Evidence for donor strand complementation in the biogenesis of Haemophilusinfluenzae haemagglutinating pili

Haemophilus influenzae haemagglutinating pili are surface appendages that p romote attachment to host cells and facilitate respiratory tract colonizati on, 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 a ssembled by a pathway that involves a periplasmic chaperone and an outer me mbrane usher, analogous to the prototype pathway involved in the biogenesis of Escherichia coli P pili. In this study, we performed site-directed muta genesis 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 bindi ng 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 betw een HifA and HifB, we examined the interchangeability of pilus gene cluster components from H. influenzae type b strain Eagan (hifA-hifE(Eag)) and the related H. influenzae biogroup aegyptius strain F3031 (hifA-hifE(F3031)). Functional pili were assembled both with HifA(Eag) and the strain F3031 gen e cluster and with HifA(F3031) and the strain Eagan gene cluster, underscor ing the flexibility of the H. influenzae chaperone/usher pathway in incorpo rating HifA subunits with significant sequence diversity. To gain additiona l insight into the interactive surfaces of HifA and HifB, we aligned HifA s equences from 20 different strains and then modelled the HifA structure bas ed on the recently crystallized PapD-PapK complex. Analysis of the resultin g structure revealed high levels of sequence conservation in regions predic ted to interact with HifB, and maximal sequence diversity in regions potent ially 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 s trand exchange. In addition, they provide insight into the structure of Hif A and suggest a basis for antigenic variation in H. influenzae haemagglutin ating pili.