Single-residue changes in the C-terminal disulfide-bonded loop of the Pseudomonas aeruginosa type IV pilin influence pilus assembly and twitching motility

Abstract

PilA, the major pilin subunit of Pseudomonas aeruginosa type IV pili (T4P), is a principal structural component. PilA has a conserved C-terminal disulfide-bonded loop (DSL) that has been implicated as the pilus adhesinotope. Structural studies have suggested that DSL is involved in intersubunit interactions within the pilus fiber. PilA mutants with single-residue substitutions, insertions, or deletions in the DSL were tested for pilin stability, pilus assembly, and T4P function. Mutation of either Cys residue of the DSL resulted in pilins that were unable to assemble into fibers. Ala replacements of the intervening residues had a range of effects on assembly or function, as measured by changes in surface pilus expression and twitching motility. Modification of the C-terminal P-X-X-C type II beta-turn motif, which is one of the few highly conserved features in pilins across various species, caused profound defects in assembly and twitching motility. Expression of pilins with suspected assembly defects in a pilA pilT double mutant unable to retract T4P allowed us to verify which subunits were physically unable to assemble. Use of two different PilA antibodies showed that the DSL may be an immunodominant epitope in intact pili compared with pilin monomers. Sequence diversity of the type IVa pilins likely reflects an evolutionary compromise between retention of function and antigenic variation. The consequences of DSL sequence changes should be evaluated in the intact protein since it is technically feasible to generate DSL-mimetic peptides with mutations that will not appear in the natural repertoire due to their deleterious effects on assembly.

FIG. 1.

The DSL region of PilA. Panel A (left) shows a high-confidence model of the full-length PAO1 pilin generated by the Phyre algorithm (35) based on the strain PAK pilin, PDB file 1OQW (18), with the DSL region in green and the Cys residues forming the disulfide bond in yellow. At right is a detailed view of the DSL region showing the residues mutated in this study. The figure was generated using Pymol (Delano Scientific). Panel B summarizes the mutations of the PAO1 PilA DSL that were generated for this study.

FIG. 2.

Stability, assembly, and twitching motility of mutant PilA proteins. The ability of each of the mutant pilins to complement twitching motility of the NP mutant at 0.2% arabinose was tested in an agar subsurface assay; the mean area of the twitching zones (average of at least three assays, with at least six individual zones) is shown in panel A. Strains with statistically significant differences in motility relative to the wild type (using a pairwise 2-tailed Student's t test) are indicated by an asterisk. The amount of recoverable surface pili from each strain was compared on Coomassie blue-stained SDS-PAGE gels (B) while the amount of pilin present in whole-cell lysates was determined by Western blotting with two different anti-PilA antibodies: anti-PilA.WP (C) and anti-PilA.CT (D). The levels of recoverable surface pilin (PilA) in panel B were first normalized to the amount of flagellin (FliC) in each sample and then reported as a percentage of the positive control, the complemented NP mutant (NP+PilA). (W. S. Rasband, ImageJ, U.S. National Institutes of Health, Bethesda, MD; http://rsb.info.nih.gov/ij/).

FIG. 3.

Effect of mutating D133 on pilus assembly and function. The amount of recoverable surface pili (Coomassie brilliant blue-stained 15% SDS-PAGE) (A) and whole-cell pilin levels (Western blotting with anti-PilA.CT antibodies) (B) of each of the D133 variants is shown. The levels of recoverable surface pilin (PilA) are reported as a percentage of the positive control, the complemented NP mutant (NP+PilA), as described in the legend of Fig. 2.

FIG. 4.

Effect of mutating residues in the P-X-X-C motif on pilus assembly and function. The amount of recoverable surface pili (Coomassie brilliant blue-stained 15% SDS-PAGE) (A) and whole-cell pilin levels (Western blotting with anti-PilA.CT antibodies) (B) of the P138 variants, as well as the insertion/deletion mutants of the P-X-X-C motif are shown. The levels of recoverable surface pilin (PilA) are reported as a percentage of the positive control, the complemented NP mutant (NP+PilA), as described in the legend of Fig. 2.

FIG. 5.

Expression of mutant pilins in the pilA pilT double-mutant background. The ability of mutant pilins to assemble into surface-recoverable pili when expressed in a strain unable to retract its pili was tested by comparing the levels of sheared surface pili using Coomassie brilliant blue-stained 15% SDS-PAGE gels. The levels of recoverable surface pilin (PilA) are reported as a percentage of the positive control, the complemented NP mutant (NP+PilA), as described in the legend of Fig. 2. Note that the expression of PilA in trans in the pilT background results in a twofold increase in recoverable surface pilin compared with the level recovered from a pilT mutant that expresses PilA from the chromosome.

FIG. 6.

Dominant-negative effects of expressing mutant pilins in the PAO1 wild-type strain. The ability of the mutant pilins to disrupt twitching motility in the wild type was tested by comparing motility on LB plates (white bars) versus LB supplemented with 0.2% l-arabinose plates (black bars). Induction of expression of the wild-type pilA allele in PAO1 caused a small but statistically insignificant decrease in twitching motility (using a pairwise two-tailed Student's t test), while induction of expression of any of the mutant pilins tested caused a significant decrease in motility. Assays were repeated at least three times, with at least six replicates per assay.

FIG. 7.

Congruence of PAK PilA X-ray crystal and NMR structures. The full-length (purple) and truncated (yellow) PAK pilin structures have a Cα root mean square deviation of <1 Å and are thus superimposable. Panel B shows a closer view of the DSL region of the two PilA crystal structures. In contrast, the NMR structure of a disulfide-bonded peptide whose sequence corresponds to the DSL of the PAK PilA protein (rainbow coloring) could not be fitted onto the corresponding region of the crystal structure by automated algorithms. Instead, the NMR structure was manually fitted to the crystal structure by triangulation of the alpha carbons of C129, F137 (shown in stick form for both the crystal and NMR structures), and C141. Figures were generated in Pymol (Delano Scientific) using PDB files 1DZO, 1OQW, and 1NIL (11, 18, 28).