Bacterial strains, tissue cultures and growth conditions
M. pneumoniae M129 strain was grown in cell culture flasks containing SP4 medium and incubated at 37°C and 5% CO2. Surface-attached mycoplasmas were harvested using a cell scraper and resuspended in SP4 medium. To grow mycoplasma cells on IBIDI 8-well chamber slides, each well was seeded with about 105 CFUs and incubated for 12–24 h in 200 μL SP4 supplemented with 3% gelatin.
NSI myeloma cells(23) were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 50 μg mL-1 gentamycin (complete RPMI). Hybridomas were selected in complete RPMI supplemented with HAT media and BM-Condimed (Sigma Aldrich, St. Louis, USA).
Cloning, expression, and purification of P116 constructs
Regions corresponding to the MPN213 gene from M. pneumoniae were amplified from synthetic clones using different primers for each construct: P116F30 and P116R957 for P116(30–957); P116F13 and P116R957 for P116(13–957); P116F212 and P116R862 for P116(212–862); and P116W681 to introduce mutation W681A PCR fragments were cloned into the expression vector pOPINE (gift from Ray Owens; plasmid #26043, Addgene, Watertown, USA) to generate constructs, with a C-terminal His-tag. Recombinant proteins were obtained after expression at 22°C in B834 (DE3) cells (Merck , Darmstadt, Germany), upon induction with 0.6 mM IPTG at 0.8 OD600. Cells were harvested and lysed by French press in binding buffer (20 mM TRIS-HCl pH: 7.4, 40 mM imidazole and 150 mM NaCl) and centrifuged at 49,000 × g at 4°C. Supernatant was loaded onto a HisTrap 5 ml column (GE Healthcare , Chicago, USA) that was pre-equilibrated in binding buffer and elution buffer (20 mM TRIS-HCl pH: 7.4, 400 mM imidazole and 150 mM NaCl). Soluble aliquots were pooled and loaded onto a Superdex 200 GL 10/300 column (GE Healthcare, Chicago, USA) in a protein buffer (20 mM TRIS-HCl pH 7.4 and 150 mM NaCl).
To obtain empty P116, 2.6% Triton X-100 was added to the protein sample and incubated for 1.5 h at room temperature. Subsequent purification followed the same methodology described above, but also included a wash step with the binding buffer plus 1.3% Triton X-100, followed by extensive washing with at least 20 column volumes of wash buffer (20 mM TRIS-HCl pH: 7.4, 20 mM imidazole) before eluting the samples from the column. P116 was concentrated with Vivaspin 500 centrifugal concentrators (10,000 MWCO PES, Sartorius, Göttingen, Germany) to a final concentration of >0.5 mg/mL.
To refill P116 with lipids, the empty protein was incubated with approximately 1 ml FBS per mg P116 for 2 h at 30°C while still bound on the column. After extensive washing with at least 40 column volumes of wash buffer, elution and concentration were performed as described above.
HDL isolation and determination of cholesterol transfer rate
Human HDL (density 1.063–1.210 g/mL) was isolated from plasma of healthy donors via sequential gradient density ultracentrifugation, using potassium bromide for density adjustment, at 100,000 g for 24 h with an analytical fixed-angle rotor (50.3, Beckman Coulter, Fullerton, CA, USA). The amount of cholesterol and apolipoprotein A1 were determined enzymatically and by an immunoturbidimetric assay, respectively, using commercial kits adapted for a COBAS 6000 autoanalyzer (Roche Diagnostics, Rotkreuz, Switzerland). Radiolabeled HDLs were prepared as previously described (24). Briefly, 10 μCi of either [1,2-3H(N)] free cholesterol or [1,2-3H(N)]cholesteryl oleate (Perkin Elmer, Boston, MA) were mixed with absolute ethanol, and the solvent was dried under a stream of N2. HDL (0.5 mL, 2.25 g/L of ApoA1) was added to the tubes containing the radiotracers, as appropriate, and then incubated for 16 h in a 37°C bath. The labeled HDLs (both 3H-cholesterol-containing and 3H-cholesteryl oleate-containing HDLs) were re-isolated by gradient density ultracentrifugation at 1.063–1.210 g/mL and dialyzed against PBS via gel filtration chromatography. Specific activities of 3H-cholesterol-containing and 3H-cholesteryl oleate-containing HDLs were 1221 and 185 counts per minute (cpm)/nmol, respectively. The cholesterol transfer to P116 (1 g/L) was measured after adding either [3H] free cholesterol-containing or [3H]cholesteryl oleate-containing HDL (0.5 g/L of APOA1) and incubating for 2 h at 37°C. HDL and P116 were separated by a HisTrap HP affinity column. The radioactivity associated with each P116 and HDL fraction was measured via liquid scintillation counting. The percentage of [3H]cholesterol transferred per mL was determined for each condition. The specific activities for each radiotracer were used to calculate the amount of free cholesterol and cholesteryl ester transferred from HDL to P116.
Size exclusion chromatography and multi-angle light scattering (SEC-MALS)
Molecular weights were measured from P116 samples using a Superose 6 10/300 GL (GE Healthcare, Chicago, USA) column in a Prominence liquid chromatography system (Shimadzu, Kyoto, Japan) connected to a DAWN HELEOS II multi-angle light scattering (MALS) detector and an Optilab T-REX refractive index (dRI) detector (Wyatt Technology, Santa Barbara, USA). ASTRA 7 software (Wyatt Technology) was used for data processing and analysis. An increment of the specific refractive index in relation to concentration changes (dn/dc) of 0.185 mL/g (typical of proteins) was assumed for calculations.
Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-TOF)
All samples were mixed in a 1:1 ratio with either DHB or sDHB (Bruker Daltonics, Germany) matrix solution (50 mg·ml-1 in 50% Acetonitrile (ACN), 50% water and 0.1% TFA). Subsequently 1 μl aliquots of the mixture were deposited on a BigAnchor MALDI target (Bruker Daltonics, Germany) and allowed to dry and crystallize at ambient conditions. Unless stated otherwise, all reagents and solvents were obtained from Sigma Aldrich, Germany.
MS spectra were acquired on a rapifleX MALDI-TOF/TOF (Bruker Daltonics, Germany) in the mass range from 20.000-120.000 m/z in linear positive mode and in the mass range from 100-1600 m/z in reflector positive mode. The Compass 2.0 (Bruker, Germany) software suite was used for spectra acquisition and processing.
Lipidomics analysis (LC-TIMS-MS/MS)
Samples were extracted using a modified MTBE/Methanol extraction protocol, and submitted to LC-nanoESI-IMS-MS/MS analysis using a Bruker NanoElute UHPLC coupled to a Bruker TimsTOF Pro 2 mass spectrometer operated in DDA-PASEF mode. In brief, 40 min gradients on PepSep C18 columns (1.9A, 75µm ID, 15cm length) were recorded in positive and negative ion mode. Data were analysed using the MS-DIAL pipeline (version 4.9).
Single-particle cryoEM
For single-particle cryoEM, a 3.5 µl drop of purified P116 (100–400 µg/mL in 20 mM Tris, pH 7.4 buffer or 600 µg/mL in 20 mM Tris, 2 mM CHAPSO, pH 7.4 buffer) or P116 mixed with HDL (250 µg/mL P116 and 1116 µg/mL HDL in 20 mM Tris, pH 7.4 buffer) was applied to a (45 s) glow-discharged R1.2/1.3 C-flat grid (Electron Microscopy Science, Hatfield, USA), and plunge-frozen in liquid ethane (Vitrobot Mark IV, Thermo Scientific, Waltham, USA) at 100% relative humidity, 4 °C, nominal blot force –3, wait time 45 s, with a blotting time of 12 s. Before freezing, Whatman 595 filter papers were incubated for 1 h in the Vitrobot chamber at 100% relative humidity and 4°C.
Dose-fractionated Movies of P116, P116 refilled and P116 mixed with HDL were collected with SerialEM v3.8 (25) at a nominal magnification of 130,000x (1.05 Å per pixel) in nanoprobe EFTEM mode at 300 kV with a Titan Krios (Thermo Scientific, Waltham, USA) electron microscope equipped with a GIF Quantum S.E. post-column energy filter in zero loss peak mode and a K2 Summit detector (Gatan Inc., Pleasanton, USA). For P116, P116 refilled and P116 with HDL a total of 4376, 4019 and 3114 micrographs with 34, 29 and 30 frames per micrograph and a frame time of 0.2 s were collected. The camera was operated in dose-fractionation counting mode with a dose rate of ~8 electrons per Å 2 s-1, resulting in a total dose of 50 electrons per Å 2 s-1. Defocus values ranged from –1 to –3.5 µm.
For P116 empty, dose-fractionated Movies were collected using EPU 2.12 (Thermo Scientific, Waltham, USA) at a nominal magnification of 105,000x (0.831 Å per pixel) in nanoprobe EFTEM mode at 300 kV with a Titan Krios G2 electron microscope (Thermo Scientific, Waltham, USA), equipped with a BioQuantum-K3 imaging filter (Gatan Inc., Pleasanton, USA), operated in zero loss peak mode with 20 eV energy slit width. In total 15,299 micrographs with 50 frames per micrograph and frame time of 0.052 s were collected. The K3 camera was operated in counting mode with a dose rate of ~ 16 electrons per A2 s-1, resulting in a total dose of 50 electrons per Å 2 s-1. Defocus values ranged from -0.8 to -3.5 µm.
CryoSPARC v3.2 (26) was used to process the cryoEM data, unless stated otherwise. Beam-induced motion correction and CTF estimation were performed using CryoSPARC’s own implementation. Particles were initially clicked with the Blob picker using a particle diameter of 200–300 Å. Particles were then subjected to unsupervised 2D classification. For the final processing, the generated 2D averages were taken as templates for the automated particle picking, for the processing of P116 with HDL no template picking was performed. In total, 3,463,490, 4,532,601 particles, 2,930,863 particles and 262,981 particles were picked and extracted with a binned box size of 256 pixels for P116, P116 empty, P116 refilled and P116 with HDL respectively. False-positive picks were removed by two rounds of unsupervised 2D classification. The remaining 1,324,330 particles (P116), 1,140,275 particles (P116 empty), 1,311,526 particles (P116 refilled) and 46,277 particles (P116 with HDL) were used to generate an ab initio reconstruction with three classes followed by a subsequent heterogeneous refinement with three classes. For the final processing, 1,315,362 particles (P116), 633,332 particles (P116 empty), 1,311,526 particles (P116 refilled) and 46,277 particles (P116 with HDL) were used. For the remaining particles, the beam-induced specimen movement was corrected locally.
The CTF was refined per group on the fly within the non-uniform refinement. The obtained global resolution of the homodimer was 3.3 Å (P116), 4 Å (P116 empty), and 3.5 Å (P116 refilled) (Supplementary Figure 2 & 8 and Supplementary Table II). To analyze the sample in regard to its flexibility the particles were subjected to the 3D variability analysis of cryoSPARC which was used to display the continuous movements of the protein.
Cryo-electron tomography of M. pneumoniae
M. pneumoniae M129 cells of an adherently growing culture were scraped in a final volume of 1 ml of SP4 medium and washed three times in PBS. This solution was mixed with fiducial markers (Protein A conjugated to 5 nm colloidal gold: Cell biology department, University Medical Center Utrecht, The Netherlands). From this stock a 3.5 µl drop was applied to a (45 s) glow-discharged R1.2/1.3 C-flat grid (Electron Microscopy Science, Hatfield, USA), and plunge-frozen in liquid ethane (Vitrobot Mark IV, Thermo Scientific, Waltham, USA) at 100% relative humidity, 4 °C, nominal blot force –1, with a blotting time of 10 s.
Tilt-series were recorded using SerialEM v3.8 (25) at a nominal magnification of 105,000x (1.3 Å per pixel) in nanoprobe EFTEM mode at 300 kV with a Titan Krios (Thermo Scientific, Waltham, USA) electron microscope equipped with a GIF Quantum S.E. post-column energy filter in zero loss peak mode and a K2 Summit detector (Gatan Inc., Pleasanton, USA). The total dose per tomogram was 120 e-/ Å 2 , the tilt series covered an angular range from -60° to 60° with an angular increment of 3° and a defocus set at -3 µm. Tomograms were reconstructed by super-sampling SART (27) with a 3D CTF correction (28).
P116 model building and refinement
The initial tracing of the core domain was performed manually with Coot (29). It contained numerous gaps and ambiguities that were slowly polished by alternating cycles of refinement using the “Real Space” protocol in the program Phenix (30, 31) and manual reinterpretation and rebuilding with Coot. The tracing and assignment of specific residues in the N-terminal domain were very difficult due to the low local resolution of the map for this domain, and only a partial interpretation was achieved. Using Robetta and AlphaFold (14) we obtained different predictions of the N-terminal domain structure using different parts of the sequence. The highest ranked predictions, selected using the partial experimental structure already available, were obtained with AlphaFold for residues 81–245, which allowed us to complete the building of the N-terminal domain according to the cryoEM map. The RMS deviation between the AlphaFold prediction and the experimental model was 2.6 Å for 104 (63%) structurally equivalent residues. Some residues at the N-end of the N-terminal domain were difficult to identify and were represented as alanines in the final model. The whole P116 model was then refined using Phenix, and the final refined structure was deposited in the EMDB with code XXXX (Supplementary Table II).
Polyclonal and monoclonal antibody generation
Two BALB/C mice were serially immunized with four intraperitoneal injections, each one containing 150 μg of recombinant P116 ectodomain (residues 30–957) in 200 μL of PBS with no adjuvants. The last injection was delivered four days before splenectomy. Isolated B lymphocytes from the immunized mice were fused to NSI myeloma cells (23) to obtain stable hybridoma cell lines producing monoclonal antibodies, as previously described (32) .Supernatants from hybridoma cell lines derived from single fused cells were first investigated by indirect ELISA screening against the recombinant P116 ectodomain. Positive clones were also tested by Western blot against protein profiles from M. pneumoniae cell lysates and by immunofluorescence using whole, non-permeabilized M. pneumoniae cells (see below). Only those clones with supernatants revealing a single 116 kDa band in protein profiles and also exhibiting a consistent fluorescent staining of M. pneumoniae cells were selected and used in this work. Polyclonal sera were obtained by cardiac puncture of properly euthanized mice just before splenectomy and titred using serial dilutions of the antigen. The titer of each polyclonal serum was determined as the IC50 value from four parameter logistic plots and found to be approximately 1/4000 for both sera. Polyclonal anti-P1 antibodies were obtained by immunizing two BALB/C mice with recombinant P1 proteins (33), respectively, as described above. The titers obtained for polyclonal anti-P1 antibodies were approximately 1/2500 and 1/3000, respectively.
Immunofluorescence microscopy
The immunofluorescence staining of mycoplasma cells on chamber slides was similar to previously described (34), with several modifications. Cells were washed with PBS containing 0.02% Tween 20 (PBS-T) prewarmed at 37°C, and each well was fixed with 200 μL of 3% paraformaldehyde (wt/vol) and 0.1% glutaraldehyde. Cells were washed three times with PBS-T, and slides were immediately treated with 3% BSA in PBS-T (blocking solution) for 30 min. The blocking solution was removed, and each well was incubated for 1 h with 100 μL of the primary antibodies diluted in blocking solution. For P116 polyclonal sera, we used a 1/2000 dilution; a 1/10 dilution was used for monoclonal antibodies from hybridoma supernatants. Wells were washed three times with PBS-T and incubated for 1 h with a 1/2000 dilution of a goat anti-mouse Alexa 555 secondary antibody (Invitrogen, Waltham, USA) in blocking solution. Wells were then washed three times with PBS-T and incubated for 20 min with 100 μL of a solution of Hoechst 33342 10 μg/μL in PBS-T. Wells were finally washed once with PBS-T and replenished with 100 μL of PBS before microscopic examination. Cells were observed by phase contrast and epifluorescence in an Eclipse TE 2000-E inverted microscope (Nikon, Tokyo, Japan). Phase contrast images, 4',6-diamidino-2-phenylindole (DAPI, excitation 387/11 nm, emission 447/60 nm) and Texas Red (excitation 560/20 nm, emission 593/40 nm) epifluorescence images were captured with an Orca Fusion camera (Hamamatsu, Hamamatsu, Japan) controlled by NIS-Elements BR software (Nikon, Tokyo, Japan).
Time-lapse microcinematography
The effect of anti-P116 antibodies and anti-P1 polyclonal serum on mycoplasma cell adhesion was investigated by time-lapse cinematography of M. pneumoniae cells growing on IBIDI 8-well chamber slides. Before observation, medium was replaced with PBS containing 10% FBS and 3% gelatin prewarmed at 37°C. A similar medium has been used to test the effect of P1 antibodies on mycoplasma adhesion and gliding motility(35). After incubation for 10 min at 37°C and 5% CO2, the slide was placed in a Nikon Eclipse TE 2000-E inverted microscope equipped with a Microscope Cage Incubation System (Okolab, Pozzuoli, Italy) at 37°C. Images were captured at 0.5 s intervals for a total observation time of 10 min. After the first 60 s of observation, the different antibodies were dispensed directly into the wells. The frequencies of motile cells and detached cells before the addition of antibodies were calculated from the images collected between 0 and 60 s of observation. The frequencies of motile cells and detached cells after the addition of antibodies were calculated from the images collected in the last minute of observation.