Molecular basis of usher pore gating in Escherichia coli pilus biogenesis

Screen of PapC PLUG Residues Involved in Pore Gating.

Based on the crystal structure of the PapC TD/PLUG (3), residues were identified for mutagenesis to investigate the pore gating function of PapC. Given the close proximity of the PLUG domain to the β-barrel lumen and the luminal α-helix in the apo PapC structure (3) (Fig. 1A), we hypothesized that residues at these interfaces would be involved in positioning of the PLUG in the inactive usher. PLUG residues Q279, R303, and R305 contain charged side chains that extend from the PLUG domain toward a luminal TD β5–6-hairpin loop (Q279) and a luminal α-helix (R303 and R305). P289 lies at the opposite side of the PLUG from Q279, R303, and R305 and forms a kink between two β-strands within the PLUG domain. D234, which resides within the β5–6-hairpin loop, and E467, which extends from the base of the α-helix, both extend toward the PLUG domain. V322 is situated in the core of the PLUG domain facing the PLUG’s own β-strands. Mutations were made in each of these residues to investigate their role in pore gating. Mutation of residue V322 was carried out to investigate any effects of internal changes to the PLUG domain β-strand packing on pore gating. In addition, deletions of the entire PapC PLUG domain, β5–6-hairpin loop, and α-helix were constructed.

Gram-negative bacteria are normally resistant to the antibiotic Erm owing to their OM impermeability (26). Thus, an Erm sensitivity assay was used to detect changes in OM permeability and to test the impact of mutated residues on the pore gating function of the PLUG domain. Expression of WT PapC from pTrc-PapC_His6 in C600 cells caused an increase in Erm sensitivity [∼0.5–0.6 cm inhibition zone radius (izr)], compared with the empty vector pTrc99a control (∼0.2 cm izr), likely owing to spontaneous opening and closing of the PLUG domain that was observed in planar lipid bilayer electrophysiology studies (25). Most of the PapC point mutants had Erm inhibition zones similar to WT levels (∼0.5–0.6 cm izr) (Fig. 1B). The most drastic effect on pore permeability was observed when the PLUG domain was deleted (∼1.2 cm izr) (Fig. 1B). Deleting the α-helix and β5–6-hairpin loop significantly increased pore permeability (∼0.8–0.9 cm izr and ∼0.7 cm izr; P = 0.0048 and P = 0.013 by Mann–Whitney test, respectively) compared with the WT PapC, indicating their importance in stabilizing the PLUG domain in the closed position in the PapC β-barrel, as already suggested from electrophysiology (25). The R305A PLUG mutant had the most significant effect on pore gating of a single amino acid change (∼0.8–0.9 cm izr), suggesting that this residue plays a critical role in stabilizing the PLUG within the TD pore. The effect of the R305A PapC point mutant is comparable to that of deleting the entire α-helix and slightly larger than deleting the β5–6 hairpin (P = 0.053, Mann–Whitney test). Similar results were obtained with an R305P mutation. Mutation of a neighboring arginine residue, which also extends from the PLUG toward the TD, to alanine (R303A) did not cause any significant changes to Erm sensitivity (Fig. 1B), highlighting the specificity of residue R305 in regulating pore gating. Further, creating the double-mutant R305A+R303A did not further increase Erm sensitivity (Fig. 1B), indicating that R305 is a key residue involved in stability of the PLUG domain in the PapC pore. The OM protein expression levels of the PapC point mutants and deletion constructs are shown in Fig. S1.

P pili agglutinate human erythrocytes owing to the ability of the tip adhesin, PapG, to bind to Galα1–4Gal on their surface, providing a measurement of the presence of functional pili. Despite causing an open PapC pore, the R305 point mutants did not negatively affect functional pilus biogenesis (Table S1), indicating that the pore gating function of the PLUG domain is distinct from its involvement in the catalysis of pilus assembly. Importantly, the Erm sensitivity phenotypes of the PapC point mutants R305A, R305A+R303A, and R305P were reversed under pilus-inducing conditions, likely owing to mature/polymerizing pili blocking the PapC pore and preventing entry of the antibiotic Erm. However, the PapC PLUG deletion mutant remained Erm-sensitive, consistent with its inability to assemble pili. Interestingly, α-helix and β5–6-hairpin loop deletion constructs remained Erm-sensitive even under pilus-inducing conditions (Fig. 1C). This may suggest that the α-helix and β5–6-hairpin loop are involved in retaining pili on the cell surface or that their deletion may cause a change in the occluded shape/size of the transmembrane barrel such that Erm can still be transported even when a polymerizing pilus fiber is transiting the channel.

Electrophysiology Experiments Suggest a Possible Destabilization of the PLUG Domain in the R305A Mutant.

As previously documented (25), the WT PapC channel is characterized by many short openings of various sizes, seen as downward spikes in (Fig. 2A). Few of these transitions have a conductance greater than 1 ns, and those that do are extremely transient (a few milliseconds). These small transitions have been interpreted as originating from the “jiggling” of the PLUG domain and/or the dynamic interference of the N- and C-terminal domains with the ion conduction pathways identified at the interface of the PLUG and the TD. However, in 7 out of 10 experiments performed on R305A PapC the channels displayed occasional large transitions that lasted for several seconds (3–30 s) (Fig. 2B), in addition to the openings of small conductance similar to those of the WT channel. These large openings have a conductance of ∼4 ns, which is similar to the monomeric conductance of the PLUG-less PapC mutant (25), and thus could represent PLUG release. Just as was observed for the PLUG-less mutant (25), numerous flickers to lower conductance levels occur within the large openings, probably representing a combination of the dynamic behavior of the released PLUG and the mobility of various other elements (loops or the β-barrel itself) within the conductance pathway. Therefore, although the behavior is not consistently observed in every sweep, the R305A channels seem to be more prone to spontaneously adopt a state in which the PLUG domain has been displaced and the pore is fully open for a significant amount of time.

Salt Bridge Between the Usher PLUG and α-Helix Is Important for Stable Positioning of the PLUG in the PapC Pore.

In the PapC crystal structure, the PLUG residue R305 forms a salt bridge with residue E467, which extends from the base of the α-helix toward the PLUG domain (Fig. 1A). An E467A mutation increased Erm sensitivity equivalent to R305A or deletion of the α-helix, suggesting that much of the α-helix’s contribution to PLUG’s positioning in the pore is mediated through this salt bridge (Fig. 3). Consistent with this hypothesis, mutation of R305 to the acidic residue glutamate similarly increased Erm sensitivity (∼0.8–0.9cm izr), suggesting that the observed changes in pore gating are likely due to charge–charge interactions. We thus switched the locations of the charged residues by creating the R305E+E467R double mutant. This double mutant restored Erm sensitivity to WT levels (∼0.5–0.6 cm izr) (Fig. 3), revealing that reestablishment of the charge–charge interaction between these two residues restores stability of the PLUG in the closed usher state.

Usher Periplasmic Loop β12–13 Is Involved in Pore Gating and Pilus Assembly.

In the crystal structure of the FimD usher in complex with the FimCH chaperone-adhesin complex, the usher periplasmic loop residues S421 and L422, found between FimD’s β-barrel strands 12 and 13, stack against the PLUG domain, suggesting that translocation of the PLUG to the periplasm may be stabilized by these interactions. To test this hypothesis, we replaced the mature PapC β12–13 loop residues 430–433 with a glycine and expressed the mutant PapC from the arabinose-inducible plasmid pNH305. This resulted in a decreased Erm inhibition zone radius (∼0.1–0.2 cm izr) compared with the corresponding WT PapC, which had an izr of ∼0.6, indicating a reduction in the open conformation of the PLUG. This effect was not due to a defect in PapC expression because immunoblots of isolated OM carrying WT PapC or β12–13 loop mutant with anti-PapC antisera revealed similar expression levels (Fig. 4C). The mutation did not affect the ability of the usher to form a stable β-barrel in the OM as determined by heat-modifiable mobility assay for β-barrel stability (27) (data not shown). This mutation decreased the efficiency of P pilus biogenesis as determined by HA titers (Fig. 4D) and by analysis of pilus preparations, which showed reduced levels of polymerized major pilus rod subunit PapA (Fig. S2). Our results suggest that the usher periplasmic β12–13 loop is important to open the PLUG and/or stabilize it in an open state required for efficient pilus assembly.

Usher PLUG Domain and NTD Work in Synergy.

Biophysical studies suggested that the translocation of the PLUG to the periplasm, where it subsequently serves to recruit further chaperone-subunit complexes, is stabilized by an interaction with NTD (23). In the FimDCH crystal structure, NTD residue T114 and PLUG residue P281 are in close proximity (5) (Fig. 5A). Thus, we probed the NTD/PLUG interaction by mutating these residues to cysteines, hypothesizing that this would potentiate an open conformation. The point mutants did not affect FimD OM expression levels as determined by immunoblotting with anti-FimD antisera (Fig. 5D). However, expression of the NTD and PLUG double cysteine mutant (P281C+T114C) resulted in a dramatic increase in Erm sensitivity (∼1 cm izr) (Fig. 5B) compared with the WT or a P281C FimD point mutant (∼0.5–0.6 cm izr), demonstrating that the cysteine mutations resulted in an usher with a greater propensity to exist in an open pore conformation. Thereafter, we investigated the ability of P281C + T114C FimD to assemble pili. Despite the increase in pore permeability, the P281C + T114C FimD was able to assemble pili as determined by mannose-sensitive HA assays, similar to WT FimD (Fig. 5C). These results further suggest that the pore gating function of the PLUG domain can be decoupled from its function in catalysis.

Anchored, Mature Pili Are Necessary to Retain OM Integrity of E. coli.

Previous studies revealed the importance of PapH (the terminator subunit of P pili) in anchoring and terminating P pili at the OM (18, 28). Biophysical studies further revealed that the PapDH complex was specifically recruited by the PLUG domain or the intramolecular NTD/PLUG complex but did not bind to CTD2 (23). This suggested that PapH may anchor pili on the cell surface via interaction with the usher’s PLUG domain (23). Thus, we hypothesized that the PapDH–PLUG interaction may result in PLUG positioning that has ramifications on OM impermeability and thus deletion of PapH would affect OM permeability. We investigated this hypothesis by expressing the pap operon lacking papH on plasmid pEMM13 in MC1061 cells and providing either papH or an empty vector control in trans (pEVpapH and pTrc99a plasmids, respectively) in MC1061 cells and then analyzing Erm sensitivity. EM analysis showed that the papH deletion resulted in long pili that were shed, whereas providing papH in trans using pEVpapH plasmid restored assembled pili to their expected length and abundance (Fig. 6B). Erm sensitivity assays revealed that deletion of papH resulted in a significant increase in Erm sensitivity compared with WT (∼0.9 cm izr vs. ∼0.4 cm izr, respectively) (Fig. 6A). Thus, incorporation of the PapH terminator results in maintainence of OM impermeability, likely owing in part to preventing the dissociation of pili from the cell surface and leaving behind an activated, open pore (23).

Bacterial Strains, Plasmids, and Culture Conditions.

Bacterial strains were grown in LB agar, Tryptic Soy Agar (TSA), or LB broth with antibiotics as appropriate. Detailed strain and plasmid information is provided in Table S2.

Erm Sensitivity Assays.

Erm sensitivity was determined using disk diffusion assays on agar plates using discs containing Erm (15 μg; BD Biologicals). SI Materials and Methods gives further details.

HA Titer Assays.

HA titers for type 1 pili were carried out using cells after 2 × 24 h of 10 mL static bacterial growth with appropriate antibiotics as described previously. HA titers for P pili were performed on bacteria grown on TSA plates overnight as previously described (35).

Pili and protein expression/purification, electrophysiology, and pili assays were done as previously described (23, 25, 36). Details can be found in SI Materials and Methods.