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Evidence for donor strand complementation in the biogenesis of Haemophilus influenzae haemagglutinating pili
G P Krasan1, F G Sauer, D Cutter
1Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine, St. Louis Children's Hospital, St. Louis, Missouri, 63110, USA.
Abstract:
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.
Insights
Haemophilus influenzae haemagglutinating pili assembly involves a chaperone (HifB) and subunit (HifA) interaction. Mutations reveal key binding sites and flexibility in the chaperone/usher pathway, explaining pilus structure and variation.
Area of Science:
- Microbiology
- Structural Biology
- Bacterial Pathogenesis
Background:
- Haemophilus influenzae haemagglutinating pili are crucial for host cell attachment and respiratory tract colonization.
- These pili possess unique two-stranded helical structures, distinct from other characterized pili.
- Their biogenesis utilizes a pathway involving a periplasmic chaperone and an outer membrane usher, similar to Escherichia coli P pili.
Purpose of the Study:
- To investigate the structure-function relationships of H. influenzae HifB chaperone and HifA pilus subunit.
- To elucidate the molecular interactions governing haemagglutinating pilus assembly.
- To explore the flexibility of the H. influenzae chaperone/usher pathway in accommodating diverse pilus subunits.
Main Methods:
- Site-directed mutagenesis of HifB and HifA at positions analogous to those in E. coli P pili.
- Analysis of HifB-HifA complex formation and HifA oligomerization.
- Interchangeability studies using pilus gene clusters from different H. influenzae strains.
- Sequence alignment of HifA from multiple strains and structural modeling based on the PapD-PapK complex.
Main Results:
- Mutations in HifB binding pocket residues or HifA penultimate tyrosine abolished HifB-HifA complex formation.
- Mutations at HifA's -14 glycine site abrogated HifA oligomerization but not HifB-HifA interaction.
- Functional pili were assembled using components from both H. influenzae and H. aegyptius strains, demonstrating pathway flexibility.
- Structural modeling revealed conserved regions for HifB interaction and diverse surface-exposed regions in HifA, suggesting a basis for antigenic variation.
Conclusions:
- The H. influenzae chaperone/usher pathway is flexible and can incorporate HifA subunits with significant sequence diversity.
- Structure-function relationships from E. coli P pili are applicable to H. influenzae haemagglutinating pili.
- Conserved and variable regions in HifA provide insights into pilus assembly and antigenic variation mechanisms.