PilMNOPQ from the Pseudomonas aeruginosa Type IV Pilus System Form a Transenvelope Protein Interaction Network That Interacts with PilA

Bacterial strains.

Table S1 in the supplemental material summarizes the bacterial strains and vectors used in this study. The pET-Duet and pET vectors were transformed by heat shock into chemically competent Escherichia coli.

Western blot analysis.

All protein samples analyzed by Western blotting were mixed with 2× SDS-PAGE sample buffer at a 1:1 ratio, boiled for 10 min, separated on 16% SDS-PAGE minigels, and transferred to polyvinylidene difluoride (PVDF) membranes. Proteins of interest were detected using the purified rabbit polyclonal antibodies to PilMNOPQA (8). The primary antibodies were diluted in TBST (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% [vol/vol] Tween 20). The secondary anti-rabbit or anti-mouse antibodies conjugated to alkaline phosphatase were used per the manufacturer's instructions (Bio-Rad), and the blots were developed with nitroblue tetrazolium/5-bromo-4-chloro-3-indoyl-phosphate (NBT/BCIP) from BioShop Canada Inc. Detection of the biotinylated PilN_N20 peptide was performed using the streptavidin-horseradish peroxidase-conjugated reagent (Strep-HRP) from GenScript in TBST, followed by detection of HRP using the Super Signal West Pico chemiluminescent substrate from Pierce (Thermo Scientific).

Construct generation and peptide synthesis. (i) PilN N-terminal peptides.

The peptides encompassing the N-terminal 20 amino acids of PilN, MARINLLPWREELREQRKQQ (PilN_N20; 95% pure and reconstituted in water at 1 mg/ml) and the Asp5-to-Ala variant (PilN_N20_N5A; 86% purity) were synthesized by GenScript. For both wild-type and mutant PilN_N20 peptides, a lysine residue and a mini-PEG linker were added at the C terminus to facilitate the addition of a biotin molecule.

(ii) Generation of heterologous protein expression vectors.

The generation of the coexpression vector that produces N-terminally 6×His-tagged PilN_Δ44 and untagged PilO_Δ51 (PilN_Δ44/PilO_Δ51), as well as the expression vectors for the 6×His-tagged proteins PilO_Δ43 and PilM or the untagged PilP_Δ18 (PilP_Δ18T7), were reported previously (8, 11, 12). Details about these vectors are shown in Table S1 in the supplemental material. The untagged versions N- and C-terminal constructs of PilP (PilP_18-84T7 and PilP_{Δ71_TAAT}, respectively) were generated using PilP_Δ18T7 as a template to amplify the appropriate regions and subsequently ligated into the BamI and XhoI sites of pET24a. The PilQ expression constructs were amplified with specific primers containing a 5′ NcoI site and a 3′ XhoI site. Purified PCR fragments were digested and ligated into an appropriately digested pET28a vector. Clones were verified by sequencing (The Centre for Applied Genomics [TCAG] or the Analytical Genetics Technology Centre [AGTC], Toronto, Ontario, Canada).

Protein expression.

PilM, PilN_Δ44/PilO_Δ51, PilP_Δ18, PilP_Δ71, and the PilQ constructs used in pulldown studies were expressed in E. coli. The pET vectors containing the genes of interest were transformed into BL21 Codon Plus cells (Stratagene) and plated on LB plates supplemented with 50 mg/ml kanamycin. One colony was then used to inoculate 5 ml LB containing the appropriate antibiotic (termed LB-antibiotic). Each overnight culture was used to inoculate 500 ml of LB-antibiotic, and the cells were grown at 37°C until an optical density at 600 nm (OD₆₀₀) of 0.6 to 0.7, when protein expression was induced by adding isopropyl-β-d-thiogalactopyranoside (IPTG) to a final concentration of 1.0 mM. The cells were allowed to grow either overnight at 18°C or for 4 h at 37°C prior to being harvested by centrifugation (7,700 × g, 30 min, 4°C). Cell pellets were stored at −20°C until required.

Bioinformatics analyses. (i) PilQ domain identification.

Sequence alignments were performed by T-Coffee (25). Initially the sequences for the T2SS and T4P secretins were aligned separately. These individual alignments were subsequently combined using the COMBINE algorithm in the T-Coffee suite and then manipulated manually to refine the alignment in JalView (26). Secondary structure predictions were performed by JnetPRED (27) in the JalView alignment editor program (26). Figures for sequence alignments were produced in JalView. Structural analysis and alignments were performed using PyMOL (28).

(ii) PilP and GspC structural comparison.

Sequence alignments were performed as described in reference 12, and structural superpositions were performed and displayed using PyMOL (28).

PilM copurification with PilN_N20 and PilN_N20_N5A peptides.

N-terminally 6×His-tagged PilM (PilM_Nt6×His) was copurified with PilN_N20 peptides using Ni MagBeads (GenScipt). Initially, 0.2 g of dry PilM_Nt6×His pellet was solubilized in 4 ml Easy BacLysis buffer (GenScript) and 2 ml buffer A (20 mM Tris-HCl, pH 7.5, 150 mM NaCl) with 100 mg/ml lysozyme, 10 mg/ml DNase, and 10 mg/ml RNase. Cells were lysed by incubation for 1 h at 4°C on a rotator. Subsequently, 2 ml of cell lysate solution was added to each of the three falcon tubes containing 100 μl of Ni MagBeads preequilibrated in lysis buffer. After mixing for 1.5 h at room temperature on a rotator, the falcon tubes were inserted in the magnetic base and the supernatant was discarded. The MagBeads with bound PilM_Nt6×His were then incubated with either 1 ml of buffer A alone (control) or 1 ml buffer A and 100 μl of 1 mg/ml wild-type PilN_N20 or mutant PilN_N20_N5A peptide (freshly made solutions of lyophilized synthetic peptides in water). The control experiments of both PilN peptides in the absence of PilM were set up in a similar manner. After an overnight incubation at 4°C on a rotator, the Ni-NTA beads were washed in a batch-wise manner using the magnetic base, and the 1.1-ml flowthrough, together with 3-, 3-, 0.5-, 0.5-, and 0.5-ml elute fractions, containing 10, 30, 75, 150, or 400 mM imidazole, respectively, were collected and analyzed by SDS-PAGE and Western blotting using both anti-PilM antibody and a Strep-HRP reagent.

PilN/O and PilP N-terminal pulldowns.

The expression, lysis, copurification, and analysis of pulldowns were performed as described in Tammam et al. (12) for the PilN_Δ44/PilO_Δ51/PilP_{Δ18_6×His} cofractionation. In brief, the pET28a-pilP_Δ18-6His and pET-Duet-pilN_Δ44-pilO_Δ51 expression vectors were transformed separately into E. coli BL21(DE3) cells (Novagen). Overnight cultures were used to inoculate 1 liter of LB-kanamycin, and cells were grown at 37°C until an OD₆₀₀ of 0.6 to 0.7, when protein expression was induced by adding IPTG to a final concentration of 1 mM. The cells were grown for 4 h after induction at 37°C, harvested by centrifugation, and stored at −20°C until required. The frozen cells were thawed, resuspended, and then mixed together. The cells were lysed by homogenization, and the cellular debris was removed by centrifugation. The cell lysate was mixed with 2 ml of Ni-agarose (Qiagen) at 4°C for 2 h and then packed into a column and washed twice with 20 ml of Tris buffer containing, successively, 10 and 30 mM imidazole. Bound protein was eluted from the column in three 10-ml batches with Tris buffer and 75, 150, and 300 mM imidazole, respectively. The 75 mM imidazole fraction was subsequently concentrated to 2 ml (Amicon unit with 10-kDa cutoff; Millipore), to a final protein concentration of 10 to 15 mg/ml. The concentrated protein sample was applied to a Superdex S-200 column (GE Health) and eluted using 20 mM Tris-HCl, pH 7.5, and 150 mM NaCl.

PilQ pulldown experiments.

All proteins were overexpressed as described above. Each protein construct was expressed individually, and appropriate cell pellets were then mixed prior to lysis. PilP_Δ18, PilP_Δ71, PilN_Δ44/PilO_Δ51, PilQ_24-445, PilQ_24-280, PilQ_281-445, PilQ_281-410, and PilQ_281-376 were used in 0.5-liter aliquots, while 1-liter aliquots of PilQ_377-445 were used.

Individual cell pellets were initially resuspended in 10 ml of lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM imidazole, 1 mM phenylmethylsulfonyl fluoride [PMSF], 0.1 mg/ml DNase, RNase, lysozyme, and protease inhibitor cocktail [Sigma]). After mixing the appropriate resuspended pellets, cells were lysed by homogenization using an Avestin EmulsiFlex-C3 by passing the lysate three times through the machine at 15 to 20 kpsi. The insoluble debris was removed by centrifugation (39,000 × g, 30 min, 4°C). The cell lysate was then loaded on a preequilibrated Ni-NTA column, and the flowthrough was collected and loaded a second time on the column. The column was washed with 30 ml of buffer A containing 5 mM imidazole and a second wash of 10 ml buffer A with 30 mM imidazole. Bound protein was eluted from the column in three 10-ml batches with buffer A with 75, 150, and 300 mM imidazole, respectively. Fractions were analyzed by SDS-PAGE and Western blot analysis with PilN, PilO, PilP, and 6×His specific antibodies as appropriate.

Negative controls for PilP_{Δ18_TAAT}, PilP_{Δ71_TAAT}, and PilN_Δ44/PilO_Δ51 were run, where soluble lysate of cells overexpressing these constructs was loaded onto a Ni-NTA column in the absence of PilQ. Purification was carried out as described above, and the eluted fractions were analyzed by SDS-PAGE.

PilA pulldown assays.

Cell pellets containing expressed PilQ_24-445, PilQ_24-280, and PilQ_281-445 were resuspended in 10 ml lysis buffer (as described above). Cells were lysed using an Avestin EmulsiFlex-C3 (as described above), and cellular debris was then removed by centrifugation (39,000 × g, 30 min, 4°C). The soluble supernatant was mixed with 1 ml Ni-NTA agarose (Qiagen), preequilibrated in buffer A, for 2 h at 4°C. The beads were collected by centrifugation, with the supernatant fraction being the flowthrough. The beads were washed once with 10 ml buffer A for 1 h at 4°C. The beads were again collected by centrifugation, resulting in Ni-NTA beads with prebound proteins that were subsequently used to pull down PilA from PAO1 lysates prepared as described below.

PAO1 cell lysates were prepared by growing cells streaked in a grid pattern on LB agar plates overnight at 37°C. Cells were collected by scraping them off the surface, and cell pellets were lysed in 6 ml Easy BacLysis and 14 ml buffer A with 100 mg/ml lysozyme, 10 mg/ml DNase, and 10 mg/ml RNase. Cell pellets were homogenized by brief vortexing and incubation at room temperature for 30 min on a rotator. Cellular debris was then removed by centrifugation (39,000 × g, 30 min, 4°C), and 4 ml of the cell lysate supernatant was applied to the Ni-NTA beads charged with specific His-tagged protein as described above.

The PAO1 cell lysates and Ni-NTA beads prebound with PilQ_24-445, PilQ_24-280, and PilQ_281-445 were incubated at 4°C overnight. The beads were collected by centrifugation, while the supernatant contained the flowthrough fraction. The resin was washed twice using a batch method (mixed for 1 h on a rotator at 4°C and centrifuged to pellet the beads and then remove the supernatants) with 10 ml buffer A containing, successively, 10 and 30 mM imidazole, and the bound proteins were eluted from the column in three 2-ml batches, containing buffer A and 75, 150, or 400 mM imidazole, respectively. The identity of each protein was confirmed by Western blotting of the flowthrough and 400 mM fractions using PilN-, PilO-, PilP-, and PilA-specific antibodies.

The conserved N terminus of PilN is important for T4P function and interacts with PilM in vitro.

We previously identified the PilN N-terminal sequence I₄N₅L₆L₇P₈ as a signature sequence for PilN family members and predicted that this motif would be involved in interactions with PilM (11). To test this hypothesis, we examined the interaction of P. aeruginosa PilM and PilN using N-terminally tagged PilM (PilM_Nt6×His) and a biotinylated peptide of PilN that contains the first 20 N-terminal residues of the protein conjugated via a C-terminal lysine and a mini-PEG linker to biotin (PilN_N20). This 2.5-kDa peptide copurified with PilM_Nt6×His using either nickel (Fig. 1) or streptavidin affinity chromatography (see Fig. S1 in the supplemental material). When HRP-conjugated streptavidin was used to probe the fractions from the nickel affinity purification, the biotinylated PilN_N20 peptide was recovered in the 10, 30, and 75 mM imidazole washes at ∼40 kDa. Western blot analysis with an anti-6×His antibody confirmed that these bands correspond to PilM (Fig. 1A). In the presence of PilM, the PilN_N20 peptide appeared at the approximate molecular mass of PilM, indicating that this peptide forms a high-affinity interaction with PilM_Nt6×His that is not disrupted by SDS. In the absence of PilM, some peptide bound nonspecifically to the column (Fig. 1A), and under these conditions, the peptide eluted at ∼2.5 kDa. However, when PilM was present, the levels of PilN_N20 in the high imidazole fractions were greatly diminished, presumably because most of the peptide is bound to PilM. Compared to the purification profile of PilM in the absence of the PilN peptide (Fig. 1C), it is clear that the addition of the peptide does not affect binding of PilM to the column. Together, these data suggest that PilN_N20 binds to PilM in the presence of SDS.

The effects of point mutations at the highly conserved Asn5 (N5A/Q/D) of PilN and a series of N-terminal deletions (Δ2-9, Δ10-18, and Δ4-17) were tested in vivo by complementation of a P. aeruginosa pilN mutant (pilN::FRT). Constructs expressing the PilN_N5A variant or deletion constructs lacking residues 2 to 9 or 4 to 18 did not restore twitching motility, phage sensitivity, or surface PilA in a pilN mutant (see Fig. S2 in the supplemental material). Other deletions and site-specific mutations decreased T4P biogenesis and function to various degrees (see Fig. S2). Mutations that prevent association of PilM and PilN in vivo were previously shown to lead to degradation of PilNO (8), consistent with the phenotypes seen here. When the pulldown experiments were repeated with a peptide containing the N5A mutation (PilN_N20_N5A), PilM was still pulled down (Fig. 1B), despite the in vivo data suggesting that this variant was nonfunctional (see Fig. S2) (23). Together, these data suggest that Asn5 in PilN plays an additional role in communicating a signal from the cytoplasm through PilN to the periplasmic components, or that the binding of PilM to the PilN peptide in vivo is influenced by other proteins or protein interactions that may be present.

The N terminus of PilP is sufficient for the interaction with PilNO.

Previously, we showed that full-length PilP (minus its signal sequence) interacted with a stable complex of PilNO, and that the C-terminal β-sandwich domain of PilP alone could not bind PilNO (12). To directly probe the potential interaction of the N terminus of PilP with PilNO, a construct corresponding to the unstructured domain of PilP (PilP_19-84T7; 8.9 kDa) was used. Under the conditions used previously to characterize the interaction of mature PilP (PilP_{Δ18_6×His}) with the periplasmic PilNO (PilN_Δ44/PilO_Δ51) heterodimer (12), the PilP N-terminal construct cofractionated with PilNO, and this interaction was stable during size-exclusion chromatography (Fig. 2). Although the N-terminal PilP fragment was unstable when expressed in the absence of PilNO (see Fig. S3 in the supplemental material), it was protected from proteolytic degradation when in complex with PilNO. Examination of the PilNOP_19-84T7 complex by limited proteolysis suggested that the protected region of PilP spans residues 19 to 76, a region that our bioinformatics analysis predicted to be entirely disordered (12). These data support our hypothesis that the N terminus of PilP mediates the interaction with PilNO (12).

An important structural difference between PilP and T2SS GspC HR region.

Our earlier work highlighted a pair of highly conserved residues in PilP, Pro85 (found in the N-terminal disordered region) and Trp161 (found on β7), which interact and tether the N-terminal disordered region to the β-sandwich domain in solution (12). The recent release of two structures of the HR domain of the T2SS protein GspC from E. coli (GspC_Ec; Protein Data Bank [PDB] code 3OSS) and Dickeya dadantii 3937 (GspC_Dd; PDB code 2LNV) allowed us to extend our analysis of the PilP structure. The structures of PilP_Δ71 and GspC_Ec or GspC_Dd can be superimposed with root-mean-squared deviations of 0.92 and 1.5 Å over 51 and 43 C^α atoms, respectively (Fig. 3). The β-sandwich domains are very similar, but β-strands 6 and 7 in the GspC proteins are each two residues shorter than those in PilP (Fig. 3). Notably, this β6-β7 extension is the site of the highly conserved Trp residue (Trp161 in PilP) found in all PilP proteins. There is no equivalent conserved aromatic residue in the GspC family (data not shown). Mutation of Pro85 to either Ala or Glu, or the mutation of residues in the β6-β7 extension (Gly157 and Leu162), impaired P. aeruginosa twitching motility (see Fig. S4 in the supplemental material), indicating that this region of the protein is important for T4P biogenesis. However, in the case of the Pro85Val mutation, twitching was not diminished, reinforcing the notion that a hydrophobic interaction between the residue at this position and Trp161 is important for function. We propose that the interaction formed between Pro85 and Trp161 is important for stabilizing the relative orientations of the disordered N terminus and the β-sandwich domain, and that the β6-β7 extension and the Pro85-Trp161 interaction are T4P-specific adaptations for function.

The C terminus of PilP interacts with the periplasmic domain of PilQ.

Genin and Boucher (29) analyzed a number of secretin sequences from the T2SS and T3SS and identified four highly conserved regions toward the C termini of the proteins that they termed secretin consensus regions. Alignments of PilQ protein sequences from various T4P-producing bacteria show the same conserved regions (see Fig. S5 in the supplemental material). Using these sequence alignments, we identified four discrete periplasmic domains in T4P secretins. The two N-terminal β-strand-rich domains share the lowest percent identity across PilQ homologues (Table 1; also see Fig. S5); thus, we termed these regions of PilQ the species-specific (SS) domains (SS1 and SS2). C terminal to the SS domains, we predicted that there are two additional domains that were recently confirmed to be structurally homologous to N0 and N1 from T2SS secretins (6), and we use that terminology here. From this analysis, we designed and expressed six soluble constructs spanning the periplasmic region of PilQ to examine the roles of these individual domains in more detail (Fig. 4). Briefly, these soluble constructs are PilQ_24-445 (containing the entire periplasmic domain), PilQ_24-280 (SS domains), PilQ_281-445 (N0 and N1), PilQ_281-410 (N0 and approximately one-third of N1), PilQ_281-376 (N0), and PilQ_377-445 (N1).

Each of the six 6×His-tagged constructs was tested for its ability to pull down untagged PilP from E. coli lysates. Three constructs of PilP were used: mature PilP (PilP_Δ18), the N-terminal region (PilP_19-84T7), and the C-terminal region (PilP_Δ71). PilP_19-84T7 was rapidly degraded after lysis, suggesting that no PilQ fragment could stabilize this region of PilP (see Fig. S3 in the supplemental material). In contrast, PilP_Δ18 and PilP_Δ71 were pulled down by three of the six soluble PilQ constructs (Fig. 5 and Table 2), while the negative control showed that the PilP fragments did not bind to the Ni-NTA column in the absence of PilQ (data not shown). PilP_Δ18 and PilP_Δ71 interacted with PilQ_281-445, PilQ_281-410, and PilQ_281-376, the three constructs containing the N0 domain at their N termini (Table 2). Together, these data suggest that the C-terminal β-domain region of PilP is sufficient for the interaction with PilQ, and that the site of PilP interaction with PilQ is the N0 domain.

Notably, the full-length periplasmic domain of PilQ (PilQ_24-445) did not pull down either PilP_Δ18 or PilP_Δ71 from cell lysates, even though this construct contains the N0 domain (Fig. 5B). It is possible that in this context in vitro, the binding site for PilP on the N0 domain is occluded. This PilQ periplasmic fragment may be conformationally flexible when expressed as a monomer in vitro but not as an oligomer in vivo, where its orientation would be constrained.

The PilNOP heterotrimer interacts with PilQ.

Previously, we showed that PilP forms a stable heterotrimer with PilNO in vitro (12), and in this study we have shown that the extended N terminus of PilP interacts with PilNO, while its C-terminal β-domain interacts with PilQ. To test whether PilP could interact simultaneously with PilNO and PilQ, the pulldown experiments were repeated with all four proteins. PilNOP were pulled down by the same PilQ constructs that pulled down PilP_Δ18 and PilP_Δ71 (Table 2). Negative controls consisting of PilNO alone or PilNO with tagged PilQ constructs showed that PilNO was not retained on the column unless both PilP and tagged PilQ were present (Table 2). Therefore, PilP is sufficient to coordinate a stable interaction network between the inner membrane components PilNO and the outer membrane component PilQ.

PilA interacts with a PilNOPQ complex in vivo.

Bacterial two-hybrid experiments using N. meningitidis T4P components expressed in E. coli indicated potential interactions of the major pilin subunit PilE (equivalent to PilA in P. aeruginosa) with specific inner membrane proteins (23). To test for potential interactions in P. aeruginosa and to include the periplasmic domains of PilQ, we performed a series of pulldown experiments using 6×His-tagged PilQ bait to capture prey proteins from detergent-solubilized P. aeruginosa lysates. Negative controls showed no interaction of proteins from detergent-solubilized PAO1 lysates with the Ni-NTA column (Fig. 6). In contrast, after extensive washing of the column, the bound PilQ_24-445, PilQ_24-280, or PilQ_281-445 fragments captured PilA from the lysates (Fig. 6). Western blots of imidazole eluates with antibodies specific to PilN, PilO, and PilP showed that PilQ captured all three proteins. Together, these data support formation of a stable PilNOPQ complex and suggest that PilA interacts with this complex. These data support the idea that PilA and/or the pilus make extensive contacts with the PilMNOPQ transenvelope complex.