Our current data show that Hib pili are threefold symmetric about the helical axis, with a repeat distance of ≈242 Å. It was reported previously (25
), based upon fast-freeze/deep-etch electron microscopy, that the Hib pilus contained a left-handed one-start and a right-handed two-start helix, with a crossover distance of 260 Å. How can these observations be reconciled with our current description of the Hib pilus structure? Shadowed samples provide a view of a surface-coated structure, whereas negatively stained samples provide a projection image through the protein density. The apparent one-start helix seen in shadowed images may be a visualization of the ring of density formed by filling the groove between each set of three subunits that comprise a unit cell. Three-dimensional reconstruction provides information about the protein density through the interior of a macromolecular assembly. Thus, at our current resolution of better than 20 Å, additional details are now available for a more complete understanding of the Hib pilus structure.
type b pili and P-pili are fundamentally similar; they are helical adhesive filaments extending from the outer cell membrane of pathogenic gram-negative bacteria. In both H. influenzae
and E. coli
, structural pilins are transported across the periplasm by chaperone proteins and exit via a homo-oligomeric usher protein that sits at the outer membrane. The pili each have lengths on the order of 1 μm and diameters of ≈70 Å (Fig. ). In addition, the major structural pilin for each pilus, HifA for Hib pili and PapA for P-pili, is expected to be structurally similar, and there is evidence for donor strand complementation based on morphological similarities of the pili and on sequence alignment between HifA and PapK, a minor structural P-pilin (17
As seen in structures of filamentous phages, similar monomers assembled into filaments with different symmetry can produce dramatic alterations in functional properties. For example, a difference in the packing of the capsid proteins from Pf1 (19
) versus Pf3 (27
) produces phage that differ by a factor of 2.4 in their DNA-to-protein ratios. Despite their overall similarity in length and width, Hib pili and P-pili also have significant morphological differences. For example, Hib pili are not straight for extended stretches as are P-pili, necessitating the use of new image analysis methods (7
). In addition, the amino acid sequence insertions in the HifA subunit compared with the PapA subunit must alter the resultant helical filament. That is, one cannot add mass to an object without changing it. By analogy, if a building supplier delivers bricks that are 20% larger than those sent previously, a house built with the new bricks cannot be structurally identical to one constructed with the older (smaller) bricks (Fig. ).
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 (more ...)
The extra mass of HifA compared with PapA appears to be accommodated in the Hib pilus structure by a rotation of each subunit and concomitant reduction in the number of subunits per turn (3.0 compared with 3.28). While there are a variety of ways to build a filament with larger subunits, there are experimental data restricting the possibilities for Hib pili. First, the diameter of the filament could increase, making the walls thicker (r
, Fig. ). Data do not support this possibility, as both P-pili and Hib pili have diameters of ≈70 Å. Second, the cavity within the fiber could be reduced in size. Again, data do not support this possibility; the central channel in Hib pili is 20 Å in diameter, while that in P-pili has an elliptical cross-section, with major and minor axes of 25 and 15 Å, respectively, yielding an insufficient difference in volume (<7%). Third, the rise per subunit could increase (z
, Fig. ). This does occur; there is a 7.6-Å rise per subunit for P-pili, compared with a 9.0-Å rise per subunit for H. influenzae
type b (calculated from a 26.9-Å rise per three subunits). This increase could be due to an increase in the height, along the helical axis, of each subunit. To calculate the expected change, the new height for a cylinder with constant diameter (70 Å) and central channel (20 Å) and an increased volume of 20% was determined. The increase in height would be from 24.9 Å in P-pili (2
) to 29.1 Å in Hib pili. This does not fit the existing data, as the pitch in Hib is increased compared to P-pili, but only to 26.9 Å. Fourth, the subunits may tip up, creating a space for the extra mass to be inserted at the surface (shown in Fig. ). This possibility need not change the filament radius (r
), the channel size, or the rise per subunit (z
) and is consistent with our structural data. It is also consistent with biochemical and immunochemical data from other laboratories, which predict that the insertions of HifA occur at surface-exposed regions of high sequence variability (4
This predicted addition of mass at the pilus surface is also consistent with the results of our sequence alignment between the major structural pilins of Hib pili and P-pili, HifA/Eagan and PapA/J96, respectively. With biochemical, mutational, immunologic, and evolutionary data (4
), we have produced an alignment in which the absence of cross-reactivity of antibodies to HifA amino acids 62 to 72 and 97 to 102 (20
) is correlated with insertion regions of HifA compared with PapA, and the extended hypervariable region defined by Krasan et al. (17
) corresponds to the largest insertion region. This region is expected to be surface exposed. While it is not yet possible to define the boundaries between individual subunits in our reconstruction, our data suggest the location of the large HifA insert to be the mass extending farthest from the helical axis, as can be seen modeled in Fig. .
What are the structural consequences of Hib pili having threefold symmetry, with subunits oriented approximately vertically along the filament axis? Both Hib and P-pili have about 3 subunits per turn of their helix; Hib pili contain 3 subunits in each turn, and P-pili contain 3.28 subunits per turn. Thus, only Hib pili comprise a structure with threefold symmetry at all positions along the helical axis. An observed consequence of this difference in symmetry and differences between the pilin subunits (HifA compared to PapA) is that in P-pili the predominant interactions are along the one-start helix, forming a coil that can be unwound under stress. In Hib pili the predominant interactions are along each of three interwound helices. It is therefore not possible to unwind the Hib pilus into a comparable single fibrillar structure. The Hib filament can be described by imagining three elastic ropes wound about each other, each connected to the others by weak cross-bridges. If the cross-bridges begin to peel apart, separating one rope from the other two, that rope may bulge out but will not easily become a thin distinct fiber; this would require either that the damage be propagated all the way to the end of the filament or that the strand be severed, conditions that were not observed in images of thousands of Hib pili.
Overextension of the helical filament into thin fibrillae, as shown for P-pili (1
), was not observed in Hib pili. The comparative velocity of flow in each host environment provides a plausible argument for this structural difference. A cough or a sneeze can reach a velocity of 150 km/h, whereas urine flows at ≈0.002 km/h and mucociliary clearance occurs at ≈0.0005 km/h. Continued adherence of H. influenzae
to the nasopharynx most likely occurs in niches about which there is no detailed information, whereas initial adherence may occur in exposed, relatively unprotected locations. The bacteria must then maintain contact long enough to create or locate a more protected environment. Consequently, while typical environmental velocities in the urinary tract and the nasopharynx differ by a factor of only about 10, intermittent coughs and sneezes may result in an extreme environment for H. influenzae
to withstand. The precise shear pressures have not yet been calculated for these two environments, but it seems likely that an extended thin fiber 20 Å in diameter could not survive velocities 75,000 times greater than those experienced in the urinary tract.
We hypothesize that the combination of a three-stranded helix and orientation of the HifA subunits almost vertically along the helical axis provide the structural stability necessary for Hib pili to survive in the nasopharynx prior to invasion of host cells. Thus, while P-pili and Hib pili have an overall morphology of ≈70-Å-diameter helical filaments with a length on the order of 1 μm, the structure of each pilus type is specifically adapted to its local environmental niche.