{"paper_id":"0d666b81-83bb-4113-a65e-63b4e910f39c","body_text":"Physiological Sources of Essential Lipids for Mycoplasma pneumoniae via Protein P116: Innovative Biotechnological Tools for Targeting Atherosclerotic and Hepatic Lesions | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Physiological Sources of Essential Lipids for Mycoplasma pneumoniae via Protein P116: Innovative Biotechnological Tools for Targeting Atherosclerotic and Hepatic Lesions Joan Carles Escola-Gil, David Vizarraga, Marina Marcos-Silva, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5668698/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Dec, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Mycoplasma pneumoniae ( MPN ) is a bacterial pathogen in humans that primarily causes atypical pneumonia. MPN cannot synthesize several lipids crucial for its cell membrane structure and needs to extract them from the lung of the host to survive. The protein responsible for extracting essential lipids from cell membranes is P116. MPN has been detected in increased quantities within ruptured atherosclerotic plaques and the question is how MPN survives in the blood and in the plaques and obtains the lipids necessary for its membrane. Here we show that P116 can uptake essential lipids from LDL and HDL and when targeting its C-terminal domain via a monoclonal antibody there is growth inhibition in vitro . Phase contrast epifluorescence microscopy of human arteries also revealed that this antibody blocks MPN binding to human atherosclerotic lesions ex vivo . Furthermore, injection of MPN in the blood results in accumulation of MPN within the liver and atheroma plaques in a hyperlipidemic mouse model. We conclude that P116 plays a critical role in extracting essential lipids from physiological circulating lipoproteins and from host cells and regulates MPN localization to liver and atheromatous plaques. These results suggest new strategies for managing mycoplasma infections and addressing the potential complications of MPN infections in atherosclerotic lesions. They also open avenues for utilizing biotechnological tools in the treatment of atherosclerotic and hepatic lesions. Health sciences/Diseases/Cardiovascular diseases/Dyslipidaemias Biological sciences/Biochemistry/Lipids/Lipoproteins Biological sciences/Biological techniques/Microscopy/Cryoelectron microscopy Atherosclerosis Cholesterol HDL LDL M. pneumoniae Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Mycoplasma pneumoniae (MPN) is a facultative intracellular human pathogen that lacks a cell wall and causes community-acquired pneumonia, sometimes leading to severe systemic effects 1 . MPN accounts for 40% of community-acquired pneumonia cases 2 . MPN , in addition to being a respiratory pathogen, can clinically manifest at extrapulmonary sites in up to 25% of infections 3 . Unlike other significant respiratory pathogens, such as Streptococcus pneumoniae and Haemophilus influenzae , no vaccine currently exists to address both the pulmonary and extrapulmonary complications associated with MPN 4 . Prospective surveillance data indicate a re-emergence of MPN in Europe and Asia, with increasing cases reported across various regions 5 , 6 . With one of the smallest genomes of 816 kbp 7 , MPN cannot synthesize several lipids crucial for its cell membrane structure, including sphingomyelin, phosphatidylcholine, and cholesterol 8 , 9 . Unesterified cholesterol, a rare component among prokaryotes, is vital for MPN cells and constitutes one of the most abundant lipids in their membranes 10 . Our recent research has revealed the structure of the immunogenic and essential protein P116 from MPN 11 . The 116 kDa P116 subunits feature two extracellular domains—a small N-terminal domain and a large C-terminal or core domain—that are anchored to the mycoplasma membrane via a flexible linker 11 . The C-terminal domain exhibits a unique fold resembling a half-open hand, characterized by a large β-sheet, the palm, and by pairs of antiparallel amphipathic α-helices, the fingers, that collectively form a vast hydrophobic, lipid-binding, cavity. The fingers flexibility allows the cavity volume to vary according to the lipid cargo. The cavity appears to exhibit significant specificity for lipids crucial for the growth of MPN 11 . MPN and Chlamydia pneumoniae have been detected in increased quantities within ruptured atherosclerotic plaques 12 , 13 . These pathogens have been associated with adventitial inflammation and positive vessel remodeling in thrombosed coronary arteries of patients who suffered acute myocardial infarction 14 . Additionally, experimental studies have demonstrated that inoculation with both agents exacerbates atherosclerosis in the aortas of hyperlipidemic mice 15 . Low-density lipoproteins (LDLs) and high-density lipoproteins (HDLs) are characterized by their content of apolipoprotein (apo)B-100 and apoA-I respectively, and their major lipid components are cholesterol esters and phospholipids, with a smaller amount of unesterified cholesterol. The function of LDL is primarily the transport and delivery of cholesterol to cells, including peripheral tissues and the liver, while HDL transports cholesterol to steroidogenic tissues 16 . Accumulation of oxidatively modified LDL in the subendothelial space induces their uptake by macrophages, promoting foam cell formation and a wide range of bioactivities that drive the development of atherosclerotic lesions. In contrast, HDL promotes macrophage cholesterol efflux to reduce lesion formation 17 . Since, as mentioned above, MPN cannot synthesize several lipids essential for its cell membrane structure, it must obtain these lipids from the blood or arterial lining cells to survive in the bloodstream and localize at atherosclerotic plaques. In this work, we assessed the capacity of P116 to extract the MPN essential lipids from circulating human lipoproteins and from cells. We also investigated the role of P116 in mediating the localization of MPN to human atherosclerotic lesions ex vivo . Moreover, using a genetically engineered MPN chassis, we determined the bacterium's ability in vivo to reach the liver and atheroma plaques via the bloodstream in hyperlipidemic mice, unveiling a new biotechnological approach for studying and treating atherosclerotic and hepatic lesions. 2. Materials and Methods 2.1. Cloning, expression and purification of P116 constructs Regions corresponding to the mpn_213 gene from MPN were amplified from synthetic clones using different primers for each construct: P116F 30 and P116R 957 for P116(30–957); P116F 30 and P116R 845 for P116(30–845); P116F 246 and P116R 818 for P116(246–818); and P116W 681 to generate variant P116W681A. Detailed description on protein expression, purification and protein emptying has been reported previously 11 . 2.2. Determination of isotopic unesterified cholesterol transfer rate from human LDL or HDL The human plasma samples from healthy donors were obtained in accordance with the standards for medical research involving human subjects as recommended by the Declaration of Helsinki. The study protocol was approved by the Ethical Committee of Hospital de la Santa Creu i Sant Pau (protocol code IIBS-APO-2013-105). Human LDL (density 1.019–1.063 g/mL) and HDL (density 1.063–1.210 g/mL) were isolated through sequential gradient density ultracentrifugation, using potassium bromide for density adjustment, at 100,000 g for 24 hours with an analytical fixed-angle rotor (50.3, Beckman Coulter). The composition of both LDL and HDL, including total and unesterified cholesterol, triglycerides and phospholipids, was determined enzymatically, using commercial kits adapted for a COBAS 6000 autoanalyzer (Roche Diagnostics), whereas apoB and apoA1 were determined by an immunoturbidimetric assay, using commercial kits adapted for a COBAS 6000 autoanalyzer (Roche Diagnostics). Radiolabeled LDLs and HDLs were prepared in the following way: 5 µCi of [1,2- 3 H(N)] unesterified cholesterol (Perkin Elmer) was mixed with absolute ethanol, and the solvent was dried under a stream of N 2 . LDL or HDL (0.5 mL, 2.5 g/L of ApoB or ApoA1) were added to the tubes containing the radiotracers, as appropriate, and then incubated for 16 hours in a 37°C bath 11 . The labeled LDLs and HDLs were re-isolated by gradient density ultracentrifugation and dialyzed against PBS through gel filtration chromatography. Specific activities of 3 H-cholesterol-containing LDLs and HDLs were 520 and 4,750 counts per minute (cpm)/nmol of unesterified cholesterol, respectively. The cholesterol transfer to P116 (the construct spanning residues 30 to 957, 1 g/L) was measured after adding either [ 3 H] unesterified cholesterol-containing LDLs or HDL (0.5 g/L of ApoB or apoA1, respectively) and incubation at different times at 37°C. LDL, HDL and P116 were separated by a HisTrap HP affinity. The radioactivity associated with each P116 and HDL fraction was measured through liquid scintillation counting. The percentage of [ 3 H]cholesterol transferred per mL was determined for each condition. The specific activities for each radiotracer were used to calculate the amount of unesterified cholesterol from LDL or HDL to P116. Human apoB and apoA1 levels were determined in both the LDL, HDL and purified P116 fractions by the immunoturbidimetric assays in the COBAS 6000 autoanalyzer. 2.3. H1299 lung, J774.A1 macrophage and Caco-2 colorectal cell cholesterol efflux to P116 Cellular cholesterol efflux to HDL and P116 (residues 30–957) was evaluated using a radiochemical method with H1299 human epithelial-like lung cells (ATCC® CRL-5803, Manassas, VA), J774A1.1 mouse macrophages (ATCC® TIB67™) and epithelial human Caco-2 colorectal cells (ATCC® HTB-37™). For this purpose, 3x10 5 cells/well were seeded in 6-well plates and grown for 72 hours in complete Dulbecco's Modified Eagle's Medium (DMEM) high glucose with L-glutamine and sodium pyruvate (Corning, Glendale, AZ), supplemented with 10% fetal bovine serum (FBS) (Pan Biotech, Aidenbach, Germany) and 100 U/mL penicillin/streptomycin (Dominique Dutscher, Brumath, France). At that point, the cells were incubated with DMEM containing 1 µCi/well of [1α,2α(n)-3H]cholesterol (Perkin Elmer, Boston, MA) and 5% FBS for 48 hours. Subsequently, the macrophages and lung cells were equilibrated overnight with 0.2% free fatty acid bovine serum albumin (Sigma Aldrich/Merck) in DMEM. The following day, the media was removed, and the cells were incubated for 90 minutes with HDL or P116 (100 µg/mL) in medium without any cholesterol supplement. For these in vitro analyses, human HDL and P116 were isolated as described above. The percentage of cholesterol efflux from cells to the acceptors was calculated by dividing the amount of radiolabeled cholesterol in the medium by the sum of radiolabeled cholesterol in both the medium and the cells, as determined by liquid scintillation counting at the end of the experiments. 2.4. Lipid Extraction for Mass Spectrometry Analyses The lipid transfer to P116 (1 g/L) was measured after incubation with either LDL, HDL (0.5 g/L of ApoB or ApoA1, respectively), or H1299 human epithelial-like lung cells (1×10 6 ) at 37°C for 90 minutes. The different P116 fractions were separated using a HisTrap HP affinity column. Metabolite extraction was performed by fractionating the protein samples into pools of species with similar physicochemical properties using appropriate combinations of organic solvents. Two extraction methods were employed based on the chemical class of the target analytes 18 . Platform 1 (Fatty Acyls, Bile Acids, Steroids, and Lysoglycerophospholipids Profiling): 125 µL of P116 (1 g/L) was mixed with 350 µL of methanol (spiked with metabolites not detected in unspiked human serum extracts) in 1.5 mL microtubes on ice. After brief vortexing, the samples were incubated for 1 hour at -20°C. The supernatants were collected following centrifugation at 18,000×g for 15 minutes, dried, and reconstituted in 60 µL of methanol. The reconstituted samples were then centrifuged and transferred to vials for UHPLC-MS analysis. Platform 2 (Glycerolipids, Cholesteryl Esters, Sphingolipids, and Glycerophospholipids Profiling): 50 µL of P116 (1 g/L) was mixed with sodium chloride (50 mM) and chloroform/methanol (2:1) in 1.5 mL microtubes on ice. The extraction solvent was spiked with metabolites not detected in unspiked human serum extracts. After brief vortexing, the samples were incubated for 1 hour at -20°C. Following centrifugation at 16,000×g for 15 minutes, the organic phase was collected, and the solvent was removed. The dried extracts were reconstituted in 60 µL of acetonitrile/isopropanol (1:1), centrifuged (18,000×g for 5 minutes), and transferred to vials for UHPLC-MS analysis. Randomized sample injections were performed for each analytical platform, with QC calibration and validation extracts uniformly interspersed throughout the batch run. 2.5. Ultra-high-performance liquid chromatography–mass spectrometry (UHPLC-MS) analyses Different UHPLC-MS methods were used for each platform. Chromatographic separation and mass spectrometric detection conditions are summarized in Supplementary Table 1 . Chromatography was performed using ACQUITY UPLC systems (Waters Corp., Milford, USA). A LCT Premier XE Time-of-Flight (ToF) (Waters Corp.) and a Xevo G2 QTof (Waters Corp.) mass spectrometers were used for Lipidomics Platform 1 and 2, respectively. The overall quality of the analysis procedure was monitored using repeat extracts of the QC samples. Retention time stability was generally < 6 s variation (injection-to-injection), and mass accuracy was generally < 5 ppm for m/z 400–1000, and < 1.2 mDa for m/z 50–400. All data were processed using the TargetLynx application manager for MassLynx 4.1 software (Waters Corp., Milford, USA). Predefined retention time and mass-to-charge ratio pairs (Rt-m/z) corresponding to the analyzed metabolites were fed into the program. Extracted ion chromatograms (mass tolerance window = 0.05 Da) were then peak-detected and noise-reduced in both the LC and MS domains. This ensured that only true metabolite-related features were processed. Chromatographic peak areas were generated for each sample injection. For identified metabolites, representative MS detection response curves were generated using an internal standard for each chemical class included in the analysis. Assuming similar detector response levels for all metabolites within a chemical class allowed for defining a linear detection range for each variable. Maximum values were defined where the detector response became non-linear relative to the concentration of the representative internal standard. 2.6. Immunoassays Twenty-five patients with an infection by MPN based on a positive result with the Liaison MPN IgG, IgM kit (DiaSorin, Italy) were selected. Similarly, six sera from healthy donors and testing negative for the Liaison M. pneumoniae IgG, IgM kit were also selected. The serum samples were obtained in accordance with the standards for medical research involving human subjects as recommended by the Declaration of Helsinki. The study protocol was approved by the Ethical Committees of Parc Taulí (Ref 2019/664) and Vall d’Hebron University Hospitals (PR(AG)24/2020). In both cases, we used a 1/100 dilution of the patient sera. Indirect ELISA assays were performed on 96 well plates Immulon 4 HBX 96 well plates (ThermoFisher) incubating 1µg of each antigen at 4°C overnight. 1/100 dilutions of each patient sera were added to the plate and detected using and anti-human IgG antibody conjugated with HRP (Thermofisher Scientific). Upon incubation for 30 minutes with 100 µl of substrate (Thermofisher Scientific), 100 µl of sulphuric acid 25% were added to stop the reaction and absorbance was read at 450 nm on a Triturus ELISA instrument (Grifols) device. Reference filter was set at 620 nm. 2.7. Bacterial strains and culture conditions Mycoplasma strains were grown at 37°C under 5% CO 2 in tissue culture flasks (Corning) with Hayflick liquid medium. Hayflick was prepared by mixing 800 ml of non-complete medium A (20 g PPLO broth (BD Difco, Franklin Lakes, NJ), 30 g HEPES [100 mM final], 25 ml 0.5% phenol red solution (Sigma Aldrich/Merck St. Louis, MO), 200 ml heat-inactivated horse serum (Life Technologies), 20 ml sterile‐filtered 50% glucose, and 1 ml of a 100 mg/ml stock of ampicillin (final concentration 100 µg/ml, ampicillin sodium salt (Sigma Aldrich/Merck). 2.8. Polyclonal and monoclonal antibody generation and purification 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 11 . The last injection was delivered four days before splenectomy. Isolated B lymphocytes from the immunized mice were fused to NSI myeloma cells 19 to obtain stable hybridoma cell lines producing monoclonal antibodies (mAb), as previously described 20 . 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 MPN cell lysates and by immunofluorescence using whole, non-permeabilized MPN cells. Only those clones with supernatants revealing a single 116 kDa band in protein profiles and also exhibiting a consistent fluorescent staining of MPN cells were selected and used in this work. Polyclonal sera were obtained by cardiac puncture of properly euthanized mice just before splenectomy and tittered using serial dilutions of the antigen. The titer of each polyclonal serum was determined as the IC 50 value from four parameter logistic plots and found to be approximately 1/4000 for both sera. The monoclonal antibody P116 mAb-3B5 was selected by ELISA screening using P116 30–957 construct, but tested negative against the P116 central region construct (residues 246–818). The mAb-3G9 was selected by ELISA screening and showed high specificity to P116 in Western blotting analysis and immunofluorescence microscopy of living MPN cells 11 . To ensure the produced antibody was monospecific, hybridoma cells producing mAb-3G9 were cloned by limiting dilution and subjected to a second cloning step using the BD FACS Discover S8, selecting cells by size and complexity and sorting in single cell mode. As indicated in the different experiments, mAb-3G9 was used unpurified from supernatants of exponentially grow hybridoma cell cultures in Roswell Park Memorial Institute medium (RPMI) 1640 supplemented with 8% fetal bovine serum. For cryoelectron microscopy studies, mAb-3G9 was purified from 100 mL aliquots of cell culture supernatants. Briefly, supernants were diluted twice in 20 mM sodium phosphate buffer pH 7.0 and submitted to 1 mL HiTrap Protein G columns (cytiva) previously equilibrated in this buffer. After washing the column with 10 volumes of phosphate buffer, mAb was eluted in 0.1 M glycine pH 2.7 on tubes with a 1/10 volume of Tris 1 M pH 9. Pooled eluted fractions were concentrated using a 50 K Amicon Ultra 15 centrifugal filters to a 1 mL final volume and washed three times with PBS. The concentrated antibody sample was further purified using a Superose 6 Increase 10/300 GL column previously equilibrated in PBS and the eluted fractions in the range of 150 kDa were finally concentrated to a 0.5 mL volume also using a 50 K Amicon. 2.9. Monoclonal antibody sequencing Cloned hybridoma cells producing mAb-3G9 were cultured in T75 flasks using RPMI 1640 medium supplemented with 8% fetal bovine serum and grown until 50% confluence. RNA extraction was performed using the RNeaSY Mini Kit (QIAGEN) and 5 x 10 6 hybridoma cells by direct lysis of the cell pellet and omitting the optional DNase digestion step. Then, the cDNAs coding for the heavy and light chains were amplified by SMART RT-PCR following the simplified workflow described by Meyer et al 21 with few modifications. Briefly, the first strand was obtained by incubating 500 ng of total RNA, 1 µL of primers mIGK RT, mIGL RT or mIGHG RT at 10 µM in separate tubes, 1 µL of 10 mM dNTPs and 11µL of RNase free water at 65 ºC for 5 minutes. Then, 4 µL of the first strand buffer of M-MLV reverse transcriptase (Invitrogen), 2 µL 100 mM DTT, 1 µL RNaseOut and 1 µL Template-Switch primer 100 µM were added to each tube. After incubating 2 minutes at 37 ºC 1 µL of M-MLV reverse transcriptase (Invitrogen) was added to each tube, which were incubated for 50 minutes at 37 ºC and 15 minutes at 70 ºC. The touch down PCR reaction was performed as originally described using the Phusion termopolymerase (Thermo Scientific) using 3 µL of each cDNA obtained in the previous step. The resulting PCR products were subjected to a 1% agarose gel and bands corresponding to the cDNAs of the heavy and kappa light chains ( Supplementary Fig. 1A ). Next, 700 bp and 600 bp bands corresponding to the RT-PCR products of the heavy and kappa light chains, respectively were excised from the gel, and the purified DNAs were ligated to the pBSKII vector previously digested with EcoRV. Ligation reactions were then transformed to competent Escherichia coli XL1-blue cells and several colonies were selected and cultured to obtain the plasmid DNAs. A total of 9 plasmids bearing cDNAs from the heavy chain and 8 plasmids containing cDNAs from the kappa chain were submitted to Sanger sequencing on an ABI 3730 DNA Analyzer using the BigDye Terminator method and the reverse universal primer. The obtained DNA sequences were trimmed to remove the plasmid sequences using the VectorStrip application from the mEMBoss suite, translated to the six possible read frames and aligned using the ClustaX v2.1 application ( http://www.clustal.org/clustal2 ). Eight out of nine sequences for the kappa chain and five out eight sequences from the kappa light chain contained sequences to the corresponding immunoglobulin chains. Aligned amino acid sequences coding for heavy chains were 95% identical starting from the first Met residue, and sequences coding for kappa light chains were 100% identical starting from the first Met residue ( Supplementary Fig. 1B ). The consensus sequence from each alignment was chosen as the complementary determining sequence in each of the chains of the P116 mAb-3G9 antibody. 2.10. Single-particle cryoEM P116-mAb complex For single-particle cryo-electron microscopy (cryoEM), a 3 µL drop of purified P116-mAb complex (0.2 mg/mL in 20 mM Tris pH 7.4 and 150mM NaCl buffer) was applied to glow-discharged C-Flat™ holey grids (CF-1.2/1.3; 300 mesh), 5s blotting (-3 blotting force) and vitrified in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific) set to 4 ºC and 95% humidity. Cryo-EM grids were stored at liquid nitrogen temperature. Movies of P116-mAb complex were collected on a Glacios electron microscope operated at 200 kV (IBMB-CSIC CryoEM Platform). Imaging was performed using EPU at a nominal magnification of ×150,000 (0.96 Å/pixel) at 200 kV Glacios (Thermo Fisher Scientific) electron microscope equipped with Falcon 4 detector (Thermo fisher Scientific). A total of 5859 movies were collected. The camera was operated in counting mode with a dose rate of 7.24 electrons per Å 2 s − 1 , resulting in a total dose of 39.70 electrons per Å 2 . Defocus values ranged from − 1.0 to − 2.5 µm. CryoSPARC v4 22 was used to process the cryoEM data. Particles were selected with the Blob picker using a particle diameter of 150–300 Å. Particles were extracted and classified in 2D. For the final processing, the 2D particles containing the complex were selected, remaining a total of 412,947 particles which were used to generate an ab initio reconstruction with three classes followed by a subsequent non-uniform heterogeneous refinement with the largest class. Finally, a model with a resolution of 5.06 Å was obtained ( Supplementary Table 2 ). Docking, tracing and refinement of all the structures was performed alternating interactive and automatic cycles with programs Coot and Phenix. The final refined structure has been deposited in the PDB with code: 8ROR and the map in the EMDB with code: 19402 2.11. Effect of mAb-3G9 on mycoplasma cell growth Growth rates of MPN cultures were determined using an adaptation of the colorimetric protocol described by Karr and colleagues 23 . MPN M129 strain was grown to mid-log phase in 25 cm 2 flasks with 5 mL SP4 medium. Attached cells were scraped off, recovered by centrifugation at 15000 g and resuspended in 3 mL of fresh SP4 medium, SP4 medium supplemented with a 1/200 dilution of hybridoma cell culture supernatant in PBS, or SP4 medium supplemented with RPMI 1640 medium also diluted 1/200 in PBS as control. Then, 300 µL of the cellular suspension were seeded in four different wells of a 96-well plate and serially diluted 1/3 until reaching a 1/243 using the same respective media described above. The resulting 96-well plated was sealed with transparent tape, placed into a Tecan Sunrise Absorbance Microplate Reader (Tecan), and incubated at 37ºC for 8 days. During the incubation time, absorbance at 550 nm for each well was recorded each 15 minutes to quantify the medium acidification resulting from the mycoplasma growth, which turns the color of the phenol red indicator from red to yellow. Curves of absorbance vs time were plotted for each well and the inflection point of each curve was determined by iteration using the Excel application controlled by a Phyton script. Next, the inflection points were plotted, using the Napierian logarithm of the dilution as the x coordinate for each dilution. Once all the inflection points were plotted, the slope (µ, growth rate constant) was inferred by linear regression, and the doubling time (g) was obtained according to the general equation for exponential growth of bacteria (g = ln2/(1/ µ)). To visualize the effect of the mAb-3G9 addition on mycoplasma cells, MPN strain M129 was grown in IBIDI chamber slides as described above using SP4 medium supplemented with a 1/40 dilution in PBS of a supernatant of exponentially grow hybridoma cell cultures. 2.12. Interaction of MPN with human atherosclerotic plaques ex vivo Carotid endarterectomy specimens were obtained within one hour of surgical resection. The study protocol was approved by the Ethical Committee of Hospital de la Santa Creu i Sant Pau (protocol code IIBSP-LPM-2019-94). Immediately after collection, the specimens were placed in phosphate-buffered saline for processing. The specimens were then evaluated under a dissecting microscope and cut into small pieces. Fragments containing atherosclerotic plaque and fragments from the distal boundary of the plaque were collected for further analysis. The MPN strain M129 expressing the fluorescent protein Venus 24 was grown in cell culture flasks containing SP4 medium and incubated at 37°C and 5% CO 2 . Surface-attached mycoplasmas were collected using a cell scraper and resuspended in SP4 medium. Co-cultures were performed in IBIDI eight-well chamber slides, being each well seeded with 1 × 10 5 mycoplasma cfus in 200 µL of 0.22 µm filtered SP4 medium and a small piece of healthy carotid tissue (HT) or atherosclerotic plaque tissue (AP). Phase contrast (PhC) epifluorescence microscopy images of MPN cells grown in presence of Healthy Human Atherosclerotic tissue (H.T.) or Human Atherosclerotic plaque tissue (A.P.) were taken using an inverted Nikon Eclipse TE 2000-E microscope. After inoculation, images were taken at different times (0h, 24h and 48h) and focusing into the same regions of the human tissues in the different observation times. Phase contrast and YFP (excitation 490/510 nm, 520/550 emission) images were captured using an Orca Fusion camera (Hamamatsu) controlled by NIS-Elements BR software (Nikon). 2.13. Biodistribution of MPN into atherosclerotic mice Wild-type mice and LDL receptor knockout mice on the C57BL/6 background were purchased from Jackson Laboratories (Bar Harbor, ME; #000664 and #002207, respectively), and housed under pathogen-free conditions at the Clinica Universidad de Navarra animal facility. Animal handling and procedures followed the current European (Directive 86/609/EEC) and National (Real Decreto 53/2013) legislations as well as the FELASA and ARRIVE guidelines and obtained the approval of the Animal Experimentation Ethic Committee of Clinica Universidad de Navarra (Code: 096 − 23) and the local Government authorization. Mice were kept in a temperature-controlled (22 ºC) room with a 12-hour light/dark cycle, and food and water were provided ad libitum. We used 4-5th -month-old male mice fed with a Western-type diet (TD.88137, Harlan Teklad, Madison, WI, containing 21% fat and 0.2% cholesterol) for 8 weeks. Plasma total cholesterol and triglycerides were determined enzymatically using commercial kits adapted for a COBAS 501/6000 autoanalyzer (Roche Diagnostics, Rotkreuz, Switzerland). Liver lipids were extracted using isopropyl alcohol-hexane (2:3, v/v) and the lipid layer was collected, evaporated, and resuspended in 0.5% (w/v) sodium cholate (Serva, Heidelberg, Germany). Cholesterol and triglycerides were determined using commercial kits adapted for the COBAS 501/6000 autoanalyzer. Serial sections of the proximal aorta were stained with hematoxylin and eosin for quantification of the aortic lesion area 25 . To investigate the in vivo biodistribution of Mycoplasma using single photon emission computed tomography (SPECT/CT), the non-pathogenic MPN strain CV8 26 was cultured in Hayflick for 3–4 days in T75 flask. Cells were washed twice and scraped in 1 ml of PBS. A bacterial suspension containing 10 7 CFUs was radiolabeled by incubating it with 161.162 µCi of [¹¹¹In]-oxine at 37°C for 15 minutes. Following radiolabeling, 100 µL (18.65 ± 2.70 µC) of the radiolabeled Mycoplasma was injected in the vein tail. Six hours post-injection, images were acquired using a SPECT/CT scanner (U-SPECT6/E-class, (MILabs, Utrecht, The Netherlands). During image acquisition, the animals were placed in the prone position on the scanner bed under continuous anesthesia with isoflurane (2% in 100% O₂), and a 60-minute whole body scan was performed. After SPECT acquisition, CT scans were conducted to obtain anatomical reference, using a tube setting of 55 kV and 0.33 mA. SPECT and CT images were reconstructed using the ¹¹¹In photopeaks at 170 and 245 keV with a 20% energy window. A calibration factor was applied to determine activity (MBq/mL), and attenuation correction was performed using the CT attenuation map. The animals were subsequently sacrificed, and the heart along with major vessels was excised for SPECT/CT imaging. To enhance contrast and improve anatomical visualization in CT, the hearts were immersed in a 1:1 mixture of radiographic contrast agent (Omnipaque) and formalin and positioned in a well plate. Experiments were conducted in a blind manner concerning the origin of the specimens to reduce bias. 2.14. Statistical methods Data are presented as the mean ± standard error of mean (SEM). Unpaired t tests were used to compare the differences between two groups. Multiple t-tests were used to compare differences in the amount of each lipid species between groups. A Kruskal-Wallis test, followed by Dunn’s multiple comparison test, was performed to compare the growth of cultured mycoplasma cells under different conditions and the reactivity of human sera against P116 constructs. A chi-square test was used to compare the distribution of human antibody reactivity against different P116 constructs. GraphPad Prism version 8.0.2 for Windows (GraphPad Software, San Diego, CA) was used to perform all statistical analyses. A P - value ≤ 0.05 was considered statistically significant. 2.15. Data availability The data, analytical methods, and study materials will be available to other researchers for purposes of reproducing the results or replicating the procedure upon reasonable request. Source data are provided with this paper. 3. Results 3.1. Human circulating LDL is a major source of unesterified cholesterol for MPN P116 Since both LDL and HDL are major cholesterol-carrying lipoproteins in human plasma and are responsible for supplying cholesterol to tissues, we initially measured the rate of radiolabeled unesterified cholesterol transfer from LDL or HDL to MPN P116 (see the schematic diagram of the experimental design in Fig. 1 A). The rate of cholesterol transfer from these lipoproteins to P116 was measured at equal concentrations of their main apolipoproteins (see Supplementary Fig. 2 for lipoprotein composition). A time-dependent experiment was performed in which LDL containing radiolabeled cholesterol was incubated with unlabeled P116 for up to 90 minutes. Under these conditions, the quantity of labeled cholesterol in isolated P116 increased rapidly during the incubation, reaching a maximum plateau after 45 minutes (Fig. 1 B). In all experiments, the main LDL protein (ApoB100) was not detected, cross-checked by immunoturbidimetric detection, verifying that no LDL had contaminated the purified P116 samples. When radiolabeled HDL was incubated with P116, a significant amount of the HDL-derived radiotracer was transferred to the post-incubated, isolated P116; however, this transfer occurred with lower efficiency compared to LDL (Fig. 1 B and C ). The presence of the main HDL protein (apoA1) was not detected in any of the P116 fractions. The specificity of this uptake was validated by mutating Trp681 to Ala, which is located on the P116 surface and may participate in the interaction with LDL and HDL. We found that the amount of LDL-derived radiotracer detected in the post-incubated and isolated P116 mutant was 60% lower compared to wild-type P116 ( Supplementary Fig. 3 ). 3.2. Human circulating LDL and HDL differentially transfer multiple lipids to the P116 We conducted a detailed ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) analysis of the lipid species in the isolated P116 fraction after incubation with human LDL or HDL under the same experimental conditions described for the isotopic method (Fig. 1 A ) . We identified 370 lipid species in the isolated P116 fractions. In empty P116, most of these lipid species were not detected, except for some triacylglycerol species with low relative intensity ( Supplementary Table 3 ). In the P116 samples refilled from LDL, we found a striking accumulation of sphingomyelins, phosphatidylcholines and triacylglycerols, although the two latter were even higher in P116 refilled with HDL ( Fig. 1 D). Additionally, significant levels of esterified cholesterol, phosphatidylethanolamines and lysophosphatidylcholines were found in the isolated P116 fraction after incubation with both human LDL and HDL, with the former one being higher in P116 refilled with HDL (Fig. 1 D). 3.3. P116 uptakes lipids from H1299 lung cells with low efficiency We then adapted a method used for evaluating unesterified cholesterol efflux stimulated by HDL from macrophages to evaluate the potential of P116 to uptake unesterified cholesterol from the human pulmonary cell membrane (see the schematic diagram of the experimental design in Fig. 2 A). When radiolabeled pulmonary cells were incubated with P116 in the media, a significant fraction of radiotracer was detected in the isolated P116, although with lower efficiency compared to that stimulated by the main physiological acceptor, HDL (Fig. 2 B). The analysis of the lipid species in the isolated P116 fraction after incubation with human pulmonary cells also identified 276 lipid species, but their relative intensity in P116 was very low in comparison with that of the isolated P116 incubated with lipoproteins (Fig. 2 C and Supplementary Table 3 ). We also evaluated the potential of P116 to uptake unesterified cholesterol from cholesterol-loaded macrophages and colorectal cells (Fig. 2 D). As observed with pulmonary cells, a portion of the radiotracer was detected in the isolated P116 after incubation with cholesterol-loaded macrophages and with cholesterol-loaded colorectal cells ( Fig. 2 E and F) , indicating that P116 can extract cholesterol from a diversity of cell types. 3.4. Antibodies targeting the C-terminal domain of P116 interfere both cholesterol uptake and mycoplasma cell growth To dissect the immunogenic activity of P116, we produced three different constructs: P116/30–957 (residues 30 to 957), a derivative lacking the C-term region (P116/30–845) and a derivative lacking also the N-terminal domain (P116/246–818) 11 . The three constructs were tested with total sera from twenty-five patients with positive diagnostic for MPN based on serological tests. All patients were positive for the P116/30–957 construct ( Supplementary Table 4 ). We obtained identical results when we used the P116/30–845 construct. However, the P116/246–818 construct resulted in a significant decrease in positive sera (72%) compared to the P116/30–957 and P116/30–845 constructs ( Supplementary Table 4 ). Moreover, the overall signal of the P116/30–957 and P116/30–845 constructs was higher than the obtained with P116/246–818 (Fig. 3 A). These results also indicate that targeting the C-terminal domain of P116 in mycoplasma infections has potential therapeutic applications. Then, a mAb-3G9 raised against P116 was selected. The structure of the P116-mAb-3G9 complex determined by Cryo-EM ( Supplementary Fig. 4 ), showed that the epitope is located in four α-helices forming two of the fingers that define the hydrophobic cavity of P116 (Fig. 3 B), suggesting that mAb-3G9 hinders the fingers flexibility required by the functioning of P116. Importantly, pre-incubation of P116 with mAb-3G9 and then incubation with radiolabeled LDL, resulted in a notable reduction in the amount of radiolabeled cholesterol found in the post-incubated and isolated P116 fraction (Fig. 3 C). MAb-3G9 also interfered with the growth of cultured mycoplasma cells (Figs. 3 D). 3.5. Antibodies targeting the C-terminal domain of P116 impair the localization of MPN to human atherosclerotic plaques ex vivo We assessed the ability of MPN to adhere to human carotid atherosclerotic plaques using cell culture invasion assays. For this purpose, we used the MPN strain M129 expressing the fluorescent protein Venus 24 . Cells were cultured in the presence of human atherosclerotic carotid tissue fragments, with distal healthy tissue as a control (see the schematic diagram of the experimental design in Fig. 4 A). After inoculation, growth was monitored at 0-48h by phase contrast and epifluorescence microscopy. Although the tissue exhibited some autofluorescence, the presence of mycoplasma cells could be properly detected. Compared to the control healthy tissue, mycoplasma cell localization and growth was significantly higher in the vicinity of the atherosclerotic tissue (Fig. 4 B). In line with the cell growth inhibition by mAb-3G9, this antibody that interacts with the C-terminal domain of P116, also interfered with the presence of MPN cells in the atherosclerotic tissue (Fig. 4 B and Supplementary Fig. 5 ). This effect was not observed when MPN cells were incubated with P116 mAb-3B5, which exhibits no specificity to the central region of P116 (residues 246–818, Fig. 4 B and Supplementary Fig. 5 ). 3.6. Biotransfer of MPN in hyperlipidemic mice in vivo We further investigated whether MPN could colonize various organs in vivo using SPECT/CT imaging. This was achieved by intravenously injecting a radiolabeled non-pathogenic MPN chassis into C57BL/6 wild-type mice and a hyperlipidemic mouse model (see the schematic diagram of the experimental design in Fig. 5 A). We specifically used LDL receptor knockout mice on a C57BL/6 background, which develop hyperlipidemia when fed a Western-type diet ( Supplementary Fig. 6 ). These mice are also prone to developing fatty liver and extensive atherosclerosis in the proximal aorta, compared to wild-type mice fed with a Western-type diet (Fig. 5 B and C ). The biodistribution of radiolabeled MPN chassis revealed unexpectedly strong signal intensity in the livers of wild-type mice, with even higher signal levels observed in knockout mice, consistent with liver cholesterol data (Fig. 5 D). Notably, images of the heart also detected the presence of radiolabeled MPN in the aortic root of knockout mice, but not in wild-type mice (Fig. 5 E). In contrast, no signal from the radiolabeled MPN chassis was detected in the lungs. These observations confirm the effective tropism of MPN toward cholesterol-rich livers and atherosclerotic plaques. 4. Discussion MPN cannot synthesize cholesterol de novo and must acquire it from the host, underscoring its dependence on host resources for survival 27 . P116 in MPN is an essential and highly immunogenic protein reported to play also a role in the adherence of the bacterium to host cells 28 . We recently demonstrated that the P116 structure features a novel fold, including an unusually large hydrophobic cavity filled with ligands 11 . Mass spectrometry and radioactivity transfer experiments confirmed the ability of P116 to extract various lipids from fetal bovine serum and cholesterol from HDLs 11 . In the present work, we demonstrate that human LDLs can be a major source of unesterified cholesterol for P116, with a very efficient transfer rate compared with that of HDLs, at least at equal major protein concentrations, i.e., at an equal number of lipoproteins. The amount of unesterified cholesterol per particle and its location on the surface of the lipoprotein could explain this accelerated transfer from LDLs to P116. Furthermore, LDLs also efficiently transfer a large variety of sphingomyelin species. Importantly, phosphatidylcholine species, which are also located on the surface of lipoproteins, were the major lipids found in P116 after being incubated with either LDLs or HDLs. The presence of the hydrophobic cavity in P116 and its structure allows the interaction with the amphipathic phospholipids, facilitating the uptake and exchange of these lipid species. In contrast, the elongated shape of P116 could explain its effectiveness, when incubated with HDLs, in the up taking of triacylglycerol and esterified cholesterol species, which are found in the core of HDL 29 . In summary, this work demonstrates, for the first time to our knowledge, that human LDLs serve as a key source of unesterified cholesterol, phosphatidylcholines, and sphingomyelins to P116 from MPN. In contrast, HDLs primarily provide phosphatidylcholines, esterified cholesterol, and triacylglycerols to P116. P116 is also capable of inducing the efflux of unesterified cholesterol from lung and colorectal cells and from macrophages although with lower efficiency compared to HDL particles. It should be noted that HDLs are complex lipoproteins with a larger surface area relative to volume and contains apoA-I, which facilitate cholesterol and phospholipid efflux from cells by interacting with specific transporters on the cell membrane 30 . Our lipidomic analyses confirmed that P116 could uptake a variety of lung cell phosphatidylcholines, but again with much lower efficiency compared to LDL and HDL. Overall, these results demonstrate the ability of mycoplasmas to acquire essential lipids from diverse sources, enabling the colonization of various tissues. A P116 variant of a tryptophan residue at the protein surface, W681A, accumulated 60% less LDL-derived unesterified cholesterol compared to wild-type P116. Surface tryptophan residues are known to play critical roles in the functioning of the extensively investigated cholesteryl ester transfer proteins 31 , which suggests that related mechanisms for cholesterol uptake and release might be at work in cholesteryl ester transfer protein and P116. The mAb-3G9, raised against P116, was found to target an epitope within the C-terminal domain. This antibody interfered with the ability of P116 to uptake unesterified cholesterol and directly inhibited the growth of cultured mycoplasma cells. Furthermore, our serological studies in patients infected with P. pneumoniae indicate a significant reduction in the percentage of antibodies generated against the N-terminal domain of P116. Therefore, an antigen derived exclusively from the C-terminal domain of P116 could serve as a promising candidate for the development of vaccines against MPN . Some studies have found a higher prevalence of MPN in patients with cardiovascular diseases compared to healthy controls 32 , 33 , although a direct causal link has not been demonstrated in humans. Given that ruptured atherosclerotic plaques have shown increased quantities of MPN 12 , 13 , we further investigated the potential localization of MPN in atherosclerotic lesions ex vivo . Compared to healthy control tissue, mycoplasma cell localization was notably increased around lipid-rich atherosclerotic tissue. Consistent with its observed inhibition of cell growth and cholesterol uptake, mAb-3G9 impaired the binding and proliferation of MPN cells in atherosclerotic tissue, highlighting its potential for treating both pulmonary and extrapulmonary MPN infections. SPECT/CT analysis in biotransfer assays in vivo revealed a significant localization of MPN in the liver and in the aortic atheroma plaques following introduction into the bloodstream of a mouse model of hyperlipidemia and atherosclerosis. MPN primarily colonized the liver, even in wild-type mice, likely due to elevated lipid levels in the liver. MPN infection has been associated with liver disease, particularly in children 34 . Thus, the availability of nutrients directing MPN cells toward specific tissues may partly explain the organism’s tissue colonization and its association with various non-respiratory symptoms and conditions 35 . Our findings underscore the concept that P116 plays a critical role in extracting essential lipids from physiological circulating lipoproteins and from a diversity of cell types, which can explain the colonization of different tissues by MPN . In addition, a mAb targeting the C-terminal domain of P116, which reduces cholesterol extraction, inhibits mycoplasma growth in culture, and blocks MPN binding to human lipid-rich atherosclerotic lesions ex vivo , demonstrates therapeutic potential. By limiting MPN colonization in vulnerable areas, this mAb could help reduce the risk of infections or complications associated with atherosclerosis. The presence of MPN in the liver and atheroma plaques in vivo suggests the potential use of the MPN chassis, engineered as a genetically modified biological pill 36 , as an innovative biotechnological tool for studying and treating liver diseases, such as fatty liver and liver cancer, as well as atherosclerotic lesions. Declarations Acknowledgements This work was partly funded by the Instituto de Salud Carlos III and FEDER \"Una manera de hacer Europa\" grant PI2300232 (to J.E-G). N.R was funded by Agencia Estatal de Investigación (AEI/10.13039/501100011033 and CNS2023-144119) within the Subprograma Ramón y Cajal (RYC-201722879). CIBERDEM is an Instituto de Salud Carlos III project. I.F. and J.P. were funded by MICINN-Spain grant PID2021-125632OB-C21 and PID2021-125632OB-C22. The authors acknowledge funding from Project, IU16-014045 (CRYO-TEM) from Generalitat de Catalunya and by “ERDF A way of making Europe”, by the European Union. Authorship contribution statement David Vizarraga: Conceptualization, Methodology, Validation, Formal analysis, Writing – review & editing. Marina Marcos: Conceptualization, Methodology, Validation, Formal analysis, Writing – review & editing. Noemi Rotllan: Conceptualization, Methodology, Validation, Formal analysis, Funding, Writing – review & editing. Jesús Martín: Methodology, Validation, Formal analysis. David Santos: Methodology. Mercedes Camacho: Methodology, Writing – review & editing. Pablo Guerra: Methodology, Validation, Formal analysis. Félix Pareja: Methodology, Validation, Formal analysis. María Collantes: Methodology, Validation, Formal análisis, Writing – review & editing. Wanlu Wu: Methodology, Validation, Formal analysis. Irene Rodríguez-Arce: Methodology, Writing – review & editing , Luis Serrano: Funding, Writing – review & editing. Jaume Piñol: Conceptualization, Methodology, Validation, Formal analysis, Funding, Writing – original draft, Writing – review & editing. Ignacio Fita: Conceptualization, Methodology, Validation, Formal analysis, Funding, Writing – original draft, Writing – review & editing. Joan Carles Escolà-Gil: Conceptualization, Methodology, Validation, Formal analysis, Funding, Writing – original draft, Writing – review & editing. Declaration of Interest All authors declare that they have no relationships relevant to the contents of this paper to disclose and have approved the final version of the article. References Tsiodras S, Kelesidis I, Kelesidis T, Stamboulis E, Giamarellou H (2005) Central nervous system manifestations of Mycoplasma pneumoniae infections. J Infect 51:343–354 Ferwerda A, Moll HA, de Groot R (2001) Respiratory tract infections by Mycoplasma pneumoniae in children: a review of diagnostic and therapeutic measures. Eur J Pediatr 160:483–491 Atkinson TP, Balish MF, Waites KB (2008) Epidemiology, clinical manifestations, pathogenesis and laboratory detection of Mycoplasma pneumoniae infections. FEMS Microbiol Rev 32:956–973 Parrott GL, Kinjo T, Fujita J (2016) A Compendium for Mycoplasma pneumoniae. Front Microbiol 7:513 Meyer Sauteur PM et al (2024) Mycoplasma pneumoniae: delayed re-emergence after COVID-19 pandemic restrictions. Lancet Microbe 5:e100–e101 Nordholm AC et al (2023), December,. Mycoplasma pneumoniae epidemic in Denmark, October to Euro Surveill 29(2024). Himmelreich R, Plagens H, Hilbert H, Reiner B, Herrmann R (1997) Comparative analysis of the genomes of the bacteria Mycoplasma pneumoniae and Mycoplasma genitalium. Nucleic Acids Res 25:701–712 Lluch-Senar M et al (2015) Defining a minimal cell: essentiality of small ORFs and ncRNAs in a genome-reduced bacterium. Mol Syst Biol 11:780 Gaspari E et al (2020) Model-driven design allows growth of Mycoplasma pneumoniae on serum-free media. NPJ Syst Biol Appl 6:33 Dahl J (1993) The role of cholesterol in mycoplasma membranes. Subcell Biochem 20:167–188 Sprankel L et al (2023) Essential protein P116 extracts cholesterol and other indispensable lipids for Mycoplasmas. Nat Struct Mol Biol 30:321–329 Higuchi ML et al (2000) Detection of Mycoplasma pneumoniae and Chlamydia pneumoniae in ruptured atherosclerotic plaques. Braz J Med Biol Res 33:1023–1026 Higuchi Mde L et al (2003) Coinfection with Mycoplasma pneumoniae and Chlamydia pneumoniae in ruptured plaques associated with acute myocardial infarction. Arq Bras Cardiol 81(12–22):11–11 Ramires JA, Higuchi Mde L (2002) [Mycoplasma pneumoniae and Chlamydia pneumoniae are associated to inflammation and rupture of the atherosclerotic coronary plaques]. Rev Esp Cardiol 55(Suppl 1):2–9 Damy SB et al (2009) Mycoplasma pneumoniae and/or Chlamydophila pneumoniae inoculation causing different aggravations in cholesterol-induced atherosclerosis in apoE KO male mice. BMC Microbiol 9:194 Feingold KR (2000) Introduction to Lipids and Lipoproteins. In: Feingold KR et al (eds) Endotext. South Dartmouth (MA) Linton MF et al (2000) The Role of Lipids and Lipoproteins in Atherosclerosis. In: Feingold KR et al (eds) Endotext. South Dartmouth (MA) Barr J et al (2010) Liquid chromatography-mass spectrometry-based parallel metabolic profiling of human and mouse model serum reveals putative biomarkers associated with the progression of nonalcoholic fatty liver disease. J Proteome Res 9:4501–4512 Kohler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495–497 Guasch A et al (2020) Structure of P46, an immunodominant surface protein from Mycoplasma hyopneumoniae: interaction with a monoclonal antibody. Acta Crystallogr D Struct Biol 76:418–427 Meyer L et al (2019) A simplified workflow for monoclonal antibody sequencing. PLoS ONE 14:e0218717 Punjani A, Rubinstein JL, Fleet DJ, Brubaker MA (2017) cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat Methods 14:290–296 Karr JR et al (2012) A whole-cell computational model predicts phenotype from genotype. Cell 150:389–401 Mariscal AM et al (2018) Tuning Gene Activity by Inducible and Targeted Regulation of Gene Expression in Minimal Bacterial Cells. ACS Synth Biol 7:1538–1552 Rotllan N et al (2022) Antagonism of miR-148a attenuates atherosclerosis progression in APOB(TG)Apobec(-/-)Ldlr(+/-) mice: A brief report. Biomed Pharmacother 153:113419 Montero-Blay A et al (2023) Bacterial expression of a designed single-chain IL-10 prevents severe lung inflammation. Mol Syst Biol 19:e11037 Razin S, Yogev D, Naot Y (1998) Molecular biology and pathogenicity of mycoplasmas. Microbiol Mol Biol Rev 62:1094–1156 Svenstrup HF, Nielsen PK, Drasbek M, Birkelund S, Christiansen G (2002) Adhesion and inhibition assay of Mycoplasma genitalium and M. pneumoniae by immunofluorescence microscopy. J Med Microbiol 51:361–373 Qiu X et al (2007) Crystal structure of cholesteryl ester transfer protein reveals a long tunnel and four bound lipid molecules. Nat Struct Mol Biol 14:106–113 Phillips MC (2014) Molecular mechanisms of cellular cholesterol efflux. J Biol Chem 289:24020–24029 Koivuniemi A, Vuorela T, Kovanen PT, Vattulainen I, Hyvonen MT (2012) Lipid exchange mechanism of the cholesteryl ester transfer protein clarified by atomistic and coarse-grained simulations. PLoS Comput Biol 8:e1002299 Momiyama Y, Ohmori R, Taniguchi H, Nakamura H, Ohsuzu F (2004) Association of Mycoplasma pneumoniae infection with coronary artery disease and its interaction with chlamydial infection. Atherosclerosis 176:139–144 Chung WS, Hsu WH, Lin CL, Kao CH (2015) Mycoplasma pneumonia increases the risk of acute coronary syndrome: a nationwide population-based cohort study. QJM 108:697–703 Poddighe D (2020) Mycoplasma pneumoniae-related hepatitis in children. Microb Pathog 139:103863 Hu J et al (2022) Insight into the Pathogenic Mechanism of Mycoplasma pneumoniae. Curr Microbiol 80:14 Mazzolini R et al (2023) Engineered live bacteria suppress Pseudomonas aeruginosa infection in mouse lung and dissolve endotracheal-tube biofilms. Nat Biotechnol 41:1089–1098 Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryFiguresandtables12172024.docx Supplementary Figures and Tables Cite Share Download PDF Status: Published Journal Publication published 16 Dec, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-5668698\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Article\",\"associatedPublications\":[],\"authors\":[{\"id\":398311060,\"identity\":\"aeb76dee-06c6-4e30-9682-f0667688d9f1\",\"order_by\":0,\"name\":\"Joan Carles 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Fita\",\"email\":\"\",\"orcid\":\"https://orcid.org/0000-0002-7521-2679\",\"institution\":\"nstituto de Biología Molecular de Barcelona (IBMB-CSIC), Parc Científic de Barcelona\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Ignacio\",\"middleName\":\"\",\"lastName\":\"Fita\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2024-12-18 10:50:52\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-5668698/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-5668698/v1\",\"draftVersion\":[],\"editorialEvents\":[{\"content\":\"https://doi.org/10.1038/s41467-025-66129-5\",\"type\":\"published\",\"date\":\"2025-12-16T05:00:00+00:00\"}],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":73303697,\"identity\":\"08baf314-46cd-441d-9d09-1efbb6076e16\",\"added_by\":\"auto\",\"created_at\":\"2025-01-08 16:32:50\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":82660,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eHuman circulating LDLs and HDLs are the main sources of unesterified cholesterol and multiple essential lipids for P116. \\u003c/strong\\u003e(A) \\u003cstrong\\u003eSchematic representation of isotopic unesterified cholesterol transfer and lipidomic analyses. \\u003c/strong\\u003eLDL or HDL (0.5 mg/mL) were radiolabeled with [1,2-³H(N)] unesterified cholesterol and incubated with P116 (0.25 mg/mL) at 37°C. P116 was subsequently isolated using a HisTrap HP affinity column, and the amount of radiolabeled cholesterol was quantified via liquid scintillation counting. In separate experiments, non-radiolabeled HDL or LDL were incubated with P116, and the relative lipid species content was analyzed using ultra-high-performance liquid chromatography–mass spectrometry (UHPLC–MS). (B) Time-course analyses of the transfer of radiolabeled unesterified cholesterol from LDL and HDL (0.5 mg/mL) to P116 (0.25 mg/mL) at 37°C. (C) Relative transfer of radiolabeled unesterified cholesterol from LDL and HDL (0.5 mg/mL) to P116 (0.25 mg/mL) for 90 minutes at 37°C. (D) Main lipid classes identified in ultra-high performance liquid chromatography–mass spectrometry (UHPLC-MS) analyses. The panels show the distribution of lipid classes in P116 and P116 post-incubated with LDL or HDL. The sum of the normalized areas of all the metabolites with the same chemical characteristics is shown for two independent samples of each group.\\u003c/p\\u003e\\n\\u003cp\\u003eStatistics: (C) Values are expressed as the mean ± SEM of three independent experiments. Unpaired t-test was performed for comparing the relative transfer of radiolabeled unesterified cholesterol from LDL and HDL. (D) Multiple unpaired t tests were performed to compare differences in each lipid species amount between groups. **, *** are P≤0.01 and 0.001 vs. control and *, ** are P≤0.05 and 0.01 vs. transfer from LDL.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5668698/v1/c2bbf6d1d47e495160e3cdb0.png\"},{\"id\":73303698,\"identity\":\"d19a2bb0-2844-449a-97f5-6cd569e18325\",\"added_by\":\"auto\",\"created_at\":\"2025-01-08 16:32:50\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":108224,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eP116 exhibits low efficiency in lipid uptake from H1299 lung cells. \\u003c/strong\\u003e(A) Schematic representation of isotopic unesterified cholesterol transfer and lipidomic analyses. H1299 lung cells (3x10\\u003csup\\u003e5\\u003c/sup\\u003e) were radiolabeled with [1,2-³H(N)] unesterified cholesterol and incubated with HDL (0.1 mg/mL) or P116 (0.1 mg/mL) for 90 minutes at 37°C. P116 was subsequently isolated using a HisTrap HP affinity column, and the amount of radiolabeled cholesterol was quantified via liquid scintillation counting. In separate experiments, H1299 lung cells were incubated with P116 and the relative lipid species content was analyzed using ultra-high-performance liquid chromatography–mass spectrometry (UHPLC–MS). (B) \\u003cem\\u003eIn vitro\\u003c/em\\u003e cholesterol efflux from human H1299 lung cells stimulated by HDL or P116 added to the culture media. (C) Main lipid classes identified in ultra-high performance liquid chromatography–mass spectrometry (UHPLC-MS) analyses in P116 and P116 post-incubated with H1299 lung. The sum of the normalized areas of all the metabolites with the same chemical characteristics is shown for two independent samples of each group. (D) Schematic representation of isotopic unesterified cholesterol transfer. J774.A1 mouse macrophages and intestinal caco-2 human cells (3x10\\u003csup\\u003e5\\u003c/sup\\u003e) were radiolabeled with [1,2-³H(N)] unesterified cholesterol and incubated with HDL (0.1 mg/mL) or P116 (0.1 mg/mL) for 90 minutes at 37°C. \\u003cem\\u003eIn vitro\\u003c/em\\u003e cholesterol efflux from J774.A1 mouse macrophages (E) and intestinal caco-2 human cells (F) stimulated by HDL or P116.\\u003c/p\\u003e\\n\\u003cp\\u003eStatistics: Values are expressed as the mean ± SEM of three independent experiments. (B, E and F) Unpaired t-test was performed for comparing cholesterol efflux stimulated by HDL or P116. (C) Multiple unpaired t tests were performed to compare differences in each lipid species amount between groups. *, **, *** are P≤0.05, 0.01, and 0.001 vs. control or HDL, respectively.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5668698/v1/4ba3200aa59322e695952f45.png\"},{\"id\":73303699,\"identity\":\"abbe2740-838c-4dd8-b0b7-ab6e47b95a15\",\"added_by\":\"auto\",\"created_at\":\"2025-01-08 16:32:51\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":137981,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eMonoclonal antibodies targeting the C-terminal domain of P116 interfered in both mycoplasma cell growth and cholesterol uptake. \\u003c/strong\\u003e(A)\\u003cstrong\\u003e \\u003c/strong\\u003eReactivity of \\u003cem\\u003eP. pneumoniae\\u003c/em\\u003e-infected patient’s sera against P116 constructs; the distribution of antibody reactivity in the ELISA is shown (n= 25 human subjects). (B) The Cryo-EM structure of the complex between P116 and mAb-3G9 shows that the epitope involves exclusively four amphipathic α-helices, corresponding to two fingers that form the hydrophobic, lipid-binding, cavity of P116. (C) Relative transfer of radiolabeled unesterified cholesterol from LDL to P116 after being incubated with mAb-3G9 (1:1 molar ratio) following the conditions of Figure 1. (D) The mAb-3G9 antibody directly inhibited the growth of cultured cells of \\u003cem\\u003eMPN\\u003c/em\\u003e strain M129 in SP4 medium. This inhibition was evident when compared to the growth rate of mycoplasma cells cultured in SP4 medium alone or in SP4 medium supplemented with RPMI 1640 (n = 12 independent replicates per group).\\u003c/p\\u003e\\n\\u003cp\\u003eStatistics: (A and D) The Kruskal-Wallis test followed by Dunn’s multiple comparison test was performed to compare the growth of cultured mycoplasma cells and the reactivity of human sera against P116 constructs between groups. *** indicates P≤0.001 vs. P116 246-818 or MPN129 + SP4 medium + mAb-3G9, respectively.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5668698/v1/3d12dc95d70665470539195b.png\"},{\"id\":73303702,\"identity\":\"972a1876-3cb4-4630-a83c-6817da59ad76\",\"added_by\":\"auto\",\"created_at\":\"2025-01-08 16:32:51\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":395254,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eMonoclonal antibodies targeting the C-terminal domain of P116 reduce the localization of \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003eMPN\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003eto human atherosclerotic plaques \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003eex vivo\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003e. \\u003c/strong\\u003e(A) Experimental workflow to evaluate the localization of \\u003cem\\u003eMPN\\u003c/em\\u003e strain M129.cells expressing the fluorescent protein Venus Carotid sections were used to obtain fragments of healthy human non-atherosclerotic tissue (HT) or human atherosclerotic plaque tissue (AP). These tissue fragments were subsequently incubated with \\u003cem\\u003eMPN\\u003c/em\\u003e cells and analyzed using phase contrast (PhC) and epifluorescence microscopy. (B) Epifluorescence microscopy (Venus), PhC, and merged images of \\u003cem\\u003eMPN\\u003c/em\\u003e cells were captured in the presence of healthy human non-atherosclerotic tissue (HT) or human atherosclerotic plaque tissue (AP) before and after incubation with the mAb-3G9 or mAb-3B5. Images were acquired at 0 h and 48 h following inoculation with \\u003cem\\u003eMPN\\u003c/em\\u003e Venus strain cells (for complete time-lapse image sequences, see Supplementary Figure 5).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5668698/v1/441ef1ab8356b964e49b8583.png\"},{\"id\":73304509,\"identity\":\"6a6015d4-af9e-4d74-beb6-9fdd51f4fa7f\",\"added_by\":\"auto\",\"created_at\":\"2025-01-08 16:40:51\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":318911,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eBiodistribution of [¹¹¹In]In-oxine radiolabeled \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003eMPN chassis\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003e.\\u003c/strong\\u003e (A) The \\u003cem\\u003ein vivo\\u003c/em\\u003e biodistribution of the non-pathogenic \\u003cem\\u003eMPN\\u003c/em\\u003e strain CV8 was evaluated using SPECT/CT imaging. Radiolabeled cells (\\u003csup\\u003e111\\u003c/sup\\u003eIn-oxine) were injected intravenously, followed by whole-body imaging and \\u003cem\\u003eex vivo\\u003c/em\\u003e analysis of hearts to provide quantitative and anatomical insights. (B) Cholesterol levels in the livers of wild-type and LDL receptor knockout mice were quantified after an 8-week Western-type diet. (3 mice per group). (C) The extent of atherosclerosis lesion, measured as mean plaque area in the aortic root, was assessed in the same groups following the same dietary regimen. (D) Representative \\u003cem\\u003ein vivo\\u003c/em\\u003e SPECT/CT coronal slices of LDL receptor knockout and wild-type mice obtained 6 hours post-administration. (E) SPECT/CT images of the heart. The left column shows the CT images of the excised hearts. In the right column, the SPECT image is overlaid, displaying the signal from radiolabeled Mycoplasma. * Proximal aorta.\\u003c/p\\u003e\\n\\u003cp\\u003eStatistics: (A and B) An unpaired t-test was performed to compare the different parameters between the two groups. ** P≤0.01 vs. wild-type mice.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5668698/v1/d0a7e7fe6611e76ab1841b6c.png\"},{\"id\":98382577,\"identity\":\"ae09c189-2b56-4bc5-8968-1e490c612968\",\"added_by\":\"auto\",\"created_at\":\"2025-12-17 08:07:48\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":2819676,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5668698/v1/b574f5b2-18fe-4dc6-b119-4faf462ee812.pdf\"},{\"id\":73303704,\"identity\":\"1c71e679-816a-4ff8-8829-067296d67a6e\",\"added_by\":\"auto\",\"created_at\":\"2025-01-08 16:32:51\",\"extension\":\"docx\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":3051887,\"visible\":true,\"origin\":\"\",\"legend\":\"Supplementary Figures and Tables\",\"description\":\"\",\"filename\":\"SupplementaryFiguresandtables12172024.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5668698/v1/726ca86bfdcc754b1ebe9d76.docx\"}],\"financialInterests\":\"There is \\u003cb\\u003eNO\\u003c/b\\u003e Competing Interest.\",\"formattedTitle\":\"Physiological Sources of Essential Lipids for Mycoplasma pneumoniae via Protein P116: Innovative Biotechnological Tools for Targeting Atherosclerotic and Hepatic Lesions\",\"fulltext\":[{\"header\":\"1. Introduction\",\"content\":\"\\u003cp\\u003e \\u003cem\\u003eMycoplasma pneumoniae (MPN)\\u003c/em\\u003e is a facultative intracellular human pathogen that lacks a cell wall and causes community-acquired pneumonia, sometimes leading to severe systemic effects \\u003csup\\u003e\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e\\u003c/sup\\u003e. \\u003cem\\u003eMPN\\u003c/em\\u003e accounts for 40% of community-acquired pneumonia cases \\u003csup\\u003e\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e\\u003c/sup\\u003e. \\u003cem\\u003eMPN\\u003c/em\\u003e, in addition to being a respiratory pathogen, can clinically manifest at extrapulmonary sites in up to 25% of infections \\u003csup\\u003e\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e\\u003c/sup\\u003e. Unlike other significant respiratory pathogens, such as \\u003cem\\u003eStreptococcus pneumoniae\\u003c/em\\u003e and \\u003cem\\u003eHaemophilus influenzae\\u003c/em\\u003e, no vaccine currently exists to address both the pulmonary and extrapulmonary complications associated with \\u003cem\\u003eMPN\\u003c/em\\u003e \\u003csup\\u003e\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e\\u003c/sup\\u003e. Prospective surveillance data indicate a re-emergence of \\u003cem\\u003eMPN\\u003c/em\\u003e in Europe and Asia, with increasing cases reported across various regions \\u003csup\\u003e\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e\\u003c/sup\\u003e. With one of the smallest genomes of 816 kbp \\u003csup\\u003e\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e\\u003c/sup\\u003e, \\u003cem\\u003eMPN\\u003c/em\\u003e cannot synthesize several lipids crucial for its cell membrane structure, including sphingomyelin, phosphatidylcholine, and cholesterol \\u003csup\\u003e\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e\\u003c/sup\\u003e. Unesterified cholesterol, a rare component among prokaryotes, is vital for \\u003cem\\u003eMPN\\u003c/em\\u003e cells and constitutes one of the most abundant lipids in their membranes \\u003csup\\u003e\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e\\u003c/sup\\u003e. Our recent research has revealed the structure of the immunogenic and essential protein P116 from \\u003cem\\u003eMPN\\u003c/em\\u003e \\u003csup\\u003e\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e\\u003c/sup\\u003e. The 116 kDa P116 subunits feature two extracellular domains\\u0026mdash;a small N-terminal domain and a large C-terminal or core domain\\u0026mdash;that are anchored to the mycoplasma membrane via a flexible linker \\u003csup\\u003e\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e\\u003c/sup\\u003e. The C-terminal domain exhibits a unique fold resembling a half-open hand, characterized by a large β-sheet, the palm, and by pairs of antiparallel amphipathic α-helices, the fingers, that collectively form a vast hydrophobic, lipid-binding, cavity. The fingers flexibility allows the cavity volume to vary according to the lipid cargo. The cavity appears to exhibit significant specificity for lipids crucial for the growth of \\u003cem\\u003eMPN\\u003c/em\\u003e \\u003csup\\u003e\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003e \\u003cem\\u003eMPN\\u003c/em\\u003e and \\u003cem\\u003eChlamydia pneumoniae\\u003c/em\\u003e have been detected in increased quantities within ruptured atherosclerotic plaques \\u003csup\\u003e\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e\\u003c/sup\\u003e. These pathogens have been associated with adventitial inflammation and positive vessel remodeling in thrombosed coronary arteries of patients who suffered acute myocardial infarction \\u003csup\\u003e\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e\\u003c/sup\\u003e. Additionally, experimental studies have demonstrated that inoculation with both agents exacerbates atherosclerosis in the aortas of hyperlipidemic mice \\u003csup\\u003e\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eLow-density lipoproteins (LDLs) and high-density lipoproteins (HDLs) are characterized by their content of apolipoprotein (apo)B-100 and apoA-I respectively, and their major lipid components are cholesterol esters and phospholipids, with a smaller amount of unesterified cholesterol. The function of LDL is primarily the transport and delivery of cholesterol to cells, including peripheral tissues and the liver, while HDL transports cholesterol to steroidogenic tissues \\u003csup\\u003e\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e\\u003c/sup\\u003e. Accumulation of oxidatively modified LDL in the subendothelial space induces their uptake by macrophages, promoting foam cell formation and a wide range of bioactivities that drive the development of atherosclerotic lesions. In contrast, HDL promotes macrophage cholesterol efflux to reduce lesion formation \\u003csup\\u003e\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e\\u003c/sup\\u003e. Since, as mentioned above, \\u003cem\\u003eMPN\\u003c/em\\u003e cannot synthesize several lipids essential for its cell membrane structure, it must obtain these lipids from the blood or arterial lining cells to survive in the bloodstream and localize at atherosclerotic plaques.\\u003c/p\\u003e \\u003cp\\u003eIn this work, we assessed the capacity of P116 to extract the \\u003cem\\u003eMPN\\u003c/em\\u003e essential lipids from circulating human lipoproteins and from cells. We also investigated the role of P116 in mediating the localization of \\u003cem\\u003eMPN\\u003c/em\\u003e to human atherosclerotic lesions \\u003cem\\u003eex vivo\\u003c/em\\u003e. Moreover, using a genetically engineered \\u003cem\\u003eMPN\\u003c/em\\u003e chassis, we determined the bacterium's ability in vivo to reach the liver and atheroma plaques via the bloodstream in hyperlipidemic mice, unveiling a new biotechnological approach for studying and treating atherosclerotic and hepatic lesions.\\u003c/p\\u003e\"},{\"header\":\"2. Materials and Methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.1. Cloning, expression and purification of P116 constructs\\u003c/h2\\u003e \\u003cp\\u003eRegions corresponding to the \\u003cem\\u003empn_213\\u003c/em\\u003e gene from \\u003cem\\u003eMPN\\u003c/em\\u003e were amplified from synthetic clones using different primers for each construct: P116F\\u003csub\\u003e30\\u003c/sub\\u003e and P116R\\u003csub\\u003e957\\u003c/sub\\u003e for P116(30\\u0026ndash;957); P116F\\u003csub\\u003e30\\u003c/sub\\u003e and P116R\\u003csub\\u003e845\\u003c/sub\\u003e for P116(30\\u0026ndash;845); P116F\\u003csub\\u003e246\\u003c/sub\\u003e and P116R\\u003csub\\u003e818\\u003c/sub\\u003e for P116(246\\u0026ndash;818); and P116W\\u003csub\\u003e681\\u003c/sub\\u003e to generate variant P116W681A. Detailed description on protein expression, purification and protein emptying has been reported previously \\u003csup\\u003e\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.2. Determination of isotopic unesterified cholesterol transfer rate from human LDL or HDL\\u003c/h2\\u003e \\u003cp\\u003eThe human plasma samples from healthy donors were obtained in accordance with the standards for medical research involving human subjects as recommended by the Declaration of Helsinki. The study protocol was approved by the Ethical Committee of Hospital de la Santa Creu i Sant Pau (protocol code IIBS-APO-2013-105). Human LDL (density 1.019\\u0026ndash;1.063 g/mL) and HDL (density 1.063\\u0026ndash;1.210 g/mL) were isolated through sequential gradient density ultracentrifugation, using potassium bromide for density adjustment, at 100,000\\u003cem\\u003eg\\u003c/em\\u003e for 24 hours with an analytical fixed-angle rotor (50.3, Beckman Coulter). The composition of both LDL and HDL, including total and unesterified cholesterol, triglycerides and phospholipids, was determined enzymatically, using commercial kits adapted for a COBAS 6000 autoanalyzer (Roche Diagnostics), whereas apoB and apoA1 were determined by an immunoturbidimetric assay, using commercial kits adapted for a COBAS 6000 autoanalyzer (Roche Diagnostics). Radiolabeled LDLs and HDLs were prepared in the following way: 5 \\u0026micro;Ci of [1,2-\\u003csup\\u003e\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e\\u003c/sup\\u003eH(N)] unesterified cholesterol (Perkin Elmer) was mixed with absolute ethanol, and the solvent was dried under a stream of N\\u003csub\\u003e2\\u003c/sub\\u003e. LDL or HDL (0.5 mL, 2.5 g/L of ApoB or ApoA1) were added to the tubes containing the radiotracers, as appropriate, and then incubated for 16 hours in a 37\\u0026deg;C bath \\u003csup\\u003e\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e\\u003c/sup\\u003e. The labeled LDLs and HDLs were re-isolated by gradient density ultracentrifugation and dialyzed against PBS through gel filtration chromatography. Specific activities of \\u003csup\\u003e\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e\\u003c/sup\\u003eH-cholesterol-containing LDLs and HDLs were 520 and 4,750 counts per minute (cpm)/nmol of unesterified cholesterol, respectively. The cholesterol transfer to P116 (the construct spanning residues 30 to 957, 1 g/L) was measured after adding either [\\u003csup\\u003e\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e\\u003c/sup\\u003eH] unesterified cholesterol-containing LDLs or HDL (0.5 g/L of ApoB or apoA1, respectively) and incubation at different times at 37\\u0026deg;C. LDL, HDL and P116 were separated by a HisTrap HP affinity. The radioactivity associated with each P116 and HDL fraction was measured through liquid scintillation counting. The percentage of [\\u003csup\\u003e\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e\\u003c/sup\\u003eH]cholesterol transferred per mL was determined for each condition. The specific activities for each radiotracer were used to calculate the amount of unesterified cholesterol from LDL or HDL to P116. Human apoB and apoA1 levels were determined in both the LDL, HDL and purified P116 fractions by the immunoturbidimetric assays in the COBAS 6000 autoanalyzer.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.3. H1299 lung, J774.A1 macrophage and Caco-2 colorectal cell cholesterol efflux to P116\\u003c/h2\\u003e \\u003cp\\u003eCellular cholesterol efflux to HDL and P116 (residues 30\\u0026ndash;957) was evaluated using a radiochemical method with H1299 human epithelial-like lung cells (ATCC\\u0026reg; CRL-5803, Manassas, VA), J774A1.1 mouse macrophages (ATCC\\u0026reg; TIB67\\u0026trade;) and epithelial human Caco-2 colorectal cells (ATCC\\u0026reg; HTB-37\\u0026trade;). For this purpose, 3x10\\u003csup\\u003e5\\u003c/sup\\u003e cells/well were seeded in 6-well plates and grown for 72 hours in complete Dulbecco's Modified Eagle's Medium (DMEM) high glucose with L-glutamine and sodium pyruvate (Corning, Glendale, AZ), supplemented with 10% fetal bovine serum (FBS) (Pan Biotech, Aidenbach, Germany) and 100 U/mL penicillin/streptomycin (Dominique Dutscher, Brumath, France). At that point, the cells were incubated with DMEM containing 1 \\u0026micro;Ci/well of [1α,2α(n)-3H]cholesterol (Perkin Elmer, Boston, MA) and 5% FBS for 48 hours. Subsequently, the macrophages and lung cells were equilibrated overnight with 0.2% free fatty acid bovine serum albumin (Sigma Aldrich/Merck) in DMEM. The following day, the media was removed, and the cells were incubated for 90 minutes with HDL or P116 (100 \\u0026micro;g/mL) in medium without any cholesterol supplement. For these \\u003cem\\u003ein vitro\\u003c/em\\u003e analyses, human HDL and P116 were isolated as described above. The percentage of cholesterol efflux from cells to the acceptors was calculated by dividing the amount of radiolabeled cholesterol in the medium by the sum of radiolabeled cholesterol in both the medium and the cells, as determined by liquid scintillation counting at the end of the experiments.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.4. Lipid Extraction for Mass Spectrometry Analyses\\u003c/h2\\u003e \\u003cp\\u003eThe lipid transfer to P116 (1 g/L) was measured after incubation with either LDL, HDL (0.5 g/L of ApoB or ApoA1, respectively), or H1299 human epithelial-like lung cells (1\\u0026times;10\\u003csup\\u003e6\\u003c/sup\\u003e) at 37\\u0026deg;C for 90 minutes. The different P116 fractions were separated using a HisTrap HP affinity column. Metabolite extraction was performed by fractionating the protein samples into pools of species with similar physicochemical properties using appropriate combinations of organic solvents. Two extraction methods were employed based on the chemical class of the target analytes \\u003csup\\u003e\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e\\u003c/sup\\u003e. Platform 1 (Fatty Acyls, Bile Acids, Steroids, and Lysoglycerophospholipids Profiling): 125 \\u0026micro;L of P116 (1 g/L) was mixed with 350 \\u0026micro;L of methanol (spiked with metabolites not detected in unspiked human serum extracts) in 1.5 mL microtubes on ice. After brief vortexing, the samples were incubated for 1 hour at -20\\u0026deg;C. The supernatants were collected following centrifugation at 18,000\\u0026times;g for 15 minutes, dried, and reconstituted in 60 \\u0026micro;L of methanol. The reconstituted samples were then centrifuged and transferred to vials for UHPLC-MS analysis. Platform 2 (Glycerolipids, Cholesteryl Esters, Sphingolipids, and Glycerophospholipids Profiling): 50 \\u0026micro;L of P116 (1 g/L) was mixed with sodium chloride (50 mM) and chloroform/methanol (2:1) in 1.5 mL microtubes on ice. The extraction solvent was spiked with metabolites not detected in unspiked human serum extracts. After brief vortexing, the samples were incubated for 1 hour at -20\\u0026deg;C. Following centrifugation at 16,000\\u0026times;g for 15 minutes, the organic phase was collected, and the solvent was removed. The dried extracts were reconstituted in 60 \\u0026micro;L of acetonitrile/isopropanol (1:1), centrifuged (18,000\\u0026times;g for 5 minutes), and transferred to vials for UHPLC-MS analysis. Randomized sample injections were performed for each analytical platform, with QC calibration and validation extracts uniformly interspersed throughout the batch run.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec7\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.5. Ultra-high-performance liquid chromatography\\u0026ndash;mass spectrometry (UHPLC-MS) analyses\\u003c/h2\\u003e \\u003cp\\u003eDifferent UHPLC-MS methods were used for each platform. Chromatographic separation and mass spectrometric detection conditions are summarized in \\u003cb\\u003eSupplementary Table\\u0026nbsp;1\\u003c/b\\u003e. Chromatography was performed using ACQUITY UPLC systems (Waters Corp., Milford, USA). A LCT Premier XE Time-of-Flight (ToF) (Waters Corp.) and a Xevo G2 QTof (Waters Corp.) mass spectrometers were used for Lipidomics Platform 1 and 2, respectively. The overall quality of the analysis procedure was monitored using repeat extracts of the QC samples. Retention time stability was generally\\u0026thinsp;\\u0026lt;\\u0026thinsp;6 s variation (injection-to-injection), and mass accuracy was generally\\u0026thinsp;\\u0026lt;\\u0026thinsp;5 ppm for m/z 400\\u0026ndash;1000, and \\u0026lt;\\u0026thinsp;1.2 mDa for m/z 50\\u0026ndash;400. All data were processed using the TargetLynx application manager for MassLynx 4.1 software (Waters Corp., Milford, USA). Predefined retention time and mass-to-charge ratio pairs (Rt-m/z) corresponding to the analyzed metabolites were fed into the program. Extracted ion chromatograms (mass tolerance window\\u0026thinsp;=\\u0026thinsp;0.05 Da) were then peak-detected and noise-reduced in both the LC and MS domains. This ensured that only true metabolite-related features were processed. Chromatographic peak areas were generated for each sample injection. For identified metabolites, representative MS detection response curves were generated using an internal standard for each chemical class included in the analysis. Assuming similar detector response levels for all metabolites within a chemical class allowed for defining a linear detection range for each variable. Maximum values were defined where the detector response became non-linear relative to the concentration of the representative internal standard.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.6. Immunoassays\\u003c/h2\\u003e \\u003cp\\u003eTwenty-five patients with an infection by \\u003cem\\u003eMPN\\u003c/em\\u003e based on a positive result with the Liaison \\u003cem\\u003eMPN\\u003c/em\\u003e IgG, IgM kit (DiaSorin, Italy) were selected. Similarly, six sera from healthy donors and testing negative for the Liaison \\u003cem\\u003eM. pneumoniae\\u003c/em\\u003e IgG, IgM kit were also selected. The serum samples were obtained in accordance with the standards for medical research involving human subjects as recommended by the Declaration of Helsinki. The study protocol was approved by the Ethical Committees of Parc Taul\\u0026iacute; (Ref 2019/664) and Vall d\\u0026rsquo;Hebron University Hospitals (PR(AG)24/2020). In both cases, we used a 1/100 dilution of the patient sera. Indirect ELISA assays were performed on 96 well plates Immulon 4 HBX 96 well plates (ThermoFisher) incubating 1\\u0026micro;g of each antigen at 4\\u0026deg;C overnight. 1/100 dilutions of each patient sera were added to the plate and detected using and anti-human IgG antibody conjugated with HRP (Thermofisher Scientific). Upon incubation for 30 minutes with 100 \\u0026micro;l of substrate (Thermofisher Scientific), 100 \\u0026micro;l of sulphuric acid 25% were added to stop the reaction and absorbance was read at 450 nm on a Triturus ELISA instrument (Grifols) device. Reference filter was set at 620 nm.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec9\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.7. Bacterial strains and culture conditions\\u003c/h2\\u003e \\u003cp\\u003eMycoplasma strains were grown at 37\\u0026deg;C under 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e in tissue culture flasks (Corning) with Hayflick liquid medium. Hayflick was prepared by mixing 800 ml of non-complete medium A (20 g PPLO broth (BD Difco, Franklin Lakes, NJ), 30 g HEPES [100 mM final], 25 ml 0.5% phenol red solution (Sigma Aldrich/Merck St. Louis, MO), 200 ml heat-inactivated horse serum (Life Technologies), 20 ml sterile‐filtered 50% glucose, and 1 ml of a 100 mg/ml stock of ampicillin (final concentration 100 \\u0026micro;g/ml, ampicillin sodium salt (Sigma Aldrich/Merck).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec10\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.8. Polyclonal and monoclonal antibody generation and purification\\u003c/h2\\u003e \\u003cp\\u003eTwo BALB/C mice were serially immunized with four intraperitoneal injections, each one containing 150 \\u0026micro;g of recombinant P116 ectodomain (residues 30\\u0026ndash;957) in 200 \\u0026micro;L of PBS with no adjuvants \\u003csup\\u003e\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e\\u003c/sup\\u003e. The last injection was delivered four days before splenectomy. Isolated B lymphocytes from the immunized mice were fused to NSI myeloma cells \\u003csup\\u003e\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e\\u003c/sup\\u003e to obtain stable hybridoma cell lines producing monoclonal antibodies (mAb), as previously described \\u003csup\\u003e\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e\\u003c/sup\\u003e. 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 \\u003cem\\u003eMPN\\u003c/em\\u003e cell lysates and by immunofluorescence using whole, non-permeabilized \\u003cem\\u003eMPN\\u003c/em\\u003e cells. Only those clones with supernatants revealing a single 116 kDa band in protein profiles and also exhibiting a consistent fluorescent staining of \\u003cem\\u003eMPN\\u003c/em\\u003e cells were selected and used in this work. Polyclonal sera were obtained by cardiac puncture of properly euthanized mice just before splenectomy and tittered using serial dilutions of the antigen. The titer of each polyclonal serum was determined as the IC\\u003csub\\u003e50\\u003c/sub\\u003e value from four parameter logistic plots and found to be approximately 1/4000 for both sera. The monoclonal antibody P116 mAb-3B5 was selected by ELISA screening using P116 30\\u0026ndash;957 construct, but tested negative against the P116 central region construct (residues 246\\u0026ndash;818). The mAb-3G9 was selected by ELISA screening and showed high specificity to P116 in Western blotting analysis and immunofluorescence microscopy of living \\u003cem\\u003eMPN\\u003c/em\\u003e cells \\u003csup\\u003e\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e\\u003c/sup\\u003e. To ensure the produced antibody was monospecific, hybridoma cells producing mAb-3G9 were cloned by limiting dilution and subjected to a second cloning step using the BD FACS Discover S8, selecting cells by size and complexity and sorting in single cell mode.\\u003c/p\\u003e \\u003cp\\u003eAs indicated in the different experiments, mAb-3G9 was used unpurified from supernatants of exponentially grow hybridoma cell cultures in Roswell Park Memorial Institute medium (RPMI) 1640 supplemented with 8% fetal bovine serum. For cryoelectron microscopy studies, mAb-3G9 was purified from 100 mL aliquots of cell culture supernatants. Briefly, supernants were diluted twice in 20 mM sodium phosphate buffer pH 7.0 and submitted to 1 mL HiTrap Protein G columns (cytiva) previously equilibrated in this buffer. After washing the column with 10 volumes of phosphate buffer, mAb was eluted in 0.1 M glycine pH 2.7 on tubes with a 1/10 volume of Tris 1 M pH 9. Pooled eluted fractions were concentrated using a 50 K Amicon Ultra 15 centrifugal filters to a 1 mL final volume and washed three times with PBS. The concentrated antibody sample was further purified using a Superose 6 Increase 10/300 GL column previously equilibrated in PBS and the eluted fractions in the range of 150 kDa were finally concentrated to a 0.5 mL volume also using a 50 K Amicon.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.9. Monoclonal antibody sequencing\\u003c/h2\\u003e \\u003cp\\u003eCloned hybridoma cells producing mAb-3G9 were cultured in T75 flasks using RPMI 1640 medium supplemented with 8% fetal bovine serum and grown until 50% confluence. RNA extraction was performed using the RNeaSY Mini Kit (QIAGEN) and 5 x 10\\u003csup\\u003e6\\u003c/sup\\u003e hybridoma cells by direct lysis of the cell pellet and omitting the optional DNase digestion step. Then, the cDNAs coding for the heavy and light chains were amplified by SMART RT-PCR following the simplified workflow described by Meyer et al \\u003csup\\u003e\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e\\u003c/sup\\u003e with few modifications. Briefly, the first strand was obtained by incubating 500 ng of total RNA, 1 \\u0026micro;L of primers mIGK RT, mIGL RT or mIGHG RT at 10 \\u0026micro;M in separate tubes, 1 \\u0026micro;L of 10 mM dNTPs and 11\\u0026micro;L of RNase free water at 65 \\u0026ordm;C for 5 minutes. Then, 4 \\u0026micro;L of the first strand buffer of M-MLV reverse transcriptase (Invitrogen), 2 \\u0026micro;L 100 mM DTT, 1 \\u0026micro;L RNaseOut and 1 \\u0026micro;L Template-Switch primer 100 \\u0026micro;M were added to each tube. After incubating 2 minutes at 37 \\u0026ordm;C 1 \\u0026micro;L of M-MLV reverse transcriptase (Invitrogen) was added to each tube, which were incubated for 50 minutes at 37 \\u0026ordm;C and 15 minutes at 70 \\u0026ordm;C. The touch down PCR reaction was performed as originally described using the Phusion termopolymerase (Thermo Scientific) using 3 \\u0026micro;L of each cDNA obtained in the previous step. The resulting PCR products were subjected to a 1% agarose gel and bands corresponding to the cDNAs of the heavy and kappa light chains (\\u003cb\\u003eSupplementary Fig.\\u0026nbsp;1A\\u003c/b\\u003e). Next, 700 bp and 600 bp bands corresponding to the RT-PCR products of the heavy and kappa light chains, respectively were excised from the gel, and the purified DNAs were ligated to the pBSKII vector previously digested with EcoRV. Ligation reactions were then transformed to competent \\u003cem\\u003eEscherichia coli\\u003c/em\\u003e XL1-blue cells and several colonies were selected and cultured to obtain the plasmid DNAs. A total of 9 plasmids bearing cDNAs from the heavy chain and 8 plasmids containing cDNAs from the kappa chain were submitted to Sanger sequencing on an ABI 3730 DNA Analyzer using the BigDye Terminator method and the reverse universal primer. The obtained DNA sequences were trimmed to remove the plasmid sequences using the VectorStrip application from the mEMBoss suite, translated to the six possible read frames and aligned using the ClustaX v2.1 application (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttp://www.clustal.org/clustal2\\u003c/span\\u003e\\u003cspan address=\\\"http://www.clustal.org/clustal2\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e). Eight out of nine sequences for the kappa chain and five out eight sequences from the kappa light chain contained sequences to the corresponding immunoglobulin chains. Aligned amino acid sequences coding for heavy chains were 95% identical starting from the first Met residue, and sequences coding for kappa light chains were 100% identical starting from the first Met residue (\\u003cb\\u003eSupplementary Fig.\\u0026nbsp;1B\\u003c/b\\u003e). The consensus sequence from each alignment was chosen as the complementary determining sequence in each of the chains of the P116 mAb-3G9 antibody.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.10. Single-particle cryoEM P116-mAb complex\\u003c/h2\\u003e \\u003cp\\u003eFor single-particle cryo-electron microscopy (cryoEM), a 3 \\u0026micro;L drop of purified P116-mAb complex (0.2 mg/mL in 20 mM Tris pH 7.4 and 150mM NaCl buffer) was applied to glow-discharged C-Flat\\u0026trade; holey grids (CF-1.2/1.3; 300 mesh), 5s blotting (-3 blotting force) and vitrified in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific) set to 4 \\u0026ordm;C and 95% humidity. Cryo-EM grids were stored at liquid nitrogen temperature.\\u003c/p\\u003e \\u003cp\\u003eMovies of P116-mAb complex were collected on a Glacios electron microscope operated at 200 kV (IBMB-CSIC CryoEM Platform). Imaging was performed using EPU at a nominal magnification of \\u0026times;150,000 (0.96 \\u0026Aring;/pixel) at 200 kV Glacios (Thermo Fisher Scientific) electron microscope equipped with Falcon 4 detector (Thermo fisher Scientific). A total of 5859 movies were collected. The camera was operated in counting mode with a dose rate of 7.24 electrons per \\u0026Aring;\\u003csup\\u003e\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e\\u003c/sup\\u003e s\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e, resulting in a total dose of 39.70 electrons per \\u0026Aring;\\u003csup\\u003e\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e\\u003c/sup\\u003e. Defocus values ranged from \\u0026minus;\\u0026thinsp;1.0 to \\u0026minus;\\u0026thinsp;2.5 \\u0026micro;m.\\u003c/p\\u003e \\u003cp\\u003eCryoSPARC v4 \\u003csup\\u003e22\\u003c/sup\\u003e was used to process the cryoEM data. Particles were selected with the Blob picker using a particle diameter of 150\\u0026ndash;300 \\u0026Aring;. Particles were extracted and classified in 2D. For the final processing, the 2D particles containing the complex were selected, remaining a total of 412,947 particles which were used to generate an ab initio reconstruction with three classes followed by a subsequent non-uniform heterogeneous refinement with the largest class. Finally, a model with a resolution of 5.06 \\u0026Aring; was obtained (\\u003cb\\u003eSupplementary Table\\u0026nbsp;2\\u003c/b\\u003e).\\u003c/p\\u003e \\u003cp\\u003eDocking, tracing and refinement of all the structures was performed alternating interactive and automatic cycles with programs Coot and Phenix. The final refined structure has been deposited in the PDB with code: 8ROR and the map in the EMDB with code: 19402\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.11. Effect of mAb-3G9 on mycoplasma cell growth\\u003c/h2\\u003e \\u003cp\\u003eGrowth rates of \\u003cem\\u003eMPN\\u003c/em\\u003e cultures were determined using an adaptation of the colorimetric protocol described by Karr and colleagues \\u003csup\\u003e\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e\\u003c/sup\\u003e. \\u003cem\\u003eMPN\\u003c/em\\u003e M129 strain was grown to mid-log phase in 25 cm\\u003csup\\u003e\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e\\u003c/sup\\u003e flasks with 5 mL SP4 medium. Attached cells were scraped off, recovered by centrifugation at 15000 \\u003cem\\u003eg\\u003c/em\\u003e and resuspended in 3 mL of fresh SP4 medium, SP4 medium supplemented with a 1/200 dilution of hybridoma cell culture supernatant in PBS, or SP4 medium supplemented with RPMI 1640 medium also diluted 1/200 in PBS as control. Then, 300 \\u0026micro;L of the cellular suspension were seeded in four different wells of a 96-well plate and serially diluted 1/3 until reaching a 1/243 using the same respective media described above. The resulting 96-well plated was sealed with transparent tape, placed into a Tecan Sunrise Absorbance Microplate Reader (Tecan), and incubated at 37\\u0026ordm;C for 8 days. During the incubation time, absorbance at 550 nm for each well was recorded each 15 minutes to quantify the medium acidification resulting from the mycoplasma growth, which turns the color of the phenol red indicator from red to yellow. Curves of absorbance vs time were plotted for each well and the inflection point of each curve was determined by iteration using the Excel application controlled by a Phyton script. Next, the inflection points were plotted, using the Napierian logarithm of the dilution as the x coordinate for each dilution. Once all the inflection points were plotted, the slope (\\u0026micro;, growth rate constant) was inferred by linear regression, and the doubling time (g) was obtained according to the general equation for exponential growth of bacteria (g\\u0026thinsp;=\\u0026thinsp;ln2/(1/ \\u0026micro;)).\\u003c/p\\u003e \\u003cp\\u003eTo visualize the effect of the mAb-3G9 addition on mycoplasma cells, \\u003cem\\u003eMPN\\u003c/em\\u003e strain M129 was grown in IBIDI chamber slides as described above using SP4 medium supplemented with a 1/40 dilution in PBS of a supernatant of exponentially grow hybridoma cell cultures.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.12. Interaction of MPN with human atherosclerotic plaques ex vivo\\u003c/h2\\u003e \\u003cp\\u003eCarotid endarterectomy specimens were obtained within one hour of surgical resection. The study protocol was approved by the Ethical Committee of Hospital de la Santa Creu i Sant Pau (protocol code IIBSP-LPM-2019-94). Immediately after collection, the specimens were placed in phosphate-buffered saline for processing. The specimens were then evaluated under a dissecting microscope and cut into small pieces. Fragments containing atherosclerotic plaque and fragments from the distal boundary of the plaque were collected for further analysis.\\u003c/p\\u003e \\u003cp\\u003eThe \\u003cem\\u003eMPN\\u003c/em\\u003e strain M129 expressing the fluorescent protein Venus \\u003csup\\u003e\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e\\u003c/sup\\u003e was grown in cell culture flasks containing SP4 medium and incubated at 37\\u0026deg;C and 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e. Surface-attached mycoplasmas were collected using a cell scraper and resuspended in SP4 medium. Co-cultures were performed in IBIDI eight-well chamber slides, being each well seeded with 1 \\u0026times; 10\\u003csup\\u003e5\\u003c/sup\\u003e mycoplasma cfus in 200 \\u0026micro;L of 0.22 \\u0026micro;m filtered SP4 medium and a small piece of healthy carotid tissue (HT) or atherosclerotic plaque tissue (AP).\\u003c/p\\u003e \\u003cp\\u003ePhase contrast (PhC) epifluorescence microscopy images of \\u003cem\\u003eMPN\\u003c/em\\u003e cells grown in presence of Healthy Human Atherosclerotic tissue (H.T.) or Human Atherosclerotic plaque tissue (A.P.) were taken using an inverted Nikon Eclipse TE 2000-E microscope. After inoculation, images were taken at different times (0h, 24h and 48h) and focusing into the same regions of the human tissues in the different observation times. Phase contrast and YFP (excitation 490/510 nm, 520/550 emission) images were captured using an Orca Fusion camera (Hamamatsu) controlled by NIS-Elements BR software (Nikon).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec15\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.13. Biodistribution of MPN into atherosclerotic mice\\u003c/h2\\u003e \\u003cp\\u003eWild-type mice and LDL receptor knockout mice on the C57BL/6 background were purchased from Jackson Laboratories (Bar Harbor, ME; #000664 and #002207, respectively), and housed under pathogen-free conditions at the Clinica Universidad de Navarra animal facility. Animal handling and procedures followed the current European (Directive 86/609/EEC) and National (Real Decreto 53/2013) legislations as well as the FELASA and ARRIVE guidelines and obtained the approval of the Animal Experimentation Ethic Committee of Clinica Universidad de Navarra (Code: 096\\u0026thinsp;\\u0026minus;\\u0026thinsp;23) and the local Government authorization. Mice were kept in a temperature-controlled (22 \\u0026ordm;C) room with a 12-hour light/dark cycle, and food and water were provided ad libitum. We used 4-5th -month-old male mice fed with a Western-type diet (TD.88137, Harlan Teklad, Madison, WI, containing 21% fat and 0.2% cholesterol) for 8 weeks. Plasma total cholesterol and triglycerides were determined enzymatically using commercial kits adapted for a COBAS 501/6000 autoanalyzer (Roche Diagnostics, Rotkreuz, Switzerland). Liver lipids were extracted using isopropyl alcohol-hexane (2:3, v/v) and the lipid layer was collected, evaporated, and resuspended in 0.5% (w/v) sodium cholate (Serva, Heidelberg, Germany). Cholesterol and triglycerides were determined using commercial kits adapted for the COBAS 501/6000 autoanalyzer. Serial sections of the proximal aorta were stained with hematoxylin and eosin for quantification of the aortic lesion area \\u003csup\\u003e\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eTo investigate the \\u003cem\\u003ein vivo\\u003c/em\\u003e biodistribution of Mycoplasma using single photon emission computed tomography (SPECT/CT), the non-pathogenic \\u003cem\\u003eMPN\\u003c/em\\u003e strain CV8 \\u003csup\\u003e26\\u003c/sup\\u003e was cultured in Hayflick for 3\\u0026ndash;4 days in T75 flask. Cells were washed twice and scraped in 1 ml of PBS. A bacterial suspension containing 10\\u003csup\\u003e7\\u003c/sup\\u003e CFUs was radiolabeled by incubating it with 161.162 \\u0026micro;Ci of [\\u0026sup1;\\u0026sup1;\\u0026sup1;In]-oxine at 37\\u0026deg;C for 15 minutes. Following radiolabeling, 100 \\u0026micro;L (18.65\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.70 \\u0026micro;C) of the radiolabeled Mycoplasma was injected in the vein tail. Six hours post-injection, images were acquired using a SPECT/CT scanner (U-SPECT6/E-class, (MILabs, Utrecht, The Netherlands). During image acquisition, the animals were placed in the prone position on the scanner bed under continuous anesthesia with isoflurane (2% in 100% O₂), and a 60-minute whole body scan was performed. After SPECT acquisition, CT scans were conducted to obtain anatomical reference, using a tube setting of 55 kV and 0.33 mA. SPECT and CT images were reconstructed using the \\u0026sup1;\\u0026sup1;\\u0026sup1;In photopeaks at 170 and 245 keV with a 20% energy window. A calibration factor was applied to determine activity (MBq/mL), and attenuation correction was performed using the CT attenuation map. The animals were subsequently sacrificed, and the heart along with major vessels was excised for SPECT/CT imaging. To enhance contrast and improve anatomical visualization in CT, the hearts were immersed in a 1:1 mixture of radiographic contrast agent (Omnipaque) and formalin and positioned in a well plate.\\u003c/p\\u003e \\u003cp\\u003eExperiments were conducted in a blind manner concerning the origin of the specimens to reduce bias.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec16\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.14. Statistical methods\\u003c/h2\\u003e \\u003cp\\u003eData are presented as the mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;standard error of mean (SEM). Unpaired t tests were used to compare the differences between two groups. Multiple t-tests were used to compare differences in the amount of each lipid species between groups. A Kruskal-Wallis test, followed by Dunn\\u0026rsquo;s multiple comparison test, was performed to compare the growth of cultured mycoplasma cells under different conditions and the reactivity of human sera against P116 constructs. A chi-square test was used to compare the distribution of human antibody reactivity against different P116 constructs. GraphPad Prism version 8.0.2 for Windows (GraphPad Software, San Diego, CA) was used to perform all statistical analyses. A P\\u003cem\\u003e-\\u003c/em\\u003evalue\\u0026thinsp;\\u0026le;\\u0026thinsp;0.05 was considered statistically significant.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec17\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.15. Data availability\\u003c/h2\\u003e \\u003cp\\u003eThe data, analytical methods, and study materials will be available to other researchers for purposes of reproducing the results or replicating the procedure upon reasonable request. Source data are provided with this paper.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"3. Results\",\"content\":\"\\u003cdiv id=\\\"Sec19\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.1. Human circulating LDL is a major source of unesterified cholesterol for MPN P116\\u003c/h2\\u003e \\u003cp\\u003eSince both LDL and HDL are major cholesterol-carrying lipoproteins in human plasma and are responsible for supplying cholesterol to tissues, we initially measured the rate of radiolabeled unesterified cholesterol transfer from LDL or HDL to \\u003cem\\u003eMPN\\u003c/em\\u003e P116 (see the schematic diagram of the experimental design in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA). The rate of cholesterol transfer from these lipoproteins to P116 was measured at equal concentrations of their main apolipoproteins (see \\u003cb\\u003eSupplementary Fig.\\u0026nbsp;2\\u003c/b\\u003e for lipoprotein composition). A time-dependent experiment was performed in which LDL containing radiolabeled cholesterol was incubated with unlabeled P116 for up to 90 minutes. Under these conditions, the quantity of labeled cholesterol in isolated P116 increased rapidly during the incubation, reaching a maximum plateau after 45 minutes (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB). In all experiments, the main LDL protein (ApoB100) was not detected, cross-checked by immunoturbidimetric detection, verifying that no LDL had contaminated the purified P116 samples. When radiolabeled HDL was incubated with P116, a significant amount of the HDL-derived radiotracer was transferred to the post-incubated, isolated P116; however, this transfer occurred with lower efficiency compared to LDL (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB \\u003cb\\u003eand C\\u003c/b\\u003e). The presence of the main HDL protein (apoA1) was not detected in any of the P116 fractions.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eThe specificity of this uptake was validated by mutating Trp681 to Ala, which is located on the P116 surface and may participate in the interaction with LDL and HDL. We found that the amount of LDL-derived radiotracer detected in the post-incubated and isolated P116 mutant was 60% lower compared to wild-type P116 (\\u003cb\\u003eSupplementary Fig.\\u0026nbsp;3\\u003c/b\\u003e).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec20\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.2. Human circulating LDL and HDL differentially transfer multiple lipids to the P116\\u003c/h2\\u003e \\u003cp\\u003eWe conducted a detailed ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) analysis of the lipid species in the isolated P116 fraction after incubation with human LDL or HDL under the same experimental conditions described for the isotopic method (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA\\u003cb\\u003e)\\u003c/b\\u003e. We identified 370 lipid species in the isolated P116 fractions. In empty P116, most of these lipid species were not detected, except for some triacylglycerol species with low relative intensity (\\u003cb\\u003eSupplementary Table\\u0026nbsp;3\\u003c/b\\u003e). In the P116 samples refilled from LDL, we found a striking accumulation of sphingomyelins, phosphatidylcholines and triacylglycerols, although the two latter were even higher in P116 refilled with HDL \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eD). Additionally, significant levels of esterified cholesterol, phosphatidylethanolamines and lysophosphatidylcholines were found in the isolated P116 fraction after incubation with both human LDL and HDL, with the former one being higher in P116 refilled with HDL (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eD).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec21\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.3. P116 uptakes lipids from H1299 lung cells with low efficiency\\u003c/h2\\u003e \\u003cp\\u003eWe then adapted a method used for evaluating unesterified cholesterol efflux stimulated by HDL from macrophages to evaluate the potential of P116 to uptake unesterified cholesterol from the human pulmonary cell membrane (see the schematic diagram of the experimental design in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA). When radiolabeled pulmonary cells were incubated with P116 in the media, a significant fraction of radiotracer was detected in the isolated P116, although with lower efficiency compared to that stimulated by the main physiological acceptor, HDL (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB). The analysis of the lipid species in the isolated P116 fraction after incubation with human pulmonary cells also identified 276 lipid species, but their relative intensity in P116 was very low in comparison with that of the isolated P116 incubated with lipoproteins (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC \\u003cb\\u003eand Supplementary Table\\u0026nbsp;3\\u003c/b\\u003e). We also evaluated the potential of P116 to uptake unesterified cholesterol from cholesterol-loaded macrophages and colorectal cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eD). As observed with pulmonary cells, a portion of the radiotracer was detected in the isolated P116 after incubation with cholesterol-loaded macrophages and with cholesterol-loaded colorectal cells \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eE \\u003cb\\u003eand F)\\u003c/b\\u003e, indicating that P116 can extract cholesterol from a diversity of cell types.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec22\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.4. Antibodies targeting the C-terminal domain of P116 interfere both cholesterol uptake and mycoplasma cell growth\\u003c/h2\\u003e \\u003cp\\u003eTo dissect the immunogenic activity of P116, we produced three different constructs: P116/30\\u0026ndash;957 (residues 30 to 957), a derivative lacking the C-term region (P116/30\\u0026ndash;845) and a derivative lacking also the N-terminal domain (P116/246\\u0026ndash;818) \\u003csup\\u003e\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e\\u003c/sup\\u003e. The three constructs were tested with total sera from twenty-five patients with positive diagnostic for \\u003cem\\u003eMPN\\u003c/em\\u003e based on serological tests. All patients were positive for the P116/30\\u0026ndash;957 construct (\\u003cb\\u003eSupplementary Table\\u0026nbsp;4\\u003c/b\\u003e). We obtained identical results when we used the P116/30\\u0026ndash;845 construct. However, the P116/246\\u0026ndash;818 construct resulted in a significant decrease in positive sera (72%) compared to the P116/30\\u0026ndash;957 and P116/30\\u0026ndash;845 constructs (\\u003cb\\u003eSupplementary Table\\u0026nbsp;4\\u003c/b\\u003e). Moreover, the overall signal of the P116/30\\u0026ndash;957 and P116/30\\u0026ndash;845 constructs was higher than the obtained with P116/246\\u0026ndash;818 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA). These results also indicate that targeting the C-terminal domain of P116 in mycoplasma infections has potential therapeutic applications.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eThen, a mAb-3G9 raised against P116 was selected. The structure of the P116-mAb-3G9 complex determined by Cryo-EM (\\u003cb\\u003eSupplementary Fig.\\u0026nbsp;4\\u003c/b\\u003e), showed that the epitope is located in four α-helices forming two of the fingers that define the hydrophobic cavity of P116 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB), suggesting that mAb-3G9 hinders the fingers flexibility required by the functioning of P116. Importantly, pre-incubation of P116 with mAb-3G9 and then incubation with radiolabeled LDL, resulted in a notable reduction in the amount of radiolabeled cholesterol found in the post-incubated and isolated P116 fraction (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eC). MAb-3G9 also interfered with the growth of cultured mycoplasma cells (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eD).\\u003c/p\\u003e \\u003cp\\u003e \\u003cem\\u003e3.5. Antibodies targeting the C-terminal domain of P116 impair the localization of MPN to human atherosclerotic plaques ex vivo\\u003c/em\\u003e \\u003c/p\\u003e \\u003cp\\u003eWe assessed the ability of \\u003cem\\u003eMPN\\u003c/em\\u003e to adhere to human carotid atherosclerotic plaques using cell culture invasion assays. For this purpose, we used the \\u003cem\\u003eMPN\\u003c/em\\u003e strain M129 expressing the fluorescent protein Venus \\u003csup\\u003e\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e\\u003c/sup\\u003e. Cells were cultured in the presence of human atherosclerotic carotid tissue fragments, with distal healthy tissue as a control (see the schematic diagram of the experimental design in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA). After inoculation, growth was monitored at 0-48h by phase contrast and epifluorescence microscopy. Although the tissue exhibited some autofluorescence, the presence of mycoplasma cells could be properly detected. Compared to the control healthy tissue, mycoplasma cell localization and growth was significantly higher in the vicinity of the atherosclerotic tissue (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eB). In line with the cell growth inhibition by mAb-3G9, this antibody that interacts with the C-terminal domain of P116, also interfered with the presence of \\u003cem\\u003eMPN\\u003c/em\\u003e cells in the atherosclerotic tissue (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eB \\u003cb\\u003eand Supplementary Fig.\\u0026nbsp;5\\u003c/b\\u003e). This effect was not observed when \\u003cem\\u003eMPN\\u003c/em\\u003e cells were incubated with P116 mAb-3B5, which exhibits no specificity to the central region of P116 (residues 246\\u0026ndash;818, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eB \\u003cb\\u003eand Supplementary Fig.\\u0026nbsp;5\\u003c/b\\u003e).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec23\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.6. Biotransfer of MPN in hyperlipidemic mice in vivo\\u003c/h2\\u003e \\u003cp\\u003eWe further investigated whether \\u003cem\\u003eMPN\\u003c/em\\u003e could colonize various organs in vivo using SPECT/CT imaging. This was achieved by intravenously injecting a radiolabeled non-pathogenic \\u003cem\\u003eMPN\\u003c/em\\u003e chassis into C57BL/6 wild-type mice and a hyperlipidemic mouse model (see the schematic diagram of the experimental design in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA). We specifically used LDL receptor knockout mice on a C57BL/6 background, which develop hyperlipidemia when fed a Western-type diet (\\u003cb\\u003eSupplementary Fig.\\u0026nbsp;6\\u003c/b\\u003e). These mice are also prone to developing fatty liver and extensive atherosclerosis in the proximal aorta, compared to wild-type mice fed with a Western-type diet (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eB \\u003cb\\u003eand C\\u003c/b\\u003e). The biodistribution of radiolabeled \\u003cem\\u003eMPN\\u003c/em\\u003e chassis revealed unexpectedly strong signal intensity in the livers of wild-type mice, with even higher signal levels observed in knockout mice, consistent with liver cholesterol data (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eD). Notably, images of the heart also detected the presence of radiolabeled \\u003cem\\u003eMPN\\u003c/em\\u003e in the aortic root of knockout mice, but not in wild-type mice (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eE). In contrast, no signal from the radiolabeled \\u003cem\\u003eMPN\\u003c/em\\u003e chassis was detected in the lungs. These observations confirm the effective tropism of \\u003cem\\u003eMPN\\u003c/em\\u003e toward cholesterol-rich livers and atherosclerotic plaques.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"4. Discussion\",\"content\":\"\\u003cp\\u003e \\u003cem\\u003eMPN\\u003c/em\\u003e cannot synthesize cholesterol \\u003cem\\u003ede novo\\u003c/em\\u003e and must acquire it from the host, underscoring its dependence on host resources for survival \\u003csup\\u003e\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e\\u003c/sup\\u003e. P116 in \\u003cem\\u003eMPN\\u003c/em\\u003e is an essential and highly immunogenic protein reported to play also a role in the adherence of the bacterium to host cells \\u003csup\\u003e\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e\\u003c/sup\\u003e. We recently demonstrated that the P116 structure features a novel fold, including an unusually large hydrophobic cavity filled with ligands \\u003csup\\u003e\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e\\u003c/sup\\u003e. Mass spectrometry and radioactivity transfer experiments confirmed the ability of P116 to extract various lipids from fetal bovine serum and cholesterol from HDLs \\u003csup\\u003e\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e\\u003c/sup\\u003e. In the present work, we demonstrate that human LDLs can be a major source of unesterified cholesterol for P116, with a very efficient transfer rate compared with that of HDLs, at least at equal major protein concentrations, i.e., at an equal number of lipoproteins. The amount of unesterified cholesterol per particle and its location on the surface of the lipoprotein could explain this accelerated transfer from LDLs to P116. Furthermore, LDLs also efficiently transfer a large variety of sphingomyelin species. Importantly, phosphatidylcholine species, which are also located on the surface of lipoproteins, were the major lipids found in P116 after being incubated with either LDLs or HDLs. The presence of the hydrophobic cavity in P116 and its structure allows the interaction with the amphipathic phospholipids, facilitating the uptake and exchange of these lipid species. In contrast, the elongated shape of P116 could explain its effectiveness, when incubated with HDLs, in the up taking of triacylglycerol and esterified cholesterol species, which are found in the core of HDL \\u003csup\\u003e\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e\\u003c/sup\\u003e. In summary, this work demonstrates, for the first time to our knowledge, that human LDLs serve as a key source of unesterified cholesterol, phosphatidylcholines, and sphingomyelins to P116 from MPN. In contrast, HDLs primarily provide phosphatidylcholines, esterified cholesterol, and triacylglycerols to P116.\\u003c/p\\u003e \\u003cp\\u003eP116 is also capable of inducing the efflux of unesterified cholesterol from lung and colorectal cells and from macrophages although with lower efficiency compared to HDL particles. It should be noted that HDLs are complex lipoproteins with a larger surface area relative to volume and contains apoA-I, which facilitate cholesterol and phospholipid efflux from cells by interacting with specific transporters on the cell membrane \\u003csup\\u003e\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e\\u003c/sup\\u003e. Our lipidomic analyses confirmed that P116 could uptake a variety of lung cell phosphatidylcholines, but again with much lower efficiency compared to LDL and HDL. Overall, these results demonstrate the ability of mycoplasmas to acquire essential lipids from diverse sources, enabling the colonization of various tissues. A P116 variant of a tryptophan residue at the protein surface, W681A, accumulated 60% less LDL-derived unesterified cholesterol compared to wild-type P116. Surface tryptophan residues are known to play critical roles in the functioning of the extensively investigated cholesteryl ester transfer proteins \\u003csup\\u003e\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e\\u003c/sup\\u003e, which suggests that related mechanisms for cholesterol uptake and release might be at work in cholesteryl ester transfer protein and P116. The mAb-3G9, raised against P116, was found to target an epitope within the C-terminal domain. This antibody interfered with the ability of P116 to uptake unesterified cholesterol and directly inhibited the growth of cultured mycoplasma cells. Furthermore, our serological studies in patients infected with \\u003cem\\u003eP. pneumoniae\\u003c/em\\u003e indicate a significant reduction in the percentage of antibodies generated against the N-terminal domain of P116. Therefore, an antigen derived exclusively from the C-terminal domain of P116 could serve as a promising candidate for the development of vaccines against \\u003cem\\u003eMPN\\u003c/em\\u003e. Some studies have found a higher prevalence of \\u003cem\\u003eMPN\\u003c/em\\u003e in patients with cardiovascular diseases compared to healthy controls \\u003csup\\u003e\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e\\u003c/sup\\u003e, although a direct causal link has not been demonstrated in humans. Given that ruptured atherosclerotic plaques have shown increased quantities of \\u003cem\\u003eMPN\\u003c/em\\u003e \\u003csup\\u003e\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e\\u003c/sup\\u003e, we further investigated the potential localization of \\u003cem\\u003eMPN\\u003c/em\\u003e in atherosclerotic lesions \\u003cem\\u003eex vivo\\u003c/em\\u003e. Compared to healthy control tissue, mycoplasma cell localization was notably increased around lipid-rich atherosclerotic tissue. Consistent with its observed inhibition of cell growth and cholesterol uptake, mAb-3G9 impaired the binding and proliferation of \\u003cem\\u003eMPN\\u003c/em\\u003e cells in atherosclerotic tissue, highlighting its potential for treating both pulmonary and extrapulmonary \\u003cem\\u003eMPN\\u003c/em\\u003e infections. SPECT/CT analysis in biotransfer assays \\u003cem\\u003ein vivo\\u003c/em\\u003e revealed a significant localization of \\u003cem\\u003eMPN\\u003c/em\\u003e in the liver and in the aortic atheroma plaques following introduction into the bloodstream of a mouse model of hyperlipidemia and atherosclerosis. \\u003cem\\u003eMPN\\u003c/em\\u003e primarily colonized the liver, even in wild-type mice, likely due to elevated lipid levels in the liver. \\u003cem\\u003eMPN\\u003c/em\\u003e infection has been associated with liver disease, particularly in children \\u003csup\\u003e\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e\\u003c/sup\\u003e. Thus, the availability of nutrients directing \\u003cem\\u003eMPN\\u003c/em\\u003e cells toward specific tissues may partly explain the organism\\u0026rsquo;s tissue colonization and its association with various non-respiratory symptoms and conditions \\u003csup\\u003e\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eOur findings underscore the concept that P116 plays a critical role in extracting essential lipids from physiological circulating lipoproteins and from a diversity of cell types, which can explain the colonization of different tissues by \\u003cem\\u003eMPN\\u003c/em\\u003e. In addition, a mAb targeting the C-terminal domain of P116, which reduces cholesterol extraction, inhibits mycoplasma growth in culture, and blocks \\u003cem\\u003eMPN\\u003c/em\\u003e binding to human lipid-rich atherosclerotic lesions \\u003cem\\u003eex vivo\\u003c/em\\u003e, demonstrates therapeutic potential. By limiting \\u003cem\\u003eMPN\\u003c/em\\u003e colonization in vulnerable areas, this mAb could help reduce the risk of infections or complications associated with atherosclerosis. The presence of \\u003cem\\u003eMPN\\u003c/em\\u003e in the liver and atheroma plaques in vivo suggests the potential use of the \\u003cem\\u003eMPN\\u003c/em\\u003e chassis, engineered as a genetically modified biological pill \\u003csup\\u003e\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e\\u003c/sup\\u003e, as an innovative biotechnological tool for studying and treating liver diseases, such as fatty liver and liver cancer, as well as atherosclerotic lesions.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis work was partly funded by the Instituto de Salud Carlos III and FEDER \\u0026quot;Una manera de hacer Europa\\u0026quot; grant PI2300232 (to J.E-G). N.R was funded by Agencia Estatal de Investigaci\\u0026oacute;n (AEI/10.13039/501100011033 and CNS2023-144119) within the Subprograma Ram\\u0026oacute;n y Cajal (RYC-201722879). CIBERDEM is an Instituto de Salud Carlos III project. I.F. and J.P. were funded by MICINN-Spain grant PID2021-125632OB-C21 and PID2021-125632OB-C22. The authors acknowledge funding from Project, IU16-014045 (CRYO-TEM) from Generalitat de Catalunya and by \\u0026ldquo;ERDF A way of making Europe\\u0026rdquo;, by the European Union.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthorship contribution statement\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eDavid Vizarraga:\\u0026nbsp;\\u003c/strong\\u003eConceptualization, Methodology, Validation, Formal analysis,\\u0026nbsp;Writing \\u0026ndash; review \\u0026amp; editing.\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003eMarina Marcos:\\u0026nbsp;\\u003c/strong\\u003e Conceptualization, Methodology, Validation, Formal analysis, Writing \\u0026ndash; review \\u0026amp; editing.\\u003cstrong\\u003e\\u0026nbsp;Noemi Rotllan:\\u0026nbsp;\\u003c/strong\\u003eConceptualization, Methodology, Validation, Formal analysis, Funding, Writing \\u0026ndash; review \\u0026amp; editing.\\u003cstrong\\u003e\\u0026nbsp;Jes\\u0026uacute;s Mart\\u0026iacute;n:\\u0026nbsp;\\u003c/strong\\u003eMethodology, Validation, Formal analysis.\\u003cstrong\\u003e\\u0026nbsp;David Santos:\\u0026nbsp;\\u003c/strong\\u003eMethodology. \\u003cstrong\\u003eMercedes Camacho:\\u0026nbsp;\\u003c/strong\\u003eMethodology, Writing \\u0026ndash; review \\u0026amp; editing. \\u003cstrong\\u003ePablo Guerra:\\u0026nbsp;\\u003c/strong\\u003eMethodology, Validation, Formal analysis. \\u003cstrong\\u003eF\\u0026eacute;lix Pareja:\\u0026nbsp;\\u003c/strong\\u003eMethodology, Validation, Formal analysis.\\u003cstrong\\u003e\\u0026nbsp;Mar\\u0026iacute;a Collantes:\\u003c/strong\\u003e Methodology, Validation, Formal an\\u0026aacute;lisis, Writing \\u0026ndash; review \\u0026amp; editing.\\u003cstrong\\u003e\\u0026nbsp;Wanlu Wu:\\u0026nbsp;\\u003c/strong\\u003eMethodology, Validation, Formal analysis. \\u003cstrong\\u003eIrene Rodr\\u0026iacute;guez-Arce:\\u003c/strong\\u003e Methodology, Writing \\u0026ndash; review \\u0026amp; editing\\u003cstrong\\u003e, Luis Serrano:\\u0026nbsp;\\u003c/strong\\u003eFunding,\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003eWriting \\u0026ndash; review \\u0026amp; editing. \\u003cstrong\\u003eJaume Pi\\u0026ntilde;ol:\\u0026nbsp;\\u003c/strong\\u003eConceptualization, Methodology, Validation, Formal analysis, Funding, Writing \\u0026ndash; original draft, Writing \\u0026ndash; review \\u0026amp; editing. \\u003cstrong\\u003eIgnacio Fita:\\u0026nbsp;\\u003c/strong\\u003eConceptualization, Methodology, Validation, Formal analysis, Funding, Writing \\u0026ndash; original draft, Writing \\u0026ndash; review \\u0026amp; editing. \\u003cstrong\\u003eJoan Carles Escol\\u0026agrave;-Gil:\\u0026nbsp;\\u003c/strong\\u003eConceptualization, Methodology, Validation, Formal analysis, Funding, Writing \\u0026ndash; original draft, Writing \\u0026ndash; review \\u0026amp; editing.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eDeclaration of Interest\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll authors declare that they have no relationships relevant to the contents of this paper to disclose and have approved the final version of the article.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eTsiodras S, Kelesidis I, Kelesidis T, Stamboulis E, Giamarellou H (2005) Central nervous system manifestations of Mycoplasma pneumoniae infections. J Infect 51:343\\u0026ndash;354\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eFerwerda A, Moll HA, de Groot R (2001) Respiratory tract infections by Mycoplasma pneumoniae in children: a review of diagnostic and therapeutic measures. Eur J Pediatr 160:483\\u0026ndash;491\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eAtkinson TP, Balish MF, Waites KB (2008) Epidemiology, clinical manifestations, pathogenesis and laboratory detection of Mycoplasma pneumoniae infections. FEMS Microbiol Rev 32:956\\u0026ndash;973\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eParrott GL, Kinjo T, Fujita J (2016) A Compendium for Mycoplasma pneumoniae. Front Microbiol 7:513\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eMeyer Sauteur PM et al (2024) Mycoplasma pneumoniae: delayed re-emergence after COVID-19 pandemic restrictions. Lancet Microbe 5:e100\\u0026ndash;e101\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eNordholm AC et al (2023), \\u003cem\\u003eDecember,. Mycoplasma pneumoniae epidemic in Denmark, October to Euro Surveill 29(2024).\\u003c/em\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eHimmelreich R, Plagens H, Hilbert H, Reiner B, Herrmann R (1997) Comparative analysis of the genomes of the bacteria Mycoplasma pneumoniae and Mycoplasma genitalium. Nucleic Acids Res 25:701\\u0026ndash;712\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLluch-Senar M et al (2015) Defining a minimal cell: essentiality of small ORFs and ncRNAs in a genome-reduced bacterium. Mol Syst Biol 11:780\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eGaspari E et al (2020) Model-driven design allows growth of Mycoplasma pneumoniae on serum-free media. NPJ Syst Biol Appl 6:33\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eDahl J (1993) The role of cholesterol in mycoplasma membranes. Subcell Biochem 20:167\\u0026ndash;188\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSprankel L et al (2023) Essential protein P116 extracts cholesterol and other indispensable lipids for Mycoplasmas. Nat Struct Mol Biol 30:321\\u0026ndash;329\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eHiguchi ML et al (2000) Detection of Mycoplasma pneumoniae and Chlamydia pneumoniae in ruptured atherosclerotic plaques. Braz J Med Biol Res 33:1023\\u0026ndash;1026\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eHiguchi Mde L et al (2003) Coinfection with Mycoplasma pneumoniae and Chlamydia pneumoniae in ruptured plaques associated with acute myocardial infarction. Arq Bras Cardiol 81(12\\u0026ndash;22):11\\u0026ndash;11\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eRamires JA, Higuchi Mde L (2002) [Mycoplasma pneumoniae and Chlamydia pneumoniae are associated to inflammation and rupture of the atherosclerotic coronary plaques]. Rev Esp Cardiol 55(Suppl 1):2\\u0026ndash;9\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eDamy SB et al (2009) Mycoplasma pneumoniae and/or Chlamydophila pneumoniae inoculation causing different aggravations in cholesterol-induced atherosclerosis in apoE KO male mice. BMC Microbiol 9:194\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eFeingold KR (2000) Introduction to Lipids and Lipoproteins. In: Feingold KR et al (eds) Endotext. South Dartmouth (MA)\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLinton MF et al (2000) The Role of Lipids and Lipoproteins in Atherosclerosis. In: Feingold KR et al (eds) Endotext. South Dartmouth (MA)\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBarr J et al (2010) Liquid chromatography-mass spectrometry-based parallel metabolic profiling of human and mouse model serum reveals putative biomarkers associated with the progression of nonalcoholic fatty liver disease. J Proteome Res 9:4501\\u0026ndash;4512\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKohler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495\\u0026ndash;497\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eGuasch A et al (2020) Structure of P46, an immunodominant surface protein from Mycoplasma hyopneumoniae: interaction with a monoclonal antibody. Acta Crystallogr D Struct Biol 76:418\\u0026ndash;427\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eMeyer L et al (2019) A simplified workflow for monoclonal antibody sequencing. PLoS ONE 14:e0218717\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003ePunjani A, Rubinstein JL, Fleet DJ, Brubaker MA (2017) cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat Methods 14:290\\u0026ndash;296\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKarr JR et al (2012) A whole-cell computational model predicts phenotype from genotype. Cell 150:389\\u0026ndash;401\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eMariscal AM et al (2018) Tuning Gene Activity by Inducible and Targeted Regulation of Gene Expression in Minimal Bacterial Cells. ACS Synth Biol 7:1538\\u0026ndash;1552\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eRotllan N et al (2022) Antagonism of miR-148a attenuates atherosclerosis progression in APOB(TG)Apobec(-/-)Ldlr(+/-) mice: A brief report. Biomed Pharmacother 153:113419\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eMontero-Blay A et al (2023) Bacterial expression of a designed single-chain IL-10 prevents severe lung inflammation. Mol Syst Biol 19:e11037\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eRazin S, Yogev D, Naot Y (1998) Molecular biology and pathogenicity of mycoplasmas. Microbiol Mol Biol Rev 62:1094\\u0026ndash;1156\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSvenstrup HF, Nielsen PK, Drasbek M, Birkelund S, Christiansen G (2002) Adhesion and inhibition assay of Mycoplasma genitalium and M. pneumoniae by immunofluorescence microscopy. J Med Microbiol 51:361\\u0026ndash;373\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eQiu X et al (2007) Crystal structure of cholesteryl ester transfer protein reveals a long tunnel and four bound lipid molecules. Nat Struct Mol Biol 14:106\\u0026ndash;113\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003ePhillips MC (2014) Molecular mechanisms of cellular cholesterol efflux. J Biol Chem 289:24020\\u0026ndash;24029\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKoivuniemi A, Vuorela T, Kovanen PT, Vattulainen I, Hyvonen MT (2012) Lipid exchange mechanism of the cholesteryl ester transfer protein clarified by atomistic and coarse-grained simulations. PLoS Comput Biol 8:e1002299\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eMomiyama Y, Ohmori R, Taniguchi H, Nakamura H, Ohsuzu F (2004) Association of Mycoplasma pneumoniae infection with coronary artery disease and its interaction with chlamydial infection. Atherosclerosis 176:139\\u0026ndash;144\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eChung WS, Hsu WH, Lin CL, Kao CH (2015) Mycoplasma pneumonia increases the risk of acute coronary syndrome: a nationwide population-based cohort study. QJM 108:697\\u0026ndash;703\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003ePoddighe D (2020) Mycoplasma pneumoniae-related hepatitis in children. Microb Pathog 139:103863\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eHu J et al (2022) Insight into the Pathogenic Mechanism of Mycoplasma pneumoniae. Curr Microbiol 80:14\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eMazzolini R et al (2023) Engineered live bacteria suppress Pseudomonas aeruginosa infection in mouse lung and dissolve endotracheal-tube biofilms. Nat Biotechnol 41:1089\\u0026ndash;1098\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":true,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"nature-portfolio\",\"isNatureJournal\":true,\"hasQc\":false,\"allowDirectSubmit\":false,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Nature Portfolio\",\"twitterHandle\":\"\",\"acdcEnabled\":false,\"dfaEnabled\":false,\"editorialSystem\":\"ejp\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false},\"keywords\":\"Atherosclerosis, Cholesterol, HDL, LDL, M. pneumoniae\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-5668698/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-5668698/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003e \\u003cem\\u003eMycoplasma pneumoniae\\u003c/em\\u003e (\\u003cem\\u003eMPN\\u003c/em\\u003e) is a bacterial pathogen in humans that primarily causes atypical pneumonia. \\u003cem\\u003eMPN\\u003c/em\\u003e cannot synthesize several lipids crucial for its cell membrane structure and needs to extract them from the lung of the host to survive. The protein responsible for extracting essential lipids from cell membranes is P116. MPN has been detected in increased quantities within ruptured atherosclerotic plaques and the question is how \\u003cem\\u003eMPN\\u003c/em\\u003e survives in the blood and in the plaques and obtains the lipids necessary for its membrane. Here we show that P116 can uptake essential lipids from LDL and HDL and when targeting its C-terminal domain via a monoclonal antibody there is growth inhibition \\u003cem\\u003ein vitro\\u003c/em\\u003e. Phase contrast epifluorescence microscopy of human arteries also revealed that this antibody blocks \\u003cem\\u003eMPN\\u003c/em\\u003e binding to human atherosclerotic lesions \\u003cem\\u003eex vivo\\u003c/em\\u003e. Furthermore, injection of \\u003cem\\u003eMPN\\u003c/em\\u003e in the blood results in accumulation of \\u003cem\\u003eMPN\\u003c/em\\u003e within the liver and atheroma plaques in a hyperlipidemic mouse model. We conclude that P116 plays a critical role in extracting essential lipids from physiological circulating lipoproteins and from host cells and regulates \\u003cem\\u003eMPN\\u003c/em\\u003e localization to liver and atheromatous plaques. These results suggest new strategies for managing mycoplasma infections and addressing the potential complications of \\u003cem\\u003eMPN\\u003c/em\\u003e infections in atherosclerotic lesions. They also open avenues for utilizing biotechnological tools in the treatment of atherosclerotic and hepatic lesions.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Physiological Sources of Essential Lipids for Mycoplasma pneumoniae via Protein P116: Innovative Biotechnological Tools for Targeting Atherosclerotic and Hepatic Lesions\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-01-08 16:32:46\",\"doi\":\"10.21203/rs.3.rs-5668698/v1\",\"editorialEvents\":[],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"nature-communications\",\"isNatureJournal\":true,\"hasQc\":false,\"allowDirectSubmit\":false,\"externalIdentity\":\"NCOMMS\",\"sideBox\":\"Learn more about [Nature Communications](http://www.nature.com/ncomms/)\",\"snPcode\":\"\",\"submissionUrl\":\"https://mts-ncomms.nature.com/\",\"title\":\"Nature Communications\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"ejp\",\"reportingPortfolio\":\"Nature Communications\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false}}],\"origin\":\"\",\"ownerIdentity\":\"41424f8a-26e9-4d5a-a41b-21390d6e73be\",\"owner\":[],\"postedDate\":\"January 8th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[{\"id\":42430428,\"name\":\"Health sciences/Diseases/Cardiovascular diseases/Dyslipidaemias\"},{\"id\":42430430,\"name\":\"Biological sciences/Biochemistry/Lipids/Lipoproteins\"},{\"id\":42430431,\"name\":\"Biological sciences/Biological techniques/Microscopy/Cryoelectron microscopy\"}],\"tags\":[],\"updatedAt\":\"2025-12-17T08:07:36+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-5668698\",\"link\":\"https://doi.org/10.1038/s41467-025-66129-5\",\"journal\":{\"identity\":\"nature-communications\",\"isVorOnly\":false,\"title\":\"Nature Communications\"},\"publishedOn\":\"2025-12-16 05:00:00\",\"publishedOnDateReadable\":\"December 16th, 2025\"},\"versionCreatedAt\":\"2025-01-08 16:32:46\",\"video\":\"\",\"vorDoi\":\"10.1038/s41467-025-66129-5\",\"vorDoiUrl\":\"https://doi.org/10.1038/s41467-025-66129-5\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-5668698\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-5668698\",\"identity\":\"rs-5668698\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}