Structure and function of Hib pili from Haemophilus influenzae type b

Abstract

Pathogenic bacteria are specifically adapted to bind to their customary host. Disease is then caused by subsequent colonization and/or invasion of the local environmental niche. Initial binding of Haemophilus influenzae type b to the human nasopharynx is facilitated by Hib pili, filaments expressed on the bacterial surface. With three-dimensional reconstruction of electron micrograph images, we show that Hib pili comprise a helix 70 A in diameter with threefold symmetry. The Hib pilus filament has 3.0 subunits per turn, with each set of three subunits translated 26.9 A along and rotated 53 degrees about the helical axis. Amino acid sequence analysis of pilins from Hib pili and from P-pili expressed on uropathogenic Escherichia coli were used to predict the physical location of the highly variable and immunogenic region of the HifA pilin in the Hib pilus structure. Structural differences between Hib pili and P-pili suggest a difference in the strategies by which bacteria remain bound to their host cells: P-pili were shown to be capable of unwinding to five times their original length (E. Bullitt and L. Makowski, Nature 373:164-167, 1995), while damage to Hib pili occurs by slight shearing of subunits with respect to those further along the helical axis. This capacity to resist unwinding may be important for continued adherence of H. influenzae type b to the nasopharynx, where the three-stranded Hib pilus filaments provide a robust tether to withstand coughs and sneezes.

FIG. 1.

Alignment of HifA and PapA after accounting for data from previous studies (10, 12, 13, 17, 20, 22, 26). HifA from two strains of H. influenzae, Eagan and M43p+, have 82% identity and 95% similarity or 76% identity and 91% similarity, excluding and including, respectively, the highly variable regions of HifA. HifA/Eagan (from H. influenzae) and PapA/J96 (from E. coli) have 21% identity and 58% similarity or 17% identity and 48% similarity, excluding and including, respectively, the amino acid insertions of HifA with respect to PapA. The alignment of HifA/Eagan and PapK/J96 of Krasan et al. (17) is included.

FIG. 2.

Images of isolated, negatively stained H. influenzae type b pili. These pili are straight for short distances along the filament (black arrow in A). When damaged, Hib pili do not unwind into thin fibrillar structures but appear to be sheared perpendicular to the filament axis (black arrowhead in B). Along the length of the filament, there are regions in which a low-density channel is barely visible (white arrowhead) and regions in which the channel appears as a zigzag along the filament axis (white arrow). Bars: 100 Å (A and B), 50 Å (inset).

FIG. 3.

Images of Hib pili treated as single particles. (A) Filaments were selected and boxed as overlapping segments; shown are 36 of the ≈5,000 selected areas that were used in the data analysis. (B) The same 36 images after in-plane rotation and translational alignment to a reference particle. Bar, 100 Å.

FIG. 4.

Results of three-dimensional reconstruction of Hib pili. (A) In projection, the central channel around the helical axis has a zigzag appearance along part of its length (arrow) and regions in which the central channel is indistinct (arrowhead), as is observed in electron micrographs of negatively stained Hib pili (see Fig. 2). (B) Cross section views show the threefold symmetry about the helical axis and a central channel straight up the filament axis. Cross-sections shown are spaced 2.7 Å apart. (C) Surface of the three-dimensional reconstruction of Hib pili. (D) For comparison, the surface of the three-dimensional reconstruction of P-pili; see also reference . Bar: 50 Å (A and B), 25 Å (C and D).

FIG. 5.

Possible ways for a helix to accommodate a 20% increase in mass. (A) Initial rectangular “subunit,” oriented approximately horizontally, with radius r and rise per subunit z. The helix is viewed after slicing it down the back and laying it flat on the page. (B) The subunit could increase in thickness, thereby increasing the filament radius, r. (C) The subunit could increase in height, increasing the rise per subunit, z. (D) As appears to occur in Hib pili compared with P-pili, the subunits could tip up, making space for the additional mass without increasing z or r; the number of subunits per turn is reduced.

FIG. 6.

Three-dimensional reconstruction and model of Hib pilus. (A) Three-dimensional reconstruction computed from electron microscope data of H. influenzae type b pilus. Note the strong connections running along the left-handed three-start helix. The filament is ≈7 nm in diameter, with scalloped edges. (B) One model of the Hib pilus constructed from subunits of the homologous minor P-pilin PapK. The PapK structure was taken from PDB file 1PDK B (23) and placed on the Hib pilus helix. (C) An enlarged view of the three-dimensional reconstruction from the boxed region in A, with a cartoon of the PapK monomer included to illustrate the proposed position of the extra density of HifA compared to PapK. In our model, the extra 20% mass of HifA is seen as the surface-exposed region that extends furthest from the helical axis (arrow in C), corresponding to regions of highly variable amino acid sequence of the HifA subunit.