Sources of essential lipids for Mycoplasma pneumoniae via P116 to target liver and atherosclerotic lesions

Domain: biology
Published: 2025-12-16
Editorial outcome: minor_revision

Abstract
Mycoplasma pneumoniae (MPN) is a bacterial pathogen that primarily causes atypical pneumonia. It cannot synthesize certain essential lipids and therefore relies on the host for their acquisition to survive. MPN has been detected in increased amounts within ruptured atherosclerotic plaques. In this work, we show that the protein P116 facilitates cholesterol acquisition from LDL, HDL and various cell types. Targeting P116’s C-terminal domain with a monoclonal antibody inhibits cholesterol acquisition and bacterial growth in vitro. Phase contrast epifluorescence microscopy of human arteries reveals that this antibody blocks MPN binding to atherosclerotic lesions ex vivo. Additionally, an MPN chassis injected into hyperlipidemic female mice localizes to the liver and atherosclerotic plaques. Here, we report that P116 plays a role in extracting essential lipids from lipoproteins and host cells and regulates MPN localization to atheromatous plaques. The study highlights MPN’s potential as a tool for targeting atherosclerotic lesions and fatty liver.

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 effects1. MPN accounts for 40% of community-acquired pneumonia cases2. MPN, in addition to being a respiratory pathogen, can clinically manifest at extrapulmonary sites in up to 25% of infections3. 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 MPN4. Prospective surveillance data indicate a re-emergence of MPN in Europe and Asia, with increasing cases reported across various regions5,6. With one of the smallest genomes of 816 kbp7, MPN cannot synthesize several lipids crucial for its cell membrane structure, including sphingomyelin, phosphatidylcholine, and cholesterol8,9. Unesterified cholesterol, a rare component among prokaryotes, is vital for MPN cells and constitutes one of the most abundant lipids in their membranes10. Our recent research has revealed the structure of the immunogenic and essential protein P116 from MPN11. 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 linker11. 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 MPN11.

MPN and Chlamydia pneumoniae have been detected in increased quantities within ruptured atherosclerotic plaques12,13. These pathogens have been associated with adventitial inflammation and positive vessel remodeling in thrombosed coronary arteries of patients who suffered acute myocardial infarction14. Additionally, experimental studies have demonstrated that inoculation with both agents exacerbates atherosclerosis in the aortas of hyperlipidemic mice15.

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 cholesteryl 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 tissues16. Accumulation of oxidatively modified LDL in the subendothelial space induces its 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 formation17. 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 biotechnological approach for studying and treating atherosclerotic and hepatic lesions.

Results
Human LDL is a 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 and the protein P116 (see the schematic diagram of the experimental design in Fig. 1a). The rate of cholesterol transfer from these lipoproteins to MPN and P116 was measured at equal concentrations of their main apolipoproteins (see supplementary Fig. 1 for lipoprotein composition). In all experiments, the main LDL protein (ApoB100) and HDL protein (apoA1) were not detected, cross-checked by immunoturbidimetric detection, verifying that no LDL and HDL had contaminated the purified MPN and P116 samples. When LDL containing radiolabeled unesterified cholesterol was incubated with MPN, a significant portion of the LDL-derived radiotracer was transferred to the subsequently isolated MPN, whereas transfer was less efficient when incubating with HDL (Fig. 1b). Similar patterns were observed when LDL or HDL were incubated directly with P116 (Fig. 1c). A time-dependent experiment was performed in which LDL containing radiolabeled cholesterol was incubated with unlabeled P116 for up to 90 min. Under these conditions, the quantity of labeled cholesterol in isolated P116 increased rapidly during the incubation, reaching a maximum plateau after 45 min (Fig. 1c, inset).

Human lipoproteins transfer multiple lipids to 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. 1d). 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 Data). In P116 samples refilled with LDL or HDL, we observed a striking accumulation of phosphatidylcholines, triacylglycerols, and sphingomyelins, with triacylglycerols being relatively more abundant in HDL-refilled P116, whereas sphingomyelins were lower (Fig. 1e). Unesterified sterols (cholesterol and derivatives) were higher in P116 after incubation with LDL, in contrast to esterified cholesterol, which was detected in significant amounts after incubation with both LDL and HDL, and was particularly enriched in HDL-refilled P116 (Fig. 1e).

P116 acquires cholesterol from diverse cell types
We then adapted a method used for evaluating unesterified cholesterol efflux stimulated by HDL and lipid-free apoA1 from macrophages to evaluate the potential of P116 to extract unesterified cholesterol from the human pulmonary cell membrane (see the schematic diagram of the experimental design in Fig. 2a). When pulmonary cells containing radiolabeled unesterified cholesterol were incubated with P116 in the media, a significant fraction of radiotracer was detected in the isolated P116, although the transfer was significantly lower to that to the main physiological acceptor, HDL (Fig. 2a). We also evaluated the potential of P116 to acquire unesterified cholesterol from cholesterol-loaded Chinese hamster ovary (CHO) cells, CHO cells overexpressing scavenger receptor type B1 (SR-BI), and macrophages before and after activation of the cholesterol efflux transporters ATP-binding cassette (ABC)A1 and ABCG1 (Fig. 2b, c). As observed with pulmonary cells, a portion of the radiotracer was recovered in isolated P116 following incubation with cholesterol-loaded CHO cells and macrophages (Fig. 2b, c). In contrast to HDL, P116-mediated cholesterol uptake was unaffected by SR-BI expression (Fig. 2b). Furthermore, while activation of ABCA1 and ABCG1 promotes cholesterol efflux to classical acceptors such as HDL or lipid-free apoA-I, upregulation of these transporters did not enhance cholesterol transfer to P116 (Fig. 2c). Notably, the ability of P116 to acquire unesterified cholesterol from cell membranes was highly correlated with the cellular cholesterol content (Supplementary Fig. 2). These findings suggest that P116 bypasses canonical receptor-mediated uptake or efflux mechanisms and instead captures freely available or loosely bound unesterified cholesterol. Furthermore, inhibition of lipase activity with orlistat also had no effect on P116-mediated uptake (Supplementary Fig. 3).

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. 2d and supplementary Data).

Effects of antibodies targeting the C-terminal domain of P116
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 and six uninfected subjects. Sera from uninfected individuals exhibited very low reactivity against any of the P116 constructs compared with infected patients (Supplementary Fig. 4). All infected patients were positive for the P116/30-957 construct (Supplementary Table 1). 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 1). Importantly, the overall signal of the P116/30-957 and P116/30-845 constructs was higher than that obtained with P116/246-818 (Fig. 3a).

A panel of antibodies against P116 (residues 30–957) was tested, and all except one targeted the core domain of P116 and showed inhibitory effects on MPN growth and adhesion. Based on these results, monoclonal antibody (mAb) 3G9 was selected for further experiments as it produced the strongest inhibitory effect. We also used mAb 3B5, which lacks specificity for the central region of P116 and exhibits lower affinity. The structure of the P116-mAb-3G9 complex determined by Cryo-EM (Supplementary Fig. 5), showed that the epitope is located in four α-helices forming two of the fingers that define the hydrophobic cavity of P116 (Fig. 3b and Supplementary Fig. 6)11, and indicates that mAb-3G9 hinders the fingers flexibility required by the functioning of P116. Importantly, pre-incubation of P116 with mAb-3G9, followed by incubation with radiolabeled LDL, resulted in a notable reduction in the amount of radiolabeled cholesterol recovered in the post-incubated, isolated P116 fraction (Fig. 3c). This effect was not observed when P116 was incubated with mAb-3B5. Furthermore, mAb-3G9 not only blocked cholesterol transfer but also significantly inhibited the growth of cultured mycoplasma cells compared with mAb-3B5 (Fig. 3d). These results suggest that targeting the C-terminal domain of P116 in MPN infections could represent a potential therapeutic strategy.

Antibody effects on MPN localization to atherosclerotic plaques
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 Venus18. 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. 4a). After inoculation, growth was monitored at 0–48 h by phase contrast and epifluorescence microscopy. Although the tissue exhibited some autofluorescence, the presence of mycoplasma cells could be detected. Compared to the control healthy tissue, mycoplasma cell localization and growth were significantly higher in the vicinity of the atherosclerotic tissue (Fig. 4b).

In line with the cell growth inhibition by mAb-3G9, this antibody, which interacts with the C-terminal domain of P116, also interfered with the presence of MPN cells in the atherosclerotic tissue (Fig. 4b and Supplementary Fig. 7). 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). Importantly, we quantified the percentage increase in MPN-positive area from 0 to 48 h across four independent carotid artery specimens (Fig. 4c). Incubation with mAb-3G9 caused a significant reduction in MPN growth compared with atherosclerotic plaques incubated without antibody or with mAb-3B5, with values comparable to those observed in healthy tissue.

Biotransfer of MPN in hyperlipidemic mice
We further investigated whether MPN could colonize various organs in vivo using single-photon emission computed tomography (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. 5a). We specifically used female LDL receptor knockout mice (LDLR−/−) on a C57BL/6 background, which develop hyperlipidemia and atherosclerosis when fed a Western-type diet, a high-fat, cholesterol-rich regimen (Supplementary Fig. 8). The biodistribution of radiolabeled MPN chassis revealed strong signal intensity in the livers of both female wild-type and LDLR−/− mice (Fig. 5b). As expected, wild-type mice accumulated substantial amounts of cholesterol in the liver after being fed a Western diet, but this accumulation was significantly greater in LDLR−/− mice (Fig. 5c). Consistent with the biodistribution data, the total hepatic MPN load—measured by both radioactivity and qPCR—was comparable between the two groups (Fig. 5d, e). No significant correlation was observed between liver cholesterol content and radiolabeled MPN abundance (Fig. 5f), indicating that liver accumulation does not scale directly with hepatic cholesterol levels.

Using SPECT/CT imaging, we observed that MPN chassis localized specifically to areas containing atherosclerotic lesions, but only in LDLR−/− mice, which develop atherosclerosis under high-fat diet conditions (Fig. 5g–i). Finally, no signal from the radiolabeled MPN chassis was detected in the lungs. These observations confirm the effective tropism of MPN toward livers and atherosclerotic plaques.

Methods
This research complies with all relevant ethical regulations. Human plasma samples were obtained in accordance with the Declaration of Helsinki and institutional ethical guidelines. Eligible donors for lipoprotein collection were normolipidemic and normoglycemic, with no personal or family history of premature coronary artery disease, major cardiovascular risk factors, or known infectious or inflammatory conditions. All participants provided written informed consent before participation. The study protocol was approved by the Ethical Committee of Hospital de la Santa Creu i Sant Pau (protocol code IIBS-APO-2013-105). The protocols for studies involving infected and uninfected patients were approved by the Ethical Committees of Parc Taulí (Ref 2019/664) and Vall d’Hebron University Hospitals (PR(AG)24/2020). The study involving carotid endarterectomy specimens was approved by the Ethical Committee of Hospital de la Santa Creu i Sant Pau (protocol code IIBSP-LPM-2019-94).

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).

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 and P116R for P116(30–957); P116F and P116R for P116(30–845); P116F and P116R for P116(246–818); and P116W to generate variant P116W681A. Detailed description on protein expression, purification and protein emptying has been reported previously11.

Isotopic unesterified cholesterol transfer from lipoproteins
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 x g for 24 h 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 6000/501c autoanalyzer (Roche Diagnostics), whereas apoB and apoA1 were determined by an immunoturbidimetric assay, using commercial kits adapted for a COBAS 6000/501c autoanalyzer. Radiolabeled LDLs and HDLs were prepared in the following way: 5 μCi of [1,2-3H(N)] unesterified cholesterol (Perkin Elmer) was mixed with absolute ethanol, and the solvent was dried under a stream of N2. 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 h in a 37°C bath11. The labeled LDLs and HDLs were re-isolated by gradient density ultracentrifugation and dialyzed against phosphate-buffered saline (PBS) through gel filtration chromatography. Specific activities of 3H-cholesterol-containing LDLs and HDLs were 520 and 4,750 counts per minute (cpm)/nmol of unesterified cholesterol, respectively.

MPN M129 was grown to mid-log phase in 25 cm2 cell culture flasks containing 5 mL SP4 medium and incubated at 37°C with 5% CO2. The medium was then replaced with 5 mL of SP4 lacking fetal bovine serum (FBS) and supplemented with [³H] unesterified cholesterol–containing LDL or HDL (0.1 g/L apoB or apoA1, respectively). Cultures were incubated for 90 min, after which the medium was recovered, and surface-attached mycoplasmas were washed once with PBS, collected using a cell scraper, and resuspended in PBS. Radioactivity associated with both medium and cell fractions was measured by liquid scintillation counting, and cholesterol transfer per mL was determined, including control samples without cells.

The cholesterol transfer to P116 (the construct spanning residues 30 to 957, 1 g/L) was measured after adding either [3H] unesterified cholesterol-containing LDLs or HDL (0.5 g/L of apoB or apoA1, respectively) and incubation at different times at 37°C. Where indicated, mAb-3G9 and mAb-3B5 were added at a final concentration of 3 µg/mL. 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 [3H] 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 or MPN. Human apoB and apoA1 levels were determined in both the LDL, HDL and purified P116 or MPN fractions by the immunoturbidimetric assays in the COBAS 6000/501c autoanalyzer.

Cholesterol efflux from cells 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), CHO cells, CHO cells expressing SR-B1 (provided by Dr. Joerg Heere) and J774A.1 mouse macrophages (ATCC® TIB67™). For this purpose, 3×105 cells/well were seeded in 6-well plates and grown for 72 h in complete Dulbecco’s Modified Eagle’s Medium (DMEM) high glucose with L-glutamine and sodium pyruvate (Corning, Glendale, AZ), supplemented with 10% FBS (PanBiotech, 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 h. Subsequently, the macrophages and lung cells were equilibrated overnight with 0.2% free fatty acid bovine serum albumin (Sigma Aldrich/Merck) in DMEM. This experiment was also conducted in macrophages, where efflux pathways were stimulated under controlled conditions. To activate the ABCA1/ABCG1-dependent cholesterol efflux, cells were treated during the equilibration period with 2 µM of the liver X receptor agonist T0901317 (Cayman Chemicals, Ann Arbor, MI)36. In separate experiments, 20 µM of the lipase inhibitor orlistat (Sigma-Aldrich/Merck) was applied to assess the potential contribution of lipase activity37. The following day, the media was removed, and the cells were incubated for 90 min with HDL, lipid-free apoA1 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.

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 (1×106) at 37°C for 90 min. 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 analytes38. 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 h at −20°C. The supernatants were collected following centrifugation at 18,000 x g for 15 min, 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 h at −20°C. Following centrifugation at 16,000 x g for 15 min, 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 x g for 5 min), 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.

UHPLC-MS analyses
The lipidomic workflow consisted of the following steps: sample extraction → chromatographic separation → mass spectrometric detection → data processing → normalization and statistical analysis. Lipidomic profiling was carried out using two ultra-high-performance liquid chromatography–mass spectrometry (UHPLC–MS) platforms, each optimized for specific lipid classes. Chromatographic separation was performed on ACQUITY UPLC systems (Waters Corp., Milford, USA). Mass spectrometric detection was carried out on a LCT Premier XE Time-of-Flight (ToF) (Waters Corp.) for Lipidomics Platform 1 and on a Xevo G2 QTof (Waters Corp.) for Lipidomics Platform 2. The full chromatographic and MS conditions are summarized in Supplementary Table 2.

To ensure analytical robustness, pooled quality control (QC) extracts were injected regularly throughout the batch sequence. Retention time stability was generally <6 s (injection-to-injection), and mass accuracy was maintained at <5 ppm for ions in the m/z 400–1000 range and <1.2 mDa for ions in the m/z 50–400 range.

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 (Rt–m/z) pairs corresponding to known metabolites were entered into the software, which extracted ion chromatograms (mass tolerance window = 0.05 Da). These chromatograms were peak-detected and noise-reduced in both the LC and MS domains, ensuring that only true metabolite-related signals were included.

Chromatographic peak areas were generated for each metabolite in each sample. For normalization, representative internal standards were included for every major lipid class analyzed. MS detection response curves were constructed using these standards to define linear detection ranges. Assuming similar ionization and detector responses within a lipid class, all metabolites were normalized against their respective class-specific internal standards. Final normalized peak areas were expressed relative to the sum of all detected features per sample to account for potential variability in sample preparation and instrument response. Normalized data were then classified according to their chemical properties following the comprehensive system proposed by Fahy et al.39 under the International Lipid Classification and Nomenclature Committee and expressed in the LIPID MAPS initiative (http://www.lipidmaps.org).

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 MPN IgG, IgM kit were also selected. No sex, gender, race, ethnicity, or other socially relevant group information was collected; only clinical diagnosis and IgG/IgM anti-MPN values were recorded. 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 (Thermo Fisher) incubating 1 µg 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 min 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.

Bacterial strains and culture conditions
The MPN M129 and MPN-Venus strains18 were grown in cell culture flasks containing SP4 medium and incubated at 37°C with 5% CO2. Surface-attached mycoplasmas were collected using a cell scraper and resuspended in SP4 medium.

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 adjuvants11. The last injection was delivered four days before splenectomy. Isolated B lymphocytes from the immunized mice were fused to NSI myeloma cells40 to obtain stable hybridoma cell lines producing mAb, as previously described41. 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 IC50 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 cells11. 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, supernatants 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 50K 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 50K Amicon.

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 5x106 hybridoma cells by directly siso 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.42 with few modifications. Briefly, the first strand was obtained by incubating 500 ng of total RNA, 1 µL of primers mIGKRT, mIGLRT or mIGHGRT at 10 µM in separate tubes, 1 µL of 10 mM dNTPs and 11 µL of RNase-free water at 65°C for 5 min. Then, 4 µL of the first strand buffer of M-MLV reverse transcriptase (Invitrogen), 2 µL 100 mM DTT, 1 µL RNase Out and 1 µL Template-Switch primer 100 µM were added to each tube. After incubating 2 min at 37°C 1 µL of M-MLV reverse transcriptase (Invitrogen) was added to each tube, which were incubated for 50 min at 37°C and 15 min at 70°C. The touchdown PCR reaction was performed as originally described using the Phusion thermo polymerase (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. 9a). Next, 700 bp and 600 bp band 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. 9b). The consensus sequence from each alignment was chosen as the complementary determining sequence in each of the chains of the P116 mAb-3G9 antibody.

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 150 mM NaCl buffer) was applied to glow-discharged C-FlatTM holey grids (CF-1.2/1.3; 300 mesh), 5 s blotting (−3 blotting force) and vitrified in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific) set to 4°C 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 Å2 s−1, resulting in a total dose of 39.70 electrons per Å2. Defocus values ranged from −1.0 to −2.5 µm.

CryoSPARC v443 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 3).

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: 9GVG and the map in the EMDB with code: 51635.

Effect of mAb on mycoplasma cell growth
Growth rates of MPN cultures were determined using an adaptation of the colorimetric protocol described by Karr and colleagues44. MPN M129 strain was grown to mid-log phase in 25 cm2 flasks with 5 mL SP4 medium. Attached cells were scraped off, recovered by centrifugation at 15,000 x g and resuspended in 3 mL of fresh SP4 medium, SP4 medium supplemented with a 1/200 dilution of P116 mAb-3G9 or mAb-3B5 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 37°C for 8 days. During the incubation time, absorbance at 550 nm for each well was recorded each 15 min 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 = ln 2 / (1/µ)).

Interaction of MPN with human atherosclerotic plaques ex vivo
Carotid endarterectomy specimens were obtained within one hour of surgical resection. 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 Venus18 was grown in cell culture flasks containing SP4 medium and incubated at 37°C and 5% CO2. 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 1×105 mycoplasma cfu in 200 µL of 0.22 µm filtered SP4 medium and a small piece of healthy carotid tissue (HT) or atherosclerotic plaque tissue (AP). In some experiments, mAb-3G9 and mAb-3B5 were added at a final concentration of 3 µg/mL at the time of inoculation. Phase contrast (PhC) and epifluorescence microscopy images of MPN cells incubated with human carotid tissue fragments were acquired using an inverted Nikon Eclipse TE2000-E microscope. HT and AP were analyzed in parallel. After inoculation with MPN Venus strain cells, images were acquired at 0 h, 24 h, and 48 h, focusing on the same tissue regions across time points. YFP fluorescence (excitation 490/510 nm, emission 520/550 nm) was detected with an Orca Fusion camera (Hamamatsu) controlled by NIS-Elements BR software (Nikon).

Quantification of MPN localization and proliferation was performed by measuring the percentage increase in fluorescent area from 0 h to 48 h using four independent biological replicates.

Biodistribution of MPN into atherosclerotic mice
Female wild-type mice and LDLR−/− 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. Mice were kept in a temperature-controlled (22°C) room with a 12-hour light/dark cycle, and food and water were provided ad libitum. We used 4–5th-month-old mice fed with a Western-type diet (TD.88137, Harlan Teklad, Madison, WI, containing 21% fat and 0.2% cholesterol) for 8 weeks.

To investigate the in vivo biodistribution of Mycoplasma using single SPECT/CT, the non-pathogenic MPN strain CV845 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 107 CFUs was radiolabeled by incubating it with 5.96 MBq of [¹¹¹In]In-oxine at 37°C for 15 min. Following radiolabeling, 100 µL (3.34 ± 0.03 MBq) 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, exsanguinated, and perfused with saline, after which the heart and major vessels were excised for SPECT/CT imaging. Liver-associated activity was quantified by measuring Bq of [¹¹¹In]In-oxine with a gamma counter.

Experiments were conducted in a blind manner concerning the origin of the specimens to reduce bias. Plasma total cholesterol and triglycerides were determined enzymatically using commercial kits adapted for a COBAS 6000/501c 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 6000/501c autoanalyzer.

Liver samples stored at −80°C were homogenized with an Ultra-Turrax (IKA), and total DNA was extracted using the DNeasy® UltraClean® Microbial Kit (Qiagen) according to the manufacturer’s instructions. For standard curve generation, the MPN CV8 strain was cultured for 72 h. Cells were harvested, and one aliquot was plated on Hayflick agar to estimate CFU/mL. The remaining culture was centrifuged (15,000 x g, 5 min), and the pellet was processed for DNA extraction. DNA concentration was measured using a NanoDrop One spectrophotometer (Thermo Scientific), and integrity was assessed by 1% agarose gel electrophoresis. Quantitative PCR (qPCR) was performed with SYBR Premix Ex Taq II (Tli RNaseH Plus; Takara) on an AriaMx Real-Time PCR System (Agilent Technologies). The following primers targeting the adk gene (mpn185) were used: forward 5′-CCGTTTGGTTTGTCCTAGTTGTGGTAGTG-3′ and reverse 5′-GATTACCGCTTCCGTGTCATCATTGC-3′. Murine hearts were fixed in 4% paraformaldehyde for 4 h at 4°C, washed in PBS for 1 h, and incubated overnight in 30% sucrose. Tissues were embedded in OCT and immediately frozen. Hearts were sectioned transversely at 6 µm using a Leica CM1950 cryostat (Leica Biosystems, Wetzlar, Germany). To quantify aortic lesion size, serial sections were stained with Oil Red O (Sigma-Aldrich). Plaque areas were outlined, and the mean lesion size was calculated across at least five sections per mouse46.

Statistical methods
Data are presented as the mean ± standard deviation (SD). Unpaired t-tests were used to compare the differences between two groups. Differences between two groups were analyzed using unpaired t-tests, while comparisons across more than two groups were performed using one-way ANOVA followed by Tukey’s multiple comparisons post-test. Associations between variables were assessed using Pearson’s correlation coefficient. 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.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "David Vizarraga, Marina Marcos, Noemi Rotllan and colleagues previously identified P116 as a critical protein for MPN to extract lipids for its cell membrane and survival. In the current manuscript the authors argue that MPN can also cause systemic effects because P116 it is able to extract lipids from LDL, HDL and cells of lipid-rich tissues including the liver and atherosclerotic plaques. The results are convincing and the manuscript well-written. However, I do have some methodological concerns. In addition, I am missing some mechanistical insight and experiments that show the application of MPN antibodies in liver diseases or atherosclerosis.\nMajor remarks:\n1. The number of replicates for some experiments is very low. Fig 1B/C is based on n=3, and unclear why Fig 1D is only conducted in an n=2. Same for Fig 2, and Fig 3C appears the results of a single measurement. Data of Fig 4 should be quantified, and repeated if this is (again) based on only n=2 per group.\n2. The choice for the statistical comparisons is concerning. Multiple t-tests are used when three or more groups are compared, which does not seem appropriate. I’m not a statistical expert, but for example in Fig 1D t-tests are definitely not appropriate for various reasons, 1) only n=2 which precludes testing for normal distribution, and 2) with three groups and at least 11 lipid species that are being analyzed there is a very high chance of false positive results. Showing SEMs instead of SDs does also not seem appropriate with these low number of replicates.\n3. The authors need to show evidence for targeting P116 in atherosclerosis or hepatic lesions, or remove this suggestion from title and abstract.\n4. What does P116 expression result in uptake of lipids by MPN? I am missing experimental evidence or a discussion on the mechanism; does the protein become part of the cell membrane or is it secreted, does it bind to apolipoproteins, how does it scavenge such a variety of lipid species, does it have any lipase activity or requires other cellular proteins?\n5. In relation to the previous question, the authors suggest MPN to be attracted to lipid-rich tissues, but why does it specifically end up in liver or atherosclerotic plaques? Is there a correlation between infection and certain gene/protein expression, or is this simply the result of acceptable endothelium (i.e. damaged in case of plaques and fenestrated in liver). Is there any relationship between liver lipids or atherosclerotic lesion size and MPN colonization and growth?\n6. A short discussion on how MPN acerbates atherosclerosis or hepatic lesions would be appropriate. And why were the developed antibodies not tested in this context, or at least in combination with the SPECT/CT assessment.\nMinor suggestions:\n1. Provide a brief description of the characteristics of the patients with infectious diseases, as well as the HDL/LDL donors.\n2. The schematic overviews of the experiments are not very helpful. I find them redundant and often not clear. For example in Fig 2A it should become clear that binding of lipids to P116 is compared to uptake by HDL, and what the control is in the assessment of the lipidome.\n3. Many typos and formatting errors throughout the manuscript. Some examples:\na. Fig 2: Different colors are used to mark the P116 group.\nb. Figure 5C: Typo in the y axis.\nc. Line 261: Briefly, supernants were diluted twice in 20 mM… Typo.\nd. Line 285: described using the Phusion termopolymerase (Thermo Scientific) using..", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "It was my pleasure to review the enclosed manuscript titled “\"Physiological Sources of Essential Lipids for Mycoplasma pneumoniae via Protein P116: Innovative Biotechnological Tools for Targeting Atherosclerotic and Hepatic Lesions.” This study investigates the role of the Mycoplasma pneumoniae (MPN) protein P116 in lipid uptake, particularly its ability to extract essential lipids from human lipoproteins (LDL and HDL) and host cells. The authors demonstrate that P116 facilitates MPN survival by acquiring cholesterol, phosphatidylcholine, and sphingomyelin, which are crucial for its membrane structure. The study also explores the interaction of MPN with atherosclerotic plaques and the liver, highlighting the potential of monoclonal antibodies targeting P116 to inhibit cholesterol uptake and bacterial localization. The findings provide insights into MPN pathophysiology and open avenues for developing therapeutic interventions against MPN infections and related cardiovascular complications. The manuscript presents a compelling study on P116-mediated lipid acquisition and its implications for MPN pathogenesis. With additional discussion of lipid metabolism, mechanistic details of P116 function, and therapeutic relevance, the manuscript will be significantly strengthened.\nCmments\n1. Discussion of Mycoplasma Lipids and Membranes: The manuscript focuses on lipid acquisition but provides limited discussion on the lipid composition of Mycoplasma membranes. Expanding on how Mycoplasma membranes differ from other bacterial membranes, particularly in their reliance on cholesterol, would provide better context. The authors could also discuss the functional significance of phosphatidylcholine and sphingomyelin in Mycoplasma membranes and whether their uptake by P116 aligns with known Mycoplasma lipidomics.\nIn general the discussion of lipids, and in particular mycoplasma lipids is rather thin. There have been a number of papers on mycoplasma lipidomes and membranes in the past few years that are not discussed and should be to connect this study with the state-of-the-art. Also a large body of literature from the 1960s-80s, that could provide some useful basis for discussion. Given what is known about Mycoplasma membrane adaptation and lipidome remodeling, it would be interesting for the authors to discuss how P116 could support selective lipid uptake in response to temperature adaptation, for example?\n2. Mechanism of Lipid Uptake: The study suggests that P116 is essential for lipid acquisition, but the exact molecular mechanism remains unclear. Are there structural analogies between P116 and other lipid transporters? Additional discussion on whether P116 functions as a passive lipid-binding protein or actively mediates lipid transfer would provide some clues about mechanism. Is there any evidecne for selective uptake when comparing the lipid composition of the serum vs. the lipids taken up in the experiments? This would be interesting to discuss as well.\n3. Relevance to Atherosclerosis and Liver Localization: The study suggests that MPN preferentially localizes in lipid-rich environments, but a more detailed discussion of the underlying physiological basis (e.g., lipid metabolism in these tissues) would strengthen the discussion.\n4. Monoclonal Antibody Therapeutics: The data indicate that monoclonal antibodies targeting P116 reduce lipid uptake and bacterial colonization. However, it is unclear whether these antibodies could serve as viable therapeutic agents in vivo. Could they be used prophylactically or as adjuncts to antibiotics? It would be interesting to compare the inhibitory effects of these antibodies with other known antimicrobial strategies targeting Mycoplasma.\n5. The study relies on in vitro assays and ex vivo tissue models. Additional discussion on how these findings might translate to in vivo infection models or clinical scenarios is warranted.\n6. Given the importance of lipidomic analyses, a more explicit discussion of the UHPLC-MS workflow and data normalization methods would improve reproducibility. For example, what standards were used to quanitify which lipids, were they added before or after lipid extraction, concentrations, etc…?", "reviewer_4": "This study provides new insights into the role of the P116 protein in M. pneumoniae lipid uptake and its potential impact on bacterial survival in the bloodstream. The authors present compelling evidence that P116 helps extract essential lipids from LDL and HDL, a key adaptation for M. pneumoniae in lipid-rich environments. These findings are particularly important as they link M. pneumoniae colonization in liver and atherosclerotic lesions to its ability to acquire essential lipids. To further improve the study’s impact and reproducibility, consider addressing the following points:\nIn Figures 1 and 2, where human circulating LDLs and HDLs are identified as primary lipid sources for P116, including at least three replicates would strengthen statistical robustness and reproducibility.\nThe same applies to Figure 3C on lipid uptake. Testing the mAb-3B5 antibody (which does not target the central region of P116) would further confirm that only antibodies targeting the C-terminal domain interfere with lipid acquisition and M. pneumoniae presence in atherosclerotic tissue.\nSince M. pneumoniae lacks traditional energy-dependent lipid transporters, diffusion is likely the mechanism by which P116 mediates cholesterol and neutral lipid uptake. Investigating whether cellular transporters contribute to this process would provide valuable mechanistic insights into P116’s function.\nOther points:\nIn Figures 4 and 5, including quantitative measurements of signal intensities would provide an objective assessment of the observed visual changes.\nEstablishing a numerical correlation between liver lipid content and M. pneumoniae presence would further validate the physiological impact of P116-mediated lipid uptake.", "reviewer_5": "The manuscript describes a series of experiments designed to substantiate a hypothesis, as written in l609-611, 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’. Unfortunately, there are several weaknesses in the design and interpretation of the data which limit the conclusions which can be drawn from this work.\nMajor points\n1. The model for the function of P116 is unclear; the structure suggests it is a lipid-binding protein attached to the mycoplasma membrane by a flexible linker (l69). But is it acting as an adhesin or as part of an uptake system for lipid nutrients- the authors seem to suggest the latter (l62)? However, interpretation of the data in the manuscript does not draw a clear distinction between these two models for the role of P116.\n2. The heading for section 3.1 over-states the conclusion which can be drawn from these experiments ‘Human circulating LDL is a major source of unesterified cholesterol for MPN P116’ (l436). All that can be said about the results from Fig 1 is that cholesterol from LDLs or HDLs in vitro binds to P116. This may or may not happen in vivo. It is clear from the results in section 3.2 that the specificity of P116 is broad.\n3. The description of the conclusion in section 3.3 implies an active function of P116 in lipid acquisition- ‘P116 uptakes lipids from H1299 lung cells with low efficiency’- which is not necessarily warranted by the data. If P116 is simply a lipid-binding protein, it will presumably bind free cholesterol in solution. Cells with lower cholesterol content and/or less propensity to shed cholesterol will transfer less of it to P116. This is more a property of the cells studied than of P116 itself.\n4. The data on serum reactivity against different forms of P116 lacked suitable controls from uninfected patients (Supplementary Table 4). Some bacterial antigens exhibit a high degree of reactivity against serum IgG, even in individuals with no history of infection by that organism. At the very least, a statistical comparison is needed between infected and uninfected groups to establish the conclusion proposed. It is a stretch to suggest ‘..C-terminal domain of P116 could serve as a promising candidate for the development of vaccines against MPN’ (l591). What reason is there to think that it will elicit a protective immune response?\n5. Fig 3C Is the result from one experiment? Is there a control with an antibody without specificity for P116?\n6. Fig 3D: why would MAb-3G9 inhibit growth? Does this imply that P116 plays a role in lipid nutrient uptake?\n7. The microscopy data in Fig 4B are unclear; limited samples are shown and there is no quantification of fluorescence, which would enable a statistical analysis of the effects of both antibodies. Without that, the conclusions are qualitative at best and appear tenuous.\n8. The interpretation of the [¹¹¹In]In-oxine radiolabelling experiments depends on the in vivo fate of the ¹¹¹In label. [¹¹¹In]In-oxine is a neutral, lipophilic complex which passively diffuses through cell membranes. Inside the cell, the isotope binds to intracellular macromolecules. In the literature, [¹¹¹In]In-oxine has been described as a label for leukocytes but I am not aware of any details about Mycoplasma labelling. The labelling period is only 15 minutes (l402)- how do we know that any labelling of the bacteria has occurred at all? Has the isotope been taken up into the bacteria? Could the results in Figure 5 be explained by the lipophilic nature of the complex, preferentially targeting to the fat-enriched livers of the LDL-knockout mice? More data are needed to exclude these explanations.\nMinor\nFig 1B why do the lines on this plot not extend to zero? Presumably isolated P116 at t=0 contains no radioactive label?"}, "author_response": "We sincerely thank the reviewer for the thoughtful and constructive evaluation of our manuscript. In response to the methodological and mechanistic concerns raised, we have made substantial revisions throughout the manuscript, including additional experiments, improved statistical analyses, expanded mechanistic insight, and clarification of translational claims. We believe these changes have significantly strengthened the rigor, scope, and relevance of the study. Below, we provide detailed responses to each of the reviewer’s points.\nMajor remarks:\n1. The number of replicates for some experiments is very low. Fig 1B/C is based on n=3, and unclear why Fig 1D is only conducted in an n=2. Same for Fig 2, and Fig 3C appears the results of a single measurement. Data of Fig 4 should be quantified, and repeated if this is (again) based on only n=2 per group.\nIn response to this comment, we have ensured that a minimum of three biological replicates is included for each experiment in Figures 1–3. Specifically, Fig 1e and Fig 2d have now been repeated to achieve n=3. We have also included a new experiment that evaluated the transfer of unesterified cholesterol from LDL/HDL to MPN (new Figure 1b) to strength data from Figure 1c. In addition, Fig 3c has been expanded to include triplicate measurements to strengthen the robustness of the data. For Figure 4, we have now quantified the results in four independent samples and included the corresponding statistical analyses. The revised figures and updated descriptions can be found in the revised manuscript. Importantly, the inclusion of additional replicates and new experiments did not change the outcomes of our original findings.\n2. The choice for the statistical comparisons is concerning. Multiple t-tests are used when three or more groups are compared, which does not seem appropriate. I’m not a statistical expert, but for example in Fig 1D t-tests are definitely not appropriate for various reasons, 1) only n=2 which precludes testing for normal distribution, and 2) with three groups and at least 11 lipid species that are being analyzed there is a very high chance of false positive results. Showing SEMs instead of SDs does also not seem appropriate with these low number of replicates.\nTo address this concern, we have revised our statistical approach as follows:\n- We have increased the number of biological replicates to at least n=3 for all lipidomic analyses, including those previously shown in Figs 1D and 2B (now the revised Fig 1e and Fig 2d and supplementary data), as detailed in our previous response.\n- Instead of multiple t-tests, we have now applied a one-way ANOVA to compare groups, both for the overall comparisons and for individual lipid species.\n- Furthermore, SDs are now reported instead of SEMs to more accurately reflect variability within small sample sizes.\nThese adjustments are incorporated into the revised manuscript and reflected in the updated figures and figure legends and supplemental Tables. The inclusion of additional replicates and new experiments did not change the outcomes of our original findings.\n3. The authors need to show evidence for targeting P116 in atherosclerosis or hepatic lesions, or remove this suggestion from title and abstract.\nWe acknowledge that while our in vivo experiments in mice demonstrate the accumulation of the MPN chassis in the liver and in atherosclerotic plaques, they do not establish direct mechanistic targeting by P116 alone in vivo. Additionally, we would like to highlight that our study includes ex vivo experiments using human atherosclerotic plaques, in which we observed P116-dependent binding of MPN (updated Figure 4), supporting the relevance of our findings beyond the murine model. Accordingly, we have revised the title and abstract to more precisely reflect our findings without overinterpreting the data.\n4. What does P116 expression result in uptake of lipids by MPN? I am missing experimental evidence or a discussion on the mechanism; does the protein become part of the cell membrane or is it secreted, does it bind to apolipoproteins, how does it scavenge such a variety of lipid species, does it have any lipase activity or requires other cellular proteins?\nWe thank the reviewer for these questions regarding the mechanism by which P116 mediates lipid uptake in MPN. Clarifying this process has been a major focus of our previous work (Nature Structural & Molecular Biology, 2023). P116 is a membrane-anchored protein in MPN and is not secreted. We demonstrated via single-particle cryo-electron microscopy that P116 presents a previously undescribed fold, generating a large, solvent-accessible hydrophobic cavity. This unique structural feature is consistent with its role in binding and extracting lipids from the extracellular environment and resembles lipid transfer proteins. These findings are now complemented by new functional data presented in the revised Figure 2. To further investigate the mechanism of P116-mediated cholesterol uptake from host cells, we conducted additional experiments using CHO cells and CHO cells expressing scavenger receptor type BI (SR-B1), H1299 pulmonary epithelial cells, and J774 macrophages. Interestingly, P116-mediated cholesterol uptake was not affected by the presence or activity of SR-BI. Furthermore, while the activation of the cholesterol efflux transporters ATP-binding cassette (ABC)A1 and ABCG1 promotes cholesterol efflux to classical acceptors such as HDL or lipid-free apoA-I, our data show that upregulation of ABCA1 and ABCG1 did not enhance cholesterol transfer to P116. This suggests that P116 does not rely on canonical receptor-mediated uptake or efflux mechanisms, but rather exploits freely available or loosely bound lipids.\nWe also tested whether P116 binds directly to lipoproteins. Incubation of P116 with HDL or LDL followed by immunoturbidimetric assays for apoA-I and apoB showed no evidence of stable or permanent binding, further supporting a model of direct lipid extraction without forming permanent binding. This argues against a mechanism involving apolipoprotein recognition. Additionally, inhibition of lipase activity using orlistat has no effect on P116-mediated uptake, excluding a lipase-dependent mechanism (new supplementary Figure 3). In this specific case, the lipase action would be found in the membrane from MPN, which can rearrange the acyl chains of phospholipids (Biochim Biophys Acta . 1995 May 4;1235(2):369-77. doi: 10.1016/0005-2736(95)80026-c and NPJ Syst Biol Appl. 2020 Oct 23;6:33. doi: 10.1038/s41540-020-00153-7. Additionally, the ability of P116 to acquire unesterified cholesterol from cell membranes was highly correlated with the cellular cholesterol content (new Supplementary Figure 2). Taken together, these data reinforce the view that P116 operates via a passive, diffusion-driven process, leveraging its hydrophobic cavity to directly acquire lipids from the surrounding milieu. We propose that P116 functions as a surface-exposed lipid sink, facilitating the scavenging of diverse lipid species, including cholesterol, phosphatidylcholines, and neutral lipids, without requiring energy input, or host receptor engagement.\nWe have included these new findings in the revised Figure 2 and the new Supplementary Figures 2 and 3, and expanded the Discussion section to integrate these mechanistic insights and highlight the novelty of P116 as a unique microbial lipid acquisition system (page 13, second paragraph).\n5. In relation to the previous question, the authors suggest MPN to be attracted to lipid-rich tissues, but why does it specifically end up in liver or atherosclerotic plaques? Is there a correlation between infection and certain gene/protein expression, or is this simply the result of acceptable endothelium (i.e. damaged in case of plaques and fenestrated in liver). Is there any relationship between liver lipids or atherosclerotic lesion size and MPN colonization and growth?\nThe primary recognized route of entry for MPN is through the respiratory tract, with lung tissue as the initial target. However, it is well established that MPN in some cases can disseminate to other organs, particularly the vascular system. In this study, we investigated the distribution of MPN following direct introduction into the bloodstream. Our data indicate that MPN preferentially localizes to lipid-rich tissues, particularly the liver and, when present, atherosclerotic plaques. We believe localization of MPN is primarily driven by the availability of lipids rather than specific gene or protein expression or receptor-mediated mechanisms.\nLiver accumulation was observed in both LDLR-deficient (LDLR-/-) and wild-type mice, consistent with the liver’s role as a major site of lipid processing and cholesterol synthesis. Interestingly, although LDLR-/- mice exhibited larger and more cholesterol-rich livers than their wild-type counterparts, the total MPN load detected by radioactivity and qPCR across the entire liver was comparable between the two groups (see new data in Figure 5d-e). We also analyzed the physiological correlation between MPN abundance and liver cholesterol content (new Figure 5f). Interestingly, no significant correlation was observed between liver cholesterol content and MPN abundance. This indicates that while liver accumulation reflects a general tropism, it does not scale directly with hepatic cholesterol levels—likely due to diffuse distribution throughout liver tissue. It should be noted that hepatic lipid content is elevated in both mouse models, which may facilitate the lipid-tropism of MPN by providing an excess lipid environment regardless of LDLR status.\nGiven that the 6-hour post-injection time point used in our study precedes any potential replication of the MPN chassis, we interpret these findings as reflective of initial biodistribution and lipid-driven accumulation rather than proliferation.\nUsing SPECT/CT imaging, we observed that MPN chassis localize specifically to the areas where atherosclerotic lesions are present—but only in LDLR-/- mice, which develop atherosclerosis under high-fat diet conditions (see revised Figure 5g-i). This targeted accumulation was not observed in wild-type mice, which do not develop significant plaques, suggesting that lesion-associated features (e.g., increased lipid content and altered endothelium) are necessary for specific MPN association with vascular tissue.\nWe have expanded Figure 5 with these new relevant data and revised the Results and Discussion sections to clarify this interpretation and better contextualize the role of lipid availability and tissue structure in MPN localization (pages 10 and 11, last paragraph on page 15 and first paragraph on page 16).\n6. A short discussion on how MPN acerbates atherosclerosis or hepatic lesions would be appropriate. And why were the developed antibodies not tested in this context, or at least in combination with the SPECT/CT assessment.\nWhile our current study does not directly address whether the MPN chassis exacerbates atherosclerosis or hepatic lesions, we agree that this is an important topic for future exploration. We have now included a brief discussion in the revised manuscript addressing the potential for MPN to influence disease progression, particularly through preferential localization to lipid-rich tissues and potential interactions with inflammatory microenvironments. However, we emphasize that the main focus of this study is to characterize lipid uptake mechanisms of MPN and the biodistribution of the engineered chassis.\nIn response to the reviewer’s suggestion regarding the use of antibodies, we tested the anti-P116 monoclonal antibody 3G9 in LDLR-deficient mice, combined with SPECT/CT imaging. Interestingly, MPN chassis were still detected in the liver despite the presence of the antibody. The persistent liver accumulation in vivo may reflect the high systemic lipid availability and the liver’s central role in cholesterol metabolism, which could partially bypass the antibody’s capacity to block cholesterol access. Additionally, the liver’s unique vascular architecture—characterized by a fenestrated endothelium and absence of a basement membrane—may further facilitate passive localization of lipid-seeking organisms or particles. Indeed, dissemination of bacteria to organs such as the liver and spleen has been demonstrated in mice orally administered Salmonella enterica serotype Typhimurium (EMBO Rep. 2025 Feb;26(3):656-689. doi: 10.1038/s44319-024-00328-x. Notably, Salmonella reroutes host lysosomal cholesterol via peroxisomes as a bridge, and free cholesterol is essential for the integrity of Salmonella-containing vacuoles and for bacterial proliferation. However, this does not appear to be the case in human atherosclerotic arteries, where the 3G9 antibody effectively blocked MPN binding, supporting the notion of specific interactions. Consistent with this, MPN was detected in atherosclerotic plaques of LDLR-deficient mice fed a high-fat diet, but not in wild-type controls that do not develop significant atherosclerotic lesions, indicating that lesion formation is required for vascular association. This supports our interpretation that P116-mediated lipid acquisition promotes selective MPN localization in lipid-rich, structurally compromised vascular lesions.\nThese findings underscore the challenges of translating ex vivo blocking efficacy into in vivo systems, where multiple physiological factors influence distribution and access to lipids. We have expanded the Discussion section to highlight these points (second paragraph on page 16).\nMinor suggestions:\n1. Provide a brief description of the characteristics of the patients with infectious diseases, as well as the HDL/LDL donors.\nAs requested, we have now included a brief description of the patient cohort and lipoprotein donors used in our study. Specifically, we analyzed the immunogenic activity of various P116 constructs using sera from 25 individuals with serologically confirmed Mycoplasma pneumoniae infection. To provide appropriate controls, sera from five individuals who tested negative for M. pneumoniae by serology were also included in the analysis.\nHDL and LDL used in lipid uptake assays were obtained from healthy donors who were normolipidemic and normoglycemic, with no personal or family history of premature coronary artery disease, major cardiovascular risk factors, or known infectious or inflammatory conditions. This information has now been added to the revised Methods section (pages 18 and 23) to clarify the clinical and biochemical context of the biological materials used.\n2. The schematic overviews of the experiments are not very helpful. I find them redundant and often not clear. For example in Fig 2A it should become clear that binding of lipids to P116 is compared to uptake by HDL, and what the control is in the assessment of the lipidome.\nWe thank the reviewer for this helpful feedback regarding the clarity of the schematic overviews. We agree that the current diagrams could be more informative and we have revised them accordingly in the updated version of the manuscript. Specifically, we have improved Figure 2 to clearly indicate the comparison of lipid uptake by P116, HDL, and lipid-free apoA-I across all tested cell types, including CHO-SR-BI, H1299, and J777 cells. In addition, we have explicitly defined the control used in the lipidomic analyses: P116 protein that was isolated and incubated under identical conditions but without LDL, HDL, or pulmonary cells. This control accounts for background lipids or contaminants potentially associated with P116 during purification and ensures that measured lipid uptake reflects genuine extraction from the tested biological sources. We have updated both the Figures 1 and 2 and their legend to reflect these clarifications and have streamlined the schematic to improve its clarity and usefulness.\n3. Many typos and formatting errors throughout the manuscript. Some examples:\na. Fig 2: Different colors are used to mark the P116 group.\nb. Figure 5C: Typo in the y axis.\nc. Line 261: Briefly, supernants were diluted twice in 20 mM… Typo.\nd. Line 285: described using the Phusion termopolymerase (Thermo Scientific) using.\nWe have now carefully revised the entire manuscript to correct typographical and formatting mistakes, including:\nEnsuring consistent color coding in Figure 2, with a clear and uniform representation of the P116 group across all panels.\nCorrecting the typo in the y-axis label of Figure 5C.\nFixing the spelling error in Line 261 (\"supernants\" → \"supernatants\").\nCorrecting Line 285 to read \"Phusion thermopolymerase (Thermo Scientific)\".\nWe have also performed a thorough review to identify and correct additional inconsistencies and errors throughout the text and figures.\nReviewer #2 (Remarks to the Author)\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nWe acknowledge and appreciate the reviewer’s participation in the Nature Communications co-review initiative and thank both the primary and co-reviewers for their time and constructive evaluation. As there were no additional comments from the co-reviewer, we have focused our responses on the points raised in the main review.\nReviewer #3 (Remarks to the Author)\nIt was my pleasure to review the enclosed manuscript titled “\"Physiological Sources of Essential Lipids for Mycoplasma pneumoniae via Protein P116: Innovative Biotechnological Tools for Targeting Atherosclerotic and Hepatic Lesions.” This study investigates the role of the Mycoplasma pneumoniae (MPN) protein P116 in lipid uptake, particularly its ability to extract essential lipids from human lipoproteins (LDL and HDL) and host cells. The authors demonstrate that P116 facilitates MPN survival by acquiring cholesterol, phosphatidylcholine, and sphingomyelin, which are crucial for its membrane structure. The study also explores the interaction of MPN with atherosclerotic plaques and the liver, highlighting the potential of monoclonal antibodies targeting P116 to inhibit cholesterol uptake and bacterial localization. The findings provide insights into MPN pathophysiology and open avenues for developing therapeutic interventions against MPN infections and related cardiovascular complications. The manuscript presents a compelling study on P116-mediated lipid acquisition and its implications for MPN pathogenesis. With additional discussion of lipid metabolism, mechanistic details of P116 function, and therapeutic relevance, the manuscript will be significantly strengthened.\nWe sincerely appreciate Reviewer’s insightful suggestions, particularly regarding lipid metabolism, P116’s mechanism of action, and translational implications. In response, we have substantially revised the manuscript to deepen the discussion on Mycoplasma lipidomics, incorporating both historical and recent literature to contextualize P116’s role in selective lipid uptake and membrane adaptation, including potential temperature-dependent remodeling. We have clarified the mechanism of lipid acquisition by P116, presenting new data that support a passive, transporter-independent uptake model via a hydrophobic cavity, and highlighted enrichment patterns consistent with MPN’s membrane needs. We have also expanded on MPN’s tropism for lipid-rich tissues and discussed the therapeutic potential of anti-P116 monoclonal antibodies, outlining their limitations and future directions in prophylactic or adjunctive settings.\nComments\n1. Discussion of Mycoplasma Lipids and Membranes: The manuscript focuses on lipid acquisition but provides limited discussion on the lipid composition of Mycoplasma membranes. Expanding on how Mycoplasma membranes differ from other bacterial membranes, particularly in their reliance on cholesterol, would provide better context. The authors could also discuss the functional significance of phosphatidylcholine and sphingomyelin in Mycoplasma membranes and whether their uptake by P116 aligns with known Mycoplasma lipidomics.\nIn response to the reviewer’s comment, we have expanded the Discussion to better contextualize P116-mediated lipid uptake within the known lipid profile of Mycoplasma pneumoniae membranes, which are markedly different from those of most other bacteria. Unlike typical bacteria, which synthesize their own phospholipids, Mycoplasma species rely almost entirely on the host for exogenous lipids—particularly cholesterol, phosphatidylcholine (PC), and sphingomyelin (SM) (J. Bacteriol. 1970;102:306–310. doi:10.1128/jb.102.2.306-310.1970; Biochim. Biophys. Acta. 1980;604:65–90. doi:10.1016/0005-2736(80)90585-4; J. Bacteriol. 1986;167:299–304. doi:10.1128/JB.167.1.299-304.1986; Science. 2009;326:1263–1268. doi:10.1126/science.1177263). Cholesterol can constitute 30–50% of Mycoplasma membrane lipids—far higher than in most bacteria—where it plays a critical role in stabilizing and rigidifying the membrane (Biochim. Biophys. Acta. 1980;604:65–90. doi:10.1016/0005-2736(80)90585-4). PC helps maintain membrane fluidity, while SM contributes to lipid raft formation, potentially facilitating host–pathogen interactions (Biochim. Biophys. Acta. 1980;604:65–90. doi:10.1016/0005-2736(80)90585-4; J. Bacteriol. 1986;167:299–304. doi:10.1128/JB.167.1.299-304.1986; J. Lipids. 2012;2012:640762. doi:10.1155/2012/640762).\nThese characteristics align closely with our lipidomic findings, which show that P116 can acquire not only cholesterol but also PC and SM. The revised Discussion (page 14) now explicitly links these uptake patterns to the established Mycoplasma lipid dependency, reinforcing the concept that P116 functions as a lipid scavenger that supports the organism’s unique membrane composition (npj Syst. Biol. Appl. 2020;6:33. doi:10.1038/s41540-020-00153-7).\nIn general the discussion of lipids, and in particular mycoplasma lipids is rather thin. There have been a number of papers on mycoplasma lipidomes and membranes in the past few years that are not discussed and should be to connect this study with the state-of-the-art. Also a large body of literature from the 1960s-80s, that could provide some useful basis for discussion. Given what is known about Mycoplasma membrane adaptation and lipidome remodeling, it would be interesting for the authors to discuss how P116 could support selective lipid uptake in response to temperature adaptation, for example?\nWe fully agree that situating our findings within the broader context of Mycoplasma membrane biology strengthens the manuscript. In response, we have substantially expanded the Discussion to incorporate both foundational studies and recent lipidomic analyses (page 14).\nHistorical studies established that Mycoplasma species maintain membrane homeostasis almost exclusively through uptake of exogenous lipids, especially cholesterol and saturated phospholipids, by modulating the cholesterol-to-phospholipid ratio and the degree of lipid saturation (Microbiol Rev. 1978 Jun;42(2):414–470. doi: 10.1128/mr.42.2.414-470.1978 and Bio-chim Biophys Acta. 1976 Mar 5;426(2):258-70. doi: 10.1016/0005-2736(76)90336-9 J Bacteriol. 1981 Jun;146(3):1124-34. doi: 10.1128/jb.146.3.1124-1134.1981 Membrane Fluidity. Biomembranes, 1984 vol 12. Springer, Boston, MA. https://doi.org/10.1007/978-1-4684-4667-8_7). Recent high-resolution lipidomics has confirmed the remarkable plasticity of these membranes, showing that lipid composition can shift dynamically in response to environmental stressors such as temperature changes (Cell Rep . 2024 Jul 23;43(7):114435. doi: 10.1016/j.celrep.2024.114435). These findings are consistent with lipidomic profiles reported for M. pneumoniae (npj Syst Biol Appl 6, 33 (2020). https://doi.org/10.1038/s41540-020-00153-7).\nIn light of this, we now discuss how P116 could contribute to such adaptive remodeling. Its broad lipid-binding capacity and selective uptake of key membrane lipids suggest that P116 may act as a molecular gatekeeper, enabling M. pneumoniae to fine-tune its lipidome in response to temperature shifts or other physiological stresses. While direct evidence for temperature-dependent lipid selectivity remains to be established, our results provide a conceptual framework for future studies exploring this mechanism.\n2. Mechanism of Lipid Uptake: The study suggests that P116 is essential for lipid acquisition, but the exact molecular mechanism remains unclear. Are there structural analogies between P116 and other lipid transporters? Additional discussion on whether P116 functions as a passive lipid-binding protein or actively mediates lipid transfer would provide some clues about mechanism. Is there any evidence for selective uptake when comparing the lipid composition of the serum vs. the lipids taken up in the experiments? This would be interesting to discuss as well.\nWe thank the reviewer for the insightful comments regarding the molecular mechanism of lipid uptake by P116. In our previous study (Nature Structural & Molecular Biology, 2023), we determined the structure of P116 using single-particle cryo-electron microscopy and identified a unique architecture featuring a previously uncharacterized fold. This structure revealed a large, solvent-accessible hydrophobic cavity, consistent with a role in lipid binding and capable of accommodating multiple lipid molecules. However, structural comparisons using the DALI server did not reveal any significant analogies between P116 and other experimentally determined protein structures in the Protein Data Bank, including known lipid transporters or carrier proteins.\nTo further explore its mechanism, we have now performed new lipid uptake assays using CHO cells overexpressing SR-BI, H1299 pulmonary epithelial cells, and J774 macrophages (see restructured Figure 2). P116-mediated cholesterol uptake was unaffected by SR-BI activity, indicating that it does not rely on this pathway.\nFurthermore, while the activation of the cholesterol efflux transporters ABCA1 and ABCG1 promotes cholesterol efflux to classical acceptors such as HDL or lipid-free apoA-I, our data show that upregulation of ABCA1 and ABCG1 did not enhance cholesterol transfer to P116. This suggests that, unlike classical acceptors, P116 does not benefit from transporter-mediated efflux. We also investigated whether P116 binds directly to lipoproteins. Incubation with HDL or LDL followed by analysis of the P116 fraction via immunoturbidimetric assays for apoA-I and apoB revealed no evidence of stable binding. Additionally, inhibition of lipase activity using orlistat does not impair P116-mediated lipid uptake, excluding a lipase-dependent mechanism (Supplementary Figure 3). These findings support a model in which P116 mediates lipid acquisition through a passive, diffusion-driven process. Its hydrophobic cavity enables direct extraction of lipids from the environment, without requiring stable interactions with carrier proteins.\nLipidomic analyses reveal that P116 facilitates the uptake of a broad range of host-derived lipids, yet there is a notable enrichment of phosphatidylcholine, sphingomyelin, and cholesterol—three key lipid classes known to be essential for Mycoplasma pneumoniae membrane integrity and function. While this enrichment suggests a degree of compatibility with the organism’s membrane requirements, P116 does not exhibit strict selectivity for these lipids, as it also mediates the uptake of other lipid species. The mechanisms underlying this preferential accumulation remain to be elucidated.\nWe have now added the new findings, along with updated lipidomic comparisons and a more detailed discussion of potential mechanistic models, in the revised manuscript (Discussion section, second paragraph on page 13, and revised Figures 1 and 2 and supplementary Figures 2 and 3). These additions provide a more comprehensive understanding of P116’s function as a novel lipid acquisition system.\n3. Relevance to Atherosclerosis and Liver Localization: The study suggests that MPN preferentially localizes in lipid-rich environments, but a more detailed discussion of the underlying physiological basis (e.g., lipid metabolism in these tissues) would strengthen the discussion.\nWe agree that elaborating on the physiological basis of MPN localization enhances the interpretation of our findings. Our data indicate that MPN preferentially localizes to lipid-rich tissues, specifically the liver and atherosclerotic plaques, likely driven by the local availability of lipids. Using SPECT/CT imaging, we observed that MPN chassis accumulated specifically in atherosclerotic lesions in LDLR-deficient mice fed a high-fat diet, but not in wild-type mice, indicating that the presence of lesions—characterized by lipid accumulation and altered endothelial integrity—is required for vascular association. In contrast, the liver consistently showed MPN accumulation in both mouse models, which is consistent with its central role in lipid and cholesterol metabolism. Interestingly, although LDLR-deficient livers were larger and richer in lipids, the MPN signal per gram of tissue was lower than in wild-type mice; however, total MPN levels across the whole liver were similar (see restructured Figure 5). These observations suggest a distribution proportional to organ size. It should be noted that hepatic lipid content is elevated in both mouse models, which may facilitate the lipid-tropism of MPN by providing an excess lipid environment regardless of LDLR status. Since the 6-hour post-injection time point used precedes any potential replication, we interpret this as initial lipid-driven accumulation rather than proliferation. Indeed, dissemination of bacteria to organs such as the liver and spleen has been demonstrated in mice orally administered Salmonella enterica serotype Typhimurium (EMBO Rep. 2025 Feb;26(3):656-689. doi: 10.1038/s44319-024-00328-x. Notably, Salmonella reroutes host lysosomal cholesterol via peroxisomes as a bridge, and free cholesterol is essential for the integrity of Salmonella-containing vacuoles and for bacterial proliferation.\nWe have expanded the Results and Discussion sections (restructured Figure 5 and last paragraph on page 15 and first paragraph on page 16) to include this mechanistic interpretation and to better contextualize MPN localization within tissue lipid metabolism and structure.\n4. Monoclonal Antibody Therapeutics: The data indicate that monoclonal antibodies targeting P116 reduce lipid uptake and bacterial colonization. However, it is unclear whether these antibodies could serve as viable therapeutic agents in vivo. Could they be used prophylactically or as adjuncts to antibiotics? It would be interesting to compare the inhibitory effects of these antibodies with other known antimicrobial strategies targeting Mycoplasma.\nWe thank the reviewer for this insightful comment regarding the translational potential of monoclonal antibodies targeting P116. Currently, treatment options for MPN infections are limited. The organism lacks a cell wall, rendering β-lactam antibiotics ineffective, and macrolide resistance is an increasing concern globally. As such, there is an unmet need for novel therapeutic strategies targeting essential bacterial processes. P116 is essential for MPN survival due to its role in lipid acquisition, and our results support its candidacy as a target for intervention. In our study, we evaluated the 3G9 monoclonal antibody against P116 in LDLR-deficient mice using SPECT/CT imaging. Interestingly, despite antibody administration, MPN cells were still detected in the liver up to 6 hours after injection. This contrasts with our ex vivo findings, where 3G9 effectively inhibited the growth of MPN within human atherosclerotic arteries. These results suggest that in vivo dynamics are substantially more complex than ex vivo conditions, likely due to factors such as tissue-specific lipid availability, systemic distribution, or immune compartmentalization, which may influence the accessibility or efficacy of antibody targeting. While our data validate P116 as a potentially druggable target, they also highlight the need for further studies to optimize antibody-based approaches. This includes evaluating pharmacokinetics, in vivo biodistribution, delivery platforms, and combination therapies with existing antibiotics. In particular, prophylactic or adjunctive use of anti-P116 antibodies may offer synergistic benefits in settings of antibiotic resistance or immune compromise.\nWe now include this clarification and discussion (second paragraph on page 16) in the revised manuscript to better frame the limitations and future directions for P116-based interventions.\n5. The study relies on in vitro assays and ex vivo tissue models. Additional discussion on how these findings might translate to in vivo infection models or clinical scenarios is warranted.\nTo address the clinical relevance of our findings, we have expanded the Discussion to emphasize their implications at three different levels:\n- Therapeutic targeting and vaccine development: P116 is essential for MPN viability, and antibodies raised against its C-terminal domain exhibit strong growth-inhibitory effects in vitro. This suggests that P116, particularly its C-terminal region, could be a promising candidate for vaccine development or targeted immunotherapy.\n- Extrapulmonary manifestations of MPN: While MPN is primarily associated with respiratory infections, it has also been implicated in extrapulmonary pathologies, including liver involvement. Reports have described elevated liver enzymes, and in rare cases, acute hepatitis or cholestasis—especially in pediatric patients (Microb Pathog 2020 Feb:139:103863. doi: 10.1016/j.micpath.2019.103863). Our findings offer a possible mechanistic explanation for these clinical observations. The P116-mediated lipid scavenging mechanism may drive MPN accumulation in lipid-rich tissues such as the liver and atherosclerotic plaques. While our current ex vivo models support tissue-specific localization based on lipid content, we agree that additional in vivo studies are essential to confirm the role of P116 in organ-specific colonization and pathogenesis.\n- Biotechnological applications: The MPN chassis is being explored as a delivery platform for therapeutic use in lung diseases. Our discovery that the MPN chassis can also efficiently target the liver and atheromatous plaques opens new possibilities for engineering targeted delivery vehicles to non-pulmonary tissues, expanding its potential in translational biotechnology.\nThese perspectives have now been incorporated into the revised manuscript to better contextualize the broader significance of our findings and highlight future directions for both research and therapeutic application (pages 15 and 16).\n6. Given the importance of lipidomic analyses, a more explicit discussion of the UHPLC-MS workflow and data normalization methods would improve reproducibility. For example, what standards were used to quanitify which lipids, were they added before or after lipid extraction, concentrations, etc…?\nWe appreciate the reviewer’s comment highlighting the importance of detailed lipidomic methodology to ensure reproducibility. In response, we have expanded the Methods section to include a more explicit description of the UHPLC-MS workflow and data normalization procedures (page 22 and the first paragraph on page 23).\nReviewer #4 (Remarks to the Author):\nThis study provides new insights into the role of the P116 protein in M. pneumoniae lipid uptake and its potential impact on bacterial survival in the bloodstream. The authors present compelling evidence that P116 helps extract essential lipids from LDL and HDL, a key adaptation for M. pneumoniae in lipid-rich environments. These findings are particularly important as they link M. pneumoniae colonization in liver and atherosclerotic lesions to its ability to acquire essential lipids. To further improve the study’s impact and reproducibility, consider addressing the following points:\nWe thank the reviewer for their positive assessment and valuable suggestions. In response, we have strengthened the study by increasing biological replicates and applying more rigorous statistical analyses to enhance reproducibility. We have incorporated new quantitative measurements of MPN chassis accumulation in liver and atherosclerotic tissues using both imaging and qPCR, establishing correlations with tissue lipid content and lesion severity. Additional mechanistic insights were gained from lipid uptake assays in multiple cell types, clarifying that P116 mediates lipid acquisition via a passive, diffusion-driven process. These comprehensive updates deepen our understanding of P116’s role in MPN survival and its localization in lipid-rich tissues, thereby improving the study’s overall impact.\nIn Figures 1 and 2, where human circulating LDLs and HDLs are identified as primary lipid sources for P116, including at least three replicates would strengthen statistical robustness and reproducibility.\nWe appreciate the reviewer’s suggestion. In the revised manuscript, we have increased the number of biological replicates to at least n=3 for all lipidomic analyses, including those previously shown in Figures 1D and 2B (now included into the updated Figures 1 and 2d, and supplementary data). To ensure appropriate statistical analysis, we replaced multiple t-tests with one-way ANOVA for both overall group comparisons and individual lipid species. Additionally, we now report standard deviations (SDs) instead of standard errors of the mean (SEMs) to more accurately represent variability within small sample sizes. The inclusion of additional replicates and new experiments did not change the outcomes of our original findings.\nThe same applies to Figure 3C on lipid uptake. Testing the mAb-3B5 antibody (which does not target the central region of P116) would further confirm that only antibodies targeting the C-terminal domain interfere with lipid acquisition and M. pneumoniae presence in atherosclerotic tissue.\nIn response, we have performed additional experiments to directly assess the ability of mAb-3G9 to inhibit cholesterol transfer from LDL to P116, comparing the results with those obtained using the antibody mAb-3B5. As shown in the revised Figure 3c, only mAb-3G9 significantly reduced cholesterol transfer to P116, whereas mAb-3B5 had no effect. These findings confirm that inhibition of lipid acquisition is specific to antibodies targeting the C-terminal domain of P116.\nSince M. pneumoniae lacks traditional energy-dependent lipid transporters, diffusion is likely the mechanism by which P116 mediates cholesterol and neutral lipid uptake. Investigating whether cellular transporters contribute to this process would provide valuable mechanistic insights into P116’s function.\nTo further investigate whether host cellular transporters contribute to the lipid acquisition mechanism of P116, we have performed new cholesterol uptake assays using CHO cells overexpressing SR-BI, H1299 pulmonary epithelial cells, and J774 macrophages. Our results show that P116-mediated cholesterol uptake is unaffected by SR-BI expression or activity, indicating that P116 does not rely on this receptor-mediated pathway (new restructured Figure 2b). Furthermore, while the activation of the cholesterol efflux transporters ABCA1 and ABCG1 promotes cholesterol efflux to classical acceptors such as HDL or lipid-free apoA-I, our data show that upregulation of ABCA1 and ABCG1 did not enhance cholesterol transfer to P116 (new Figure 2c). This suggests that, unlike classical acceptors, P116 does not benefit from transporter-mediated efflux. These findings support a model in which P116 functions as a passive lipid-binding protein, directly capturing cholesterol and other essential lipids from LDL, HDL and host membranes.\nOther points:\nIn Figures 4 and 5, including quantitative measurements of signal intensities would provide an objective assessment of the observed visual changes.\nEstablishing a numerical correlation between liver lipid content and M. pneumoniae presence would further validate the physiological impact of P116-mediated lipid uptake.\nWe thank the reviewer for this constructive suggestion. In response, we have performed quantitative analyses to objectively assess the observed visual changes in Figures 4 and 5. For Figure 4 (ex vivo imaging), we have quantified signal intensities corresponding to MPN accumulation in four independent tissues. In the restructured Figure 5, we have quantified liver-associated signals using radioactivity measurements from SPECT/CT imaging, enabling an objective assessment of MPN localization in this organ. In addition, the presence of MPN in livers has been confirmed and quantified by qPCR, which has been included as a complementary measurement to the imaging data.\nTo address the reviewer’s request for physiological correlation, we analyzed the relationship between MPN abundance and tissue lipid content. Specifically, we correlated radioactivity- and qPCR-based MPN quantification with hepatic cholesterol levels and proximal aortic atherosclerosis. Notably, no significant correlation was found between liver cholesterol content and MPN abundance (Figure 5f). This suggests that while liver accumulation may reflect a general lipid-driven tropism, it does not correlate directly with hepatic lipid levels—likely because MPN is diffusely distributed throughout the liver tissue. The elevated hepatic lipid levels in both mouse models may create a permissive environment that facilitates MPN tropism, independently of LDLR status, particularly at the 6-hour post-injection time point before any bacterial replication occurs. Consistent with this, MPN was detected in atherosclerotic plaques of LDLR-deficient mice fed a high-fat diet, but not in wild-type controls that do not develop significant atherosclerotic lesions, indicating that lesion formation is required for vascular association. This supports our interpretation that P116-mediated lipid acquisition promotes selective MPN localization in lipid-rich, structurally compromised vascular lesions.\nThese new quantitative data and correlations have been incorporated into the revised Figures 4c and 5f, and are discussed in the updated Results and Discussion sections (pages 10, 11, 15 and 16) to better contextualize the physiological relevance of MPN distribution.\nReviewer #5 (Remarks to the Author):\nThe manuscript describes a series of experiments designed to substantiate a hypothesis, as written in l609-611, 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’. Unfortunately, there are several weaknesses in the design and interpretation of the data which limit the conclusions which can be drawn from this work.\nWe appreciate the careful evaluation of our hypothesis and the detailed critique of our experimental design and data interpretation. In response, we have performed several additional experiments, clarified key mechanistic points, revised overstated conclusions, and improved the overall presentation of the data. These changes have strengthened the rigor of our findings and helped refine our model of P116 as a lipid acquisition factor in MPN. We believe that the revised manuscript more clearly and accurately conveys our conclusions, and we are grateful for the reviewer’s insights, which have significantly improved the quality of the work.\nMajor points\n1. The model for the function of P116 is unclear; the structure suggests it is a lipid-binding protein attached to the mycoplasma membrane by a flexible linker (l69). But is it acting as an adhesin or as part of an uptake system for lipid nutrients- the authors seem to suggest the latter (l62)? However, interpretation of the data in the manuscript does not draw a clear distinction between these two models for the role of P116.\nWe agree that clearly distinguishing between potential functions of P116 is critical for interpreting its biological relevance. Based on our structural, biochemical, and in vivo data, we favor a model in which P116 primarily functions as part of a lipid acquisition system. P116 is a membrane-anchored protein connected by a flexible linker, as we previously reported (Nature Structural & Molecular Biology, 2023 30, 321–329, https://doi.org/10.1038/s41594-023-00922-y). The structure revealed a unique fold with a large, solvent-accessible hydrophobic cavity, consistent with a lipid-binding role. P116 can bind to lipoproteins and host cell membranes to extract essential lipids from them; however, it is not the main adhesin, as previous studies have shown that the Nap complex (composed of the P1 and P40/P90 proteins) is the primary adhesion mechanism of M. pneumoniae and M. genitalium (Nat Commun 2020 11, 5188, https://doi.org/10.1038/s41467-020-18777-y) To further investigate whether host cellular transporters contribute to the lipid acquisition mechanism of P116, we have performed new cholesterol uptake assays using CHO cells overexpressing scavenger receptor type BI (SR-BI), H1299 pulmonary epithelial cells, and J774 macrophages. Our results show that P116-mediated cholesterol uptake is unaffected by SR-BI expression, indicating that P116 does not rely on this receptor-mediated pathway (new restructured Figure 2b). Furthermore, while the activation of the cholesterol efflux transporters ATP-binding cassette (ABC)A1 and ABCG1 promotes cholesterol efflux to classical acceptors such as HDL or lipid-free apoA-I, our data show that upregulation of ABCA1 and ABCG1 did not enhance cholesterol transfer to P116 (new Figure 2c). This suggests that, unlike classical acceptors, P116 does not benefit from transporter-mediated efflux.\nThis disrupted pattern of cholesterol acquisition when P116’s lipid-binding ability is blocked by antibodies targeting its C-terminal domain (see updated Figure 3) and the lack of stable complexes with lipoproteins, further support its role in nutrient uptake rather than adhesion. Overall, we have not observed evidence of host receptor-dependent binding or adhesion mediated by P116 in any of our previous (Nature Structural & Molecular Biology, 2023) or present assays. Unlike typical adhesins, P116 does not show high-affinity or saturable binding to cellular surfaces. These observations strongly support the idea that P116 functions primarily in lipid scavenging rather than adhesion. We have now clarified this distinction in the revised Discussion section (second paragraph on page 13 and page 14) to better articulate the mechanistic model and distinguish between these two possible functions.\n2. The heading for section 3.1 over-states the conclusion which can be drawn from these experiments ‘Human circulating LDL is a major source of unesterified cholesterol for MPN P116’ (l436). All that can be said about the results from Fig 1 is that cholesterol from LDLs or HDLs in vitro binds to P116. This may or may not happen in vivo. It is clear from the results in section 3.2 that the specificity of P116 is broad.\nWe agree that the original heading may have overstated the relevance of the findings presented in Section 3.1. We have modified the heading to: 'Human LDL is a source of unesterified cholesterol for MPN P116' to better reflect the experimental context and avoid overinterpretation. To support the relevance of these findings, we have conducted additional experiments to assess whether cholesterol is specifically transferred from lipoproteins to MPN (see updated Figure 1b). These results suggest that LDL can serve as a viable cholesterol source under controlled conditions, although we refrain from making in vivo claims at this stage (second paragraph on page 12 and first paragraph on page 13).\nThe text, section heading, and figure legends have all been revised accordingly to clarify that our conclusions are based on controlled in vitro experiments.\n3. The description of the conclusion in section 3.3 implies an active function of P116 in lipid acquisition- ‘P116 uptakes lipids from H1299 lung cells with low efficiency’- which is not necessarily warranted by the data. If P116 is simply a lipid-binding protein, it will presumably bind free cholesterol in solution. Cells with lower cholesterol content and/or less propensity to shed cholesterol will transfer less of it to P116. This is more a property of the cells studied than of P116 itself.\nWe agree that the original sentence in Section 3.3 may imply a level of active function by P116 that goes beyond what the data directly support. As the reviewer points out, P116 can be described as a high-capacity lipid-binding protein that passively acquires lipids from its environment. Our revised interpretation acknowledges that the efficiency of cholesterol acquisition by P116 in co-culture assays largely reflects the lipid availability of the host cells (see restructured Figure 2). Indeed, the observed differences in cholesterol transfer from H1299 lung cells compared to other cell types are likely due to lower cellular cholesterol content (new supplementary Figure 2).\nThe structure-function relationship supports a model in which P116 passively captures cholesterol by a mechanism driven by favorable thermodynamics rather than receptor-mediated efflux. We have now revised the relevant section and figure legends to clarify this interpretation and avoid overstating the active role of P116. The revised Discussion emphasizes that P116 functions as a passive lipid scavenger, with the extent of lipid uptake primarily dictated by the properties of the surrounding cells and prevailing lipid gradients (page 13).\n4. The data on serum reactivity against different forms of P116 lacked suitable controls from uninfected patients (Supplementary Table 4). Some bacterial antigens exhibit a high degree of reactivity against serum IgG, even in individuals with no history of infection by that organism. At the very least, a statistical comparison is needed between infected and uninfected groups to establish the conclusion proposed. It is a stretch to suggest ‘..C-terminal domain of P116 could serve as a promising candidate for the development of vaccines against MPN’ (l591). What reason is there to think that it will elicit a protective immune response?\nFollowing your recommendation, we have now included a statistical comparison of serum reactivity between MPN-infected and uninfected individuals (Supplementary Figure 4). This analysis shows that sera from uninfected individuals exhibited very low reactivity against all P116 constructs compared with infected patients, confirming the specificity of the immune recognition to MPN infection.\nAs detailed in the Results, all infected patients tested positive for the P116/30–957 and P116/30–845 constructs, whereas removal of both terminal regions (P116/246–818) resulted in a marked reduction in reactivity. Overall, P116/30–957 and P116/30–845 showed stronger reactivity than P116/246–818 (Fig. 3a, Supplementary Table 1).\nIn addition, a panel of antibodies against P116 (residues 30–957) was tested, and all except one targeted the core domain and showed inhibitory effects on MPN growth and adhesion. Based on these results, mAb 3G9 was selected for further experiments as it exhibited the strongest inhibitory effect. We also included mAb 3B5, which lacks specificity for the central region and exhibits lower affinity.\nWe agree with the reviewer that the ability of the C-terminal domain of P116 to elicit a protective immune response remains to be determined. Our data support that this domain is immunogenic and specifically recognized during infection, and that antibodies targeting epitopes in this region (e.g., mAb-3G9) can interfere with P116 function, cholesterol uptake, and MPN growth in vitro (Fig. 3b–d). While these findings suggest that the C-terminal domain may represent a promising candidate for future vaccine or therapeutic development, we have revised the manuscript to present this as a possibility rather than a definitive conclusion (first paragraph on page 15).\n5. Fig 3C Is the result from one experiment? Is there a control with an antibody without specificity for P116?\nWe have performed additional experiments to directly evaluate the ability of mAb-3G9 to inhibit cholesterol transfer from LDL to P116 and compared these results with the effects of non-inhibitory antibody mAb-3B5, which targets a different region of P116. The revised Figure 3c now includes three independent biological replicates and includes mAb-3B5. As shown in the restructured Figure 3c, only mAb-3G9 significantly reduced cholesterol uptake, while mAb-3B5 had no effect. These findings confirm that interference with lipid acquisition is specific to antibodies targeting the C-terminal domain of P116. This additional evidence further supports the functional importance of the C-terminal domain in lipid binding and uptake, and reinforces the specificity of mAb-3G9 as a mechanistic probe.\n6. Fig 3D: why would MAb-3G9 inhibit growth? Does this imply that P116 plays a role in lipid nutrient uptake?\nAs shown in the revised Figure 3, treatment with mAb-3G9 significantly inhibits both cholesterol transfer to P116 and MPN growth. This effect is consistent with mAb-3G9 binding to the C-terminal domain of P116, which appears to modulate the size of its hydrophobic cavity to accommodate lipid cargo (updated Figure 3b). These results support the interpretation that P116 facilitates cholesterol acquisition, and that mAb-3G9 inhibits growth by functionally blocking this essential activity. We have revised the text to clarify this mechanism and its implications for MPN lipid acquisition (last paragraph on page 14 and first paragraph on page 15).\n7. The microscopy data in Fig 4B are unclear; limited samples are shown and there is no quantification of fluorescence, which would enable a statistical analysis of the effects of both antibodies. Without that, the conclusions are qualitative at best and appear tenuous.\nAs requested, we have performed quantitative analysis of the fluorescence microscopy data shown in Figure 4. Specifically, we have quantified fluorescence intensities from four independent biological replicates to assess the effects of mAb-3G9 and mAb-3B5 on MPN adherence to human carotid atherosclerotic plaques. The analysis shows that treatment with mAb-3G9 leads to a significant reduction in fluorescence intensity, consistent with impaired lipid acquisition and reduced MPN viability, while mAb-3B5 has no significant effect. These quantitative results are now included in the revised Figure 4c, with full statistical details provided in the figure legend and described in the Methods section.\n8. The interpretation of the [¹¹¹In]In-oxine radiolabelling experiments depends on the in vivo fate of the ¹¹¹In label. [¹¹¹In]In-oxine is a neutral, lipophilic complex which passively diffuses through cell membranes. Inside the cell, the isotope binds to intracellular macromolecules. In the literature, [¹¹¹In]In-oxine has been described as a label for leukocytes but I am not aware of any details about Mycoplasma labelling. The labelling period is only 15 minutes (l402)- how do we know that any labelling of the bacteria has occurred at all? Has the isotope been taken up into the bacteria? Could the results in Figure 5 be explained by the lipophilic nature of the complex, preferentially targeting to the fat-enriched livers of the LDL-knockout mice? More data are needed to exclude these explanations.\nPrior to the in vivo imaging studies, we confirmed efficient incorporation of [¹¹¹In]In-oxine into both the CV8 MPN chassis and wild-type MPN cells (WT) via in vitro imaging, demonstrating successful bacterial labeling. Although [¹¹¹In]In-oxine is a neutral, lipophilic complex capable of passive diffusion, we took care to verify that the radiolabel was indeed internalized by the bacteria rather than remaining unbound (figure for reviewer). To address the concern that the liver signal might result from nonspecific accumulation of free tracer due to its lipophilicity, we performed qPCR analyses on liver tissue, which detected significant MPN DNA loads consistent with bacterial presence (new Figure 5e). This confirms that the in vivo signal corresponds to labeled bacteria rather than free or unincorporated isotope. Representative images from the in vitro uptake experiments have now been included for review, and we have expanded the Methods section to detail these validation procedures (the last paragraph on page 31 and the first paragraph on page 32).\nMinor\nFig 1B why do the lines on this plot not extend to zero? Presumably isolated P116 at t=0 contains no radioactive label?\nThank you for your observation. Yes, the isolated P116 at time point zero did not contain any radioactive label. We have now adjusted the plot in Figure 1c inset so that the lines correctly extend to zero, reflecting the absence of radioactivity at the starting point of the experiment."}, {"round": 2, "reviewer_comments": {"reviewer_1": "My concerns have been addressed. I have no further questions.", "reviewer_3": "The authors have addressed my concerns.", "reviewer_4": "The authors have adequately addressed the comments raised, and I consider the revised version of the manuscript suitable for publication.", "reviewer_5": "The authors have responded well to the points made in my previous review- detailed below.\n1. General hypothesis: changes to the Discussion and elsewhere now clarify the proposed mechanism for P116.\n2. Changes to the manuscript clarify that the reported observations relate to in vitro experiments.\n3. This point relates to point 1- now acknowledged in the revised manuscript.\n4. The comparison between sera from MPN-infected and uninfected now makes this point more strongly.\n5. The updated antibody control data makes this conclusion stronger.\n6. Point is now addressed.\n7. Point is now addressed.\n8. Point is now addressed."}, "author_response": ""}]

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