The Pilin N-terminal Domain Maintains <i>Neisseria gonorrhoeae</i> Transformation Competence during Pilus Phase Variation

The obligate human pathogen Neisseria gonorrhoeae is the sole aetiologic agent of the sexually transmitted infection, gonorrhea. Required for gonococcal infection, Type IV pili (Tfp) mediate many functions including adherence, twitching motility, defense against neutrophil killing, and natural transformation. Critical for immune escape, the gonococcal Tfp undergoes antigenic variation, a recombination event at the pilE locus that varies the surface exposed residues of the major pilus subunit PilE (pilin) in the pilus fiber. This programmed recombination system has the potential to produce thousands of pilin variants and can produce strains with unproductive pilin molecules that are completely unable to form Tfp. Saturating mutagenesis of the 3’ third of the pilE gene identified 68 unique single nucleotide mutations that each resulted in an underpiliated colony morphology. Notably, all isolates, including those with undetectable levels of pilin protein and no observable surface-exposed pili, retained an intermediate level of transformation competence not exhibited in ΔpilE strains. Site-directed, nonsense mutations revealed that only the first 38 amino acids of the mature pilin N-terminus (the N-terminal domain or Ntd) are required for transformation competence, and microscopy, ELISAs and pilus purification demonstrate that extended Tfp are not required for competence. Transformation in strains producing only the pilin Ntd has the same genetic determinants as wild-type transformation. The Ntd corresponds to the alternative product of S-pilin cleavage, a specific proteolysis unique to pathogenic Neisseria. Mutation of the S-pilin cleavage site demonstrated that S-pilin cleavage mediated release of the Ntd is required for competence when a strain produces unproductive pilin molecules that cannot assemble into a Tfp through mutation or antigenic variation. We conclude that S-pilin cleavage evolved as a mechanism to maintain competence in nonpiliated antigenic variants and suggest there are alternate forms of the Tfp assembly apparatus that mediate various functions including transformation.专性人类病原体淋病奈瑟菌（Neisseria gonorrhoeae）是性传播感染淋病（gonorrhea）的唯一致病原。作为淋球菌感染所必需的结构，IV型菌毛（Type IV pili, Tfp）介导诸多生理功能，包括黏附、抽动运动、抵御中性粒细胞杀伤以及自然转化。对于免疫逃逸至关重要的是，淋球菌IV型菌毛会发生抗原变异（antigenic variation）：这是一类发生于pilE基因座（pilE locus）的重组事件，可改变菌毛纤维中主要菌毛亚基PilE（pilin，菌毛蛋白）的表面暴露残基。这一程序化重组系统可产生数千种菌毛蛋白变体，还可能生成携带无效菌毛蛋白的菌株——这类蛋白完全无法组装形成IV型菌毛。对pilE基因3'端三分之一区域进行饱和诱变（saturating mutagenesis），共鉴定出68种独特的单核苷酸突变（single nucleotide mutation），每一种突变均可导致菌落呈现菌毛发育不全的形态。值得注意的是，所有分离株——即便那些菌毛蛋白水平无法检测、且未观察到表面暴露菌毛的分离株——均保留了中等水平的转化能力，而这一能力在ΔpilE（pilE缺失）菌株中并未出现。定点诱变与无义突变（nonsense mutation）实验表明，仅需成熟菌毛蛋白N端的前38个氨基酸（N端结构域，N-terminal domain, Ntd）即可赋予转化能力；显微镜观察、酶联免疫吸附测定（Enzyme-Linked Immunosorbent Assay, ELISA）以及菌毛纯化实验均证实，完整延伸的IV型菌毛并非转化能力所必需。仅表达菌毛蛋白N端结构域的菌株，其转化过程的遗传决定因子与野生型菌株完全一致。该N端结构域对应S-菌毛蛋白（S-pilin）裂解的可变产物，而这一特异性蛋白水解过程仅存在于致病性奈瑟菌属物种中。对S-菌毛蛋白裂解位点进行诱变的实验证实：当菌株因突变或抗原变异而产生无法组装为IV型菌毛的无效菌毛蛋白时，通过S-菌毛蛋白裂解释放N端结构域的过程是转化能力所必需的。本研究得出结论：S-菌毛蛋白裂解是一种在无菌毛的抗原变异株中维持转化能力的进化机制；同时提示，IV型菌毛组装装置存在多种亚型，可介导包括转化在内的多种生理功能。