Single-Residue Changes in the C-Terminal Disulfide-Bonded Loop of the Pseudomonas aeruginosa Type IV Pilin Influence Pilus Assembly and Twitching Motility
Single-Residue Changes in the C-Terminal Disulfide-Bonded Loop of the Pseudomonas aeruginosa Type IV Pilin Influence Pilus Assembly and Twitching Motility
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DOI：
10.1128/jb.00943-09
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发表时间：
2009-11-01
影响因子：
3.2
通讯作者：
Burrows, Lori L.
中科院分区：
文献类型：
--
作者：
Harvey, Hanjeong;Habash, Marc;Burrows, Lori L.
Pila是铜绿假单胞菌IV型菌毛(T4P)的主要菌毛蛋白亚基，是其主要的结构成分。PILA有一个保守的C-末端二硫键环(DSL)，被认为是粘附素表位。结构研究表明，DSL参与了毛状肌纤维内亚单位间的相互作用。对DSL中具有单个残基替换、插入或缺失的PILA突变体进行了菌毛稳定性、菌毛组装和T4P功能测试。DSL的任一半胱氨酸残基的突变导致菌毛蛋白无法组装成纤维。ALA取代中间残基对组装或功能有一系列影响，通过表面菌毛表达和抽动运动的变化来衡量。C-末端的P-X-X-CII型β转角基序是不同物种菌毛中为数不多的高度保守的特征之一，它的修饰导致了组装和抽动运动的严重缺陷。在不能收回T4P的Pila PILT双重突变体中表达具有可疑组装缺陷的菌毛蛋白，使我们能够验证哪些亚基在物理上不能组装。使用两种不同的PilA抗体表明，与Pilin单体相比，DSL可能是完整菌毛的免疫优势表位。IVA型菌毛蛋白的序列多样性可能反映了功能保留和抗原变异之间的进化妥协。DSL序列改变的后果应该在完整的蛋白质中进行评估，因为产生DSL模拟多肽在技术上是可行的，这些突变不会出现在自然谱系中，因为它们对组装有有害影响。
PilA, the major pilin subunit of Pseudomonas aeruginosa type IV pili (T4P), is a principal structural component. PilA has a conserved C-terminal disulfide-bonded loop (DSL) that has been implicated as the pilus adhesinotope. Structural studies have suggested that DSL is involved in intersubunit interactions within the pilus fiber. PilA mutants with single-residue substitutions, insertions, or deletions in the DSL were tested for pilin stability, pilus assembly, and T4P function. Mutation of either Cys residue of the DSL resulted in pilins that were unable to assemble into fibers. Ala replacements of the intervening residues had a range of effects on assembly or function, as measured by changes in surface pilus expression and twitching motility. Modification of the C-terminal P-X-X-C type II beta-turn motif, which is one of the few highly conserved features in pilins across various species, caused profound defects in assembly and twitching motility. Expression of pilins with suspected assembly defects in a pilA pilT double mutant unable to retract T4P allowed us to verify which subunits were physically unable to assemble. Use of two different PilA antibodies showed that the DSL may be an immunodominant epitope in intact pili compared with pilin monomers. Sequence diversity of the type IVa pilins likely reflects an evolutionary compromise between retention of function and antigenic variation. The consequences of DSL sequence changes should be evaluated in the intact protein since it is technically feasible to generate DSL-mimetic peptides with mutations that will not appear in the natural repertoire due to their deleterious effects on assembly.