Sophie et al. 10.1073/pnas.0707581104. |
Fig. 7. Hydrophobic packing within the PilX globular domain. (A) Hydrophobic residues including the three Met comprise the well-packed interface between the central sheet and a1C. Trp115, donated by b3, is at the center of a smaller hydrophobic core that stabilizes the D-region interaction with the central sheet. (B)Viewed from roughly the same orientation but translated toward the reader, the packing of a1 against a2 highlights the malleable role of Phe50. In two chains P43 A (cyan) and P43 B (not shown) a1 unwinds by one residue and Ile51 moves into its position. P43 C (green) and C2221 A (magenta) and C2221 B (violet) are also shown, while for clarity P43 chain D is not.
Fig. 8. PilX colocalizes with the pilus filaments in the WT and pilX/pilXWT strains. In confocal IF microscopy, strains were doubly stained for PilX (green) and Tfp (red). Merged images reveal colocalization (orange) of PilX with Tfp. (Scale bars: 10 mm.) The anti-PilX antibody is specific, as revealed by the absence of signal in the pilX mutant.
Fig. 9. Modulating the expression of the WT gene in strain pilX/pilXWT leads to different levels of aggregation and adhesion. (A) Quantification of aggregation by measuring the decrease in OD600 that occurs upon sedimentation of bacterial aggregates in static liquid cultures. Values are means of two to three independent experiments. (B) Quantification of the adhesive abilities. After a 30-min contact during which bacteria adhered to the HUVEC, cells were incubated with regular hourly washes and the numbers of adherent CFU after 4 h of infection were recovered by scraping the wells and counted. Values are the means ± standard deviations of three to four independent assays.
Table 1. Crystallographic data collection and analysis
Data collection | Native PilX | SeMet-PilX peak | SeMet-PilX inflection | Native PilX | Native PilX |
Beamline | Synchrotron APS 32ID-B | In-house | |||
Spacegroup | P43 | P43 | P43 | P43 | C2221 |
Wavelength, Å | 1.0001 | 0.97869 | 0.9788 | 1.54178 | 1.54178 |
Resolution*, Å | 25.0-2.25 (2.33-2.25) | 50.0-2.6 (2.69-2.60) | 50.0-2.75 (2.85-2.75) | 25.0-2.4 (2.49-2.40) | 25.0-2.5 (2.59-2.50) |
Unique reflections | 24,568 (2,435) | 31,259 (3,151) | 26,513 (2,655) | 20,280 (1794) | 10,281 (999) |
Redundancy† | 4.2 (4.2) | 7.5 (7.2) | 3.9 (3.8) | 6.4 (3.1) | 8.6 (7.1) |
Rsymm§, % | 5.3 (24.2) | 10.4 (47.5) | 7.6 (35.4) | 7.2 (24.3) | 7.4 (27.7) |
Completeness (%) | 99.9 (100) | 99.5 (99.7) | 99.5 (99.6) | 98.6 (87.2) | 99.8 (100) |
Average I/sI | 27.9 (4.9) | 20.7 (4.5) | 18.3 (4.0) | 22.3 (3.7) | 21.3 (6.3) |
Refinement | |||||
mol/au | 4 | 2 | |||
Resolution*, Å | 25.0-2.4 | 25.0-2.5 | |||
No. protein atoms | 3622 | 1908 | |||
No. solvent atoms | 181 | 75 | |||
R work/Rfree§ | 20.8/27.9 (21.7/29.0) | 20.4/23.6 (25.0/42.5) | |||
Wilson B, Å2 | 44.3 | 58.6 | |||
Avg. B overall | 47.5 | 52.3 | |||
Protein atoms | 47.1 | 52.0 | |||
Solvent atoms | 56.0 | 60.8 | |||
rmsd | |||||
Bond lengths, Å | 0.018 | 0.016 | |||
Bond angles, º | 1.49 | 1.64 | |||
e.s.u., Å | 0.31 | 0.27 | |||
*Numbers in parentheses apply to the highest resolution shell.
†
Anomalous pairs were not merged in processing the first three data sets listed.‡
Rsymm = ∑[|I - <I>|] / ∑I with single measurements excluded.§
Rwork = ∑|Fo - Fc| / ∑Fo and Rfree is calculated for the 5% of data not used during refinement.Bacterial Strains.
The portion of the pilX gene encoding residues 29 to 152 was amplified by PCR using the primers pilX-p2X-F 5'-GAATTCGAAAAAGGCTATCAGTCCCA-3' and pilX-p2X-R 5'-CTGCAGCTATTTTTTACGATTAGAGAAAG-3', which contained overhangs (italics) corresponding to restriction sites for EcoRI and PstI, respectively. This PCR fragment was cloned into pCRII-TOPO (Invitrogen) to generate plasmid pYU13. After digesting pYU13 by EcoRI and PstI, the fragment was subcloned in the pMAL-p2X vector (New England Biolabs) digested with the same enzymes. The insert in the resulting plasmid named pYU15 was sequenced and found to contain no errors.In order, to test their functionality, pilX alleles altered in vitro by site-directed mutagenesis were ectopically expressed in the N. meningitidis pilX mutant. The variant alleles were generated with the QuikChange Site-Directed Mutagenesis Kit (Stratagene), using as a template a vector previously constructed that contains the pilX gene flanked by PacI restriction sites (1) and specific pairs of complementary primers. Variant PilX135Ser was generated using L145S #1 5'-CTGCCCGAGCCCATTCGGAGACCTTGTCCTC-3' and L145S #2 5'-GAGGACAAGGTCTCCGAATGGGCTCGGGCAG-3' (the point mutations are underlined). Variant PilX138Ser was generated using L148S #1 5'-CCCATTTGGAGACCTCGTCCTCAGATGTCGG-3' and L148S #2 5'-CCGACATCTGAGGACGAGGTCTCCAAATGGG-3'. Variant PilX142Ser was generated using V152S #1 5'-GACCTTGTCCTCAGATTCCGGCTGTGAAGCTTTCTC-3' and V152S #2 5'-GAGAAAGCTTCACAGCCGGAATCTGAGGACAAGGTC-3'. Variant PilXD126-131 was generated using D102-107#1 5'-GACGGATACAAATGCCGAGCCCATTTGGAG-3' and D102-107#2 5'-CTCCAAATGGGCTCGGCATTTGTATCCGTC-3'. Variant PilXD127-138 was generated using D103-114#1 5'-GGATACAAATGCCGTTCCTCAGATGTCGGC-3' and D103-114#2 5'-GCCGACATCTGAGGAACGGCATTTGTATCC-3'. Variant PilXD132-137 was generated using D108-113#1 5'-GATGCCGCTTCTGCCTTGTCCTCAGATGTC-3' and D108-113#2 5'-GACATCTGAGGACAAGGCAGAAGCGGCATC-3'. Variant PilXD136-142 was generated using D112-118 #1 5'-GCCCGAGCCCATTTGGGCTGTGAAGCTTTC-3' and D112-118 #2 5'-GAAAGCTTCACAGCCCAAATGGGCTCGGGC-3'. Variant PilXD138-142 was generated using D114-118#1 5'-GCCCATTTGGAGACCGGCTGTGAAGCTTTC-3' and D114-118#2 5'-GAAAGCTTCACAGCCGGTCTCCAAATGGGC-3'. Plasmids containing inserts with the desired mutations, which were identified by sequencing, were then digested by PacI and the variant pilX alleles were subcloned into PacI-cut pGCC4 vector (2), which placed them under the transcriptional control of an IPTG-inducible promoter within a DNA fragment corresponding to a region of the gonococcal chromosome conserved in N. meningitidis. Finally, the recombinant plasmids were linearized by ClaI and used to transform the pilX mutant, which led to their integration in the chromosome by homologous recombination. The transformants thus obtained contained the desired mutation as verified by sequencing after amplifying the variant alleles by PCR using primers 1110IndF 5'-CCTTAATTAAGGAGTAATTTTATGATGAGTAATAAAATGGAACA-3' and lctPavtPvuI-3 5'-CCGCTGCTTGCCCTGATGGGTGCGC-3'.
PilX Purification.
One ml of an overnight culture of E. coli BL21(DE3), or its methionine auxotroph, carrying pYU15 was used to inoculate 1 liter of LB-ampicillin (100 mg/ml). When this culture reached an OD600 of 0.6, it was transferred to 20°C. After equilibration, IPTG was added to a final concentration of 1 mM and, if needed, selenomethionine was added as described previously (3, 4). Expression was then continued overnight. Cells were then harvested and frozen in liquid nitrogen. A 5 g cell pellet was thawed and lysed by two passages through a French pressure cell. Clarified cell lysate was passed at 1 ml/min over a 60 ml amylose resin column (New England Biolabs) preequilibrated with 10 mM Tris pH 7.2 (column buffer). The amylose resin was washed extensively with the same buffer and the MBP-PilX fusion protein eluted with 10 mM maltose in column buffer. After concentration to ~30 mg/ml, 50 units of Factor Xa (New England Biolabs) were added to cleave the MBP from the truncated PilX protein and this reaction was dialyzed against 1 mM CaCl2 in PBS. The separation of PilX from MBP and uncut fusion was accomplished on HiTrap SP Sepharose resin (Pharmacia Biotech). PilX eluted cleanly at ≈300 mM NaCl. Pooled PilX fractions were further concentrated to 7 mg/ml and dialyzed exhaustively against 25 mM KCl, 50 mM Hepes pH 7.5. Purified PilX was vitrified as droplets in liquid nitrogen and stored at -80°C.Cryoprotection of PilX Crystals.
For cryoprotection, form I crystals were equilibrated in 27% PEG 8000, 80 mM LiSO4, 100 mM KCl, 50 mM Bis-Tris (pH 6.5) and transferred in five incremental steps, over a period of 1 hr, to 25% PEG 8000, 80 mM LiSO4, 200 mM KCl, 50 mM Bis-Tris (pH 6.5), 20% ethylene glycol. Form II crystals were vitrified directly from mother liquor.Structure Determination.
All data sets were integrated and scaled with HKL2000 (5) (SI Table 1). After scaling the peak and inflection data sets, the data were submitted for structure determination in the Automated Crystallographic System (ACrS) (6). The best solution resulted from the HySS (7), SHARP (8), Resolve density modification with NCS averaging (9), and ARP/wARP pathway (10). Eight selenium sites were identified and led to phases with an overall FOM of 0.48 and phasing power of 2.38, with a FOM after density modification of 0.80. The starting model traced automatically by ARP/Warp (10) contained the correct residues for a1 in two monomers and polyalanine for ≈50% of the backbone. This model was improved manually with XFIT (11) and refined with REFMAC5 (12). Radiation damage was apparent, as two of four disulfide bridges in the asymmetric unit were oxidized. Therefore, when this model had been refined to R/Rfree values of 24.2/29.4%, it was used in PHASER (13) as the molecular replacement search model for crystal form II (C2221) data collected on a Bruker Proteum CCD detector using radiation generated by a Bruker HiStar rotating anode (SI Table 1). The refinement and fitting of the C2221 structure led to a final model with R/Rfree values of 20.4/23.6% (SI Table 1) in which residues 27-147 are ordered for each of the two PilX monomers in the asymmetric unit. One monomer from the completed C2221 structure was then used as the molecular replacement model for the 2.4 Å resolution in-house P43 data set. This refinement was well-behaved for the four PilX molecules in the asymmetric unit (SI Table 1), the data showing no signs of radiation damage, although two loops are missing in monomers C and D.Aggregation and Adhesion Assays.
N. meningitidis ability to form aggregates was monitored by phase-contrast microscopy and quantified by measuring changes in OD600 that occur in static cultures upon sedimentation of the aggregates, as previously described (1). Adhesion to HUVEC was quantified as previously described (1). After a 30-min contact during which bacteria adhered to the HUVEC, cells were incubated with regular hourly washes and the numbers of adherent bacteria after 4 h of infection were recovered by scraping the wells and counted. When pilX/pilXvariant strains were tested, they were grown in the presence of 0.1 mM IPTG because this led to comparable aggregative and adhesive abilities for the pilX/pilXWT strain and the WT strain.Pilus Purification.
For purified pilus preparations, bacteria from one Petri dish inoculated with 108 colony-forming units (CFU) the previous day were resuspended in 1 ml of 0.15 M ethanolamine (pH 10.5), adjusted to a given OD600 and vortexed for 2 min to resuspend cells. Cells and large debris were removed by centrifugation at 16,000 ´g for 7 min at 4°C. One hundred microliters of ammonium sulfate-saturated solution (in ethanolamine) was then added to 900 ml of the supernatant, mixed and incubated at room temperature for 45 min. Pilus filaments were pelleted by centrifugation at 16,000 ´g for 5 min at 4°C. They were resuspended in 0.05 M Tris-buffered saline (pH 7.5) by vortexing, and pelleted again by centrifugation at 16,000 ´g for 5 min at 4°C. After repeating this step, the pellet was centrifuged at 16,000 ´ g for 3 min at 4°C and all traces of the supernatant were removed. The pilus preparations were resuspended in 50 ml of water.Purity of pilus preparations was assessed by Coomassie staining (Bio-Rad) of SDS/PAGE gels. Proteins were quantified using the Bradford reagent (Sigma), as suggested by the manufacturer. The total amount of pilus proteins was estimated by subtracting the protein background in pilus preparations obtained with a non-piliated mutant, which represents a small fraction of the proteins in the pilus preparation of the WT strain.
Immunoblotting.
PilX was detected using a rabbit polyclonal serum previously described (1) used at 1/2,500 dilution, whereas PilE was detected using the 5C5 mouse monoclonal serum at 1/10,000 dilution. The secondary antibodies were anti-rabbit or anti-mouse horseradish peroxidase-linked IgG (Amersham Pharmacia Biosciences).To estimate the relative amount of PilX in purified pilus preparations, we used the protein that was used for crystallization to standardize the above antibody reactivity. We then determined the amount of PilX in a defined amount of purified pilus preparation.
Localization of PilX.
Strains were grown, when necessary, in the presence of 0.01 mM IPTG. For confocal microscopy, bacteria were resuspended in PBS and fixed on coverslips for 20 min with a solution of PBS-2.5% paraformaldehyde. After 5 min of incubation with PBS-0.1 M glycine, samples were incubated with PBS-0.2% gelatin. PilX was stained with the newly produced antiserum used at 1/20 dilution. The secondary antibody, used at 1/100 dilution, was a goat anti-rabbit antibody IgG conjugated with Alexa Fluor 488. Tfp were stained using a 1/500 dilution of the 20D9 mouse monoclonal antibody that specifically recognize the fibers of N. meningitidis strain 8013. The secondary antibody was a 1/100 dilution of goat anti-mouse IgG conjugated with Alexa 568 (Molecular Probes). Samples were observed using a Zeiss 510 confocal microscope.1. Helaine S, Carbonnelle E, Prouvensier L, Beretti JL, Nassif X, Pelicic V (2005) Mol Microbiol 55:65-77.
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