Acinetobacter baumannii. For CRE, the MIC50 and MIC90 for S-F and S-D were 2 and 4 μM, and 0.25 and 0.5 μM, respectively. S-F and nourseothricin showed rapid, bactericidal activity. S-F and S-D both showed approximately 40-fold greater selectivity for prokaryotic than eukaryotic ribosomes in in vitro translation assays. In vivo, delayed renal toxicity occurred at >10-fold higher doses of S-F compared with S-D. Substantial treatment effect of S-F in the murine thigh model was observed against the otherwise pandrug-resistant, NDM-1-expressing Klebsiella pneumoniae Nevada strain with minimal or no toxicity. Cryo-EM characterization of S-F bound to the A. baumannii 70S ribosome defines extensive hydrogen bonding of the S-F steptolidine moiety, as a guanine mimetic, to the 16S rRNA C1054 nucleobase (Escherichia coli numbering) in helix 34, and the carbamoylated gulosamine moiety of S-F with A1196, explaining the high-level resistance conferred by corresponding mutations at the residues identified in single rrn operon E. coli. Structural analysis suggests that S-F probes the A-decoding site, which potentially may account for its miscoding activity. Based on unique and promising activity, we suggest that the streptothricin scaffold deserves further preclinical exploration as a potential therapeutic for drug-resistant, gram-negative pathogens.The streptothricin natural product mixture  was discovered in the early 1940s, generating intense initial interest because of excellent gram-negative activity. Here, we establish the activity spectrum of nourseothricin and its main components, streptothricin F  and streptothricin D , purified to homogeneity, against highly drug-resistant, carbapenem-resistant Enterobacterales (CRE) and  Streptothricins were discovered in the early 1940’s to have excellent gram-negative antibiotic activity, but how do they work? A combination of microbiological and structural biology studies reveals their mechanism of action, highlighting unique interactions with the 30S ribosomal subunit. Acinetobacter baumannii are of particular concern. Almost all approved antimicrobials that can overcome the gram-negative permeability barrier are natural products or synthetic or semisynthetic derivatives of natural products. Small molecules commonly available in high-throughput screening libraries rarely share similar physicochemical properties associated with gram-negative penetrance and activity [The rapid emergence of antimicrobial resistance presents a significant challenge for treatment of bacterial infections. Carbapenem-resistant Enterobacterales (CRE) and activity . Therefoactivity . As a reMoreover, there is little doubt that resistance will emerge to agents currently in the pipeline. We are therefore clearly in need of several new gram-negative agents that are unique in terms of antimicrobial class and potential vulnerabilities, and which can diversify our antimicrobial therapeutic portfolio .Actinomyces [Mycobacterium tuberculosis [Brucella abortus infection in guinea pigs [Salmonella paratyphi B infection in mice [Here, we further considered the properties of a historic antibiotic scaffold. Streptothricin was originally isolated by Waksman and Woodruff in 1942 from a soil inomyces . It genenea pigs  and othe in mice , infecti in mice , the strStreptothricin is now known to be a natural product mixture, currently referred to as nourseothricin. For clarity, we will henceforth refer to the natural product mixture as nourseothricin and individual components by their specific streptothricin designation. Each streptothricin in nourseothricin consists of 3 linked components: (a) a streptolidine, a guanidine-lactam bicyclic 5–6 ring system; (b) a carbamoylated gulosamine; and (c) a homopolymer consisting of varying numbers of β-lysine residues, linked head to tail. The individual streptothricins are designated by letter suffixes (A-F and X), based on the number of lysine residues, with the most abundant streptothricins in the natural product, streptothricin F (S-F) and streptothricin D (S-D), having 1 and 3 lysine moieties, respectively see .50 of 300 mg/kg for S-F with 1 lysine compared to an LD50 of approximately 10 mg/kg for S-D and S-C with 3 and 4 β-lysines, respectively [50 is 52 mg/kg IV for gentamicin [Initial studies on toxicity in animals from the 1940s used only semiquantitative activity measures and impure compound with large doses presumably exceeding 100 mg/kg  and are ectively ,9. For cntamicin ; and 260ntamicin . Streptontamicin , has notntamicin . Of notentamicin , highligStudies 40 years ago using in vitro translation systems determined that S-F both inhibited prokaryotic translation and induced concentration-dependent ribosomal miscoding activity, similar to aminoglycosides such as gentamicin . NotablyBased on potentially insufficiently explored therapeutic potential, we sought to further characterize the properties of S-F and S-D, the major constituents of nourseothricin, purified to homogeneity using modern techniques and authenticated by several complementary state-of-the-art methods. This analysis included in vitro and in vivo analysis and mechanism of action studies combining use of both mutational resistance and cryo-EM–based structural analysis. Taken together, our data establish S-F as a potently bactericidal natural product scaffold with a unique mechanism of action with what we believe are compelling properties for future medicinal chemistry exploration. To this end, we also separately established a diversity-enabling total synthesis for S-F, which is reported elsewhere .A. baumannii. We, therefore, adapted a previously reported method to purify S-F (1 β-lysine) and S-D (3 β-lysines) to homogeneity using Sephadex chromatography [Our original studies with streptothricins were motivated by the observation that a nourseothricin resistance cassette served as a reliable genetic marker for experiments in otherwise highly drug-resistant gram-negative pathogens ,17. Thistography . ConfirmKlebsiella pneumoniae Nevada strain, AR-0663 [n = 39), MIC50 and MIC90 for S-F, S-D, and nourseothricin, respectively, were 2 and 4 (range 1 to 4) μM; 0.25 and 0.5 (range 0.25 to 2) μM; and 0.5 and 1 (range 0.25 to 2) μM. The average MIC was 5.6-fold greater for S-F than for S-D and 4.2-fold greater for S-F than for nourseothricin. We tested 4 A. baumannii strains in parallel with S-F, S-D, and nourseothricin (FDA-CDC #399–402) and found that the MIC for each was 4-fold greater for S-F than for S-D and nourseothricin. Data for individual isolates are listed in A. baumannii strain set (n = 104), many isolates from wounded US soldiers [50 = 2 μM; MIC90 = 32 μM, MIC99 = 32 μM; range <0.125 to 64 μM.Using broth microdilution minimal inhibitory concentration (MIC) testing, we determined the MIC of S-F, S-D, and nourseothricin for a large group of multidrug-resistant CRE. These included strains expressing metallo- and/or serine carbapenemases and the pandrug-resistant, NDM-1–expressing  AR-0663 . For thesoldiers : MIC50 =An LC–MS analysis of commercially purchased nourseothricin showed a composition of 65.5% S-F, 29.6% S-D, and 4.9% S-E (2 β-lysines). Therefore, the somewhat enhanced activity of S-D (mean MIC 0.44 μM) compared with nourseothricin (mean MIC 0.58 μM) for CRE likely reflects a combinatorial effect of the more potent S-D and less potent S-F, as well as contributions from other minor streptothricin congeners in the nourseothricin natural product mixture.Escherichia coli and Micrococcus lysodeikticus after 17 and 65 hours of exposure without reference to an underlying MIC [K. pneumoniae Nevada isolate (AR-0636). Notably, we observed rapid bactericidal activity for both within 2 hours of exposure at 4X MIC without regrowth  and eukaryotic (rabbit reticulocyte) in vitro coupled transcription–translation systems with readout using nanoluciferase gene constructs  using commercially (NEB) available prokaryotic . Nourseothricin was used as a surrogate for S-D, based on the difficulty of isolation of the latter in quantities needed for the experiments. Over the next 72 hours, no signs of distress were observed with dosing up to 200 mg/kg of S-F and 20 mg/kg of nourseothricin, respectively. Two mice at 400 mg/kg of S-F and 1 mouse at 50 mg/kg nourseothricin died, or became moribund and were euthanized. Histology of kidneys was specifically examined for all mice based on described delayed renal toxicity in rats for the streptothricin natural product [To identify the single-dose maximum tolerated dose (MTD) of S-F and nourseothricin, CD-1 mice were injected IP with ascending doses of 0, 50, 100, 200, and 400 mg/kg S-F and 5, 10, 20, and 50 mg/kg nourseothricin, respectively . For these studies, neutropenic mice were rendered mildly renal deficient with uranyl nitrate to more closely simulate human excretion kinetics . Tissue her dose , S5 Datarrn operon, SQ110, E. coli mutant strain at a frequency of approximately 6.2 × 10−9 but not in wild-type (wt) E. coli, which has 7 rrn operons, implying mutations were recessive. In total, 54, 2, and 5 independently isolated strains containing 16S rRNA C1054A, A1196C, and A1196G mutations, respectively, were identified. All grew noticeably slower than the SQ110 parent. In addition, 16S rRNA C1054T was identified in 16 strains that proved nonviable on passaging from frozen stocks and were therefore not further analyzed. In rrnB rRNA operon into the mutant strains, in contrast to absence of effect with the empty vector control , while demonstrated the expected high MICs for tetracycline   . Bindingcryo-EM)  Figs. Nocryo-EM)  was founcryo-EM) . This sicryo-EM) . It is cThe second S-F-binding site is found to partially overlap with site 2 of S-D  Figs. ThWhile not seen in S-F, site 4 of S-D is located in the peptide exit tunnel . The S-DE. coli, Klebsiella, Enterobacter, and A. baumannii. Their activity spectra are therefore highly relevant to the emerging gap in gram-negative antimicrobial coverage, even more so since they appear to act in a unique manor to kill bacterial cells.We demonstrate that streptothricins, in particular S-F, S-D, and the natural product mixture, nourseothricin, are highly active against contemporary, carbapenem-resistant Most of the existing description of streptothricin class antibiotic activity was from several decades ago and made use of unstandardized microbiological and physicochemical methods. In general, the natural product mixture was characterized as a whole without quantitative delineation of contributions from constituent streptothricins. Instead, here, we made use of modern preparative techniques and methods to characterize the activity of S-F and S-D in comparison with the natural product mixture currently available, gaining valuable insights into their respective properties.We found that S-F maintained potent antimicrobial activity, although it was about 6-fold less potent than S-D. Importantly, S-F showed at least 10-fold lower toxicity than S-D and the natural product mixture, nourseothricin, in vitro and in vivo. Notably, in single, maximal tolerated dose experiments, treatment with the nourseothricin natural product mixture was associated with proximal tubule kidney damage at relatively low doses (10 mg/kg). In contrast, S-F showed a favorable therapeutic ratio with substantial therapeutic effect at 50 to 100 mg/kg to the point of single-dose sterilization of CRE infection with minimal or no renal histopathological toxicity.A. baumannii, and inducing translational miscoding [Biologically, streptothricins demonstrate activity similar to 2-deoxystreptamine (2-DOS) containing aminoglycosides such as gentamicin, amikacin, tobramycin, and apramycin, both inhibiting protein synthesis with rapid, potent bactericidal activity , as obseiscoding ,26. It wThe 2-DOS aminoglycosides are known to bind helix 44 in 16S rRNA (residues 1400 to 1410 and 1490 to 1500), forming a binding pocket near the ribosome decoding center –31 S14C. In doinInstead, our structural and resistance studies identified an interaction with helix 34 distinct from other known translation inhibitors. Specifically, S-F makes direct polar contacts with nucleobases, C-1054 and A-1196. This contrasts with the stacking interactions of tetracyclines  with C-1trans . Although comparative analysis between naturally occurring S-F analogs has not been systematically performed, an S-D analog with a cis-fused streptolidine lactam moiety  showed a 32-fold increase in MIC for B. subtilis [Our structural data further explain streptothricin acetyl transferase-based resistance to S-F and S-D associated with acetylation of the β-amino group of the first β-lysine attached to the gulosamine sugar. Specifically, our modeling indicates that acetylation cannot be accommodated in the S-F binding pocket without steric clash with the phosphoribose backbone of U1052 and C1054 . Finallysubtilis  comparedsubtilis . The dissubtilis , or withsubtilis , removalsubtilis , presumasubtilis , or LLBLsubtilis ), or metsubtilis  or LL-ABsubtilis ), were tE. coli  and T. PDB 4V8B , 3T1H [6PDB 4V8B , 1IBM [2PDB 4V8B , 4WQ1 [6PDB 4V8B , 6XHV [6PDB 4V8B , and 4V8PDB 4V8B ) ribosomPDB 4V8B –68 and iPDB 4V8B , supportPDB 4V8B . It shouIt was theorized previously that the observed concentration dependence of S-F induced miscoding might be a consequence of multiple binding sites with presumably synergistic or additive mechanisms of action . HoweverOf interest, the nucleotides corresponding to 16S rRNA C1054 and A1196 in human 18S rRNA  are conA. baumannii pathogens with in vivo confirmation of efficacy against an emblem of gram-negative antibiotic resistance. We also find evidence for unique mechanism of action targeting 16S rRNA helix-34 distinct from other known translation inhibitors. We therefore believe that further early-stage exploration of the historic scaffold is warranted with the ultimate goal of identification of analogs with potential for therapeutic development.In summary, we present data for compelling bactericidal activity of S-F against contemporary multidrug-resistant CRE and A. baumannii isolates were from the AR-Bank and the Multidrug Resistant Organism Repository and Surveillance Network (MRSN) at the Walter Reed Army Institute of Research (WRAIR). Strains are listed in E. coli SQ110, was from the Coli Genetics Stock Center  and was originally described by Squire and colleagues [CRE isolates were from the FDA-CDC Antimicrobial Resistance Isolate Bank (AR-Bank) including the Nevada Strain (AR-0636) and the BIDMC clinical collection sequenced as part of the CRE genomics project . A. baumlleagues .2O. The flow rate was adjusted using compressed air to 0.6 mL/min. Purifications were run in batches of approximately 300 mg of nourseothricin sulfate, which was diluted in 0.6 mL of H2O and loaded dropwise directly onto the top of the column. A mobile phase of 10% methanol/H2O was used for elution and fraction sizes of 3 mL were collected. Fractions testing positive for the ninhydrin stain were analyzed for purity by LC–MS. Pure S-D began eluting after approximately 120 mL of mobile phase, followed by mixed fractions of streptothricin D/E/F, and, finally, pure streptothricin F. Pure fractions for S-D were combined, frozen, and lyophilized to give a powdery, off-white solid. Pure fractions for S-F were combined, frozen, and lyophilized to give a powdery, off-white solid. Additional experimental details including 1H- and 13C-NMR spectra, mass spectrometry, as well as elemental analysis data of S-F and S-D can be found in the supporting information.Purification of separate streptothricin compounds from nourseothricin sulfate  was performed through modification of a previously reported method . A glass8 CFU/mL) in 0.9% NaCl solution. This suspension was diluted 1:300 into cation-adjusted Mueller–Hinton broth  to achieve a final inoculum concentration of approximately 5 × 105 CFU/mL in 100 μL well volumes in round-bottom, 96-well plates . Doubling dilutions or antimicrobials were dispensed using the D300 digital dispensing method for final doubling dilution concentrations ranging from 0.125 to 256 μg/mL [The Clinical Laboratory and Standards Institute (CLSI) broth microdilution reference method was used for MIC testing . Bacteri56 μg/mL –76. The −1 taking into account Poisson distribution considerations.Time-kill studies were performed as previously described . At timeProkaryotic and eukaryotic nanoluciferase reporter constructs were constructed as follows: For the bacterial nanoluciferase (Nluc) reporter construct, a DNA fragment containing a 516-bp fragment from pNL1.1 Promega, , including the NLuc open reading frame and Shine-Delgarno sequence and an upstream T7 promoter sequence (TAATACGACTCACTATAGG) was synthesized as a gBlock by IDT . The T7_Nluc fragment was then amplified using primer pairs, T7Nluc-F and T7NLuc-R , and ligFor the eukaryotic Nluc reporter construct, the Nluc open reading frame without the methionine initiation codon was amplified with primers, Nluc_BamHI-F and Nluc_PstI-R . The ampE. coli S30 Extract System for Circular DNA  and 100 ng of pCR XL TOPO-T7NLuc were then added to each reaction well, making use of the upstream vector, pLac promoter rather than the cloned T7 promoter for expression in the current studies. After incubation at 37°C for 60 minutes, an equal volume of PBS buffer with 1 μg/mL Nluc-specific furimazine substrate  was added to each well, and luminescent signal was detected with an Infinite M1000 Pro  plate reader. Eukaryotic in vitro translation was performed with the TnT-T7 Quick Coupled Transcription/Translation System . TnT master mix, 100 ng of pT7CFE1-NLuc plasmid DNA, and methionine were added to test wells per manufacturer’s instructions. After incubation at 30°C for 80 minutes, Nluc signal was detected as described above.Nourseothricin, S-D, and S-F were distributed to a 384-well black microplates . For bacterial in vitro translation assays, 5 cells/cm2 and approximately 7.0 × 104 cells/cm2, respectively, in M199 medium lacking phenol red supplemented with 3% porcine serum and a final concentration of 125 nM SYTOX Green. Approximately 1 day after plating (at 50% to 75% confluence), the cell lines were treated with 2-fold doubling dilutions of nourseothricin (Gold Biotechnology), S-D, and S-F, dispensed with the HP D300 digital dispensing system  using Tween-20 as the surfactant. Microplates were vortexed for 30 seconds to ensure thorough mixing within the wells prior to incubation. SYTOX Green fluorescence was read with a TECAN M1000 multi-mode reader using excitation 485 ± 7 nm and emission 535 ± 10 nm settings at indicated time points with otherwise continuous incubation at 37°C with 5% CO2 for 5 days.J774A.1 mouse macrophage and LLC-PK1 porcine renal tubule epithelial cell lines were plated in 384-well tissue culture dishes with white walls and clear bottoms  at approximately 7.0 × 10−1 of purified streptothricin-F or nourseothricin. On day 3 post dosing, mice were euthanized and kidneys fixed in 10% neutral phosphate-buffered formalin overnight, embedded in paraffin, and 10 μm tissue sections stained with hematoxylin and eosin at an institutional core facility using standard procedures as described previously [CD-1 (ICR) female mice weighing 25 to 30 g each were purchased from Charles River Laboratories . Mice were injected IP with 0, 10, 20, 50, 100, 200, and 400 mg kgeviously .6 CFU of the CRE Nevada strain AR-0636 suspended in 100 μL PBS into the thigh muscle [Mice were pretreated with cyclophosphamide and uranyl acetate and infected by injection of 10h muscle . Mice weSingle ribosomal operon strain, SQ110, was grown overnight in CAMHB at 37°C and resuspended in one-fifth volume in fresh medium. Thereafter, 200 μL of the suspension was plated on selective LB agar plates containing nourseothricin  at 32-, 64-, and 128-fold the MIC of the parent SQ100 strain and incubated for 48 to 72 hours at 35°C in ambient air until mutant colonies became visible. Selected colonies were passaged on plates with matching concentrations of nourseothricin for further analysis. Aminoglycoside resistance mutants were isolated in a similar manner selecting on apramycin.Genomic DNA from nourseothricin-resistant strains was extracted using the Wizard Genomic DNA Purification Kit  from 3 mL of overnight culture in CAMHB containing nourseothricin . Subsequent genomic DNA was amplified using primers F16S+23S, R16S+23S, and 16SR , with prA. baumannii (strain AB0057) using a sucrose cushion followed by sucrose gradient centrifugation [2 until data collection.70S ribosomes were purified from fugation  as we prfugation . Prior t2 using SerialEM [2 using SerialEM [Cryo-EM data were recorded at a defocus range of −1 to −2.5 on a Titan Krios equipped with a K3 direct electron detector (Gatan). A total of 3,054 micrographs for S-F were recorded in superresolution mode at 105K magnification resulting in a physical pixel size of 0.848 Å (0.424 Å superresolution). Each image was collected over 40 frames with a total dose of 46 e/ÅSerialEM . For S-DSerialEM .For both S-F and S-D, image stacks were aligned and binned by 0.5 (0.75 for S-D) using patch motion and the contrast transfer function (CTF) was estimated by patchCTF in cryoSPARC v3 . A subseA. baumannii ribosome [2+ ions and coordinated waters that directly interact with S-F were included in the final model. Subsequent modelling and refinement were carried out in Coot [Previously determined structures of the ribosome  were useribosome . After i in Coot  and phen in Coot . Final r in Coot . Final cS1 Results(PDF)Click here for additional data file.S1 Materials and Methods(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.S7 Table(PDF)Click here for additional data file.S8 Table(PDF)Click here for additional data file.S9 Table(PDF)Click here for additional data file.S10 Table(PDF)Click here for additional data file.S11 Table(PDF)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig1H NMR, distinct chemical shifts for protons attached to the same carbon atom are labeled Ha and Hb, respectively.The numbering scheme for streptothricin F and streptothricin D. In the (PDF)Click here for additional data file.S4 FigNourseothricin MIC was 2 μg/mL.(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S6 Fig(PDF)Click here for additional data file.S7 Fig(PDF)Click here for additional data file.S8 Fig(PDF)Click here for additional data file.S9 Fig(PDF)Click here for additional data file.S10 Fig(PDF)Click here for additional data file.S11 Fig(PDF)Click here for additional data file.S12 Fig(PDF)Click here for additional data file.S13 Fig(PDF)Click here for additional data file.S14 Fig(PDF)Click here for additional data file.S15 Fig(PDF)Click here for additional data file.S16 Fig(PDF)Click here for additional data file.S17 Fig(PDF)Click here for additional data file.S1 DataContains underlying data for (XLSX)Click here for additional data file.S2 DataContains underlying data for (XLSX)Click here for additional data file.S3 DataContains underlying data for (XLSX)Click here for additional data file.S4 DataContains underlying data for (XLSX)Click here for additional data file.S5 DataContains underlying data for (XLSX)Click here for additional data file.S6 DataContains underlying data for (XLSX)Click here for additional data file.S7 DataContains underlying data for (XLSX)Click here for additional data file.S8 DataContains underlying data for (XLSX)Click here for additional data file.


We investigated the subcellular localization of mRNAs encoding cytoplasmic EGFP, an actin marker protein Lifeact tagged with EGFP, and several EGFP-fused proteins AoSec22, AoSnc1, AoVam3, and AoUapC that localize to the endoplasmic reticulum (ER), the apical vesicle cluster Spitzenkörper, vacuolar membrane, and plasma membrane, respectively. Visualization of these mRNAs by smFISH demonstrated that each mRNA exhibited distinct localization patterns likely depending on the mRNA sequence. In particular, we revealed that mRNAs encoding Lifeact-EGFP, EGFP-AoSec22, EGFP-AoVam3, and AoUapC-EGFP, but not cytoplasmic EGFP and EGFP-AoSnc1, were preferentially localized at the apical cell, suggesting certain mechanisms to regulate the existence of these transcripts among hyphal regions. Our findings provide the distinct localization information of each mRNA in the hyphal cells of A. oryzae.Although subcellular localization analysis of proteins fused with enhanced green fluorescence protein (EGFP) has been widely conducted in filamentous fungi, little is known about the localization of messenger RNAs (mRNAs) encoding the EGFP-fused proteins. In this study, we performed single-molecule fluorescence  Aspergillus oryzae has been utilized in the fermentation and brewing industries because it can safely produce copious amounts of useful enzymes  that have a crucial role in vesicular trafficking have been identified, and enhanced green fluorescence protein (EGFP)-fused SNARE proteins have been analyzed for understanding this subcellular localization  has been widely applied to many organisms  was used, and an A. oryzae strain NSlDS1 was used as a host. A strain expressing cytoplasmic EGFP was constructed by A. oryzae transformation of pgPamyB-SmaI-egfp-TamyB. To construct a Lifeact-EGFP-expressing plasmid, pgPamyB-SmaI-egfp-TamyB was used as an expression vector. The DNA sequence of Lifeact was amplified by PCR using PrimeSTAR GXL DNA polymerase (Takara) and the primer set: YHK251, 5′- ATCAAGAAGTTCGAGTCCATCTCCAAGGTGGTGATGGTGAGCAAGGGCGAGGAG-3′; YHK252, 5′-CTCGAACTTCTTGATGAGATCGGCGACACCCATCTGTGGGGTTTATTGTTCAGAGAAGGG-3′, and then ligated with the PCR-amplified vector by In-Fusion reaction (Takara), resulting in pgLifeactegnD. For preparing an AoUapC-EGFP-expressing plasmid pgPaUAPCegnD, the abovementioned same cloning strategy with the expression vector was conducted using the primer set: AT143, 5′-TAAACCCCACAGCCCATGGATGGACCAGATCAGATTGGTCCTG-3′; AT144, 5′-CTTGCTCACCATCCCTACAATAGCCGGACCAGCCTCGG-3′. To generate strains expressing EGFP-AoSec22, EGFP-AoSnc1, or EGFP-AoVam3, plasmids pgPaGAoSec22, pgPaGAoSnc1, or pgPaGAoVam3 was introduced into the strain NSlDS1  was used. The CD liquid medium was used for microscopy, sterilized by filtering through 0.2-μm filters. Approximately 104 conidia of each strain were inoculated into 100 μl of CD liquid medium, cultured in a poly-lysine-coated glass bottom dish (Matsunami), and incubated at 30°C for 20 h. smFISH was conducted with modifications to improve the sensitivity of the previous method  with 100 mM glycine, and incubated at RT for 5 min in the second wash. To partially digest the cell wall, cells were incubated with 100 μl solution of 5 μg/μl zymolyase  in fixation buffer  at 30°C for 1.5 h. After washing twice with 150 μl of fixation buffer, cells were incubated with 100 μl solution of 0.1% Triton X-100  in fixation buffer for 10 min at RT for permeabilization. After three times wash with fixation buffer, cells were suspended in 100 μl of hybridization buffer (LGC Biosearch Technologies) containing 10% deionized formamide with or without the addition of 1 μl of 12.5 μM of the probe  overnight. After replacement of the hybridization buffer with 100 μL of buffer A (LGC Biosearch Technologies) containing 10% deionized formamide, cells were incubated with 100 μL of buffer A at 30°C for 30 min. To stain the nuclei, the cells were incubated with 100 μl of buffer A containing 0.05 ng/μl DAPI (Molecular Probes) using a stock solution of 1 mg/ml in DMSO at 30°C for 30 min. Buffer A was then replaced with 100 μl of buffer B (LGC Biosearch Technologies) and incubated at RT for 3 min. The cells were then mounted in 50 μl of Vectashield Antifade Mounting Medium (Vector Laboratories) to minimize fluorescence bleaching and subsequently observed under the fluorescence microscope. The smFISH probe (LGC Biosearch Technologies) was used according to the instructions of the manufacturer. The smFISH probe for egfp consisted of mixtures of 18–22 nt from 37 regions in 717 bp, each region was linked to CAL Fluor Red 610 . The information of the egfp probe sequence is summarized in For culturing hyphal cells of A. oryzae cells. Images were taken, merged, and processed for adjusting to the same fluorescence scaling by using the NIS Elements AR software (Nikon).An ECLIPSE Ti2-A inverted microscope (Nikon) was employed, equipped with a CFI Plan Apo Lambda 100 × objective lens , a DS-Qi2 digital camera, an LED-DA/FI/TX-A triple band filter , an LED light source X-LED1 and differential interference contrast (DIC) to observe the EGFP, CAL Fluor Red 610, and DAPI fluorescent signals and hyphal morphology of n = 10 cells from two independent experiments).For determining the relative intensity, each mRNA-derived fluorescence intensity was measured at 50 μm hyphal areas of apical (from the tip), middle (septum on the center), and basal (from the germinated conidium but excluding it) regions and corrected for the background intensity taken from the extracellular space adjacent to the cell measured. Statistical analysis was performed by Tukey–Kramer test with the quantitative data of each mRNA fluorescence image .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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Hepatitis B virus (HBV), an international public health concern, is a leading viral cause of liver disease, such as hepatocellular carcinoma. Sequence-specific ribozymes derived from ribonuclease P (RNase P) catalytic RNA are being explored for gene targeting applications. In this study, we engineered an active RNase P ribozyme, M1-S-A, targeting the overlapping region of HBV S mRNA, pre-S/L mRNA, and pregenomic RNA (pgRNA), all deemed essential for viral infection. Ribozyme M1-S-A cleaved the S mRNA sequence efficiently in vitro. We studied the effect of RNase P ribozyme on HBV gene expression and replication using the human hepatocyte HepG2.2.15 culture model that harbors an HBV genome and supports HBV replication. In these cultured cells, the expression of M1-S-A resulted in a reduction of more than 80% in both HBV RNA and protein levels and an inhibition of about 300-fold in the capsid-associated HBV DNA levels when compared to the cells that did not express any ribozymes. In control experiments, cells expressing an inactive control ribozyme displayed little impact on HBV RNA and protein levels, and on capsid-associated viral DNA levels. Our study signifies that RNase P ribozyme can suppress HBV gene expression and replication, implying the promise of RNase P ribozymes for anti-HBV therapy. E. coli comprises of a protein subunit, C5 protein, and a catalytic RNA subunit, M1 RNA -labeled oligonucleotide primers specific for the S mRNA following protocols described previously [HepG2  and HBV-infected HepG2.2.15 cells were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum as defined previously ,22. HepGeviously ,24. Primeviously ,24.The DNA sequence coding for HBV substrate s37 was constructed by PCR using pGEM3zf(+) as a template and oligonucleotides AF25 (5′-GGAATTCTAATACGACTCACTATAG-3′) and s37-3 (5′-CGGGATCCGTTCCTGGAAGTAGAGGACAAACGGGCAATCTATAGTGAGTCGTATTA-3′) as 5′ and 3′ primers, respectively.347C348 -> C347U348, C353C354C355G356 -> G353G354A355U356) at the catalytic domain (P4 helix) [Plasmid pFL117 and pC102 contain the DNA sequence coding for active ribozyme M1 RNA and inactive mutant ribozyme C102 driven by the T7 RNA polymerase promoter ,26. Plas4Cl, 100 mM CaCl2, 3% glycerol, 0.1% xylene cyanol, 0.1% bromophenol blue) [d were the averages from three independent experiments [4Cl, 100 mM MgCl2), following procedures noted previously [cat/Km)s were determined from triplicate experiments [For the binding between M1GSs and substrate s37, assays were performed in vitro in buffer E  ,27,28. Tol blue) . The valeriments ,27,28. Weviously ,30,31. Teriments ,27,28.Constructs pU6-M1-S-A, pU6-M1-S-I, and pU6-M1-P were generated by inserting the DNA sequences coding for ribozyme M1-S-A, M1-S-I, and M1-P into the expression vector pU6. Following the experimental procedures described previously ,32, HepGM1GS expression-associated cytotoxicity was assessed by an MTT assay . We grew cells in 96-well plates carrying empty vector construct pU6 and ribozyme-containing constructs . At different time points, we added 3--2,5-diphenyl tetrazolium bromide  (5 mg/mL in PBS) to each well and determined cell viability following the manufacturer’s recommendations . We thenRNA and protein samples were isolated with previously defined methods ,21,30,33v/v) were added to the lysate samples, with viral capsids precipitated via centrifugation. The capsid samples were then resuspended in buffer F  and digested with proteinase K. The viral DNAs were finally purified with phenol and chloroform extraction and ethanol precipitation [To isolate capsid-associated viral DNA, cells were lysed in buffer E  and subsequently incubated with DNase I at 37 °C for 6 h ,31,33. Epitation ,31,33.Similar experimental procedures were also used to purify extracellular capsid-associated HBV DNAs in culture media. The culture media were first digested with DNase I in buffer E and then incubated with proteinase K in buffer F to release encapsidated DNA. We finally purified the released HBV DNAs by phenol and chloroform extraction and ethanol precipitation ,31,33.7 to 100 [Quantitative PCR (qPCR) was utilized to assess capsid-associated DNA levels via an ABI 7500 device  or an iCycler Real-Time PCR Detection System . We employed TaqMan real-time PCR primers including 5′ primer P1 (5′-AGAAACAACACATAGCGCCTCAT-3′), 3′ primer P2 (5′-TGCCCCATGCTGTAGATCTTG-3′), and probe P3 (5′-TGTGGGTCACCATATTCTTGGG-3′) . The PCR7 to 100 ,31,33. TEfficient ribozyme targeting demands access to interacting with its target mRNA. We used the dimethyl sulphate (DMS)-based mapping approach ,24,37 to5-fold less active at minimum compared to M1 RNA [Active and inactive M1GS ribozymes, M1-S-A and M1-S-I, were generated by joining the 3′ end of ribozymes M1 RNA and C102 RNA to a guide sequence complementary to the target S mRNA sequence, respectively. C102 RNA is an inactive ribozyme mutant originating from M1 RNA, and contains various point mutations at the P4 catalytic domain and C012 is 1 × 10o M1 RNA . To detecat/Km)s) of the ribozymes (d) with gel-shift assays revealed that the binding affinity of M1-S-A to substrate s37 was similar to that of M1-S-I. Thus, we used M1-S-I as the antisense effect control because this ribozyme exhibited comparable affinity to s37 as M1-S-A but could not cleave the substrate due to its mutations at the P4 active domain.In vitro, active ribozyme M1-S-A efficiently cleaved substrate s37, which included a HBV S mRNA sequence of 37 nucleotides . Conversibozymes . MeasuriHepG2.2.15 cells were used as the HBV infection model system to study the effects of ribozymes on HBV gene expression and propagation in cells. These cells originated from human hepatoma HepG2 cells, containing a stable transfected full-length genome of HBV (ayw subtype). In culture, HepG2.2.15 cells produced HBV B surface (HBsAg) and e (HBeAg) antigens and engaged viral genome DNA replication .We cloned DNA sequences encoding ribozymes M1-S-A, M1-S-I, and M1-P into expression vector pU6, which is comprised of the small nuclear U6 RNA promoter for expressing M1GS and a green fluorescence protein (GFP) expression cassette. HepG2.2.15 cells were transfected with pU6 vector or pU6-M1-S-A, pU6-M1-S-I, and pU6-M1-P. After selection, those containing the transfected constructs were cloned. Northern blot analysis showed similar levels of ribozymes in these cells, with human H1 RNA as the loading control . RibozymWe studied the HBV 3.5 kb and 2.4/2.1 kb transcript levels, which represent the pre-C mRNA/pregenomic RNA and pre-S/L mRNA, respectively . We usedA decrease in HBV protein expression and production is expected due to suppression in HBV transcript levels. The levels of HBV production were assessed by studying the HBsAg and HBeAg antigen levels in cell culture supernatants . The M1-We investigated if ribozymes decreased HBV DNA replication. To achieve this, we employed quantitative PCR (qPCR) to assay intracellular capsid-associated HBV DNA levels. Cells expressing M1-S-A exhibited a decrease of 300-fold in HBV DNA levels, compared to HepG2.2.15 cells transfected with the pU6 empty vector A. MeanwhWe also studied the effect of RNase P ribozyme targeting in regard to virus particle production. Capsid-associated viral DNA levels in culture media reflect the copy numbers of viral genomic DNA found in HBV particles released in the media. Using the qPCR approach, we assayed the levels of capsid-associated HBV DNAs in media from the cell cultures without any ribozymes or with different ribozymes. The media from cell cultures with M1-S-A exhibited a decrease of about 300-fold in HBV DNA levels, compared to that from the culture of HepG2.2.15 cells with the pU6 empty vector B. The ceThe RNase P ribozyme technology is a promising strategy for gene targeting because it employs M1GS RNA to cleave a target RNA ,38. PrioMultiple lines of evidence presented here imply M1GS RNAs to be active and suppress HBV replication in cultured cells. First, the presence and expression of M1GS sequences did not substantially alter the capability and growth of HepG2.2.15 cells for up to three months. Second, the ribozymes appeared to be active in cleaving the S mRNA in cultured cells. Decreases in both HBV gene expression and viral DNA replication were observed in cultured cells that were transfected with pU6-M1-S-A but not control constructs pU6, pU6-M1-S-I, or pU6-M1-P. Third, the magnitude of decrease in the HBV transcript levels in cultured cells correlated well with that in the HBsAg/HBeAg and HBV DNA levels . Hence, E. coli) is linked to a guide sequence targeting a different region of the S mRNA sequence. M1GS ribozymes were introduced as exogenous gene-targeting agents to degrade the target HBV RNAs. Because of this, the M1GS ribozyme can be used to target both cytoplasmic and nuclear mRNAs while the EGS-mediated gene targeting action to downregulate gene expression is believed to work in the nuclei where human RNase P is primarily localized [In our previous studies, customized designed external guide sequences (EGSs) were constructed and introduced into mouse cells and human HepG2.2.15 cells to induce endogenous mouse and human RNase P, respectively, to cleave HBV RNA sequences and inhibit HBV gene expression and replication ,39. In tocalized . Moreoveocalized .As an anti-HBV therapeutic, the M1GS ribozyme approach may have several potential limitations and issues that should be addressed. First, M1GS RNA needs to be delivered to specific types of tissues and cells such as the liver and hepatocytes. New gene-transfer methods, including liposome-based or viral vector-based agents, are in development and have shown great promise for the efficient delivery of exogenous therapeutic gene-expression cassettes to different tissues and cells in vivo. These reagents can be used to deliver the expression cassettes of M1GS RNAs into the liver and hepatocytes.Second, RNase P ribozymes with better targeting activities should be developed. In vitro selection procedures have been documented to be capable of generating highly active and functional RNA molecules, including ribozymes, with enhanced activity ,42,43. WAnother potential limitation is the potential off-target effects of the M1GS ribozyme. Little is currently known about M1GS sequence specificity as a gene targeting agent. Additional studies are needed to investigate the sequence specificity and potential cytotoxicity of M1GS ribozymes in a variety of human cells and tissues as well as in animal models. These studies will reveal the safety profiles of RNase P ribozyme for its use in potential clinical applications. As observed in previous studies using mouse and human RNase P-associated EGSs, effective inhibition of HBV gene expression and replication was found in M1GS ribozyme-expressing cells in the current study. Our study here raises exciting prospects for generating M1GS ribozymes for anti-HBV therapy. There are currently several compounds that have been approved by the FDA for treatment of HBV infection and diseases ,15. Thes


Escherichia coli K-12 is a model organism for bacteriology and has served as a workhorse for molecular biology and biochemistry for over a century since its first isolation in 1922. However, Escherichia coli K-12 strains are phenotypically devoid of an O antigen (OAg) since early reports in the scientific literature. Recent studies have reported the presence of independent mutations that abolish OAg repeating-unit (RU) biogenesis in E. coli K-12 strains from the same original source, suggesting unknown evolutionary forces have selected for inactivation of OAg biogenesis during the early propagation of K-12. Here, we show for the first time that restoration of OAg in E. coli K-12 strain MG1655 synergistically sensitises bacteria to vancomycin with bile salts (VBS). Suppressor mutants surviving lethal doses of VBS primarily contained disruptions in OAg biogenesis. We present data supporting a model where the transient presence and accumulation of lipid-linked OAg intermediates in the periplasmic leaflet of the inner membrane interfere with peptidoglycan sacculus biosynthesis, causing growth defects that are synergistically enhanced by bile salts. Lastly, we demonstrate that continuous bile salt exposure of OAg-producing MG1655 in the laboratory, can recreate a scenario where OAg disruption is selected for as an evolutionary fitness benefit. Our work thus provides a plausible explanation for the long-held mystery of the selective pressure that may have led to the loss of OAg biogenesis in E. coli K-12; this opens new avenues for exploring long-standing questions on the intricate network coordinating the synthesis of different cell envelope components in Gram-negative bacteria. Escherichia coli K-12 is the most studied microorganism, widely used in laboratories for studying bacteriology and as a tool for molecular biology. E. coli K-12 strains originating from the same institute harbour independent inactivation of O antigen biosynthesis and assembly pathways, strongly suggesting that E. coli K-12 was originally producing an O antigen. However, the reason why it is devoid of O antigen, where most other E. coli lineages are not, remains a long-standing question. Our work has uncovered a previously unknown selection pressure of bile salts on bacterial O antigen biogenesis, which provides a plausible scenario for how the early propagation of E. coli K-12 strains in bile salt-containing media could have led to loss of O antigen in K-12. Our results also suggest that the accumulation of O antigen intermediates in the bacterial periplasm may interfere with bacterial cell wall synthesis, which opens a new avenue of research into the interplay of different cell envelope component synthesis pathways. Escherichia coli K-12, the most intensively studied microorganism, has served as a model organism for modern bacteriology and as a workhorse for biochemistry, genetics, and molecular biology. The wild-type strain of E. coli K-12 was first isolated in 1922 from the stool of a diphtheria patient and was soon after deposited in the strain collection of the Department of Bacteriology at Stanford University, where it was used in the teaching laboratories and maintained in stab cultures  typically found on newly isolated wild-type strains . T,2. TEsch in 1944  where it in 1944 , which h in 1997  Fig 1A)Escherich strains .E. coli is responsible both for serological specificity (due to its high antigenic variability . A . A rfb) 2 strain , confirmterature , suggestE. coli strains in the early 20th century relied on specialised culture media, including MacConkey medium . Wh. WhO16 ice to DV  despite ce to DV . Indeed,t OAgO16  and wouleriplasm  also faieriplasm . These fDOC Figs  and S4E sistance . Similar5-SΔwaaL , confirmsistance . In contP-OAgO16  in the pP-OAgO16 . Interes5-SΔwaaL , while dt to DOC . We weresistance . This suE. coli K-12 has been heavily domesticated in the laboratory, we questioned whether sensitivity to DV can also be observed in other strains, including clinically relevant isolates. Surprisingly, we found that S. flexneri 2457TΔrmlD with disrupted S. flexneri 2a-OAg rhamnose saccharide synthesis, and uropathogenic E. coli (UPEC) ST131 EC958ΔwecA with disrupted O25b-OAg and ECA synthesis all showed increased resistance to DV compared to the wild-type parent strains  sensitised them to DOC   that the production of C55-PP-OAg is the primary cause of increased sensitivity to both VBS and BS. In contrast, MG1655-SΔwzy which accumulates C55-PP-RU in the periplasm was not sensitive to BS, suggesting that the periplasmic polymerised RUs, but not single RU sensitises MG1655-S towards BS.We also observed a synergistic effect between BS and vancomycin in inhibiting the growth of MG1655-S but not MG1655. Synergistic effects were also seen in UPEC and i strain . The synon to WT , with anon to WT . HoweverrmlC, dacA, wecA, wzz and waaL, but not yfdI and wzy in MG1655-S. Disruption of the D,D-carboxypeptidase DacA (PBP5) in MG1655 was previously reported to increase vancomycin resistance [wecA would abolish the synthesis of cyclic ECA which has been demonstrated to have increased vancomycin resistance [ΔrmlC, since the disruption of rmlC would accumulate increased level of dTDP-4-keto-6-deoxy-D-Glc, which may affect homeostasis of the saccharide substrate pool for making ECA. Disruption of wzz has been shown to have effects in ECA modality through potential cross-interaction between WzzE by Wzy [ΔwaaL and MG1655-SΔwaaU is however completely unexpected. Interestingly, although deletion of wzz slightly increased vancomycin resistance in MG1655-S, it drastically reduced vancomycin resistance in MG1655-SΔwaaL, suggesting that the accumulated periplasmic OAg intermediate at the modal chain length is primarily responsible for the elevated vancomycin resistance in MG1655-SΔwaaL. Strikingly, in the absence of vancomycin, exposure to BS alone selected for mutants with increased vancomycin resistance while encouraging the inactivation of OAg production. This again suggests an intricate interplay between OAg and PG synthesis pathways or cell envelope integrity as discussed earlier. However the exact nature of this interplay remains to be determined in future work currently underway. Based on our findings, we propose a model for explaining how E. coli K-12 lost its OAg, whereby the production of OAg generates transient polymerised C55-PP-OAg intermediate in the periplasm, and upon prolonged exposure to BS this enhances interference with cell wall synthesis pathways and growth inhibition which can be alleviated by disruption of OAg synthesis.We have also observed increased vancomycin resistance upon disruption of sistance  and heresistance . This coE by Wzy  when misO16 producing E. coli K-12 (MG1655-S) at room temperature for as long as 28 days did not select for differences in sensitivity to VBS, colicin E2, bacteriophage P1kc and T4GT7, suggesting that in-lab starvation alone could not pose adequate selection pressure for MG1655-S to lose its OAg production. Only when we cultured MG1655-S in commonly used media containing BS, could we select for characteristics of OAg biosynthesis disruption in over 90% of the culture population only within 1 week. It is likely that the selection pressure posed on MG1655-S in our in-lab adaptation experiments was a combination of the effects posed by BS on the transient presence of C55-PP-OAg in the periplasm discussed above, and the oxidative stress of exposure to long-term rich media [Regardless of the complex action of VBS in selecting MG1655-S mutants with disrupted OAg biogenesis, we were able to show that the stress posed by BS on MG1655-S was aided by vancomycin killing. A common way of preserving bacterial strains in the past was through stab agar culture (master culture) maintained at room temperature . This prch media .E. coli B by Delbrück and Luria used in their early work studying interactions between bacteria and T even phage [O7 biogenesis due to the inactivation of wbbD encoding the second glycosyltransferase for synthesising OAgO7, and LPS R1 core synthesis due to the inactivation of waaT, resulted in a truncated LPS outer core. Although it is unknown how the E. coli B strain lost its O antigen production, the inactivation of waaT may not be a direct result of BS adaptation, since LPS core truncation in both a rough background  and in E. coli strains grown overnight on lysogeny broth (LB)  10g/L, Bacto Yeast Extract  5g/L, NaCl 5 g/L) [The bacterial strains, plasmids used in this work are listed in l 5 g/L)  agar (1.4 and 5 mM CaCl2) and poured onto LB agar plate followed by incubation at 37°C for 18h. The clear top soft agar layer containing the phage lysate was carefully scrapped and mixed with 2 ml of LB-Miller media , vortexed and centrifuged. The clear media containing bacteriophage were collected and sterilised by 10 μl of chloroform. Phage titres were determined by spotting 5 μl of 10-fold serially diluted phage stock onto top LB soft agar plates containing 100 μl of MG1655 mid-exponential culture. Plaque forming units (pfu) were calculated after 18 h incubation of phage infected plates at 37°C.Bacteriophage P1kc (ATCC 11303-B23) and T4GT7  lysates were prepared by adding 100 μl of mid-exponential culture of MG1655 and phage stocks at a multiplicity of infection (MOI) of ~0.5 to 3ml of LB soft agar (0.75% w/v agar) supplemented with MC salts  and BP27 (rmlCL122W) using the Q5 high-fidelity polymerase (NEB) and oligos listed in The RmlC and WaaL expression constructs used in this study were constructed by PCR amplification of the RNase I leakage assay was done exactly as described previously . BrieflySuppressor mutant selection of MG1655-S to vancomycin and deoxycholate combination treatment was done by spreading an overnight MG1655-S culture grown in LB (1 in 2000 dilution in fresh LB media) onto LB agar supplemented with 100 μg/ml vancomycin and 0.1% (w/v) sodium deoxycholate, and subsequent incubation at 37°C overnight. Colonies were carefully streaked out onto non-selective LB plates and stored at -80°C for downstream analyses.For bacterial whole genome sequencing, genomic DNA of MG1655, MG1655-S and suppressor mutants was prepared using a Qiagen QIAamp DNA blood mini kit according to the manufacturer’s protocol. Samples were prepared for DNBseq DNA library construction (BGI) followed by DNBSEQ PE150 sequencing (BGI). The processed reads for each strain were then mapped onto the NCBI MG1655 reference genome (Accession number U00096) using Geneious 8.0. Sequence changes between our laboratory MG1655 and the online reference genome, as well as within-population variations were determined using the Geneious built-in function and are excluded from the analysis in MG1655-S and its suppressor mutants .600 of 1 and spotted (4 μl) in 10-fold serial dilutions onto MacConkey agar supplemented with or without vancomycin at different concentrations. Culture dilutions were also spotted onto LB agar supplemented without or with vancomycin, deoxycholate, and crystal violet at different concentrations, as indicated in figure legends. Plates were incubated overnight at 37°C and then imaged.For vancomycin susceptibility testing, overnight cultures of bacterial strains grown in LB were adjusted to ODColicin E2 protein was purified and used to determine the susceptibility of bacterial strains as described previously . BrieflywbbL under tet promoter control, 1 ng/ml anhydrotetracycline was also supplemented in media. Microtiter plates were incubated at 37°C with aeration in a CLARIOstar plate reader  programmed to measure the optical density (O.D. 600 nm) every 6 minutes over 18 h.Growth curves for bacterial strains were generated as described previously . Briefly7 cells/ml and 10 μl samples were spotted onto MH agar plates supplemented with vancomycin at concentrations from 500 μg/ml to 7.8 μg/ml in 2-fold serial dilutions in the presence or absence of 0.1% (w/v) sodium deoxycholate. Plates were incubated overnight at 37°C. The MIC value was determined as the lowest concentration of vancomycin at which bacterial lawn growth was inhibited.Vancomycin minimum inhibitory concentrations (MIC) were determined for bacterial strains according to the Clinical and Laboratory Standards Institute guideline . Briefly10 pfu. Plates were incubated overnight at 37°C and imaged.MG1655-S grown overnight at 37°C in LB broth was sub-cultured 1:1000 in 3 ml LB broth in a 15 ml tube supplemented with or without 0.1% (w/v) sodium deoxycholate in triplicate. Tube lids were loosely attached to allow aeration and tubes were incubated at 21°C statically for 1 day, 7 days and 28 days. Cultures from different time-points were 10-fold serially diluted and spotted onto LB agar plates supplemented with or without 100 μg/ml vancomycin and 0.1% (w/v) sodium deoxycholate, 20 μg/ml colicin E2, or 200 μg/ml vancomycin, or plates pre-spread with 200 μl of either phage P1kc or T4GT7 at the titre above 109 cells) grown at mid-exponential phase were harvested via centrifugation  and lysed in 50 μl of SDS sample buffer and heated at 100°C for 10 min. Lysates were then cooled to room temperature and treated with 50 μg/ml proteinase K  for 18 h at 60°C. PK treated samples were then heated again at 100°C for 10 min, and 2–5 μl of each sample was loaded onto 10–20% SDS-tricine gels  and LPS was silver stained exactly as described previously [Bacteria cells (10eviously . WhereveS1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S1 FigwbbL allelic replacement in the rfb gene cluster in MG1655 to construct strain MG1655-S. The respective O16 repeating units that would be produced in the cytosol denote the expected LPS products by each strain. Primers P1 and P2 used to generate MG1655-S are mapped onto rfb regions by black arrows. (B) PCR amplicons of wbbL region from MG1655 and MG1655-S (using primers P3 and P4) confirming the replacement of an intact wbbL CDS in MG1655-S. (C) Colicin sensitivity assay confirming increased resistance of MG1655-S due to regained O antigen substituted LPS production. Colicin E2 was used at 1 mg/ml and in subsequent 2-fold dilutions (5 μl spots). (D&F) Silver stained SDS-PAGE of LPS samples from two sets of E. coli K-12 strains . LPS patterns confirm restored production of O antigen substituted LPS in the engineered MG1655-S and UT5600-S strains. (E) Western immunoblotting of samples as in (D) with anti-O16 antibodies showing the restoration of O16 O antigen production in MG1655-S.(A) Schematic representation of the strategy employed for (TIF)Click here for additional data file.S2 Fig600 of 1 and spotted (4 μl) in 10-fold serial dilutions (100 to 10−6) onto LB agar supplemented with 0.0001% (w/v) crystal violet (CV) or 100 μg/ml vancomycin and CV (CVV100), or 5% and 2.5% (w/v) sodium deoxycholate (DOC). (C) RNase I leakage assay of MG1655 and MG1655-S grown on LB agar supplemented without or with 0.1% (w/v) DOC.(A&B) Bacterial cultures of indicated strains grown in LB media were adjusted to OD(TIF)Click here for additional data file.S3 FigGrowth curve of MG1655, MG1655-S and BP1-BP51 suppressor mutants in LB media (A) or in LB supplemented with (B-D) 100 μg/ml vancomycin and 0.1% (w/v) DOC (DV100). E) Silver staining of SDS-PAGE of LPS samples prepared from MG1655, MG1655-S and BP1-BP51 suppressor mutants.(TIF)Click here for additional data file.S4 Fig600 of 1 and spotted (4 μl) in 10-fold serial dilutions (100 to 10−6) onto LB agar supplemented without or with 0.1% (w/v) DOC and 100 μg/ml vancomycin (DV100) in the absence or presence of 0.2% (w/v) L-arabinose (DV100ara). (D) Western immunoblotting of proteinase K-treated LPS samples with anti-O16 antibodies showing OAgO16-capped LPS in MG1655-S (red dashed box) and the accumulated C55-PP-OAgO16 intermediates (green dashed box) in MG1655-S ΔwaaL and ΔwaaU mutants.(A & C) Silver staining of SDS-PAGE of LPS samples of MG1655, MG1655-S and their mutational derivatives. Supplementation of media with 0.2% (w/v) arabinose was used in samples as indicated (+ara). Bacterial cultures of indicated strains (B & E) grown in LB media were adjusted to OD(TIF)Click here for additional data file.


Magnetic chitosan/cellulose nanofiber-Fe(III) [M-Ch/CNF-Fe(III)] composites were isolated for the elimination of Cr(VI), Cu(II), and Pb(II) from aqueous solution. Various analytical methods, such as field emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FT-IR), X-ray diffraction analysis (XRD), and thermogravimetric analysis (TGA) were employed to determine the morphological, physicochemical, and thermal properties of the isolated M-Ch/CNF-Fe(III) composites. It was found that the M-Ch/CNF-Fe(III) composites were porous materials, and they have the potential to be implemented as an adsorbent for heavy metals removal. The adsorption efficiency of M-Ch/CNF-Fe(III) composites was determined for Cr(VI), Cu(II), and Pb(II) elimination with changing pH (pH 1.0–8.0), adsorbent doses (0.05–1.0 g), time (15–90 min), and temperature (28–80 °C). In addition, isothermal and kinetics studies were conducted to assess the adsorption behavior and mass transfer phenomena of M-Ch/CNF-Fe(III) composites as an adsorbent for Cr(VI), Cu(II) and Pb(II) elimination from aqueous solution. The outcomes of the present study reveal that the M-Ch/CNF-Fe(III) composites could be utilized as an adsorbent for the Cr(VI), Cu(II), and Pb(II) elimination from industrial effluents.  A contaminant and one of the 14 toxic chemicals potentially threatening human health, even at a trace level of contamination  bio-composite. The M-Ch/CNF-Fe(III) composite was utilized as a bio-sorbent for the adsorption of Pb(II), Cr(VI), and Cu(II) and their elimination from aqueous solution. Various analytical methods were employed to determine the physicochemical, morphological, and thermal properties of the M-Ch/CNF-Fe(III) composite. In addition, the isothermal behavior of the M-Ch/CNF-Fe(III) composite was studied for the adsorptive Cr(VI), Cu(II), and Pb(II) elimination from aqueous solution.In recent years, magnetic adsorbents have gained extensive interest in the adsorptive elimination of heavy metals from industrial effluents because of their numerous advantages as adsorbents, such as thermo-mechanical strength, chemical resistance, elimination efficiency, regeneration, and environmentally friendly nature ,25. Thus3·6H2O (purity ≥ 98%) and the ionic liquid 1-butyl-3-methylimidazolim chloride (purity ≥ 99%) were acquired from Sigma Aldrich, St. Louis, MO, USA. K2Cr2O7 (purity ≥ 99%), CuSO4·5H2O (purity ≥ 98%), and Pb(NO3)2 were purchased from Merck Chemicals, Selangor, Malaysia. All other chemicals used in the present study were of analytical grade. The CNF was isolated from empty oil palm empty fruit bunch fibers following a method discussed elsewhere . FeCl3·63O4 powder was conducted using the sol-gel method as discussed elsewhere [3·6H2O were mixed with 80 mL of ethylene glycol (C2H6O2). The mixture was then mixed vigorously until the mixture became clear. Subsequently, the mixture was heated for 20 h at 100 °C to obtain a dark brown gel. The obtained gel was washed with C2H5OH and deionized H2O, followed by centrifuging at 5000 rpm for 20 min. The precipitate (Fe3O4) was collected and dried in a furnace at 300 °C for 15 min. Subsequently, the dried Fe3O4 was grinned to produce Fe3O4 powder. Approximately 5 g of chitosan and cellulose blend (1:1) was mixed with 5 mL of 1-butyl-3-methylimidazolium chloride and 2 g of Fe3O4 powder. The mixture was mixed vigorously for 15 min. Subsequently, the mixture was emulsified by adding Tween 80 (4 mL) and vacuum pump oil (80 mL) at a temperature of 100 °C and a stirring speed of 1000 rpm. The emulsified mixture was then cooled and washed with deionized water and ethanol to remove the residuals. Finally, the synthesized M-Ch/CNF-Fe(III) composite was dried in a vacuum oven and stored at 4 °C prior to characterization and utilization.The preparation of the Fe3O4, chitosan, and the isolated M-Ch/CNF-Fe(III) bio-composite were determined using SEM . In addition, the dimensions and image of CNF isolated from OP-EFB fibers were determined using transmission electron microscopy (TEM) at an accelerating voltage of 80 kV. The functional groups and chemical bonding of CNF, chitosan, and the M-Ch/CNF-Fe(III) composite were determined using Fourier transform infrared (FTIR) spectroscopy within the frequency range of 4000–400/cm. The crystallinity indexes of Fe3O4, CNF, chitosan, and the M-Ch/CNF-Fe(III) bio-composite were determined by X-ray diffraction (XRD) at 40 kv and 40 mA. The percentage crystallinity indexes (CrI %) of Fe3O4, CNF, chitosan, and the M-Ch/CNF-Fe(III) composite were calculated using Equation (1).I200 is the intensity of the crystalline and amorphous regions, and amI is the intensity of the amorphous region. The thermal stabilities of Fe3O4, chitosan, CNF, and the isolated M-Ch/CNF-Fe(III) composite were determined using thermal gravimetric analysis (TGA). Approximately 10 mg of the sample was heated under a nitrogen atmosphere at a heating rate of 10 °C/min within a heating range from 30 °C to 700 °C. The surface morphology of CNF, Fe2Cr2O7, CuSO4·5H2O, and Pb(NO3) in deionized water, respectively. A certain amount of M-Ch/CNF-Fe(III) and 50 mL of aqueous solution were placed in a 100 mL conical flask, and the mixture was mixed vigorously using a magnetic stirrer. Concentrated NaOH and H2SO4 solutions were utilized to regulate the pH. The eluent was separated after adsorption using filter paper, and the metal ions’ concentrations in the eluent were determined using atomic adsorption spectroscopy. The percentage adsorptive Cr(VI), Pb(II), and Cu(II) elimination from the aqueous solution using the M-Ch/CNF-Fe(III) composite as an adsorbent was computed as shown in Equation (2).iC and tC are the Cr(VI), Cu(II), and Pb(II) concentrations (mg/L) initially and at time ‘t’, respectively. In addition, the maximum metal ion elimination efficiency (eq) was computed using Equation (3).M is the mass of the M-Ch/CNF-Fe(III) composite being used as an adsorbent, V is the volume (L) of the aqueous solution, and eC is the Cr(VI), Pb(II), and Cu(II) concentration (mg/L) at equilibrium. The experiments were conducted in triplicate, and the results are presented in this study as mean values ± standard deviation. The adsorptive Cr(VI), Cu(II), and Pb(II) elimination was conducted using M-Ch/CNF-Fe(III) as an adsorbent with varying adsorbent doses (0.05–1.0 g), times (15–90 min), pH values (pH 1.0–8.0), and temperatures (28–80 °C). The aqueous solution containing Cr(VI), Cu(II), and Pb(II) at concentrations of 100 mg/L, 200 mg/L, and 50 mg/L, respectively, was prepared by dissolving calculated amounts of KR2) values of Langmuir and Freundlich isotherm models. The linear forms of the Langmuir and Freundlich isotherm model equations can be written as shown in Equation (4) and Equation (5), respectively [a refers to the Langmuir constant, b expresses the maximum adsorption value, fK (L/mg) denotes to the Freundlich affinity constant, and n is the Freundlich exponential constant. The Langmuir and Freundlich isotherm models were employed to determine the isothermal behavior for the adsorptive Cr(VI), Cu(II), and Pb(II) elimination from an aqueous solution using the M-Ch/CNF-Fe(III) composite. The adsorption experiments were performed at ambient temperature and pH 5.0 while varying the M-Ch/CNF-Fe(III) composite doses from 0.05 g to 0.5 g as a function of adsorption time from 5 min to 90 min. The best-fitting isotherm model for the adsorptive Cr(VI), Cu(II), and Pb(II) elimination using the M-Ch/CNF-Fe(III) composite was predicted based on the regression coefficient (ectively .(4)1qe=eq and R2 values. The linear forms of the pseudo-1st- and 2nd-order kinetics equations are presented in Equation (6) and Equation (7), respectively [eq and tq represent the adsorption efficiency at equilibrium and at adsorption time ‘t’, respectively. In addition, k1 and k2 denote the adsorption rate constant of the psuedo-1st-order and pseudo-2nd-order kinetics model equations for Cr(VI), Cu(II), and Pb(II) elimination using the M-Ch/CNF-Fe(III) composite as an adsorbent. The kinetic behavior for the Cu(II), Cr(VI), and Pb(II) elimination from aqueous solution was determined using pseudo-1st-order and pseudo-2nd-order kinetics model equations. The experiments were performed at varying temperatures from ambient temperature (28 ± 1 °C) to 70 °C as a function of time from 5 min to 60 min at pH 5.0 and adsorbent doses of 0.25 mg. The suitability of the best-fitting kinetics model equation was assessed by comparing the ectively .(6)ln(qThe reusability of the M-Ch/CNF-Fe(III) composite was determined via adsorption/desorption of Cr(VI) from aqueous solution for 6 cycles. The adsorption experiments were conducted for the elimination of Cr(VI) using the M-Ch/CNF-Fe(III) composite at a Cr(VI) concentration of 100 mg/L and adsorbent doses of 0.5 g/L, with the pH being 4.0 and at ambient temperature for an adsorption time of 30 min. After adsorption, the adsorbent was separated using a magnet, and the separated adsorbent was placed in 50 mL of 0.5 M HCl solution for the desorption of the adsorbed Cr(VI). The mixture was then vigorously stirred using a magnetic stirrer for 30 min. Subsequently, the adsorbent was separated and washed with deionized water prior to reuse for the subsequent adsorption/desorption experiments. + and SO42− during the acid hydrolysis process using sulfuric acid [4 shows a rough surface with a diameter of 100–200 nm. However, the SEM image of the M-Ch/CNF-Fe(III) composite shows the surface morphology of the isolated M-Ch/CNF-Fe(III) composite was porous and rough, with irregular shapes  composite. The broad absorption peaks obtained for chitosan and CNF approximately at 3420/cm correspond to N-H and O-H bonds, respectively [3O4 spectrum at 3420/cm reveals the existence of O-H bond. The presence of an O-H bond on the Fe3O4 spectrum was due to the presence of ethylene glycol, which was utilized in the formation of Fe3O4 from FeCl3·4H2O. The existence of the adsorption peak at 595/cm on the spectra of Fe3O4 and the M-Ch/CNF-Fe(III) composite reveals the formation of Fe-O bonds on the surface of the M-Ch/CNF-Fe(III) composite [ectively . The absectively ,33. Howeectively  obtainedomposite . Similar3O4, chitosan, CNF, and the M-Ch/CNF-Fe(III) composite are presented in 3O4, which is isolated from FeCl3·4H2O, shows peaks at 30.1°, 35.5°, 47.4°, and 53.2°. Similar peaks observed in the isolated M-Ch/CNF-Fe(III) composite, revealing that the Fe3O4 was successfully introduced to the isolated M-Ch/CNF-Fe(III) composite. Similar observations were reported by Rahimi et al. [X-ray diffraction (XRD) patterns of Fei et al.  and Ge ai et al. . 3O4, CNF, and the M-Ch/CNF-Fe(III) composite. It was observed that the thermal decomposition of the M-Ch/CNF-Fe(III) composite, CNF, and chitosan occurred at two stages. The first stage of decomposing occurred at a temperature from 28–173 °C, 28–257 °C, and 28–239 °C for the CNF, chitosan, and M-Ch/CNF-Fe(III) composite, respectively. The second stage of decomposition occurred at a temperature from 173–408 °C, 257–362 °C, and 239–328 °C for CNF, chitosan, and the M-Ch/CNF-Fe(III) composite, respectively. The onset temperature (Tonset) and the maximum degradation temperature (Tmax) for chitosan, Fe3O4, CNF, and the M-Ch/CNF-Fe(III) composite are shown in onset of the M-Ch/CNF-Fe(III) composite was lower than that of chitosan, but it was higher than that of CNF, revealing the successful incorporation of Fe3O4 particles into the M-Ch/CNF-Fe(III) composite [max for the M-Ch/CNF-Fe(III) composite was lower than those of chitosan and CNF. The weak intermolecular and intramolecular hydrogen bonding between the matrices in the M-Ch/CNF-Fe(III) composite might lessen the maximum degradation temperature. Similarly, Zhu et al. [max of the M-Ch/CNF-Fe(III) composite does not influence the adsorptive elimination of metal ions, as most of the adsorptive elimination of heavy metals and other contaminants is conducted at ambient temperature. The percentage weight loss of the M-Ch/CNF-Fe(III) composite was determined to be 50%, which is lower than the weight loss values of chitosan and cellulose, indicating that Fe3O4 was successfully incorporated with CNF and chitosan during the formation of the M-Ch/CNF-Fe(III) composite.u et al.  further + in aqueous solution at a lower pH competes with metal ions to bind to the surface of the M-Ch/CNF-Fe(III) composite, resulting in minimized Cu(II), Pb(II), and Cr(VI) removal. However, the amount of H+ decreases with increasing pH, which substantially increases deprotonation of the reactive functional groups on the surface of the M-Ch/CNF-Fe(III) composite and therefore increases the Cu(II), Pb(II), and Cr(VI) removal. However, the decreases of Pb(II) and Cr(VI) elimination over pH 4.0 and Cu(II) elimination over 6.0 can be accredited to the electrostatic repulsion between the reactive functional groups on the surface of the M-Ch/CNF-Fe(III) composite and metal ions due to the increased protonation at higher pH. The present study’s findings agree with the studies reported by Dong et al. [The adsorptive elimination of Cr(VI), Cu(II), and Pb(II) using the M-Ch/CNF-Fe(III) composite as an adsorbent was carried out with varying pH values, adsorbent doses, adsorption times, and temperatures, as shown in g et al.  and Mohag et al. .The influence of adsorbent doses on the Cu(II), Cr(VI), and Pb(II) elimination using the M-Ch/CNF-Fe(III) composite as an adsorbent was determined by varying the adsorbent doses from 0.05 g to 1.0 g at pH 4.0 and ambient temperature, using an adsorption time of 30 min, as shown in Muraya koenigii extract.Adsorptive Cr(VI), Cu(II), and Pb(II) elimination using the M-Ch/CNF-Fe(III) composite as an adsorbent was assessed with varying treatment times from 5–90 min at pH 4.0, using adsorbent doses of 0.5 g at room temperature, as presented in Penicillium simplicissimum. The effects of temperature on Cr(VI), Cu(II), and Pb(II) elimination using the M-Ch/CNF-Fe(III) composite as an adsorbent were evaluated by varying the temperature from ambient temperature to 80 °C at pH 4.0, with an adsorption time of 30 min and adsorbent doses of 0.5 g, as shown in The evaluation of the adsorption equilibrium is essential to assess the adsorption behavior for adsorptive heavy metals removal. Over the years, various isotherm model equations have been used to determine the adsorption behavior for the elimination of heavy metals ,41, dyesn), Langmuir constant (a), Freundlich affinity constant (fK), maximum adsorption (b), and correlation coefficient (R2) values were calculated for Cr(VI), Cu(II), and Pb(II) elimination from aqueous solution using the M-Ch/CNF-Fe(III) composite as an adsorbent and are shown in a) and maximum adsorption (b) values reveal the applicability of the M-Ch/CNF-Fe(III) composite as an adsorbent for the elimination of Cr(VI), Cu(II), and Pb(II) from aqueous solution. The maximum adsorption values were determined to be 1.7655 mg/mg, 0.5412 mg/mg, and 0.4232 mg/mg for the elimination of Cu(II), Cr(VI), and Pb(II), respectively. The isothermal behavior for adsorptive Cr(VI), Cu(II), and Pb(II) elimination from aqueous solution using the M-Ch/CNF-Fe(III) composite as an adsorbent was determined as shown in R2) values were utilized to determine the suitable isotherm models for assessing the adsorption behavior of the M-Ch/CNF-Fe(III) composite for Cr(VI), Cu(II), and Pb(II) removal. It was found that the R2 values for the elimination of Cu(II) and Cr(VI) were 0.8228 and 0.9677 for the Langmuir isotherm model, respectively, and 0.9378 and 0.9825 for the Freundlich isotherm model, respectively. The higher R2 values for the Freundlich isotherm model reveal that the Freundlich isotherm model is the best-fitting isotherm model for the elimination of Cu(II) and Cr(VI) using the M-Ch/CNF-Fe(III) composite as an adsorbent. The R2 values for Pb(II) elimination were determined to be 0.9967 and 0.9668 for the Langmuir and Freundlich isotherm models, respectively. Based on the R2 values, it can be postulated that both the Langmuir and Freundlich isotherm models can describe the adsorption behavior of Pb(II) using the M-Ch/CNF-Fe(III) composite because the R2 value is ˃0.96 for both isotherm models. However, the Langmuir isotherm model would be the best-fitting isotherm model for the adsorptive Pb(II) elimination because of the higher R2 value. The findings are in agreement with those reported by Razak et al. [The correlation coefficient (eq (exp)) values increased with increasing adsorption temperature from room temperature (28 ± 1 °C) to 70 °C, revealing that the exterior energy influenced the adsorption efficiency of the M-Ch/CNF-Fe(III) composite for Pb(II), Cu(II), and Cr(VI) elimination from aqueous solution [R2 values and the difference between the eq (exp) and eq  values obtained from the pseudo-first-order and pseudo-second-order kinetics model equations showed that the pseudo-second-order model was the best-fitting kinetics equation to describe the mass transfer phenomena for the elimination of Pb(II), Cr(VI), and Cu(II) using the M-Ch/CNF-Fe(III) composite as an adsorbent. Thus it can be postulated that chemisorption is a possible mechanism for the elimination of Cu(II), Pb(II), and Cr(VI) from an aqueous solution using the M-Ch/CNF-Fe(III) composite as an adsorbent. Similarly, Sun et al. [The mass transfer phenomena and adsorption mechanisms of the M-Ch/CNF-Fe(III) composite as an adsorbent for the elimination of Pb(II), Cu(II), and Cr(VI) were determined using pseudo-first-order and pseudo-second-order kinetics model equations, as depicted in solution ,42. The n et al.  found thThe reusability of the M-Ch/CNF-Fe(III) composite was determined via the adsorption and desorption of Cr(VI) from an aqueous solution, as shown in 3O4/steam-exploded rice straw composite as an adsorbent. In addition, Touihri et al. [Muraya koenigii extracts [i et al.  obtainedextracts , magnetiextracts , magnetiextracts , and magextracts . 3O4 were successfully incorporated during the formation of the M-Ch/CNF-Fe(III) composite. The pH, adsorbent doses, and adsorption time potentially influenced the adsorptive Pb(II), Cu(II), and Cr(VI) elimination using the M-Ch/CNF-Fe(III) composite as an adsorbent. Maximum values of about 86%, 100%, and 88% for Cr(VI), Pb(II), and Cu(II) elimination, respectively, were obtained at adsorbent doses of 0.5 g, pH 4.0, an adsorption time of 30 min, and ambient temperature. Isothermal studies showed that the Freundlich isotherm model was the best-fitting isotherm model for adsorptive Cu(II) and Cr(VI) elimination. The Langmuir isotherm model was the best-fitting isotherm model for removing Pb(II) from aqueous solution. Kinetics studies showed that chemisorption was the mass transfer mechanism of the M-Ch/CNF-Fe(III) composite as an adsorbent for Pb(II), Cu(II), and Cr(VI) elimination from aqueous solution. Based on the present study’s findings, it can be postulated that the isolated M-Ch/CNF-Fe(III) composite can be implemented to remove heavy metals and other contaminants from industrial effluents. The present study isolated an M-Ch/CNF-Fe(III) composite as an effective adsorbent for Pb(II), Cu(II), and Cr(VI) elimination from aqueous solution. Surface morphology analyses reveal that the isolated M-Ch/CNF-Fe(III) composite is porous and rough, with irregular shapes. FT-IR, XRD, and TEM analyses showed that chitosan, CNF, and Fe


While P2X7 receptor expression on tumour cells has been characterized as a promotor of cancer growth and metastasis, its expression by the host immune system is central for orchestration of both innate and adaptive immune responses against cancer. The role of P2X7R in anti-tumour immunity is complex and preclinical studies have described opposing roles of the P2X7R in regulating immune responses against tumours. Therefore, few P2X7R modulators have reached clinical testing in cancer patients. Here, we review the prognostic value of P2X7R in cancer, how P2X7R have been targeted to date in tumour models, and we discuss four aspects of how tumours skew immune responses to promote immune escape via the P2X7R; non-pore functional P2X7Rs, mono-ADP-ribosyltransferases, ectonucleotidases, and immunoregulatory cells. Lastly, we discuss alternative approaches to offset tumour immune escape via P2X7R to enhance immunotherapeutic strategies in cancer patients. Here, bh (AICD) –6. Althoh (AICD) , recent h (AICD) .The P2X7R is widely expressed by cells of the immune system. Because of the high threshold of ATP needed to activate the receptor, which can be found in conditions of cellular damage, it is considered a sensor for immunogenic danger signalling or a damage-associated molecular pattern (DAMP) which can trigger recruitment of various activated immune cells to the site of tissue damage, or alternatively AICD depending on ATP concentration –13. HoweDendritic cells (DCs) respond to ATP stimuli via P2X7R by activating Panx1 and causing an autocrine signalling loop that results in maturation of DCs as well as promoting cellular migration to lymph nodes facilitated by upregulation of the chemokine receptors CCR7 and CXCR4 , 19. P2XCM) and tissue-resident memory T cell (TRM) subsets were affected and lacked long-lasting protection against future reinfections. This was suggested to be due in part to metabolic impairment, specifically, a lower mitochondrial mass and a compromised spare respiratory capacity  display signs of cellular senescence while Wanhainen et al. showed that they have superior mitochondrial fitness, proliferation and apoptosis-resistance compared to P2RX7-deficient TILs , 39. A pparation , 41.While the picture of how P2X7R regulates anti-tumour immunity is complex, there are key emerging areas where tumours have been shown to skew P2X7R signalling to promote immune escape. These are summarized below and illustrated in The high concentration of extracellular ATP found in tumours is proposed to drive the expression of modified P2X7Rs in cancers to favour tumorigenesis and progression , 43. Thenull (NSG) mice, CAR-T cells were able to traffic to and infiltrate orthotopic breast and prostate tumours  for the treatment of basal cell carcinoma of the skin. The trial demonstrated that after being applied to primary lesions twice a day for 28 days (n=21), 65% of patients underwent reduction of lesion size, while 20% and 15% showed no changes, and increase in size, respectively. Although complete pathologic response was only observed in 3 of the patients after excision, treatment compliance was high, and the treatment was well tolerated . Further tumours .+), a nucleotide that can be released extracellularly after cellular damage or from activated T cells , formation of a macropore, and ultimately NAD-induced cell death (NICD)  . Importaversible . Stark a+ CD4 T cells, has been shown to MARylate immune cells in trans  patients, while in murine immune competent lung tumour models, ART1 knockdown decreased tumour growth . This has prompted testing of CD39 inhibitors in preclinical mouse models, which have shown impressive T cell and NK cell-mediated anti-tumour effects in immunogenic mouse tumour models –58. Whil+ mobilization regulating T cell activation  are recruited and polarized by tumours to blunt anti-tumour immune responses . Tregs aFurther, P2X7R is expressed on macrophages and on myeloid-derived suppressor cells (MDSCs). In a Lewis Lung carcinoma model, P2X7R expression on tumour-associated macrophages (TAMs) favoured immunosuppressive M2 polarization and anti-programmed cell death protein-1 (PD-1) resistance was overcome by administration of P2X7R inhibitors . In a muAs in other immune cells, microglial activation of the P2X7R causes a polarization of these cells towards a pro-inflammatory state, and as such the receptor has a clear role in diverse neuroinflammatory diseases such as Parkinsons and Multiple Sclerosis , 84. Mic+ TILs (In Acute Myeloid Leukemia (AML), P2X7R splice variants A and B have shown promising prognostic potential to identify patients with relapsing disease. P2X7RB has also been identified as a poor-prognosis marker in osteosarcoma, neuroblastoma, and lung adenocarcinoma , 88. In + TILs .In contrast, in hepatocellular carcinoma, intratumoral P2X7R expression did not correlate with oncologic outcomes. However, peri-tumoral expression of the receptor was inversely associated with overall survival in both an experimental and a validation cohort . SimilarContrary to the above, a study showed that in non-small cell lung cancer, overexpression of P2X7R was associated with improved overall survival . HoweverIn the preclinical setting, a variety of antagonists of P2X7R have been described in recent literature, including Brilliant Blue G (BBG), oxidized ATP (oxATP), KN-04, KN-62, A740003, and A438079, although all are not specific for P2X7 and may block other purinergic receptors , 100. Inin-vivo murine models, the anti-P2X7 agent KN-62 abrogated tumor proliferation promoted by ATP (in-vitro model, KN-62 inhibited ion currents, ethidium uptake, and calcium uptake, suggesting appropriate anti-P2X7 function (In vitro, A740003 a P2X7R blocker, has shown to reduce primary melanoma growth and to activate anti-tumor immune responses (in-vivo (in vitro and in an in-vivo zebrafish model of micrometastases (In pancreatic cancer d by ATP . Alternafunction . A similfunction . In vitresponses . In addi(in-vivo . P2X7R a(in-vivo . Adamant(in-vivo . Several(in-vivo . Emodin,tastases . The agetastases .Extracellular ATP has also been shown potential as anticancer therapy. In a study using a human prostate xenografts intraperitoneal injection of extracellular ATP resulted in significant tumour regression . HoweverIt has become clear that P2X7R is essential for innate immune cell sensing of immunogenic cell death, which plays an important role for efficient priming of tumour-specific T cells. In these T cells, P2X7R signalling is involved in orchestrating their migration, metabolic fitness, memory cell differentiation, and survival. Altogether, the demonstrated crucial role of P2X7R in initiation and maintenance of adaptive immune responses sends a clear message to approach P2X7R-inhibition in cancer with caution. While there is a strong rationale for inhibiting P2X7R in multiple cancers which exploit it to promote proliferation, invasion and metastasis, the conflicting outcomes of preclinical testing of P2X7R inhibitors in immunocompetent mouse models, and the fact that few P2X7R antagonists have made it to clinical testing in cancer patients, are further indications that more refined ways of targeting this pathway need to be devised.Nevertheless, the selective expression of nfP2X7R on tumour constitutes an attractive target that could be exploited for cancer therapy. Expression of nfP2X7R variants by tumour cells provides an explanation for how they benefit from the proliferative advantages of P2X7R activation while simultaneously avoiding NICD and AICD. Indeed, recent pre-clinical and clinical studies of nfP2X7R- targeting antibodies and CAR-T cells have generated encouraging results , 47.+-rich conditions such as the TME. Cytotoxic therapy directed at tumours may increase local levels of NAD+ and prime ART-mediated NICD in treated tumours. The recent discovery of how tumour-expressed ART1 allows tumours to co-opt the immune homeostatic mechanism of NICD suggests that targeted ART1-inhibition would counter this immune escape mechanism and maintain the viability of critical anti-tumour immune cells (Emerging findings describing immune regulation by ARTs in inflammation and cancer are rewriting the script for how P2X7R-expression shapes the TME , 117. ARne cells .It is plausible that tumour-engaged T cells rely on the ATPase activity of CD39 and NADase activity of CD38 not only to temper their activation by generating adenosine precursors but also to avoid AICD and NICD induced by sustained exposure to high concentrations of ATP and NAD+ in solid tumours . In factIn summary, new insights have provided both cautionary tales and important clues for how the dysregulated P2X7R-signalling that occurs in tumours could be alternatively targeted to optimize immunotherapeutic treatments of cancer patients.RS: Writing – original draft, Writing – review & editing, Visualization. JR-Q: Writing – original draft, Writing – review & editing. MM: Writing – original draft, Writing – review & editing. BS: Writing – original draft, Writing – review & editing. EW: Writing – original draft, Writing – review & editing, Visualization.


Polymyxin B is considered a last-line therapeutic option against multidrug-resistant gram-negative bacteria, especially in COVID-19 coinfections or other serious infections. However, the risk of antimicrobial resistance and its spread to the environment should be brought to the forefront.Pandoraea pnomenusa M202 was isolated under selection with 8 mg/L polymyxin B from hospital sewage and then was sequenced by the PacBio RS II and Illumina HiSeq 4000 platforms. Mating experiments were performed to evaluate the transfer of the major facilitator superfamily (MFS) transporter in genomic islands (GIs) to Escherichia coli 25DN. The recombinant E. coli strain Mrc-3 harboring MFS transporter encoding gene FKQ53_RS21695 was also constructed. The influence of efflux pump inhibitors (EPIs) on MICs was determined. The mechanism of polymyxin B excretion mediated by FKQ53_RS21695 was investigated by Discovery Studio 2.0 based on homology modeling.P. pnomenusa M202, isolated from hospital sewage, was 96 mg/L. GI-M202a, harboring an MFS transporter-encoding gene and conjugative transfer protein-encoding genes of the type IV secretion system, was identified in P. pnomenusa M202. The mating experiment between M202 and E. coli 25DN reflected the transferability of polymyxin B resistance via GI-M202a. EPI and heterogeneous expression assays also suggested that the MFS transporter gene FKQ53_RS21695 in GI-M202a was responsible for polymyxin B resistance. Molecular docking revealed that the polymyxin B fatty acyl group inserts into the hydrophobic region of the transmembrane core with Pi-alkyl and unfavorable bump interactions, and then polymyxin B rotates around Tyr43 to externally display the peptide group during the efflux process, accompanied by an inward-to-outward conformational change in the MFS transporter. Additionally, verapamil and CCCP exhibited significant inhibition via competition for binding sites.The MIC of polymyxin B for the multidrug-resistant bacterial strain P. pnomenusa M202 could mediate the transmission of polymyxin B resistance.These findings demonstrated that GI-M202a along with the MFS transporter FKQ53_RS21695 in The online version contains supplementary material available at 10.1007/s10123-023-00384-8. Staphylococcus aureus (MRSA), methicillin-resistant Acinetobacter baumannii (MRAB), carbapenem-resistant A. baumannii (CRAB), and carbapenem-resistant Enterobacteriaceae (CRE) among COVID-19 patients admitted to the intensive care unit . These results provide new insights into the mechanisms of dissemination of polymyxin B resistance and raise concerns regarding antibiotic therapy for bacterial infections.Polymyxins are amphipathic lipopeptide molecules with strong bactericidal activity against a range of gram-negative bacteria. Polymyxin antibiotics, polymyxin B in particular, are increasingly being used as last-line therapeutic options against MDR bacterial infections  agar plates  containing 8 mg/L polymyxin B  and then incubated at 28 °C for 24 h. All colonies with different phenotypes on plates were selected and cultivated three consecutive times on LB agar medium with antibiotics to obtain single colonies. After further purification, one single colony, named M202, was selected and grown as a pure culture. For multidrug resistance analysis, antimicrobial susceptibility tests were performed to determine the MICs for 9 antibiotics, including ampicillin, cefixime, tetracycline, ciprofloxacin, florfenicol, amikacin, polymyxin B, sulfamethoxazole, and meropenem, based on the breakpoints defined by the Clinical and Laboratory Standards Institute CLSI .P. pnomenusa M202 was extracted using the Genomic DNA Purification Kit  and analyzed using a NanoDrop UV–Vis spectrophotometer . 16S rRNA genes were amplified with the universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACCTTGTTACGACTT-3′) and sequenced. Similarity analyses of the 16S rRNA sequences were conducted for preliminary identification using BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi). A phylogenetic tree of M202 was produced by using neighbor-joining algorithms in Molecular Evolutionary Genetics Analysis 7 (MEGA 7) software based on the genome sequencing results of the 16S rDNA sequence . The M202 genome was assembled by glimmer3 (http://www.cbcb.umd.edu/software/glimmer/) with hidden Markov models . The genome alignment was performed by the PATRIC server  were analyzed by RAST and BLASTp based on the core dataset in the Antibiotic Resistance Genes Database (ARDB)  and M202 genomic DNA as a template. The promoter of the β-lactamase gene was amplified using the primers AP-F/R (with Hind III at the 5′-end and Xba I at the 3′-end)  for heterogeneous expression of FKQ53_RS21695, and the recombinant strain was named Mrc-3.The MFS transporter-encoding gene FKQ53_RS21695 in GI-M202a was amplified using the primer pair MRC-F/R (with d) Table  and the P. pnomenusa M202, E. coli M202-TC1, and E. coli Mrc-3 were harvested from broth medium at 24 h in the presence of 16 mg/L polymyxin B, and then rapidly frozen in liquid nitrogen. Total RNA was extracted using an RNA extraction kit , and then treated with Turbo DNA-free reagents . RT-qPCR assays were performed on a Roche Light Cycler 480 instrument using SYBR Green Mix . Relative quantities of cDNA were normalized to the amounts of the 16S rRNA genes. The primers used are listed in Table Total RNA isolation and RT-qPCR were performed as described previously  was also calculated. Verapamil, carbonyl cyanide (m-chlorophenyl) hydrazone (CCCP), dihydrochloride (PAβN), and reserpine (RES) were used as EPIs at final concentrations of 8.0 mg/L, 0.1 mg/L, 8.0 mg/L, and 8.0 mg/L, respectively.https://services.healthtech.dtu.dk/service.php? TMHMM-2.0). Protein homology modeling and molecular docking were performed as previously described . The binding of FKQ53_RS21695 with polymyxin B was modeled using the CDOCKER protocol of Discovery Studio 2.0 (Biovia Transmembrane (TM) regions were identified by TMHMM 2.0  was not located in any GI, it is not the reason for polymyxin B resistance transfer ability. Thus, potential mobilizable GIs harboring antibiotic resistance genes were analyzed. Two overlapping regions, the first ranging from nucleotide positions 4904719 to 4940759 and the second from positions 4924191 to 4934831, were annotated as potential antibiotic GIs by IslandPath-DIMOB and SIGI-HMM, respectively. Further alignment analysis indicated that a single GI most likely extended from position 4904719 to 4942265, and this region was named GI-M202a , and CCCP or verapamil clearly reduced the MICs of polymyxin B for the Mrc-3 strain to 2.0 mg/L, respectively  and E. coli M202-TC1 (increased 3.22-fold)  is responsible for the polymyxin B resistance of M202, the encoding gene FKQ53_RS21695 was heterogeneously expressed in _RS21695 The MFS family protein FKQ53_RS21695 in GI-M202a consists of 450 amino acid residues. Similar to previously reported MFS transporter structures xlaD(+5) rxGR(+9)kp”  , and efflux pump genes. Especially, carbapenem-hydrolyzing oxacillinase OXA-62, which was found to be a novel mechanism of carbapenem resistance in P. pnomenusa , where the cationic polypeptide portion of polymyxin electrostatically binds. LPSs are the predominant (or only) surface lipids of the outer membrane in gram-negative bacteria, and the binding is assisted by the interactions of the lipid tail with the fatty acids of the lipid A moiety of the LPS molecules