Forest K T, Bernstein S L, Getzoff E D, So M, Tribbick G, Geysen H M, Deal C D, Tainer J A
Department of Molecular Biology, Scripps Research Institute, La Jolla, California 92037, USA.
Infect Immun. 1996 Feb;64(2):644-52. doi: 10.1128/iai.64.2.644-652.1996.
The relationship between the sequence of Neisseria gonorrhoeae pilin and its quaternary assembly into pilus fibers was studied with a set of site-directed antibody probes and by mapping the specificities of antipilus antisera with peptides. Buried and exposed peptides in assembled pili were identified by competitive immunoassays and immunoelectron microscopy with polyclonal antibodies raised against 11 peptides spanning the pilin sequence. Pili did not compete significantly with pilin subunits for binding to antibodies against residues 13 to 31 (13-31) and 18-36. Pilus fibers competed well with pilin protein subunits for binding to antibodies raised against peptides 37-56, 58-78, 110-120, 115-127, 122-139, and 140-159 and competed weakly for antibodies against residues 79-93 and 94-108. Antibodies to sequence-conserved residues 37-56 and to semiconserved residues 94-108 preferentially bound pilus ends as shown by immunoelectron microscopy. The exposure of pilus regions to the immune system was tested by peptide mapping of antiserum specificities against sets of overlapping peptides representing all possible hexameric or octameric peptides from the N. gonorrhoeae MS11 pilin sequence. The immunogenicity of exposed peptides incorporating semiconserved residues 49-56 and 121-126 was revealed by strong, consistent antigenic reactivity to these regions measured in antipilus sera from rabbits, mice, and human and in sera from human volunteers with gonorrhea. The conservation and variation of antigenic responses among these three species clarify the relevance of immunological studies of other species to the human immune response against pathogens. Overall, our results explain the extreme conservation of the entire N-terminal one-third of the pilin protein by its dominant role in pilus assembly: hydrophobic residues 1-36 are implicated in buried lateral contacts, and polar residues 37-56 are implicated in longitudinal contacts within the pilus fiber.
利用一组定点抗体探针，并通过用肽定位抗菌毛抗血清的特异性，研究了淋病奈瑟菌菌毛蛋白序列与其四级组装成菌毛纤维之间的关系。通过竞争性免疫测定和免疫电子显微镜，用针对跨越菌毛蛋白序列的11种肽产生的多克隆抗体，鉴定了组装菌毛中埋藏和暴露的肽。菌毛与菌毛蛋白亚基在结合针对残基13至31（13 - 31）和18 - 36的抗体时，竞争不显著。菌毛纤维与菌毛蛋白亚基在结合针对肽37 - 56、58 - 78、110 - 120、115 - 127、122 - 139和140 - 159的抗体时竞争良好，而在结合针对残基79 - 93和94 - 108的抗体时竞争较弱。免疫电子显微镜显示，针对序列保守残基37 - 56和半保守残基94 - 108的抗体优先结合菌毛末端。通过对代表淋病奈瑟菌MS11菌毛蛋白序列中所有可能的六聚体或八聚体肽的重叠肽组的抗血清特异性进行肽图谱分析，测试了菌毛区域对免疫系统的暴露情况。在兔、小鼠和人类的抗菌毛血清以及淋病患者志愿者的血清中，对包含半保守残基49 - 56和121 - 126的暴露肽的免疫原性通过对这些区域的强烈、一致的抗原反应性得以揭示。这三个物种之间抗原反应的保守性和变异性阐明了对其他物种的免疫学研究与人类针对病原体的免疫反应的相关性。总体而言，我们的结果解释了菌毛蛋白整个N端三分之一的极端保守性，因其在菌毛组装中起主导作用：疏水残基1 - 36参与埋藏的侧向接触，而极性残基37 - 56参与菌毛纤维内的纵向接触。
