Archaeoglobus fulgidus type strain VC-16, was performed to explore how elevated pressures might affect microbial growth and physiology in the deep marine biosphere. Though commonly identified in high-temperature and high-pressure marine environments , A. fulgidus growth at elevated pressure has not been characterized previously. Here, exponential growth of A. fulgidus was observed up to 60 MPa when supported by the heterotrophic metabolism of lactate oxidation coupled to sulfate reduction, and up to 40 MPa for autotrophic CO2 fixation coupled to thiosulfate reduction via H2. Maximum growth rates for this heterotrophic metabolism were observed at 20 MPa, suggesting that A. fulgidus is a moderate piezophile under these conditions. However, only piezotolerance was observed for autotrophy, as growth rates remained nearly constant from 0.3 to 40 MPa. Experiments described below show that A. fulgidus continues both heterotrophic sulfate reduction and autotrophic thiosulfate reduction nearly unaffected by increasing pressure up to 30 MPa and 40 MPa, respectively. As these pressures encompass a variety of subsurface marine environments, A. fulgidus serves as a model extremophile for exploring the effects of elevated pressure on microbial metabolisms in the deep subsurface. Further, these results exemplify the need for high-pressure cultivation of deep-sea and subsurface microorganisms to better reflect in situ physiological conditions.High hydrostatic pressure (HHP) batch cultivation of a model extremophile,  By definition, those species that are negatively impacted by high-pressure conditions are categorized as piezosensitive, while piezotolerant species are insensitive to high pressure up to a limit; finally piezophiles grow optimally at elevated pressure and some of these are incapable of growth at ambient pressure  and sulfur compounds  was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH . Here, chemoorganoheterotrophic growth experiments contained lactate as the electron donor and carbon source coupled to sulfate reduction. The heterotrophic growth medium contained a sea salt base with a composition as follows (per liter): 0.34g KCl, 15.142g MgSO4⋅7H2O, 2.75g MgCl⋅6H2O, 0.25g NH2Cl, 0.056g CaCl2⋅2H2O, 0.0137g K2HPO4⋅3H2O, 17.8g NaCl, 0.0039g Fe(NH4)2(SO4)⋅6H2O, and 1 mL of Wolfe’s trace element solution , yeast extract , and PIPES  was added by filter sterilization in a biosafety cabinet and the pH was adjusted to 6.7. The sterile medium was aseptically distributed into sterile Balch tubes (10 mL of medium), capped with sterile butyl stoppers and crimped. Prior to inoculation, the headspace above the medium was replaced with N2 by flushing Balch tubes for 15–20 min and a 2.5% (w/v) Na2S⋅9H2O stock solution was added to a final concentration of 1 mM to obtain anoxic conditions  Na2S⋅9H2O was added to a final concentration of 1 mM to obtain anoxic conditions . After transfer, excess N2 gas was expelled from each syringe before the needle was embedded into a silicone stopper to maintain a closed system. The syringes were placed inside pre-heated stainless steel pressure vessels, filled with deionized water, and pressurized using a hydraulic screw pump (High Pressure Equipment Company©) to the desired pressure  gludaraldehyde for subsequent enumeration.For HHP batch cultivation, triplicate batch culture experiments and a single uninoculated control were conducted at 0.1–70 MPa at 10 MPa increments. For each triplicate, 30 mL of sterile, anaerobic medium was inoculated with 3% (v/v) logarithmic phase pressure . Parallepressure , or in hBecause the static pressure vessels require decompression for subsampling and such decompression can negatively impact microbial growth during high-pressure cultivation , a suiteA. fulgidus cell recovery after a 52-h exposure at 70 MPa for the heterotrophic metabolism was tested. Logarithmic-phase A. fulgidus cells were incubated in triplicate at 70 MPa and 83°C for 52 h in a single, heated pressure vessel. Because the effects of decompression on A. fulgidus cells at 70 MPa were unknown, these incubations were also decompressed and repressurized four times at 18, 24, 36, and 52 h after inoculation. Following 52 h of incubation, a final concentration of ∼1.1 × 107 cells/mL was transferred into sterile anaerobic medium in Balch tubes at 0.3 MPa and growth was monitored visually until the culture became turbid after 64 h and direct counts of triplicate experiments were taken.Lastly, 2, CO2), modifications to the standard protocol were necessary. Specifically, all HHP autotrophic experiments were carried out in glass, gas-tight syringes and additional H2 was added prior to pressurization to mitigate gas loss. The detailed experimental protocol is outlined in Similar to HHP heterotrophic growth experiments, HHP experiments exploring autotrophy were also conducted in syringes contained in static pressure vessels, with replicates distributed to minimize decompression-repressurization cycles. However, because the autotrophic metabolism depends on significant concentrations of dissolved volatiles  following methods of 2 using an over-pressurized (0.3 MPa) bottle  after incubation at 60 MPa and 83°C for 150 h. Therefore, ∼0.5 mL of 100% H2 was added to each syringe to mitigate H2 loss on pressurization  bottle , Step 2.) bottle , Step 3.r ∼5 min , Step 4.r ∼5 min , Step 5.rization , Step 6,riments” , Step 7.A. fulgidus growth in serum bottles with a ∼15 mL headspace was compared to A. fulgidus growth in 5 mL glass syringes with ∼0.5 mL gas phase incubated in the static pressure vessels. At these pressures a two-phase system is maintained in each experiment (serum bottle or glass syringe), and both phases achieve equivalent pressures. To achieve such low pressures in these vessels, an additional pressure gauge was placed onto the pressure line. This gauge was rated up to 0.7 MPa so that 0.3 MPa could be reached more precisely. Each serum bottle was subsampled six times during the course of each experiment.Two near-ambient pressure control experiments were conducted in serum bottles. First, the inoculated medium remaining in the serum bottles used to fill the glass syringes was incubated at 83°C to monitor growth under optimum conditions. Second, to observe if there was a significant difference in growth using traditional batch culture techniques versus HHP techniques at 0.3 MPa, Archaeoglobus fulgidus autotrophic HHP batch culture experiments were performed from 0.3 to 60 MPa in ∼20 MPa increments. Each HHP batch culture experiment was performed in triplicate with a negative control, along with the ambient-pressure controls described above. Five static high-pressure vessels, containing two 5 mL glass syringes (one experiment and a negative control), were pressurized and decompressed only once for sampling to minimize decompression-repressurization cycles. Vessels were decompressed at an average rate of 19 MPa/min. To obtain growth curves through the stationary phase, five 0.5 mL subsamples were taken over ∼145–215 h, fixed with 2.5% (v/v) gludaraldehyde, and later enumerated via light microscopy. Six subsamples were taken from experiments conducted in serum bottles.For heterotrophic growth, cell densities were estimated by direct counting of cells fixed in 2.5% (v/v) gludaraldehyde filtered onto 0.2 μm black polycarbonate membranes (EMD Millipore) and stained with DAPI  after 64 h of growth at 0.3 MPa , while aggregation was absent in Balch tubes . Growth Archaeoglobus fulgidus grew autotrophically from 0.3 to 40 MPa at 83°C under HHP batch cultivation , suggesting that A. fulgidus is a piezotolerant autotroph  is capable of growing at HHP conditions both heterotrophically and autotrophically and displays both piezophilic and piezotolerant behavior depending on metabolic strategy. Growing heterotrophically, A. fulgidus is a moderate piezophile, with maximum specific growth rates measured at 20 MPa for the cultivation conditions described here. On the other hand, A. fulgidus growth rates and stationary-phase cell densities were similar for autotrophy from 0.3 to 40 MPa, indicating only piezotolerance for these autotrophic conditions. Interestingly, growth rates under traditional batch culture generated different results from the HHP syringe-based techniques at 0.1 MPa or 0.3 MPa for both metabolisms, highlighting that different cultivation methods can impact microbial growth patterns. Additionally, cell clustering in experiments using the HHP batch cultivation technique was observed over most of the pressure range (0.1–50 MPa). The cause for cell clustering was not resolved during this study, however, additional analyses are underway to investigate this physiological response. Taken together, these results suggest that A. fulgidus is capable of both heterotrophic and autotrophic growth in high-pressure, subsurface, marine environments.Archaeoglobus fulgidus type strain VC-16 was originally isolated from a shallow marine hydrothermal system, and its discovery was notable as this species was the only archaeal sulfate reducer known at that time  and associated hydrothermal marine sediments, depths of many oil field reservoirs ranging from ∼1-3 km depth  over a range of pressure conditions. For instance, experiments to explore membrane structure and functionality under different pressure, decompression, and metabolic conditions may lend some insight into this species’ ability to adapt to both surface and subsurface environments. Additionally, other Archaeoglobus species that have been isolated from high-pressure environments (Overall, the high-pressure growth of ronments  are likeronments , furtherThe raw data supporting the conclusion of this article will be made available by the authors, without undue reservation, to any qualified researcher.GO, AC, and KR designed this research project, analyzed the data. GO performed the experiments and collected the data, wrote the original draft. KR and AC edited and wrote sections of the manuscript. All authors contributed to revisions of the manuscript, tables, and figures and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Escherichia coli persister cells. Using this population of persister cells, we demonstrate that cupric compounds kill E. coli persister cells. Hence, copper ions may be used in controlling the spread of important bacterial strains that withstand treatment with conventional antimicrobials by forming persister cells.Due to their reduced metabolism, persister cells can survive most antimicrobial treatments, which usually rely on corrupting active biochemical pathways. Therefore, molecules that kill bacterial persisters should function in a metabolism-independent manner. Some anti-persister compounds have been found previously, such as the DNA-crosslinkers mitomycin C and cisplatin, but more effective and lower cost alternatives are needed. Copper alloys have been used since ancient times due to their antimicrobial properties, and they are still used in agriculture to control plant bacterial diseases. By stopping transcription with rifampicin and by treating with ampicillin to remove non-persister cells, we created a population that consists solely of  E. coli, the viable but non-culturable state and persistence are equivalent [Persister cells are a subpopulation of phenotypic variants that are tolerant to antimicrobial compounds. This phenotype was discovered in the early 1940s ,3,4. Sinuivalent .H-indol-2-yl-methylamine hydrochloride was discovered that kills E. coli, Pseudomonas aeruginosa, and Staphylococcus aureus persister cells [S. aureus persisters [Despite being refractory to many antimicrobial molecules, persister cells can be killed ,9 by comer cells . Additiorsisters , and othS. aureus (MRSA) [Salmonella enterica [E. coli O157 [Copper compounds have been used for their antimicrobial properties since ancient times , and mans (MRSA) ,19, Salmenterica , and E. oli O157 , as welloli O157 ,23 and Noli O157 . Copper oli O157 , since ioli O157  or silveE. coli persister cells.The antimicrobial mechanisms of copper ions include membrane damage, oxidative stress, and protein/DNA denaturation ,26,27. CE. coli BW25113 was used throughout [roughout  and was roughout . Cisplatroughout , since Droughout . Cupric To determine copper minimum inhibitory concentration (MIC), 10 µL of culture (turbidity at 600 nm of ~1.0) was inoculated into lysogeny broth (LB) medium supplemented with a range of copper concentrations: 0, 120, 240, 480, 960, and 1920 µg/mL. Test tubes  were incubated overnight (approximately 16 h with agitation at 250 rpm), and the MIC was determined as the lowest concentration that prevented bacterial growth by visual inspection. All experiments were conducted with at least two biological replicates.E. coli culture was increased 10,000-fold, using a rifampicin pre-treatment to induce cells to enter the persister state. To form these persisters, exponentially growing cells at a turbidity of 0.8 at 600 nm were treated with rifampicin for 30 min at 100 µg/mL, harvested by centrifuging at 1600× g, and resuspended in ampicillin-supplemented LB (100 µg/mL) to kill non-persister cells for 3 h. Then, the cells were centrifuged and resuspended in LB or NaCl buffer.Using our previous method ,5, the pE. coli electro-competent cells (50 µL) were electroporated with 1 µL of pooled plasmids from a complete set of E. coli K-12 ORF archive (ASKA)  using a 0.1 cm electroporation cuvette. LB medium (900 µL) was added and after 1 h of growth (250 rpm), 990 µL of bacterial suspension was inoculated into 25 mL of LB (no antibiotics added), and cells were grown to a turbidity of approximately 0.5 at 600 nm (250 rpm). Chloramphenicol was added (30 µg/mL), and the cells grown for 1 h. After that, 25 µL of this culture were re-inoculated in LB medium supplemented with cupric sulfate , with or without IPTG (0.5 mM), and grown overnight (250 rpm). We used two different approaches to identify copper-resistant strains: LB supplemented with cupric sulfate  and cupric sulfate in NaCl 0.85% buffer. For both methods, 250 µL of the pooled KEIO mutant library was inoculated into 25 mL of LB and grown to a turbidity of approximately 0.8 (250 rpm) to let the cells revive. Cells (25 µL) were added to LB supplemented with cupric sulfate , and kept overnight (250 rpm) or 5 mL of the revived cells were centrifuged and washed once in 0.85% NaCl buffer and cupric sulfate at approximately 1/6 × MIC . This bacterial suspension was incubated for 2 h without shaking at 30 °C.E. coli exponential and persister cultures (5 mL) were centrifuged, resuspended in 5 mL containing 1 × MIC of cupric sulfate (960 µg/mL), and shaken in 125 mL Erlenmeyer flasks; then, the aliquots were used for serial dilutions. Colony-forming units (CFU) were determined after 3 h and 18 h. For cells in NaCl buffer, the cultures were centrifuged, washed 2 × in NaCl 0.85%, and resuspended in this same buffer. To each 2 mL of this bacterial suspension, cupric sulfate was added , and cells were incubated without shaking at 30 °C for 1 h.For cells in LB, Cells were grown to a turbidity of approximately 0.8 and resuspended in 0.85% NaCl with cupric sulfate (160 µg/mL). Cultures were incubated without shaking at 30 °C for 1 h in opened vials (aerobic) and in an anaerobic chamber (Coy Laboratories). Aliquots were serially diluted and plated on LB–agar plates, which were incubated under aerobic and anaerobic conditions. E. coli cells were used to confirm the PI staining of dead cells.To determine whether copper ions damage the persister cell membrane, we performed a Live/Dead assay. Persister cells (5 mL) were resuspended in 0.85% NaCl supplemented with cupric sulfate . The bacterial suspensions were incubated for 30 and 60 min at 30 °C incubator, without shaking, and washed twice in PBS buffer before the addition of propidium iodide (PI) (Invitrogen P3566) and SYTO 9 , reaching final concentrations of 60 µM and 20 µM, respectively. After a 15 min-incubation , cells were observed under a Zeiss fluorescence microscope . Pictures taken with different filters were merged using ZEN software. As a control, heat-treated g for 10 min), and the pellet was resuspended in PBS buffer. For the 100% lysed positive control, a 1 mL aliquot was sonicated , centrifuged, and the supernatant used as a positive control . For the copper-treated cultures (5 mL), cupric sulfate was added (160 µg/mL) and vials were incubated for 1 h at 30 °C; then, they were centrifuged  and the supernatants were collected. Protein detection was determined using BCA protein assay kit , following the manufacturer’s instructions. To determine if copper ions cause cell lysis, cells were grown to a turbidity of 0.8, washed twice using NaCl 0.85% , cisplatin , or the solvent control for cisplatin, sodium perchlorate (0.1 M), and incubated at 250 rpm. Aliquots for CFU assessment were obtained by serial dilution and colony counting at 0 min, 15 min, 30 min, and 60 min.t-test  versus the control conditions and considered a minimum of 0.05 (*) for the p-values.All experiments were carried out with at least two biological replicates. Median values were obtained for each time-point assessed, and the standard deviation was calculated. For statistical significance analysis, we used a cue system (Cuefflux) [cus system (Cusensing) [cue system) is involved in cytoplasm detoxification, while the cus system is involved in periplasm detoxification [cus system proteins remain functional, removing copper ions from the periplasm [pco genes) and enhance the ability of the bacterium to efflux copper ions [E. coli BW25113, contains the expected main systems for copper regulation (cus and cue) and is devoid of the plasmid-encoded pco system (E. coli and other Gram-negative bacteria. Gram-negative strains utilize the efflux)  and the sensing)  for coppfication ,37. Undeeriplasm ,37  after overnight incubation. Persister cells were formed so that they were the dominant population following the previously published methodology [The MIC of copper for hodology ,5 that ihodology  and utilhodology ,46,47,48Using the MIC for exponentially growing cells, we investigated the ability of copper ions to kill both exponential and persister cells in rich medium (LB) with 1 × MIC cupric sulfate (960 µg/mL). After 18 h of incubation, the number of viable exponentially grown cells was reduced 1000-fold more than the persister cells A; hence,Rich medium is not representative of the conditions bacteria face in natural environments, which are predominantly oligotrophic . In addiE. coli by assessing its membrane status after cupric sulfate treatment via Live/Dead staining. Live/Dead staining uses the membrane-impermeant DNA intercalator propidium iodide and the membrane-permeant DNA intercalator SYTO9 to determine the extent of cell envelope disruption; i.e., cells with membrane damage are stained red, and all cells are stained green by SYTO9. Since different mechanisms for copper killing have been suggested , includiThe microscopy results show that after 160 µg/mL of cupric sulfate in NaCl buffer for 1 h, 58% ± 8% of persister cells have damaged membranes A. The reIn addition, we hypothesized that if membrane damage was the leading cause of death by copper ions, we would be able to detect an increase in protein content outside of cells. Therefore, supernatants of the suspensions of persister cells after copper treatment (160 µg/mL of cupric sulfate in NaCl buffer for 1 h) were analyzed, and we found no additional extracellular protein after copper treatment B; hence,E. coli cells more effectively under anaerobic conditions [Since copper ions has been reported to kill nditions ,52, we tE. coli single-gene knockouts which contains all non-lethal mutations of E. coli K-12 [E. coli proteins are produced. We reasoned that either a deletion or production of a key protein would allow higher copper tolerance and that this approach could also give us additional clues about the mode of action of copper.Realizing genetic resistance could limit the use of copper as an effective antimicrobial, we assessed the likelihood of copper resistance using pooled libraries  of the 3oli K-12  as well oli K-12  where al8 cell/mL bacterial suspension to zero in 1 h in NaCl buffer , with and without 0.5 mM IPTG to induce gene expression. However, no strain capable of growing at 1 × MIC was recovered. For the pooled Keio strains, since 160 µg/mL of cupric sulfate reduces a 10l buffer B, we expl buffer . Hence, E. coli persisters. After 30 min of incubation with 2 × MIC for each of these chemicals in LB, copper ions eradicated the persister cells faster than cisplatin  was thought to be one of the main reasons for copper ions’ toxicity ,61. Indeli cells , and ourOther modes of action for copper have also been proposed, including contact killing by cell envelope-associated damage ,66. In oE coli BW25113 showed that it has no pco operon, although copper homeostasis genes were fully present. Additionally, many medically relevant E. coli strains are susceptible to copper [Unlike many antibiotics, copper resistance is unlikely to occur spontaneously , requirio copper , and eveo copper ,73,74.E. coli persister cells (Critically, one of the main results of this work is that due to the metabolism-independent membrane damage caused by copper ions, we verified that it is capable of killing er cells . Althoug


This work establishes the genetically encoded fluorescent sensor D4H to track sterol-rich membranes in yeast cells. Unexpectedly, disruption of the actin cytoskeleton leads to movement of sterols from the plasma membrane to endosomes. Internalization is independent of canonical vesicle trafficking and instead depends on the sterol transfer protein Ltc1. Schizosaccharomyces pombe. Using time-lapse and correlative light-electron microscopy, we found that inhibition of Arp2/3-dependent F-actin assembly promotes the reversible relocalization of D4H from the PM to internal sterol-rich compartments (STRIC) labeled by synaptobrevin Syb1. Retrograde sterol internalization to STRIC is independent of endocytosis or an intact Golgi, but depends on Ltc1, a LAM/StARkin-family protein localized to ER-PM contact sites. The PM in ltc1Δ cells over-accumulates sterols and upon Arp2/3 inhibition forms extended ER-interacting invaginations, indicating that sterol transfer contributes to PM size homeostasis. Anterograde sterol movement from STRIC is independent of canonical vesicular trafficking but requires Arp2/3, suggesting a novel role for this complex. Thus, transfer routes orthogonal to vesicular trafficking govern the flow of sterols in the cell.Sterols are crucial components of biological membranes, which are synthetized in the ER and accumulate in the plasma membrane (PM). Here, by applying a genetically encoded sterol biosensor (D4H), we visualize a sterol flow between PM and endosomes in the fission yeast  Sterols are critical components of biological membranes with fundamental roles in cellular physiology. They confer essential biophysical properties to biomembranes, and regulate vesicular trafficking and signal transduction. Moreover, they serve as precursors of hormones, bile acids, vitamin D, and energy storage molecules. While sterols can be either synthesized in cells or taken up from the environment, dysfunctions in their trafficking or metabolism cause severe pathologies, such as Niemann–Pick disease type C, hypercholesterolemia, or sitosterolemia. In fungi, the sterol molecule ergosterol is essential for viability and serves as a major target of antifungal drugs.Although sterols are synthesized in the ER, their concentration within the ER does not exceed 0.5–1 mol% of total lipids . InsteadHow sterol domains are formed and maintained, and how sterols are transported between different organelles, have been the subject of intense investigation. Previous work in mammalian and yeast cells showed that the transport of newly synthetized cholesterol from the ER to the PM is fast, with a half-time of a few minutes , and doe Saccharomyces cerevisiae as oxysterol-binding protein homologues (OSH). Not all proteins in this family transport sterol, but they bind phosphoinositides as common ligands, leading to the hypothesis that some ORPs  transport sterol between membranes by counter-exchange with phosphoinositides , are typically enriched at ER-organelle contact sites  or StART-like lipid-binding domain . The StAct sites . Lam1-4 ct sites ; Lam6/Ltct sites ; Lam5/Ltct sites . The mamct sites . Intriguransport .S. cerevisiae and Schizosaccharomyces pombe, have simple endomembrane systems. For instance, the trans-Golgi network (TGN) in S. cerevisiae also serves as an early and late endosomal compartment . Cells expressing this construct showed no growth problems or morphological defects , in which the ER is largely detached from the PM  or arf6, did not block CK-666–induced D4H internal movement ((PLC∂) nor GFP-LactC2, which bind phosphatidylinositol bisphosphate (PIP2) and phosphatidylserine (PS ), respectively, internalized like D4H upon CK-666 treatment . We focused on cells that contained internal and PM signal, with the latter used to align the light and EM images. The majority of D4H internal signals (144 of 154 foci in 44 cells) corresponded to membrane-enclosed compartments, which we name sterol-rich compartments (STRIC). 77.3% of these were large, often electron-dense, almost spherical organelles with a diameter of 128.5 ± 26.9 nm . The othTo examine the STRIC identity, we labeled CK-666–treated cells expressing mCherry-D4H with a large panel of organellar markers. We did not detect significant colocalization with markers labeling ER exit sites, early Golgi, prevacuolar compartment, endocytic patches, post-Golgi vesicles, lipid droplets, or mitochondria . By contBecause internal D4H dots were also observed in untreated cells . These oS. pombe, BFA blocks secretion and leads to Golgi disappearance with concomitant ER accumulation . BFA is a macrocyclic lactone that inhibits the Sec7-family of ARF exchange factors, leading to collapse of the Golgi . In S. pmulation . Indeed,mulation . 1 h BFAmulation . Howevermulation , i and/oapm1Δ, and more strongly apm1Δ exomerΔ, accumulate post-Golgi vesicles  promoter led to labeling of the entire ER . These constructs were expressed not under native promoter but under the control of either the strong act1p or the weaker pom1p promoter  domain . To detepromoter . Expressreatment . Ltc1StAltc1Δ treated with CK-666, these cells retained PM filipin staining and did not become resistant to high AmB concentrations . These observations demonstrate that D4H is a highly valuable tool to study the intracellular trafficking of sterols.D4H revealed alterations in sterol distribution for the deletion of three predicted sterol-binding LTPs: single deletions of in vitro  and is pin vitro , althougin vitro . Our iniWe discovered that inhibition of Arp2/3-mediated actin assembly triggers the movement of D4H from the PM to intracellular STRICs. These organelles are labeled by late Golgi/endosome markers, such as the Arf GEF Sec72 and the v-SNARE Synaptobrevin-like Syb1, as well as by the internalized FM4-64 dye. The morphology and electron density of the STRIC is in fact highly reminiscent of that of secretory vesicles, suggesting these may represent a recycling/sorting compartment destined to the PM. The overlay of D4H and Syb1 signals in exocyst-depleted cells, which accumulate 100-nm-diameter vesicles , furtherImportantly, the sterol-rich endosomes are distinct from early endocytic vesicles. Four lines of evidence show that sterols do not travel from the PM to endosomes in endocytic vesicles. First, sterol flow to endomembranes occurs after inhibition of Arp2/3 with CK-666, which blocks clathrin-mediated endocytosis in yeast . Second,ltc1Δ, we observed tight ER contacts with expanded regions of the PM, suggesting that these contacts exist independently of successful sterol transport. Third, we can make some rough quantitative estimates of sterol flow from PM to endosomes, which suggest there is not enough space on endosomes: these estimates make the assumption that the changes of D4H sterol detection observed here are solely or mainly due to changes in sterol membrane concentration, rather than other events that would alter D4H accessibility to sterols. Let us consider a 10-µm-long yeast cell with 30 mol% sterol at the PM (detectable by both D4 and D4H), which drop to 20 mol% (just below D4H detection levels) as they flow to 50 endosomes (upper bound of our observations) in 1 h. With a conversion of 30 mol% to 150 sterol molecules per 240 nm2 of inner membrane leaflet , 500 µM CK-666 (in DMSO), 25 µg/ml benzimidazole carbamate (MBC), 0.1 µg/ml terbinafine (in ethanol), and 300 µM BFA (in ethanol). AmB was added at a final concentration 0.2 µg/ml unless otherwise indicated. For energy depletion, cells were pelleted and dissolved in EMM-ALU containing 20 mM deoxyglucose and 10 µM antimycin B (in ethanol).Latrunculin A was ordered from Enzo Life Sciences and FM4-64 from Thermo Fisher Scientific. All other chemicals were ordered from Merck.For recovery from CK-666 treatment, cells were incubated with CK-666 for 1 h, washed twice with medium, and imaged on agarose pads immediately after washing.For filipin staining, the drug was added at final concentration of 5 µg/ml from DMSO stock to the cells diluted in EMM-ALU. Cells were imaged live within a maximum of 5 min on glass slides.nmt81-sec8 strain, precultures were grown in EMM-ALU lacking thiamine and then diluted to OD 0.025 in YE5S containing thiamine, as described  was added to 500 µl cells in EMM-ALU. Cells were harvested immediately, resuspended in residual medium, and visualized in DAPI channel. A minimum of 25 cells undergoing division was evaluated.For visualization of FM-64 uptake, cells were stained with 8 µM FM-64 dye for 2.5 min in YE5S medium and then washed three times with YE5S before microscopy.act1p promoter) or pAV747  containing at least 400 bp of homology to each of the gene flanking regions. For C-terminal tagging with fluorophores, cells were transformed with fluorophore tagging plasmids carrying at least 400 bp of homology to the C-terminal part of the gene and 3′UTR . For expMicroscopy was performed by confocal spinning disc microscopy or widefield microscopy. The majority of imaging (unless otherwise stated) was performed using a spinning-disc microscope: DMI4000B inverted microscope equipped with an HCX PL APO 6100/1.46 NA oil objective and PerkinElmer Confocal system  equipped with a cooled 14-bit frame transfer EMCCP C9100-50 camera. Stacks of z-series confocal sections were acquired at 0.4-µm intervals using the Volocity software. Unless otherwise indicated, images shown are single-plane views. All microscopy was performed at room temperature (∼23°C) unless otherwise indicated.Widefield microscopy  was perfFilipin and Calcofluor imaging  was doneImaging of cells, unless otherwise stated, was performed on EMM-ALU pads solidified with 2% agarose.sec8 mutant and FM4-64 uptake experiments was done in glass chambers coated with soybean lectin . Shortly chambers of 96-well plates  were covered with a 100 µl of filtered lectin solution (100 µg/ml in water) and kept at room temperature for 4 h. Afterward, lectin was removed, and plates were washed three times with 200 µl of water and dried. To attach cells to the bottom of glass chamber, 100 µl of cells OD 0.2 was added to plate wells and incubated for 30 min. Afterward, excess of cells was removed and wells were washed two times with medium before microscopy.Imaging of Quantification of the PM:cytosol mCherry-D4H ratio was done on single-plane images. Briefly, a segmented line was drawn along the PM, and the cytoplasm area was marked using a 4-pixel-wide polygon selection tool. Mean fluorescence value was calculated for each selection and corrected for background fluorescence intensity. For quantification of tip:side ratio in mCherry-D4H–expressing cells, cell tips or cell sides were marked using a four-pixel-wide segmented line. Mean fluorescence value was calculated for each selection and corrected for background fluorescence intensity. Quantification of Ltc1-sfGFP expression was done on sum projection images of three medial z-sections. The cell borders were outlined, and mean fluorescence intensity was measured and corrected for background fluorescence. Quantification of Ltc1 puncta intensity was done on single focal plane images. Briefly, puncta area was marked, and mean fluorescence activity was measured for each selection and corrected for background fluorescence intensity.For cell length and width measurements on calcofluor-stained cells, a line was drawn manually across the length and width of septated cells from the middle of one tip to the other, and the length measured using the Fiji Measure tool. Figures were prepared with Fiji , Plots o20, and picked up on carbon-coated 200 mesh copper grids . For light microscopy, the grid was inverted onto a 1× PBS drop on a microscope coverslip, which was mounted onto a microscope slide and imaged on a DeltaVision platform (Applied Precision) composed of a customized inverted microscope , a uPlan Apochromat 100×/1.4 NA oil objective, a 4.2Mpx PrimeBSI sCMOS camera, and a color combined unit illuminator . Images were acquired using softWoRx v.7.0 software (Applied Precision). The grid was then recovered, rinsed in H2O, and dried before post-staining with Reynolds lead citrate for 10 min. 15-nm protein A–coupled gold beads were adsorbed to the top of the section as fiducials for tomography. Transmission electron micrographs (TEM) were acquired on a FEI Tecnai 12 at 120 kV using a bottom mount FEI Eagle camera (4kx4k) at 6,800× or 9,300× magnification for correlation with the light microscopy image. For correlation, the peripheral D4H signal was used as guide to draw the cell PM on the light microscopy image, which was used to define the image magnification and rotation required to match it to the PM on corresponding TEM images. For tomographic reconstruction of regions of interest, tilt series were acquired at 18,500× magnification over a 60° to −60° tilt range where possible at 1° increments using the Serial EM software. For tomogram reconstruction, we used the IMOD software package with gold fiducial alignment  or CK-666 (500 μM). After 1 h of cultivation , AmB was added to the final concentration of 5 µg/ml, and the samples were further incubated for another hour. Afterward, cells were collected by centrifugation, washed two times with YE5S medium, and diluted to final OD of 0.00025. 100 µl of cells was plated on YE5S plates, and colonies were counted after 3 d of incubation at 30°C.Flow cytometry was performed on a BD Biosciences Fortessa analyzer. To stain for the dead cells in the population, cells were diluted in EMM-ALU to final a OD of 0.1, and 100 µl of cell suspension was mixed with 900 μl EMM-ALU containing 1 µg/ml propidium iodide. After 30 s of incubation, cells were analyzed by flow cytometry without gating during acquisition with 10,000 cells recorded for each sample. Data were analyzed using FlowJo software.Sensitivity to AmB was assessed by spotting 5 µl of yeast culture at an OD of 0.25 and its fivefold serial dilutions on plates containing EMM-ALU with indicated AmB concentration. Growth was assessed after 72 h at 30°C.S. pombe and S. cerevisiae genome were aligned using Muscle (https://www.ebi.ac.uk/Tools/msa/muscle/), and a neighbor-joining tree without distance correction was derived.Full-length sequences of all OSBP domain-containing proteins identified in the πr(l − 2r) + 4πr2 ≅ 125 µm2 for a 10-µm-long, 4-µm-wide S. pombe cell; total endosome surface, 50 × 4πr2 ≅ 2.5 µm2 for 50 endosomes of diameter 127 nm; estimated surface of a single sterol molecule, 0.25 nm2 (2 (The following considerations were made to obtain an estimate of the number of sterol molecules internalized: total PM area, 20.25 nm2 ; minimal5 nm2 (2 ; maximal5 nm2 (2 .7 sterol molecules per cell. A 10 mol% drop upon Arp2/3 inhibition thus represents ∼2.6 × 107 sterol molecules. 50 mol% sterol on endosome represents ∼2.6 × 106 molecules, i.e., 10% of the sterols depleted from the PM. As it takes ∼1h to deplete the PM from the D4H signal, this amounts to a rate of ∼7,250 molecules/s, or ∼1.4 sterol molecule per Ltc1 protein per s.If the PM contains 30 mol% sterols in the inner leaflet, this represents ∼7.8 × 10t test. Data distribution was assumed to be normal, but this was not formally tested.Statistical analysis was performed using the Excel package. Statistical significance was determined using a two-sided osh2Δ osh3Δ double mutants, and relative expression levels of Ltc1-sfGFP under native, pom1p, and act1p promoters. ltc1Δ mutant, respectively.Table S1provides the list of strains used in this study.Click here for additional data file.Table S2provides the list of plasmids used in this study.Click here for additional data file.


Pyrococcus furiosus thioredoxin (Pf Trx). Genetic fusion of the Pf Trx-L2(8x) module to P. furiosus ferritin (Pf Fe) did not interfere with ferritin self-assembly into an octahedral structure composed by 24 protomers. In guinea pigs and mice, the ferritin super-scaffolded, L2 antigen induced a broadly neutralizing antibody response covering 14 oncogenic and two non-oncogenic HPV types. Immune-responsiveness lasted for at least one year and the resulting antibodies also conferred protection in a cervico-vaginal mouse model of HPV infection. Given the broad organism distribution of thioredoxin and ferritin, we also verified the lack of cross-reactivity of the antibodies elicited against the scaffolds with human thioredoxin or ferritin. Altogether, the results of this study point to P. furiosus ferritin nanoparticles as a robust platform for the construction of peptide-epitope-based HPV vaccines.Cervical cancer remains a global health burden despite the introduction of highly effective vaccines for the prophylaxis of causative human papillomavirus infection (HPV). Current efforts to eradicate cervical cancer focus on the development of broadly protective, cost-effective approaches. HPV minor capsid protein L2 is being recognized as a promising alternative to the major capsid protein L1 because of its ability to induce responses against a wider range of different HPV types. However, a major limitation of L2 as a source of cross-neutralizing epitopes is its lower immunogenicity compared to L1 when assembled into VLPs. Various approaches have been proposed to overcome this limitation, we developed and tested ferritin-based bio-nanoparticles displaying tandemly repeated L2 epitopes from eight different HPV types grafted onto the surface of  Cervical cancer results in about 270,000 mortalities worldwide annually , 2. EvenPyrococcus furiosus thioredoxin (Pf Trx) as a scaffold for L2 epitopes presentation. This strategy allowed the generation of highly thermostable antigens capable of inducing cross-neutralizing antibody responses to multiple HPV types (The currently licensed vaccines are based on virus-like particles (VLPs) assembled by the major capsid protein L1. VLPs are highly immunogenic and induce high titers of neutralizing antibodies, which, however, act in a pronounced HPV type-restricted manner. The reason for this is the low conservation of the neutralizing epitopes located on the surface of VLPs, despite the fact that the L1 protein is overall highly conserved among different HPV types. Moreover, L1-based VLPs have a very limited thermal stability, which makes cold-chain distribution mandatory for their preservation, a requirement that substantially constrains their potential for establishment in low- and middle-income countries where nearly 90% of the deaths by cervical cancer occur , 10–13, PV types –23. A maPV types , 25. TheDue to their repetitive geometry, which allows for the ordered display of a multimeric array of immuno-epitopes, overall size and shape , bio-nanoparticle-based vaccines are being increasingly evaluated as virus-like particle mimics , 27. IndA particularly robust and well-developed “super-scaffold” is ferritin, which spontaneously assembles into octahedral nanoparticles  composed by 24 subunits organized as eight trimers (4-3-2 symmetry). Genetic and, more recently, click chemistry-based ferritin-immuno-epitope fusions have been generated and tested as innovative recombinant antigens for both prophylactic and immuno-therapeutic applications –36. MostP. furiosus ferritin (Pf Fe) as a super-scaffold. Although a clear superiority to the previous heptameric vaccine was not apparent, we found that the Pf Fe-based antigens induced robust neutralizing antibody responses against multiple HPV types both in vitro and in vivo, with a range of protection much broader than that of the licensed L1-VLP vaccines.We addressed these questions by generating and testing a set of nanoparticle HPV-L2 antigens of increasing complexity using in vitro (PBNA) and in vivo  studies was carried out to evaluate the immunogenicity of the nanoparticle vaccine in different pre-clinical settings and models of immunization/infection. Cross-reaction with human ferritin and thioredoxin was also evaluated.The goal of this study was to test the immunogenicity, thermal stability and immune-performance of HPV L2-derived peptide epitopes internally fused to thioredoxin and displayed on the surface of ferritin nanoparticles. L2-beraing ferritin nanoparticles were produced and characterized biophysically and biochemically to confirm the structural integrity of the antigen. A combination of Escherichia coli. All of constructs were confirmed by DNA sequencing.Genes encoding for ferritin fusion proteins were generated by PCR and inserted into the pFBDM baculovirus transfer vector. For details of the encoded proteins refer to E. coli DH10Bac competent cells which contained the bacmid with a mini-attTn7 target site and the helper plasmid for the site-specific transposition of nanoparticle genes from the donor vector to a bacmid DNA through lacZ gene description. The transformed cells were plated onto LB agar containing kanamycin (50 µg/ml), gentamycin (7 µg/ml), tetracycline (10 µg/ml), X-gal (100 µl/ml) and incubated at 37°C for 48 h in the presence of X-gal and IPTG. In the presence of X-gal and IPTG, the resultant recombinant bacmid produced white colonies. High molecular weight bacmids were isolated from the overnight cultures by alkaline lysis.The recombinant donor plasmids based on pFBDM were transformed into Spodoptra frugipedra (Sf9) insect cells  were grown in TNM-FH Media  supplemented with 10% fetal bovine serum (FBS) and 50 µg/ml penicillin and 50 µg/ml streptomycin (P/S) at 27°C and 150 rpm agitation. Trichoplusia ni (TN) High Five cells (Invitrogen) were routinely sub-cultured to 0.5 × 106 cells/ml every 3 days in Ex-Cell™ 405 serum free medium  containing P/S at 27°C and 150 rpm agitation in 500 ml shake flasks (10–20% working volume-w/v) at 27°C and 150 rpm in the incubator. The HeLa T cell line and 293TT cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS and P/S. The pgsa-745 cells and CHOΔfurin cells were cultured in DMEM supplemented with 10% FBS and 10 mM proline. MCF10A cells were cultured in DMEM/F12 medium supplemented with 5% horse serum, P/S, 500 µg hydrocortisone (Sigma H-0888), 10 µg/ml insulin , and 20 µg/ml epidermal growth factor .6 cells/ml. After 3 days of infection, the cells were harvested and processed using two different strategies according to the construct. For Pf Fe, Pf FeTrx3mer and Pf Fe3mer, the cell cultures were cleared by centrifugation at 1,900 rpm, 30 min at 4°C. The cell pellets were resuspended in ice-cold extraction buffer  Triton X100, and 20mM HEPES in pH 7.4) and sonicated three times for 30 seconds at 3 min interval with 50% power of the ultrasonicator and cleared by centrifugation. Cell lysates were heated in to 65°C for 30 min, following centrifugation for 1 h at 12,000 × g at 4°C. Clear lysates were loaded onto a two steps gradient with 14 ml of 40% (w/v) sucrose on top of 8 ml of 71% (w/v) CsCl solution, centrifuged for 3 h at 96,500 × g at 10°C in a Beckman SW28 rotor. The interphase was collected and transferred into a 13 ml Quick-seal tube (Beckman). A CsCl gradient was produced by 16 h centrifugation at 184,000 × g at 20°C in a Sorval TFT 65.13 rotor and 1ml fraction were collected. The purity of the collected fractions was tested by SDS-PAGE. The peak fractions were pooled, dialyzed against PBS and cleared by centrifugation at 20,000 × g at 4°C for 10 min. For Pf FeTrx8mer and Pf Fe8mer, treatment of the cell lysate at 65°C for 30 min was sufficient for purification.Nanoparticle antigens were produced in TN-High Five insect cells at 29°C in 2,000 ml shake flasks (10% w/v). Briefly, shake cultures were infected with different baculovirus at MOI 2 - 5 when the cell culture reached the concentration of 2 × 1020-38 (Nanoparticle formation was verified by transmission electron microscopy (TEM). Nanoparticle preparations were applied to carbon-coated grids and incubated at room temperature for 15 min. The grids were washed 3 times with PBS and stained with 1.5% uranyl acetate and then observed at × 80,000 nominal magnification with an electron microscope Zeiss EM912 and EM910. For the immune TEM, the nanoparticles were spread onto the surface of carbon-coated grids and incubated at room temperature for 15 min, then blocked with 1% BSA for 5 min. After washing the grid 3 times with PBS, mouse anti-HPV16 L2 monoclonal antibody 16L2-K4Size exclusion chromatography (SEC) was performed on a Superdex 200 Increase 10/300 GL column  equilibrated with PBS at a flow rate of 0.5 ml/min, using an Äkta fast protein liquid chromatography (FPLC) system (UV 280 nm). 1.5 MPa was set as the maximum pressure, carried out with Unicorn 5.0 . All elutions were carried out with 2 column volumes of running buffer. Fractions of 1ml were collected and stored at 4°C to be further analyzed for the protein content and composition with the Coomassie dye-binding assay and SDS-PAGE. The hydrodynamic sizes of nanoparticle antigens were analyzed by dynamic light scattering (DLS) with a Zetasizer Nano ZM .2.dmol-), using [θ] 5h/10 cnresl, where θ is ellipticity, c is the molar concentration of the protein, nres is the number of amino acid residues in the protein and l is the optical path length in centimeters. Thermal unfolding studies were performed in the 25–100°C temperature range, keeping the protein for 10 min at each temperature (25–30–40–50–60–70–80–90–100°C) before spectra recording. Complete protein denaturation was ensured by keeping the protein sample at 100°C for increasing lengths of time . An apparent Tm was determined based on 222 nm ellipticity values measured at different temperatures and calculated as inflection point of a sigmoid-fitted unfolding curve.Far-UV circular dichroism (CD) spectroscopy (190-260 nm), performed with a Jasco J715 Spectropolarimeter equipped with a Peltier temperature controller, was used to assess thermal stability of Pf FeTrx8mer dissolved in 0.1X PBS at a 1 µM concentration. CD spectra, acquired with the use of a 0.1 cm path length cuvette, a bandwidth of 1 nm, a data pitch of 0.5 nm, and a response time of 4 s were averaged from four scans. Following baseline correction, the measured ellipticity, h (mdeg), was converted to molar mean residue ellipticity [θ] . Ten mice per group were immunized three or four times at biweekly intervals with 20µg of different de-toxified, filter-sterilized and adjuvanted antigens in 50 µl intramuscularly. The antigens were formulated with 50% (v/v) AddaVax (InivoGen) or 50 µg aluminum hydroxide (Brenntag), and 10 µg synthetic monophosphoryl lipid A (Avanti Lipids). 350- to 400 g Outbred Hartley (Crl : HA) female guinea pigs  (Permit number: 35-9185.81/A35/12) were immunized subcutaneously four times at biweekly intervals with 30 µg of different antigens formulated with 50% (v/v) AddaVax (InivoGen) in a volume of 100 µl. Four weeks after last immunization, animals were sacrificed and blood samples were collected by cardiac puncture.Pseudovirions (PsVs) were prepared by co-transfection of the human fibroblast cell line 293TT with plasmids carrying humanized HPV L1 and L2 coding sequences plus a reporter plasmid, and purification was performed by iodixanol gradient ultracentrifugation according to previously described protocol .5 cells/ml) were added to the pseudovirion-antibody mixture and incubated at 37°C for 48 h in a humidified incubator under a 5% CO2. The amount of secreted Gaussia luciferase was determined in 10 µl of cell culture medium using the coelenterazine substrate and Gaussia glow juice , according to the instructions of the manufacturer. A microplate luminometer  was used to measure culture medium associated luminescence 15 min after substrate addition. The effective inhibitory concentration (EC50) was determined using the GraphPad Prism software program, and represent the serum dilution that was able to inhibit 50% of pseudovirion infectivity. EC50 values lower than 50 were considered non-neutralizing and, therefore, set to 0.1.Neutralizing antibody in sera against human papillomaviruses were detected by the L1 pseudovirion-based neutralization assay (L1-PBNA) as described previously , 37. Bri5 cells/ml were plated in a 96-well plate and propagated for 24 h to prepare extracellular matrix (ECM). After the incubation, the cells were washed twice with PBS and lysed with 50 µl pre-warmed lysis buffer  at 37°C for 5 min. The ECM-coated plate was washed with 100 µl PBS per well 3 times. A pseudovirion solution was prepared in conditioned medium from CHOΔfurin cells in a total volume of 120 µl/well in the presence of 5µg/ml heparin and added to the ECM-coated wells. The Pseudoviron–furin–heparin mixture was incubated overnight at 37°C overnight. The next day, the medium was removed and the wells were washed twice with PBS. Then the sera, diluted in pgsa-745 growth medium, were added to the plates and incubated at 37°C for 3 h to allow efficient antibody binding to target epitopes. Following this incubation, pgsa-745 cells were added to the antibody containing wells to a final concentration of 1.6 × 105 cells/ml. Infection was allowed to continue for 2 days at 37°C. The amount of secreted Gaussia luciferase was determined as described above.The L2 pseudovirion based neutralization assay (L2-PBNA) was performed as previously described . BrieflyThe effective inhibitory concentration (EC50) was determined using the GraphPad Prism software program, and comprehend the serum dilution that was able to inhibit 50% of pseudovirion infection. EC50 values lower than 50 were considered non-neutralizing and, therefore, set to 0.1. All L2-PBNA related cell lines  were kindly provided by John Schiller’s laboratory , and authenticity was confirmed periodically by multiplex cell authentication (MCA) assay .in vivo imaging system (IVIS) imager  was performed. Twenty µl of luciferin substrate  were instilled intravaginally, the efficiency of protection of the antibody was assessed by in vivo imaging. A region of interest (ROI) analysis was performed using living image 2.50.1 software . Background signal was obtained and subtracted by imaging each group of mice prior to installation of luciferin.The mouse cervico-vaginal challenge model was carried out as described previously  . On day Mouse and guinea pigs’ sera were analyzed in a final dilution 1:500. Antibodies against Pf ferritin, human ferritin, Pf thioredoxin and human thioredoxin were detected using GST tagged Pf ferritin, human ferritin, Pf thioredoxin and human thioredoxin Multiplex serology as described before . RecombiAnimal experimentation procedures were approved by the Regierungspräsidium Karlsruhe under permit  and were performed in accordance with the relevant guidelines and regulations. Mice and guinea pigs were kept and handled in the animal house facility of the German Cancer Research Center  under specific pathogen-free conditions, in compliance with the regulations of the Germany Animal Protection Law.Statistical significance analysis was performed with the nonparametric Mann-Whitney test performed with GraphPad Prism 8.0 ; differences between groups were considered significant at a P value of < 0.05.P. furiosus ferritin (Pf Fe) self-assembles into highly thermostable (P. furiosus thioredoxin (Pf Trx)  , 19, 37.(Pf Trx) , 25, 43.eviously , 24, 25 Starting from Pf Fe nanoparticles harboring three copies of HPV16 L2 grafted to thioredoxin, we used three different adjuvant formulations (plus a no adjuvant control) to test the immunogenicity and cross-neutralization capacity of this antigen. To this end, neutralizing antibody titers against HPV16, 18, 31, 39, 45, and 51 were evaluated by standard (L1)-PBNA. As shown in Our former studies had highlighted an absolute requirement for internal grafting to, and display on the surface of, thioredoxin in order to confer high-level immunogenicity to the L2 epitopes , 21–23. Having demonstrated the basic functionality of the ferritin superscaffold, we wished to broaden the antibody response to more HPV types. Therefore, we replaced the HPV16 L2 20-38 trimer with an L2 polytope (“8mer”) harboring the neutralization epitopes from seven oncogenic HPV types plus that of the cutaneous type HPV6 , 24, 25.Cross-neutralizing antibodies against HPV types 39, 45, 52, 56, 58, 68, 73, and 11, which are all not represented in the polytope, were also determined and similar titers were measured for the two antigens (A similar comparative (cross-) neutralization profiling (14 high-risk plus two low-risk HPVs) was performed in guinea pigs. As shown in To determine the duration of immunity induced by the two nanoparticle vaccines, mice were immunized (three times biweekly) with both antigens adjuvanted with AddaVax, followed by the assessment of antibody responses, one month and one year after the last immunization. Neutralizing antibody titers induced by Pf FeTrx8mer and Pf Trx8mer OVX313 were robust and comparable when tested one month after the last immunization Figure 5in vivo efficacy of the Pf FeTrx8mer nanoparticle vaccine, we used a mouse challenge model of HPV infection. To this end, guinea pigs were first immunized four times with the AddaVax-adjuvanted Pf FeTrx8mer antigen. One month after the last immunization, sera were collected, pooled and transferred intraperitoneally into naïve mice, which were subsequently challenged with HPV 6, 11, 16, 18, 33, 35, 39, 45, 51, and 56 pseudovirions. In vivo-determined, luciferase-associated luminescence was used as indicator of the degree of infection, which was assessed two days after pseudovirion instillation; pre-immune guinea pig sera served as controls for these experiments. A representative image of vaginal infection by HPV 6, 11, 16, and 56 pseudovirion challenge is presented in To evaluate the To follow the development of neutralizing antibodies over time, we immunized mice four times with AddaVax-adjuvanted Pf FeTrx8mer and Pf Trx8mer OVX313 antigens. As shown in Pyrococcus ferritin and thioredoxin and their human counterparts is 26.7 and 12.3%, respectively  are usually low  . These rvia ferritin nanoparticles (NP) has proven highly potent at improving induction of humoral immune responses  based nanoparticle antigens  an adequate vaccine coverage for HPV-related cancers and other pathologies is missing . Here, tAntigen formulation with adjuvants is a strategy to boost humoral and cellular immunogenicity.Three adjuvants were tested and compared with the non-adjuvanted antigen. The squalene-based adjuvant AddaVax elicited the strongest responses, but a well detectable, albeit weaker, response was also observed with the non-adjuvanted ferritin NP-based antigen. This self-adjuvancy may reflect the ability of ferritin nanoparticles to promote antigen depot but the additional contribution of ferritin-harbored T helper epitopes cannot be excluded . On the in vivo functionality of vaccine-induced neutralizing antibodies by transferring guinea pig immune sera to naïve-recipient mice and subsequent demonstration of passive immunization against the challenge with ten HPV types, including HPV56. These are important findings, since (i) in vitro neutralization not always correlates to in vivo protection, and (ii) neutralizing antibody levels are not always detectable in vitro. HPV vaccines are primarily recommended for adolescent girls; however, even adult women face the risk of HPV infection throughout their sexually active life. The duration of protection thus represents a critical issue for the overall, especially long-term, effectiveness of HPV prophylactic vaccines. Indeed, vaccine-induced antibodies peaked one month after the last immunization but could still be detected 11 years after administration of the current VLP vaccines  , 25 serv , 25 servaccines . Memory Safety is always a major concern in prophylactic vaccine development. Besides potential toxicity of antigen-adjuvant formulations, molecular mimicry, i.e., the induction of cross-reactive responses against endogenous host proteins is also a great concern –58. Our To develop a successful vaccine, the antigen should preferentially present repetitive B cell epitopes to activate B cells, and could be recognized by APCs and activate T helper cells to help B cells to produce antibody. As for the size and epitopes repetitive presenting, Pf FeTrx8mer should be more favorable, however, they share a similar immunogenicity, might be the central core domain of OVX313 contributes to bind to CD40 on B cells . In summThe datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/The animal study was reviewed and approved by the Regierungspräsidium Karlsruhe under permit .FY and MM designed research studies. FY performed the research. FM, XZ, and FY participated in animal immunizations. GS and SO performed CD analysis. FY, SO, and MM interpreted and discussed the data. FY wrote the paper. FY, FM, SO, and MM participated in manuscript revision. All authors contributed to the article and approved the submitted version.FY was supported by Chinese Scholarship Council (201506160063). XZ was supported by the Wilhelm-Sander-Stiftung (2013.136.3). Co-funding of GS fellowship by the Interuniversity Consortium for Biotechnology (CIB) is also gratefully acknowledged. This work also benefited from instrumentation facilities made available within the COMP-HUB Initiative, funded by the “Departments of Excellence” program of the Italian Ministry for Education, University and Research .At the time the research described in this manuscript was performed, FY, FM, XZ, and MM were employees of DKFZ, while GS and SO were employees of University of Parma.


DSF is widely conserved in a variety of Gram-negative bacterial pathogens and is involved in the regulation of pathogenic virulence. Quorum quenching (QQ) is a promising strategy for preventing and controlling quorum sensing (QS)-mediated bacterial infections by interfering with the QS system of pathogens. In this study, a novel DSF-degrading bacterium, Burkholderia anthina strain HN-8, was isolated and characterized for its degradation ability and potential biocontrol of black rot disease caused by Xanthomonas campestris pv. campestris (Xcc). The HN-8 strain exhibited superb DSF degradation activity and completely degraded 2 mM DSF within 48 h. In addition, we present the first evidence of bacterium having a metabolic pathway for the complete degradation and metabolism of DSF. Analysis of DSF metabolic products by gas chromatography–mass spectrometry led to the identification of dodecanal as the main intermediate product, revealing that DSF could be degraded via oxidation–reduction. Furthermore, application of strain HN-8 as a potent biocontrol agent was able to significantly reduce the severity of black rot disease in radishes and Chinese cabbage. Taken together, these results shed light on the QQ mechanisms of DSF, and they provide useful information showing the potential for the biocontrol of infectious diseases caused by DSF-dependent bacterial pathogens.Diffusible signal factor (DSF) is a type of  Xanthomonas campestris pv. campestris (Xcc), is a type of cis unsaturated fatty acid with a chemical structure of 11-methyl-2-dodecylene acid [Xcc can cause black rot disease in cruciferous vegetables, which is a global and destructive disease, generally leading to a serious decline in the quality and yield of cruciferous crops [Xcc can spread across the field to infect the crown and root of healthy plants by means of flying insects, rain, wind, and water splashes under humid conditions [Xcc is one of the most prevalent diseases in the Brassicaceae family around the world, which occurs heavily each year [Diffusible signal factor (DSF), a signal molecule produced by us crops . This dius crops ,5. Durinus crops . The gutnditions . Irreverach year ,9.To control and prevent black rot disease, agricultural and chemical measures, including strengthening field management, selecting resistant varieties, and using of pesticides, are usually taken. However, among these strategies, field management is hard to strengthen because of certain limitations of agricultural areas and the long-term survival of pathogens resistant to extreme conditions, such as dryness and low temperature . MoreoveQS plays an important role in bacterial adaptation in harsh environments. In addition, QS can also regulate functions related to pathogenicity ,17. ThroDelisea pulchra was the first natural QSI to be identified [Vibrio infection of rainbow trout [N-acyl-homoserine lactone (AHL) or N-Sulfonyl homoserine lactone derivatives were shown to act as antagonists against AHL [Xcc, the use of DSF degrading bacteria is an environmentally friendly, residue-free, and effective method. In addition, DSF has been found exist in many Gram-negative bacteria, including Burkholderia sp. and Pseudomonas aeruginosa, which are important human pathogens [cis-2-dodecenoic acid (BDSF) and -11-methyldodeca-2,5-dienoic acid (CDSF) has been identified and reported [Bacillus and Paenibacillus have been screened for their degradation ability of DSF by Newman et al. [QQ possesses two main mechanisms; one is based on QS signal molecule inhibitors (QSIs), and the other is based on QS signal molecule degradation enzymes ,22. The entified . For exaow trout ,25. Manyow trout ,28,29,30inst AHL . Howeverinst AHL ,33. Overathogens ,35. Morereported ,37,38. An et al. . HoweverBurkholderia anthina, with excellent DSF degrading activity was screened, with the particular aim to: (1) characterize the isolate HN-8 combined with cellular morphology, physio-biochemistry features, and molecular sequences; (2) investigate the degradation capacity of DSF and the mechanisms of QQ; and (3) evaluate the biocontrol effects against Xcc. This study provides information to broaden the effectiveness of isolate HN-8 for exploiting its potential role as a biocontrol agent against black rot by Xcc.In this study, the novel isolate HN-8, identified as −1. Radishes (Raphanus sativus) and Chinese cabbage (Brassica pekinensis) were obtained from a nursery , and healthy plants were selected for the biocontrol experiments.DSF (≥99%) was purchased from Shanghai UDChem Technology Co., Ltd.  and dissolved in methanol to create a stock concentration of 100 mmol·LXanthomonas campestris pv. campestris (Xcc) 8004 and Escherichia coli DH5α were provided by the Integrative Microbiology Research Centre, South China Agricultural University, Guangzhou, China. Xcc were cultured on Luria Bertani (LB) medium  with rifampicin (30 μg·mL−1) at 28 °C. E. coli DH5α was cultivated on LB medium at 37 °C. The isolates grew on LB medium or mineral salt medium (MSM) ((NH4)2SO4 2.0 g·L−1, Na2HPO4·12H2O 1.5 g·L−1, KH2PO4 1.5 g·L−1, MgSO4·7H2O 0.2 g·L−1, CaCl2·2H2O 0.01 g·L−1, FeSO4·7H2O 0.001 g·L−1, and pH 7.2) with DSF (2 mmol·L−1) at 30 °C [at 30 °C .−1 DSF as the sole source of carbon), the suspension was shaken at 30 °C and 200 rpm for 7 days, and then a series of dilutions were carried out [−1) and the plates were incubated at 30 °C for 1–2 days. The different colonies growing on the LB medium were transferred to another LB medium plate for their single pure colony culture. The different single colonies were stored at 4 °C on LB plate for further follow-up experiments in this research.Soil samples were collected from the upper layer (5–10 cm) of the rice-growing and cruciferous plant-growing fields from five different farms near South China Agricultural University, Guangzhou, China. All of the collected soil samples were stored at 4 °C in the laboratory. For the isolation of bacteria from soil samples, serial dilutions were performed for each soil sample as described below: 5 g of soil was added to 50 mL of sterile distilled MSM medium , and then the medium was sterilized at 121 °C for 20 min. When it had cooled to approximately 50 °C, it was completely mixed and immediately poured onto plates (approximately 20 mL/plate). The plate was evenly drawn into three parts with a marker pen. One part was inoculated with distilled water as the blank control, the other part was inoculated with E. coli DH5α (OD600 = 1) as the negative control, and the other part was inoculated with the DSF-degrading strain (OD600 = 1) as the test. An ultraviolet spectrophotometer  was used to determine the concentrations of the strains. After 2 days of culture, the plates were taken out to observe whether there was a colorless transparent ring around the colony. Each treatment was replicated three times. The bacterial strain HN-8 was screened and selected for further studies based on its effective DSF degradation.For the screening of the bacterial isolates, 2 mmol·LThe isolate grew on LB and was incubated at 30 °C under an alternating cycle of 12 h light and 12 h darkness. The morphological characteristics of the colony, including the color, shape, size, and edge of the colony, were observed and measured using an electron microscope  and a transmission electron microscope .The isolate HN-8 was commissioned to the Guangdong Detection Center of Microbiology  to detect physiological and biochemical characteristics by performing Biolog tests. The type of Biolog microplates used in analysis is GNIII.The isolate was cultured in 5 mL of LB medium, and then shaken at 30 °C and 200 rpm for 12–16 h. Genomic DNA was extracted using an Easy Pure Bacteria Genomic DNA Kit . The obtained genomic DNA was re-suspended in 20 μL of distilled water and stored at −20 °C. The 16S rDNA universal bacterial primer pairs 27 F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492 R (5′-GGTTACCTTGTTACGACTT-3′) were used for polymerase chain reaction (PCR) amplification of the DNA in an automated thermal cycler under the following conditions: 5 min of denaturation at 94 °C, followed by 32 cycles at 94 °C for 30 s, 55 °C for 30 s, and a final extension at 72 °C for 1 min ,44. The The sequences were analyzed by the nucleotide Basic Local Alignment Search Tool (BLASTn), compared to the genes in the GenBank database, and then deposited in GenBank (accession number: MG561937.1). Similar and relevant sequences were downloaded from the National Center for Biotechnology Information (NCBI), and were used for phylogenetic analysis and construction of the phylogenetic tree .−1), carbenicillin , streptomycin , tetracycline , kanamycin , gentamicin , neomycin sulfate , rifampicin , and chloramphenicol  at different concentrations . An ultraviolet spectrophotometer  was used to detect the growth of strain HN-8 with different concentrations of these antibiotics. Each treatment was replicated three times.Antibiotic sensitivity of HN-8 was also tested for further experiments. The isolate was cultured in 5 mL LB medium, and then shaken at 30 °C and 200 rpm for 12–16 h. The overnight cultures and the respective antibiotics were added to the LB medium, and then shaken at 30 °C and 200 rpm for 24 h. The antibiotics used in this test included ampicillin . The cultures were incubated at 30 °C and 200 rpm, and the same volume of cultures was taken out and centrifuged to obtain the supernatant, where the residual DSF was extracted from different intervals [18 reverse chromatographic column, flow rate of 1 mL·min−1, column temperature of 35 °C, mobile phase of methanol/water = 80:20 (ν:ν), detection wavelength of 210 nm, and injection quantity of 20 μL [An ultraviolet spectrophotometer and the high-performance liquid chromatography (HPLC) technique were employed to detect the growth and degradation capacity of the isolate HN-8, respectively. A two-day-old colony of DSF-degrading isolate was added into the LB medium; the suspension was shaken at 30 °C and 200 rpm for 12–16 h, and then transferred to MSM medium . As described and extracted above, samples were collected at regular intervals and mass spectrometry analyses were matched with authentic standard compounds from the National Institute of Standards and Technology  library database. The analytical conditions for GC–MS were as follows: injection amount, 1.0 μL; injection mode into the gas chromatograph, split with a 10:1 ratio; capillary column, HP-5 MS Ultra Inert column (30 m\0.25 μm\0.25 μm); column oven temperature, 60 °C for 5 min, then an increase of 10 °C/min, followed by 300 °C for 29 min; injection port temperature, 280 °C; carrier gas, helium; analytical mode, scan mode. Mass spectra were recorded in the m/z range of 40–430 [Gas chromatography–mass spectrometry (GC–MS) , which uses Agilent MassHunter Workstation software, was employed to detect the degradation products of the isolate HN-8. The HN-8 suspension, shaken at 30 °C and 200 rpm for 12–16 h, was added into the MSM medium  Makino) and Chinese cabbage (Brassica pekinensis (Lour.) Rupr.). The bacterial suspensions were inoculated on radish root slices containing suspensions of black rot pathogen Xcc (4 × 108 CFU·mL−1), suspensions of DSF-degrading strain HN-8 (2 × 108 CFU·mL−1), and suspensions of Xcc mixed with HN-8. Radish root slices inoculated only with distilled water were set as the control. Biocontrol tests were also performed on Chinese cabbage, which included four treatments, as follows: (1) cabbage treated with Xcc (4 × 108 CFU·mL−1); (2) Chinese cabbage leaves treated with Xcc mixed with HN-8; (3) Chinese cabbage leaves treated with HN-8 (2 × 108 CFU·mL−1); and (4) Chinese cabbage leaves treated with distilled water (the control). Each treatment was repeated three times. The treated plants were observed daily, and disease incidence and disease severity were recorded from the inoculation [Based on previous experimental results, the DSF-degrading isolate HN-8 was used for in vitro tests of biocontrol against black rot caused by culation . The detEscherichia coli DH5α , lysine decarboxylase, and ornithine decarboxylase, the reaction results are all positive. On the contrary, the reaction results for urease, amylolysis, nitrate reduction, hemolysis, and arginine dihydrolase are negative.The biochemical characterization  indicateBurkholderia genus, since it shares the highest sequence similarity (99%) with the 16S rRNA gene sequences of other identified Burkholderia spp. Additionally, the neighbor-joining analysis highlighted that the strain HN-8 shares about 99% of its identity with the corresponding sequences of Burkholderia anthina strain W92B (GenBank number: NR 104975.1)  . Combine−1), CARB (50 mg·mL−1), and STR (50 mg·mL−1) reached 400 μg·mL−1 or above; to KAN (50 mg·mL−1) reached 200 μg·mL−1; to TET (5 mg·mL−1) reached 50 μg·mL−1; to GEN (50 mg·mL−1) and NS (20 mg·mL−1) reached 10 μg·mL−1; and to RIF (25 mg·mL−1) and CM (30 mg·mL−1) was less than 10 μg·mL−1.The results shown in The results of the antibiotic sensitivity test show that the DSF-degrading strain HN-8 has excellent antibiotic resistance, especially to ampicillin, carboxypenicillin, and streptomycin. This result will be used as a reference in future studies with appropriate antibiotics, and its resistance to many antibiotics is a great advantage to further apply strain HN-8 as a biocontrol agent.B. anthina HN-8 at 12, 18, 24, 30, 36, 42, and 48 h was 5.6%, 8.9%, 23.5%, 52.8%, 89.0%, 97.3%, and 100%, respectively  [Paenibacillus, Bacillus, Actinobacteria, Bacteroidetes, Lactobacillus, Proteobacteria, Firmicutes, Cupriavidus, and Acinetobacter [Burkholderia anthina based on its morphological, physiological, and biochemical characteristics, as well as 16S rRNA sequence analysis. The results indicate that B. anthina HN-8 has the potential to be applied as a powerful biocontrol agent against black rot in cruciferous plants.Cruciferous plants are some of the favorite and most edible vegetables consumed extensively worldwide, greatly affected by bacterial black rot disease caused by is (Xcc) . The preis (Xcc) . In receis (Xcc) ,17,47,48is (Xcc) . Meanwhitobacter ,50,51, dtobacter ,33. ThusXcc, regulate the expression of factors that contribute to virulence and rely on cell–cell signaling [cis-unsaturated fatty acid, was first identified in the black rot disease pathogen Xanthomonas campestris [Pseudomonas aeruginosa, structurally related molecules are produced to regulate biofilm formation, virulence, and antibiotic tolerance [B. anthina HN-8, with the capacity to rapidly degrade high concentrations of DSF in a short time, may be involved in the biocontrol management of black rot caused by Xcc. To the best of our knowledge, there are few reports of DSF degradation from B. anthina. For the first time, this study proved that B. anthina can reduce the severity of black rot disease caused by Xcc by degrading the DSF signals.Many plant pathogenic bacteria, including ignaling ,52. Thermpestris . Furthermpestris ,55,56. Nolerance . Degradaolerance . Based ocis double bond of the unsaturated fatty acid was converted into a trans double bond and hydrogenated to a saturated fatty acid, which was then oxidized to aldehyde. Eventually, all intermediate products were transitory and faded away without any non-cleavable metabolites at the end of the experiment. This is the first report of a DSF metabolic pathway by oxidation–reduction in a microorganism, which we propose is of vital importance in DSF degradation.Moreover, GC–MS was employed to detect the degradation products of the isolate HN-8, and based on the chemical structures of DSF and the metabolic products formed during degradation process, the metabolic pathway of DSF in strain HN-8  was propXcc on plants was evaluated under laboratory conditions. The inoculation tests showed that B. anthina HN-8 significantly reduced the severity of black rot disease in plants compared to inoculation with the Xcc suspension. The results also showed that the strain HN-8 could not only significantly reduce the severity of black rot disease in plants, but could also avoid causing any disease symptoms when inoculated only under laboratory conditions. The main advantages of the simplified method include (1) shortening the test cycle (2) and minimizing the interference factors during the biocontrol test. However, a biocontrol test that was carried out in vitro, on detached parts of plants, cannot be considered exhaustively to assess the activity of the antagonistic microorganisms. Therefore, the biocontrol assay in this study is a preliminary test. The biocontrol efficiency of QQ bacteria maybe not be exhibited ideally in plant tests [Xcc in cruciferous plants under field conditions would be interesting.In this research, the control efficiency of the strain HN-8 against nt tests ,59. In fXcc in cruciferous plants. The strain HN-8 exhibited rapid degradation of high concentrations of DSF in a short time, which is vital to ensure an efficient prevention of pathogenic bacteria in plant assays. Additionally, the strain HN-8 had remarkable antibiotic resistance to ampicillin, carboxypenicillin, and streptomycin. The multiple beneficial abilities of the B. anthina strain HN-8 allows it to be regarded as an effective biocontrol agent, and to be considered for commercial development for management strategies of black rot caused by Xcc in cruciferous plants, similar to the Lactobacillus plantarum strain BY used for biocontrol of soft rot caused by Pectobacterium carotovorum subsp. carotovorum in Chinese cabbage under field conditions [This research of the potential biocontrol novel isolate HN-8 was mainly focused on DSF degradation and severe black rot disease caused by nditions .B. anthina HN-8, exhibiting excellent DSF degradation, and potent biocontrol activities against Xcc, was isolated and characterized. The strain HN-8 was capable of rapidly degrading DSF, and completely degraded DSF within 48 h. In addition, the application of strain HN-8 was able to significantly reduce black rot disease in radishes and Chinese cabbage. As a promising biocontrol agent, strain HN-8 could be further explored to counter other DSF-dependent bacterial pathogens such as Pseudomonas aeruginosa. Furthermore, strain HN-8 possesses a metabolic pathway for the complete degradation and metabolism of DSF. This is the first report of a pathway for DSF degradation by oxidation–reduction in a microorganism. These findings not only characterize a highly efficient DSF degradation bacterial isolate for biotechnological application, but they also shed light on the mechanisms of QQ in relation to DSF. In the future, further in-depth studies based on related enzymes and genes are needed to elaborate their role in the evolution of novel catabolic pathways and the molecular mechanisms of DSF degradation by strain HN-8.In this study, a novel QQ bacterial isolate, i.e.,


Lamtor gene expression. Furthermore, the type of functionalization was also important, with cationic MWCNT activating the transcription factor EB and inducing autophagy while the anionic MWCNT altering eukaryotic translation initiation factor 4 (EIF4) and phosphoprotein 70 ribosomal protein S6 kinase (p70S6K) signaling pathway as well as upregulation Tlr2 gene expression. This study proposes that MWCNT toxicity mechanisms are functionalization dependent and provides evidence that inflammatory response is a key event of carbon nanotubes toxicity.Functionalized multi-walled carbon nanotubes (MWCNT) have become the focus of increased research interest, particularly in their application as tools in different areas, such as the biomedical field. Despite the benefits associated with functionalization of MWCNT, particularly in overcoming issues relating to solubility, several studies have demonstrated that these functionalized nanoparticles display different toxicity profiles. For this study, we aim to compare NR8383 cells responses to three well-characterized MWCNT with varying functional groups. This study employed cytotoxicity assays, transcriptomics and proteomics to assess their toxicity using NR8383 rat alveolar macrophages as an in vitro model. The study findings indicated that all MWCNT altered ribosomal protein translation, cytoskeleton arrangement and induced pro-inflammatory response. Only functionalized MWCNT alter mTOR signaling pathway in conjunction with increased  Carbon nanotubes (CNT) are increasingly used in different sectors including the biomedical one due to their distinctive properties. We have monolayer CNT, called single walled carbon nanotubes (SWCNT) and multilayer CNT, called multi walled carbon nanotubes (MWCNT). In the last few years, 736 metric tons of these CNT, were used for energy and environmental applications, with these figures constantly increasing over time . The gloRecently, several studies have focused on carbon nanotubes functionalization. It has been demonstrated that carboxyl and amino groups can be added endowing the CNT with new characteristics and subsequently making them suitable for more applications . CarboxyDaphnia magna. However, it is important to note that there are contradictory reports, with a study by Cinzia et al. showing that carboxyl functionalization induced a toxic response in BEAS-2B cells, with increased inflammatory response and cytotoxicity in comparison to pristine CNT exposure. This finding is supported by other studies showing that carboxylic functionalization enhanced CNT cytotoxicity in HUVEC  by searching with the MaxQuant version 1.5. Modifications included C carbamylation (fixed) and M oxidation (variable). The resulting data was then analysed using online platforms such as String and PANTHER.50 of each MWCNT as well as a negative control (unexposed cells) were analysed. This corresponds to 0.8 cm2/cm2 for NM403, 6.8 cm2/cm2 for NRCWE-042 and 1.2 cm2/cm2 for NRCWE-049. Briefly, after blocking the membrane, supernatants were incubated with the membrane for 2 h at room temperature under smooth agitation. After washing, the membrane was incubated with a mixture of diluted biotinylated antibodies for 1 h at room temperature under smooth agitation. After washing, the membrane was incubated with a solution of diluted streptavidin-horseradish Peroxydase for 30 min at room temperature under smooth agitation. Finally, after washing, a mixture of H2O2 and luminol was added for chemiluminescence reaction. Revelation was performed on a ChemiDoc Touch™ . Densitometric analysis was performed on ImageLab™ .In order to verify the induction of inflammation by these MWCNT, at a protein level, a cytokine array was prepared. Supernatants from exposed cells stored at − 80 °C were analysed using Proteome Profiler Rat XL Cytokine array™  according to manufacturer’s instructions. The supernatants corresponding to cells exposed to the ¼ ICFour biological replicates are used for each experiment. Statistical differences were determined by one-way analysis of variance (ANOVA) followed by Tukey–Kramer test (Dunnett’s test) for cell viability.MWCNT mean hydrodynamic diameters, zeta potential and polydispersity index, determined by DLS were shown in Table 50 of NM403, the non-functionalized MWCNT, and the amino-group functionalized CNT NRCWE-049 were found to be similar, 3.2 cm2/cm2 and 4.8 cm2/cm2 respectively. However, the IC50 for the carboxylated CNT (NRCWE-042) was shown to be much higher at 27.2 cm2/cm2. The IC50 determined in this assay were utilized to the doses to be employed in the transcriptomics study, which was set at ¼ IC50.All The IC2/cm2 doses , at a p value < 0.001 and FC cut-off level of 1.5, for the amino functionalized CNT NRCWE-049, 3388 DEG for the carboxyl CNT NRCWE-042 and 245 for the nonfunctionalized CNT NM403. The functionalized CNT NRCWE-049 and NRCWE-042 provoked a higher number of deregulated genes compared to the nonfunctionalized NM403: respectively 6049 > 3388 > 245 genes  software but there were four pathways among the top canonical ones FC ≥ 1.5), in common between functionalized CNT NRCWE-042 and NRCWE-049 included namely mitochondrial dysfunctions, eukaryotic initiation factor-2 (EIF2) signaling, Sirtuin signaling and oxidative phosphorylation  involved in DNA damage response pathway was inhibited  oxidative stress response pathway . This analysis indicated the presence of several different subgroups within the protein network including chaperonins, i.e. CCT4, CCT8, CCT3; ribosomal proteins, i.e. RPS3, RPS12, RPL18; and blood coagulation proteins Fig. . RibosomInflammatory reaction was the main response induced by all CNT according to both transcriptomic and proteomic data. In order to prove the inflammatory potential of each CNT and to compare between them, thus deducing the role of functionalization in inflammatory reaction induction and potency, we performed a cytokine array. An analysis of 79 cytokine expression shows a global augmentation in the expression of different cytokines already expressed in our control. This result is observed after NR8383 exposure to each MWCNT. A group of the most produced cytokines was involved in inflammatory response and has been presented in Fig. Each MWCNT studied employed in this study had similar Z-averages (212.7 ± 65.6) in DMEM medium supplemented with 2% FBS. It is well known that functionalized MWCNT agglomerate due to interactions with proteins , therefoCxcl2, Ccrl2, Il6r, Il10ra, Il17re and Ilf2 for NRCWE-049 and Ccl2 and Ccl4 for NM403, and Irak2, Il7r, Il1b, Il1a, Socs3 and Ifit3 for NRCWE-042. For NM403, only proteomics analysis identified inflammatory proteins  and A2m, Agt, Ahsg, Mif and Thbs1 . These findings are consistent with a previous study of our team that showed an overexpression of TNFA and Il1b in response to exposure to pristine MWCNT NM403 [50 of each MWCNT. Expression level augmentation has notably been observed for proinflammatory cytokines such as CCL11 and CCL22 . Proteomic analysis confirmed the transcriptomics finding for pristine MWCNT NM403, identifying increased protein expression of proteins involved in Tubulin proteins synthesis, i.e. Tubb4b, Tubb5a, Tub1ab, as well as chaperonin proteins which are responsible for proper tubulin actin folding [Functionalized MWCNT gene expression analysis highlighted an overexpression of genes related to inflammatory processes, including: NT NM403 . OtherwiL22 Fig.  which ar folding , i.e. Cc folding .Grb2, Igf2bp2, Vegfb, Gfer and Pdgfa indicated a dysregulation of mTOR pathway was observed only with functionalized MWCNT, NRCWE-042 and NRCWE-049 [Lamtor4 and ribosomal genes, including Rpl39l, Rps27 and Rps14, following MWCNT exposure occurred. NRCWE-042 and NRCWE-049 overexpressed Lamtor4, a part of the Ragulator complex, that is involved in mTORC1 activation [Exposure to pristine MWCNT NM403 was shown to result in the dysregulation of ribosomal genes and lead to the unprogrammed synthesis of ribosomal proteins. We have a disruption of ribosomal proteins, proteasome proteins and chaperonin proteins which are involved in the correct folding of proteins Fig. . ConsequRCWE-049 . Indeed tivation , 52 thattivation . Therefotivation .Abcc1, Dnajc7, Maff, Mafg, Prdx1 and Txnrd1, of which expression was found to be upregulated. Figure Slc23a2 gene, both of which have been implicated in antioxidant response. Previously Vales et al. showed increased ROS production after exposure of BEAS-2B cells to the same nanomaterial [Exposure of NR8383 cells to pristine MWCNT NM403 only activatedNRF2 pathway according to transcriptomic analysis Table . NRF2 plmaterial . Taken tmaterial .Rad51, which is known to be involved in DNA repair [Pristine MWCNT NM403 exposure was also demonstrated to have a significant impact on DNA damage response signaling. Several pathways related to different factors of DNA damage response were shown to be dysregulated in response to NM403 exposure; including checkpoint kinases: CHK1 and CHK2; cell cycle: G1/S checkpoint regulation; cell cycle: G2/M DNA damage; Ataxia telangiectasia-mutated protein kinase: ATM signaling, and DNA damage proteins e.g. Brca1 , protein coding gene which acts as a positive regulator of autophagy by promoting expression of genes involved in autophagy, was shown to be overexpressed only in response to the cationic MWCNT, NRCWE-049. This finding is supported by the activation of TFEB in the presence of cations . Amino-Mutophagy . Therefoutophagy . TWEAK eutophagy , therebyutophagy , and couarticles . Therefoarticles .Rps6ka1, Rps6ka4, Mrps2 and Mrps6, as well as dysregulation of genes related to translation, such as Eef1g, Eif1b, Eif6, Eif2s2, Eif1a, Eif4ebp1, Eif1, Eif3m, Eif3k, Tpt1 and Eif3. A study by Lunova et al. [Lamtor expression, thereby activating mTOR signaling which in turn controls the translational machinery, activating p70 S6 kinase protein (p70S6k) and inhibiting the eIF-4E inhibitor, the 4E-BP1 molecule. This results in the activation of the 40S ribosomal protein S6, which is known to contribute to the translation of 5′-TOP mRNAs encoding ribosomal proteins and components of the translational apparatus [Tlr2 gene (FC = 6.8) observed in response to NRCWE-042 exposure, which is known to be implicated with interferon gamma signaling in inflammatory response induction [Our transcriptomic analysis highlighted dysregulation of the mTOR and EIF4/p70S6K signaling pathways in response to NRCWE-042 exposure (Table a et al.  showed tpparatus , 73. As nduction –77. BaseAccording to our study, the functionalization of CNT with amino groups increases their toxicity, which was not well known, to our knowledge. They have the highest inflammatory potential, they produce an activation of inflammasome, a key event which may lead to chronic inflammation as reported for silica nanoparticles . InflammAccording to the presented results of this study, all three MWCNT lead to inflammatory response and ER stress which could potentially function as a biomarker of toxicity for all MWCNT. In contrast lysosomal destabilization was shown to be a potential biomarker for the cationic MWCNT, NRCWE-049. Interestingly, the type of functionalization alters the response of macrophages to MWCNT, inducing mTOR dependent signaling pathways which can result in either autophagy or enhanced translation depending on the MWCNT charge Fig. . This st