the evidence for PilE multimerization as an essential component for competence for natural genetic transformation, this work paves the way for detailed studies of Tfp subunit-subunit interactions including self-recognition within the membrane and packing within the pilus polymer.