Evidence for donor strand complementation in the biogenesis of Haemophilus influenzae haemagglutinating pili

Abstract

SummaryHaemophilus 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±sub-unit 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 214 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-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 gene cluster and with HifA F3031 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.

The archaic chaperone–usher pathways may depend on donor strand exchange for intersubunit interactions

“…3(a), all three mutations, G142A, V148T and F154S, abolished McuA accumulation at the time points examined. This finding argues that, as in , the highly conserved glycine upstream of the C-terminal b zipper of pilus subunits is critical for subunit polymerization (Bullitt et al, 1996;Krasan et al, 2000;Soto et al, 1998;Starks et al, 2006).…”

Structure and Function of Hib Pili fromHaemophilus influenzaeType b

“…Initial alignments of pilin sequences were performed with the program ALIGN (22). These results were compared with alignments reported by Girardeau et al (13) and Krasan et al (17), alignments that were made between HifA of different strains (13), and between HifA and a minor adapter P-pilin, PapK, not the major structural pilin PapA (17). The great strength of these reference alignments is that they have taken into account additional data beyond the primary sequence, including results from biochemical, mutational, and evolutionary studies on H. influenzae pili and E. coli pili.…”

“…Each of these pilins has sequence similarity with their respective major structural pilins (FimH with FimA, PapK with PapA) as well as similarity to HifA, the major structural pilin of the Hib pilus. The sequence similarities between HifA and PapK, in combination with morphological similarities of the helical filaments that comprise the pili, provide evidence for donor strand complementation of HifA, as seen in type 1 and P-pili (17). Structural information on the similarities and differences between assembled pili can be used to investigate the range of structural polymorphism that can be accommodated by this mechanism.…”

“…influenzae type b pili have structural and gene sequence similarities (4,11,13,17) to pili described as class I (18) but not to those in the type IV pilus superfamily. In particular, strong sequence similarity is found between Hib pilins and pilins that comprise P-pili (13,17). P-pili are expressed on the surface of uropathogenic Escherichia coli that cause pyelonephritis, urinary tract infections that involve the kidneys.…”

“…Even strains of H. influenzae with 99% identity can be immunologically distinct, and researchers have constructed chimeras to enhance reactivity to conformational epitopes, thereby defining noncontiguous residues that are structurally associated (20). Studies such as these, in combination with sequence analyses that define variable regions of pilin subunits (4,17), provide data for fitting known structures from homologous pilins into new structural data.…”