Krasan G P, Sauer F G, Cutter D, Farley M M, Gilsdorf J R, Hultgren S J, St Geme J W
Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine, St. Louis Children's Hospital, St. Louis, Missouri, 63110, USA.
Mol Microbiol. 2000 Mar;35(6):1335-47. doi: 10.1046/j.1365-2958.2000.01816.x.
Haemophilus influenzae haemagglutinating pili are surface appendages that promote attachment to host cells and facilitate respiratory tract colonization, an essential step in the pathogenesis of disease. In contrast to other well-characterized forms of pili, H. influenzae haemagglutinating pili are two-stranded helical structures. Nevertheless, haemagglutinating pili are assembled by a pathway that involves a periplasmic chaperone and an outer membrane usher, analogous to the prototype pathway involved in the biogenesis of Escherichia coli P pili. In this study, we performed site-directed mutagenesis of the H. influenzae HifB chaperone and HifA major pilus subunit at positions homologous to sites important for chaperone-subunit interactions and subunit oligomerization in P pili. Mutations at putative subunit binding pocket residues in HifB or at the penultimate tyrosine in HifA abolished formation of HifB-HifA periplasmic complexes, whereas mutations at the -14 glycine in HifA had no effect on HifB-HifA interactions but abrogated HifA oligomerization. To define further the constraints of the interaction between HifA and HifB, we examined the interchangeability of pilus gene cluster components from H. influenzae type b strain Eagan (hifA-hifEEag) and the related H. influenzae biogroup aegyptius strain F3031 (hifA-hifEF3031). Functional pili were assembled both with HifAEag and the strain F3031 gene cluster and with HifAF3031 and the strain Eagan gene cluster, underscoring the flexibility of the H. influenzae chaperone/usher pathway in incorporating HifA subunits with significant sequence diversity. To gain additional insight into the interactive surfaces of HifA and HifB, we aligned HifA sequences from 20 different strains and then modelled the HifA structure based on the recently crystallized PapD-PapK complex. Analysis of the resulting structure revealed high levels of sequence conservation in regions predicted to interact with HifB, and maximal sequence diversity in regions potentially exposed on the surface of assembled pili. These results suggest broad applicability of structure-function relationships identified in studies of P pili, including the concepts of donor strand complementation and donor strand exchange. In addition, they provide insight into the structure of HifA and suggest a basis for antigenic variation in H. influenzae haemagglutinating pili.
流感嗜血杆菌血凝菌毛是一种表面附属物，可促进与宿主细胞的附着并有助于呼吸道定植，这是疾病发病机制中的关键步骤。与其他特征明确的菌毛形式不同，流感嗜血杆菌血凝菌毛是双链螺旋结构。然而，血凝菌毛是通过一种涉及周质伴侣蛋白和外膜组装分子的途径组装而成的，这类似于大肠杆菌P菌毛生物合成中涉及的原型途径。在本研究中，我们对流感嗜血杆菌HifB伴侣蛋白和HifA主要菌毛亚基进行了定点诱变，诱变位置与P菌毛中对伴侣蛋白-亚基相互作用和亚基寡聚化重要的位点同源。HifB中假定的亚基结合口袋残基或HifA中倒数第二个酪氨酸处的突变消除了HifB-HifA周质复合物的形成，而HifA中-14位甘氨酸处的突变对HifB-HifA相互作用没有影响，但消除了HifA的寡聚化。为了进一步确定HifA和HifB之间相互作用的限制因素，我们研究了b型流感嗜血杆菌伊根菌株（hifA-hifEEag）和相关的埃及流感嗜血杆菌生物群F3031菌株（hifA-hifEF3031）菌毛基因簇组件的互换性。用HifAEag和F3031菌株的基因簇以及用HifAF3031和伊根菌株的基因簇都能组装出功能性菌毛，这突出了流感嗜血杆菌伴侣蛋白/组装分子途径在整合具有显著序列多样性的HifA亚基方面的灵活性。为了进一步深入了解HifA和HifB的相互作用表面，我们比对了2０种不同菌株的HifA序列，然后基于最近结晶的PapD-PapK复合物对HifA结构进行建模。对所得结构的分析表明，在预测与HifB相互作用的区域中序列保守性很高，而在组装菌毛表面可能暴露的区域中序列多样性最大。这些结果表明，在P菌毛研究中确定的结构-功能关系具有广泛的适用性，包括供体链互补和供体链交换的概念。此外，它们为了解HifA的结构提供了线索，并为流感嗜血杆菌血凝菌毛的抗原变异提供了依据。
