Zhurongbacter thermophilus 3DAC, is a sulfur-reducing heterotroph that is phylogenetically related to Coprothermobacterota and other thermophilic bacterial groups, forming a clade that seems to represent a major, early-diverging bacterial lineage. The ancestor of this clade might be a thermophilic, strictly anaerobic, motile, hydrogen-dependent, and mixotrophic bacterium. Thus, our study provides insights into the early evolution of thermophilic bacteria.It has been proposed that early bacteria, or even the last universal common ancestor of all cells, were thermophilic. However, research on the origin and evolution of thermophily is hampered by the difficulties associated with the isolation of deep-branching thermophilic microorganisms in pure culture. Here, we isolate a deep-branching thermophilic bacterium from a deep-sea hydrothermal vent, using a two-step cultivation strategy  designed to isolate rare organisms. The bacterium, which we name  Thermophilic microorganisms can live at high temperatures, but the origin and evolution of this ability are unclear. Here, the authors isolate a thermophilic bacterium from a deep-sea hydrothermal vent, and show it belongs to a major early-diverging lineage whose ancestor was likely also a thermophilic bacterium. These thermophiles are ubiquitously distributed in natural or artificial high-temperature environments, such as terrestrial volcanic sites, hot springs, submarine hydrothermal systems, deep subseafloor, oil reservoirs, solar-heated surface soils and industrial incubators8. Some thermophiles can also live in alkaline, acidic, or high salinity conditions, highlighting their ability to tolerate multiple stressors10. These so-called “extreme environments” are generally considered analogs of conditions existing on early Earth when life originated in the late Hadean or early Archaean eon12, and multiple experimental studies14 and geological evidence15 supported the early origin of thermophiles.Organisms with optimum growth temperatures higher than 45 °C are designated as thermophiles20, other hypotheses have put forward a non-thermophilic origin of bacteria and placed bacterial lineages such as Chloroflexota or the Candidate Phyla Radiation (CPR) near the root of the bacterial phylogenomic tree23. A recent study using an outgroup-free gene tree-species tree reconciliation approach rejected previously predicted bacterial roots and placed a new robust root between two major bacterial branches, i.e., Gracilicutes and Terrabacteria, even though with uncertainties regarding the phylogenetic position of the Fusobacteriota lineage24. Nevertheless, it remains unclear when the thermophilic bacteria originated and how their metabolisms evolved.Thermophilic bacteria can be found in a wide range of bacterial phyla, yet only a few phyla contain almost exclusively thermophilic bacteria, namely, Aquificota, Dictyoglomota, Coprothermobacterota, Thermotogota, and Thermodesulfobiota. While it has been suggested that thermophily might represent an ancestral feature of bacteria or even life28. For thermophilic bacteria, several previous studies focusing on different lineages such as Thermotogota29, Aquificota30, and Coprothermobacterota31 revealed a complex evolutionary history with a high proportion of genes horizontally transferred from other prokaryotes. However, there is still a lack of an evolutionary study based on comprehensive comparative genomic analyses of major thermophilic bacterial clades. Even though cultivation-free metagenomic analyses have greatly expanded our knowledge of bacterial diversity33, research on thermophilic bacteria still requires isolated strains to provide direct evidence of their physiology, such as optimal growth temperature to confirm their thermophilic features. Yet, it is extremely difficult to obtain pure cultured novel thermophilic bacteria, especially for deep-branching clades.The predicted metabolic potential of the last bacterial common ancestor (LBCA) or early diverging bacterial lineage can shed light on the early Earth environments and early ecosystemZhurongbacter thermophilus 3DAC. Phylogenomic studies of the strain 3DAC indicate that it is closely related to major thermophilic bacterial groups, such as Coprothermobacterota, Dictyoglomota, Caldisericota, and Thermotogota. Phylogenetic ancestral analyses indicate that these closely clustered thermophilic bacterial lineages share one last common ancestor with the potential to metabolize hydrogen, sulfur compounds, and with mixotrophic carbon catabolism.Here, we report the discovery and isolation of a novel thermophilic, piezophilic, sulfur-reducing bacterial strain, To increase the probability to cultivate novel thermophilic bacteria from the rare biosphere, we designed a two-stage cultivation strategy, named “Subtraction-Suboptimal” (StS) strategy Fig. . The con34), was firstly cultivated in a chemolithoautotrophic medium  at 50 °C, then consecutively transferred for five times under autotrophic condition, reducing the microbial community complexity and the ratio of heterotrophs. The conditions of the chemoautotrophic “Subtraction” stage mimicked the natural environment in situ and established a community dominated by autotrophs (mainly Hydrogenimonas within Campylobacterota and Desulfurobacterium within Aquificota)35. It performed similarly to the traditional enrichment method by decreasing the proportion of heterotrophs, but kept the co-cultured heterotrophs with autotrophs. In general, while the community is simplified through the “Subtraction” stage, it still maintains a certain degree of diversity, providing more opportunities for the isolation of rare species supported by autotrophs.In this case, the original sample, collected from an active black smoker chimney emitting hydrothermal fluid with a temperature of 231 °C Fig. ; ref. 34Thermococcus, growth temperature ranges between 50 and 100 °C36), which were pervasive in the chimney sample, allowing the recovery of rare heterotrophic thermophilic bacteria. After a four-day incubation, growth was observed, followed by the isolation of single transparent and round colonies by using the rolling tube method37 containing a solid TRM medium.For the “Suboptimal” stage, we switched the cultivation condition from the autotrophic medium to a heterotrophic medium to provide a strong selection for heterotrophs. However, we decreased the cultivation temperature from 50 to 45 °C. While this resulted in a suboptimal growth temperature for thermophilic bacteria, it prevented the growth of heterotrophic hyperthermophilic archaea  and 0.4–0.6 μm wide . They have a single lateral flagellum and are highly motile under a light microscope. During growth, the strain 3DAC occurs mostly as single cells, but sometimes forms long chains  -glucose and pyruvate can be used as additional carbon sources  supported the rapid growth of strain 3DAC  Fig. , no growces Fig. .Fig. 2En38, it is more efficient for thermophiles to absorb amino acids directly from the environment, which is more conducive to their adaptation to the hydrothermal environment.The complete genome of strain 3DAC is a single circular chromosome of 1,588,679 bp with a GC content of 41.15%  to hydrogen sulfide (H2S) in vivo and to use reduced ferredoxin as an electron donor40. Genes in MBH of 3DAC, including the key catalytic subunit of hydrogen production (MbhL), are bacterial orthologs, as expected, while most of the MBS-coding genes (10 of 13) of 3DAC are archaeal orthologs encoding the complete membrane arm  and the key catalytic subunit of sulfur reduction (MbsL)  to hydrogen sulfide (H2S) than MBH40. In addition, two redox-balancing complexes in 3DAC, soluble hydrogenase I (SH1) and NADP-dependent ferredoxin oxidoreductase II (Nfn2), are also archaeal orthologs with a high identity (>60%) as genes reported in hyperthermophilic archaea Thermococcales43.The respiratory energetic process in strain 3DAC contains a Na 41 Fig. . MBS enal-cysteine or l-methionine, did not significantly stimulate growth  47  and AAI (48–49%)  score of strain 3DAC indicates that 3DAC might belong to a class-level lineage within the phylum Coprothermobacterota based on GTDB taxonomy53. There is a discordance between traditional classification and GTDB, hence, here we only temporally call the clade represented by 3DAC as a deep-branching lineage Zhurongbacterota until more 3DAC-related species are discovered for more accurate taxonomic assessment, as well as the prokaryotic taxonomic definition of phylum reach a consensus within the scientific community.Phylogenomic analysis based on different concatenated conserved protein sequences 47 Fig. . The mos50, strain 3DAC is most closely related to other thermophilic bacterial groups, i.e., Caldisericota55, Dictyoglomota56, Thermotogota58, and Thermodesulfobiota63   as previously described74  might have shared one potentially last common ancestor. The predicted ancestral gene set from six lineages, in general, includes genes coding for thermophilic-related components (putative heat-shock proteins), flagellar that enables its movement, hydrogenase (FeFe group type) as well as saccharide and amino acid utilization pathways  from a black smoker chimney at l-vent located at the 9°N deep-sea vent field on the East Pacific Rise . The temperature of the emitted fluid was 231 °C. More information about the fluid geochemistry and microbial communities in the chimney walls can be found in ref. 34. Pieces of the chimney were stored immediately upon retrieval in sterile anaerobic tubes, and the headspace in the tubes was filled with pure nitrogen gas with slight overpressure to prevent the entry of oxygen. The samples were then stored at 4 °C until enrichment in the laboratory.The original sample was collected with the remotely operated deep-sea research vehicle (ROV) 79, which contained the following components (L−1): NaCl, 20.0 g; MgSO4·7 H2O, 3.5 g; MgCl2·6 H2O, 2.75 g; KCl, 0.325 g; KNO3, 2.0 g; MES, 4.27 g; NaBr, 50.0 mg; H3BO3, 15.0 mg; SrCl2·6 H2O, 7.5 mg; (NH4)2SO4, 10·0 mg; KI, 0.05 mg; Na2WO2·2H2O, 0.1 mg; CaCl2·2H2O, 0.75 g; KH2PO4, 0.5 g; NiCl2·6H2O, 2.0 mg; and resazurin, 1.0 mg. The pH of the medium was adjusted to 6.0 using 1 M NaOH. After autoclaving, 1 ml of trace element mixture, 1 ml of vitamin mixture, 1 ml of thiamine solution, and 1 ml of vitamin B12 solution80 were added to the medium (L−1). Then, 50 ml aliquots of the medium were separated into 150-ml glass serum bottles and supplemented with 0.5 g of elemental sulfur and 0.5 ml of Na2S·9H2O , and the tightly stoppered bottles were pressurized with hydrogen/carbon dioxide . The cultures were incubated at 50 °C. We obtained the original enrichment in this chemolithotrophic medium, subcultured (1% inoculation) the cells in this medium for five generations, and then transferred the enrichment of the fifth generation (1% inoculation) to a heterotrophic TRM medium81 with the following composition (L−1): 1 g of yeast extract, 4 g of tryptone, 23 g of NaCl, 5 g of MgCl2·6H2O, 0.7 g of KCl, 0.5 g of (NH4)2SO4, 3.3 g of PIPES disodium salt, 1 ml of NaBr , 1 ml of SrCl2·6H2O , 1 ml of KH2PO4 , 1 ml of K2HPO4 , 1 ml of CaCl2·2 H2O , and 1 ml of Na2WO4 (10 mM). The medium was adjusted to pH 7.0, autoclaved, separated into anaerobic tubes (5 ml), and supplemented with 0.05 g of elemental sulfur and 0.05 ml of Na2S·9 H2O , and the headspace of the tubes was pressurized with pure nitrogen gas. Heterotrophic enrichment was performed at 45 °C. Colonies were obtained on solid TRM medium (1.5 % Gelrite) using the rolling tube method, which was repeated three times.The outer layer of the black chimney sample (0.25 g) was first enriched anaerobically in an SME medium82. Images were captured with a fluorescence microscope (Nikon Eclipse 90i).Fluorescent staining by SYBR Green I (Invitrogen) was carried out on cells filtered onto 0.2-μm polycarbonate GTBP membranes (Millipore) following established protocolsg for 5 min, fixed in 4% formaldehyde for 2 h at room temperature, and washed with 1× PBS (pH 7.4). Fixed cells were negatively stained with 2% (w/v) phosphotungstic acid (pH 6.5) for observation with a transmission electron microscope (FEI Tecnai G2 Spirit Biotwin) at an acceleration voltage of 120 kV.For transmission electron microscopy, cells were harvested at 3000 × 600 with a quartz colorimeter  on a spectrophotometer (HACH DR5000). The specific growth rate  was calculated according to the equation μ = ln[OD600(t2)/OD600(t1)]/(t2 − t1), where OD600(t1) and OD600(t2) are the optical densities of liquid cultures measured at 600 nm at time points t2 and t1, and t1 and t2 are the time points (in hours) of the exponential growth phases83. Growth rates were calculated using linear regression analysis of 4 to 6 points along the linear portions of the growth curves.The effects of the temperature, pH, and NaCl concentration on the growth of strain 3DAC were determined in an anaerobic TRM medium with a headspace gas composed of pure nitrogen gas at atmospheric pressure supplemented with 1% (w/v) elemental sulfur. To evaluate the optimum temperature for growth, strain 3DAC was cultivated at 25–80  °C with 5  °C intervals. The ranges of pH and NaCl concentrations for growth were determined at 45  °C. The optimal pH for growth was determined by varying the pH in the culture medium between 4.0 and 9.0 using the following buffers at a concentration of 10 mM: acetate at pH 4.0, 4.5, and 5.0; MES at pH 5.5 and 6.0; PIPES at pH 6.5 and 7.0; HEPES at pH 7.5 and 8.0; and TAPS at pH 8.5 and 9.0. The pH was adjusted at room temperature before autoclaving. To test the effect of the NaCl concentration on growth, NaCl was added to the medium at final concentrations within the range of 0.5–5.0% (w/v). All of these tests were performed in triplicate in the dark without shaking. Growth was determined by monitoring the ODThe pressure range  was tested with syringes loaded with TRM  supplemented with 1% (w/v) elemental sulfur. These syringes were placed into high-pressure and high-temperature reactors and incubated at 70 °C. Growth yields and growth rates were measured as previously described. All of these tests were performed in triplicate.l-alanine, l-arginine, l-asparagine, l-aspartic acid, l-cysteine, l-glutamine, l-glutamic acid, glycine, l-histidine, l-isoleucine, l-leucine, l-lysine, l-methionine, l-phenylalanine, l-proline, l-serine, l-threonine, l-tryptophan, l-tyrosine, and l-valine) were prepared. Then, each combination of 19 amino acid mother liquors was added to basal TRM medium  elemental sulfur) to maintain each amino acid at a final concentration of 0.1 g/L. Basal TRM medium containing all 20 amino acids and no amino acids was set as the positive and negative controls, respectively. The pH of all the tested media was adjusted to 7.0 at room temperature. For all tests, positive cultures were transferred (1% inoculum) into the tested medium at least five times for confirmation of growth. All tests were performed in duplicate at atmospheric pressure and with incubation at 70 °C.Mother liquors of 20 kinds of amino acids -glucose, d-(+)-xylose, d-(+)-maltose, sucrose, starch, glycogen, N-acetyl-d-glucosamine, d-mannitol, l-alanine, l-arginine, l-asparagine, l-aspartic acid, l-cysteine, l-glutamine, l-glutamic acid, glycine, l-histidine, l-isoleucine, l-leucine, l-lysine, l-methionine, l-phenylalanine, l-proline, l-serine, l-threonine, l-tryptophan, l-tyrosine, and l-valine. Growth was recorded after 24 and 48 h. Negative  and positive (inoculated TRM containing yeast extract and peptone) controls were prepared for each substrate. For all tests, positive cultures were transferred (1% inoculum) into the tested medium for confirmation of growth.Various single carbon sources were added to a basal TRM medium  supplemented with 1% (w/v) elemental sulfur. The following single carbon sources, all at a final concentration of 0.2% (w/v), were tested: peptone, casamino acids, beef extract, yeast extract, tryptone, sodium acetate, sodium formate, sodium citrate, sodium pyruvate, sodium oxalate, d-(+)-glucose, d-fructose, d-(+)-maltose, sucrose, starch, sodium acetate, sodium formate, and sodium pyruvate. Basal TRM medium supplemented with 20 kinds of amino acids and 1% (w/v) elemental sulfur was used as a control.To test carbon source utilization with the addition of 20 kinds of amino acids, various carbon sources at a final concentration of 0.2% (w/v) were added into a basal TRM medium  and supplemented with 20 kinds of amino acids  and 1% (w/v) elemental sulfur. The carbon sources used were as follows: l-cysteine (20 mM) and l-methionine (20 mM), were also tested.To examine the ability of the isolate to grow in the absence of elemental sulfur, cells were cultured in TRM medium  without sulfur. Other sulfur substitutes, such as sodium sulfate (20 mM), sodium sulfite (20 mM), sodium thiosulfate (20 mM), The pH of all the tested media was adjusted to 7.0 at room temperature. All tests were performed in duplicate at atmospheric pressure and with incubation at 70 °C. In cultures containing insoluble substrates, growth was monitored by microscopic examination.86 using a sulfide measurement kit (HACH) on a spectrophotometer (HACH DR5000).The sulfide concentration was measured by the methylene blue method87 and sequenced on the PacBio RSII SMRT and Illumina HiSeq X Ten sequencing platforms . The genome was assembled into one contig from the PacBio reads  by Falcon v0.3.088 and Celera Assembler v8.389. Then, the assembled genome was corrected with Illumina HiSeq data  using GATK v1.6–1390. Protein-coding genes were predicted by Glimmer v3.0291, and rRNA and tRNA annotations were performed with RNAmmer v1.292 and tRNAscan-SE v1.3.193, respectively. Predicted gene functions were annotated with databases including NCBI-nr , KEGG 94, and eggNOG-Mapper v2.0.1b-2-g816e19095. Average amino acid identity (AAI) values were calculated by CompareM v.0.0.23 (https://github.com/dparks1134/CompareM).Genomic DNA was extracted by a modified SDS-based DNA extraction method96; the X region represents the various key tags for each sample. The PCR program was 3 min at 95 °C, followed by 35 cycles of 94 °C for 40 s, 56 °C for 1 min, and 72 °C for 1 min. The final extension step was 72 °C for 7 min. The 50 µL amplification mixture contained 1 µL of each forward and reverse primer, 1 µL template DNA, 5 µL 10× ExTaq buffer, 0.5 µL ExTaq polymerase , 4 µL of 2.5 mM dNTP mix and 37.5 µL ddwater. PCR products were purified with an E.Z.N.A. Gel Extraction Kit  and sequenced on the MiSeq platform  according to the manufacturer’s instructions.For the chimney sample and chemoautotrophic enrichment, the bacterial V4 region of the 16S rRNA gene was amplified by B515F (5’-XXXXXXXXGTGCCAGCMGCCGCGGTAA-3’) with eight-nucleotide key tags for each sample and B806R (5’-GGACTACHVGGGTWTCTAAT-3’)97 and the QIIME-compatible version of the SILVA-132 database for template-based alignment and taxonomic assignment. Assembled reads that passed the chimera check were clustered into de novo operational taxonomic units (OTUs) at a cutoff of 97% sequence similarity.Data analysis of the 16S rRNA MiSeq sequences was performed using the QIIME version 1.9.1 software pipelineCells of strain 3DAC cultured with and without sulfur were harvested at the log phase by centrifugation. Three replicates under each condition were prepared for transcriptome analysis. Total RNA was extracted using a Total RNA Extractor (TRIzol) kit  according to the manufacturer’s protocol. For RNA sequencing library preparation, genomic DNA was removed with DNase I, and ribosomal RNA (rRNA) was removed with a Ribo-off rRNA Depletion Kit (bacteria) . A complementary DNA (cDNA) library was generated by a VAHTS™ Stranded mRNA-seq Library Prep Kit  following the manufacturer’s instructions. Sequencing was performed on the Illumina HiSeq 2500  platform at Sangon Biotech .https://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and all samples passed. Low-quality reads , ambiguous “N” nucleotides, adapter sequences and fragments of <35 bp were removed from raw reads with Trimmomatic98 for further analysis. rRNA was removed using a sortmerna99 with default settings. Read mapping of the non-rRNA to the genome of Zhurongbacter thermophilus 3DAC was performed using Bowtie 2100, and the relevant mapping information was summarized. The uniquely mapped reads were collected and analyzed with the DESeq2 package based on the Poisson distribution to identify the differentially expressed genes (DEGs). Estimation of gene expression levels was based on the transcript per million (TPM) values101.The sequencing quality controls of the raw reads produced by Illumina HiSeq 2500 were assessed using FastQC (102 (http://www.ncbi.nlm.nih.gov/tools/primer-blast) and are listed in Supplementary Data 2) between these two datasets were calculated . After the extraction of RNA, residual DNA was removed by a TURBO DNA-free Kit , and cDNA synthesis reverse transcription was followed by a RevertAid First Strand cDNA Synthesis Kit . The primers used for quantitative real-time PCR (RT-qPCR) were designed by Primer-BLAST103, and the full alignment was stripped of columns containing 70% or more gaps with a Perl script. The maximum-likelihood phylogeny of the 16S rRNA gene tree was inferred by IQ-TREE104 v2.0.6 with the “-MFP -B 1000” options for best-fit model selection and ultrafast bootstrap. The reference sequences of other bacteria were downloaded from the Silva database, sequences smaller than 1400 bp were filtered out, and OTUs were picked from the remaining sequences at a cutoff of 97%. The above-described representative OTU sequences were used for the tree.For the 16S rRNA gene tree, the 16S gene sequences were aligned using SINA v1.2.1124 and selected archaeal reference genomes from the different phyla were downloaded from the NCBI prokaryotic genome database (https://www.ncbi.nlm.nih.gov/assembly/). These reference genomes and the 3DAC genome from this study were used to construct a phylogenomic tree based on a concatenated alignment of three different sets of marker genes  version 0.4110. Only bacteria with complete genome information were considered in the present study because incomplete genomes would lead to biased analyses in gene gain and loss processes. Here, the phyla Synergistota and Deinococcota were used as the outgroups. The representative genomes from the phyla Aquificota, Thermosulfidibacterota and Thermodesulfobacteriota were also considered in the ALE analysis. For the CCTB, cultured bacteria with complete genomes in the phylum Dictyoglomota includes Dictyoglomus turgidum DSM6724 and Dictyoglomus thermophilum H-6-12; the phylum Thermodesulfobiota includes Thermodesulfobium acidiphilum 3127-1 GCA and Thermodesulfobium narugense DSM14796; the phylum Caldisericota includes Caldisericum exile AZM16c01; the phylum Zhurongbacter includes Zhurongbacter thermophilus 3DAC; the phylum Coprothermobacterota includes Coprothermobacter platensis DSM11748 and Coprothermobacter proteolyticus DSM5265; and the phylum Thermotogota includes Pseudothermotoga hypogea DSM11164, Thermotoga neapolitana DSM4359, Thermosipho melanesiensis BI429, Fervidobacterium pennivorans DSM9078, Kosmotoga pacifica SLHLJ1, Mesotoga prima MesG1Ag42, Defluviitoga tunisiensis L3, Petrotoga mobilis SJ95, Marinitoga piezophila KA3, and Athalassotoga saccharophila NAS-01. Ancestor metabolic reconstruction was performed with the predicted ancestral gene set (851 sequences) at the node of the CCTB with an average gene copy number larger than 0.3.A total of 43 cultured bacterial complete genomes  (performed in November 2020) and the published model of the related thermophilic bacterial strain Thermotoga maritima MSB8117. Extensive manual curations were carried out following draft model reconstruction to integrate the latest biochemical evidence of enzymatic functions in (hyper)thermophiles. Overall, literature evidence was assigned to 57 reactions in the 3DAC model. The biomass equations of 3DAC were formulated based on the biosynthesis of macromolecules, including DNA, RNA, and proteins. Their biosynthesis was defined to account for the millimole composition of each building block in assembling one gram of a given component and the associated energy cost. Simulations of carbon source utilization, electron acceptors, and gene deletion were formulated with exchange constraints that represent the corresponding culture conditions used in the experiments.A genome-scale metabolic model of Escherichia coli core model118, including DNA, RNA, and proteins that accounted for ~80% of the dry weight of cells119 .The KO terms of the predicted ancestral gene set were used to construct the initial genome-scale metabolic model of the last common ancestor of the CCTB. Reactions for each KO term were obtained from the latest KEGG database (performed in March 2022). Compounds were initially introduced from the latest KEGG database, but the physiological charges of each and the modified hydrogen number of charged compounds were added. The biomass objective function was introduced from the formulacheck, chargecheck and masscheck functions in PSAMM version 1.1.1 (https://zhanglab.github.io/psamm/)124, and the unbalanced equations were manually curated. Gap-filling processes were performed to complete the synthesis of the main biomass components  and essential cofactors  by the fastgapfill function in PSAMM. Flux variation analysis (FVA) to optimize biomass production was performed using the fva function in PSAMM with IBM ILOG CPLEX Optimizer version 12.7.1.0.For both the 3DAC model and the ancestor model, consistency checks of all reactions were computationally conducted using the Further information on research design is available in the SupplementaryDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3-11Reporting SummarySource_data


Escherichia coli single stranded (ss) DNA binding protein (SSB) plays essential roles in DNA maintenance. It binds ssDNA with high affinity through its N-terminal DNA binding core and recruits at least 17 different SSB interacting proteins (SIPs) that are involved in DNA replication, recombination, and repair via its nine amino acid acidic tip (SSB-Ct). E. coli RecO, a SIP, is an essential recombination mediator protein in the RecF pathway of DNA repair that binds ssDNA and forms a complex with E. coli RecR protein. Here, we report ssDNA binding studies of RecO and the effects of a 15 amino acid peptide containing the SSB-Ct monitored by light scattering, confocal microscope imaging, and analytical ultracentrifugation (AUC). We find that one RecO monomer can bind the oligodeoxythymidylate, (dT)15, while two RecO monomers can bind (dT)35 in the presence of the SSB-Ct peptide. When RecO is in molar excess over ssDNA, large RecO–ssDNA aggregates occur that form with higher propensity on ssDNA of increasing length. Binding of RecO to the SSB-Ct peptide inhibits RecO–ssDNA aggregation. RecOR complexes can bind ssDNA via RecO, but aggregation is suppressed even in the absence of the SSB-Ct peptide, demonstrating an allosteric effect of RecR on RecO binding to ssDNA. Under conditions where RecO binds ssDNA but does not form aggregates, SSB-Ct binding enhances the affinity of RecO for ssDNA. For RecOR complexes bound to ssDNA, we also observe a shift in RecOR complex equilibrium towards a RecR4O complex upon binding SSB-Ct. These results suggest a mechanism by which SSB recruits RecOR to facilitate loading of RecA onto ssDNA gaps. Escherichia coli single stranded (ss) DNA binding (SSB) protein is a functional homo-tetramer 65, where the subscripts denote the average number of nucleotides occluded per SSB tetramer , 35 nucleotides interact with an average of two of the four subunits with unlimited cooperativity between nearest neighbor tetramers so that long protein clusters can form  and divalent (10 mM MgCl2) salt concentrations, 65 nucleotides interact with and wrap around all four subunits of SSB with cooperativity that limits clustering to dimers of tetramers  , that arEscherichia coli RecO and RecR are essential recombination mediator proteins (RMPs) in the RecF pathway that is primarily involved in repair of single stranded DNA gaps . Spectrophotometric grade glycerol was from Alfa Aesar . Buffer BTP is 20 mM Bis–Tris propane , 50 mM NaCl unless otherwise indicated, 25% (v/v) glycerol, 1 mM TCEP. Tween-20 (0.002%)  was added to Buffer BTP in the confocal microscope imaging experiments.Escherichia coli RecO protein was overexpressed from plasmid pMCSG7 in E. coli strain BL21(DE3) pLysS  and purified using Ni-NTA affinity chromatography and a HiTrap Heparin HP affinity column  after His-tag cleavage with TEV protease as described (E. coli strain BL21(DE3) transformed with PRK793 and pRIL  and purified as described (E. coli RecR protein was overexpressed from plasmid pMCSG7 in E. coli strain BL21 Rosetta 2(DE3)pLysS (kindly provided by Dr. Sergey Korolev) and purified using Ni-NTA affinity chromatography, followed by cleavage of His-tag with TEV protease as described . The SSB-Ct peptide concentration was determined using an extinction coefficient of ϵ258 = 390 M−1 cm−1.SSB-Ct peptide, composed of the 15 C-terminal amino acids (PSNEPPMDFDDDIPF) of L, (dT)L and (((dT)3ϵdA)m(dT)3 with m = 3, 8, 17) containing the fluorescent analogue, etheno(dA) (3ϵdA)m(dT)3 for m = 3, 8, 17) were synthesized and purified as described (260 = L(8.1 × 103) M−1 cm−1 for (dT)L, and ϵ260 = 1.1 × 105, 2.6 × 105 and 5.2 × 105 M−1cm−1 for m = 3, 8, 17, respectively, for ((dT)3ϵdA)m(dT)3. Each strand of the double-stranded DNA (dsDNA) substrates were synthesized and purified as described Sedimentation experiments were performed with an Optima XL-A analytical ultracentrifuge and An50Ti or An60Ti rotors  at 25°C as described ,68. Absoin = number of moles of species ‘i’, iM = molecular weight of species ‘i’, and i’.The densities and viscosities of the buffers at 25°C were determined using SEDNTERP . The parffer BTP . The val for DNA . In expementclass2pt{minimL (0.56 and 2.24 μM DNA molecules) and mixtures of RecO (2.24 μM), RecR , and SSB-Ct (13.4 μM). Sample (380 μl) and buffer (394 μl) were loaded into each sector of an Epon charcoal-filled two-sector centerpiece. Absorbance data were collected by scanning the sample cells at intervals of 0.003 cm and analyzed using Sedfit to obtain c(s) distributions (c(s) distribution function defines the populations of species with different sedimentation rates and represents a variant of the distribution of Lamm equation solutions ibutions . The c(solutions .L (L = 15 or 35 nucleotides) (0.56 μM DNA molecules), RecO (2.24 μM), SSB-Ct (13.4 μM), and in the absence and presence of RecR (8.96 μM). Sample (110 μl) and buffer (120 μl) were loaded into each sector of an Epon charcoal-filled six-channel centerpieces. Absorbance data were collected by scanning the sample cells at intervals of 0.003 cm in the step mode with 5 averages per step. Samples were sedimented to equilibrium at the indicated rotor speeds (ranging from 18 000 to 28 000 rpm) starting with the lowest speed. The resulting absorbance profiles, rA, were analyzed by NLLS fitting to Eq. –DNA and complex species. Sedimentation equilibrium experiments were performed with 3’-Cy3-labeled (dT)s), RecO .24 μM, Sg to Eq.  to obtair is the distance from the center of rotation, 0r is an arbitrary reference radius, ω is angular velocity, T is absolute temperature, R is the gas constant, iM is the molecular weight of species ‘i’, i’, ρ is the buffer density, i’ at the reference position, and rb is a radial-dependent baseline offset. All sedimentation equilibrium experiments in this study were described by a single exponential and globally fit to a one species model . The microscope  was equipped with a piezo scanner and a high numerical aperture water immersion objective . Fluorophores were excited using a 485 nm pulsed laser  with a repetition rate of 20 MHz. Excitation power was monitored before the objective with a laser photodiode and optimized to avoid photobleaching and saturation of detectors to maintain a constant power for each set of measurements. Emitted photons were collected through the objective, passed through a dichroic mirror , and filtered by a 100 μm pinhole . Photons were separated according to polarization using a polarizer beam splitter cube  and further refined by a 642 ± 40 nm bandpass filter  in front of the SPAD detectors . Photons are counted and accumulated by a HydraHarp 400 TCSPC module  with 1 picosecond resolution (Measurements were performed in uncoated polymer coverslip cuvettes (30 μl per well) , which significantly decrease the fraction of protein adhering to the surface compared to glass cuvettes. Measurements were performed at 23 ± 1°C in a temperature-controlled room, as detected on the microscope stage.Imaging was performed using both XY and Z monodirectional scanning with 1 ms collecting steps with 256 × 256 pixels resolution. Excitation power for image collection was either 1.0 or 11 μW depending on sample concentration. Measurements were performed keeping a constant ratio between Cy3-labeled and unlabeled protein (labeled:unlabeled = 1:100) in buffer BTP with 0.002% Tween-20. Brightness thresholds were set at 50 and 1200 photons/pixel, which removed most of the background and prevented saturation in images. Images are colored in a hue scale running from blue at 100 photons/pixel to red at 800 photons/pixel.Light scattering at 90° was measured using a PTI QM-2000 fluorometer  with excitation and emission wavelengths at 350 nm. ssDNA (1.9 ml of 25 nM DNA molecules) was titrated with RecO (5 μM stock) in a 3 ml quartz cuvette in buffer BTP. Samples were stirred throughout the experiments using magnetic stir bars. For experiments in the presence of SSB-Ct, SSB-Ct was pre-mixed with ssDNA at the start of the experiments. SSB-Ct (3.8 μM) was in 6-fold molar excess of the final concentration of RecO in the cuvette at the end of titration. The stock solution of RecO was also pre-mixed with 3.8 μM of SSB-Ct to keep [SSB-Ct] inside the cuvette constant throughout the titrations. Reference titrations were also performed in which protein titrant was added to a 1.9 ml of buffer that does not contain DNA both in the absence and presence of SSB-Ct. All sedimentation experiments were performed in duplicate, with each experiment using different RecO and RecR protein preps.Light scattering intensities were normalized as in Eq. ,i,normI is the normalized scattering intensity after ‘i’th injection of titrant (RecO), iI is the scattering intensity after ‘i’th injection of titrant, and I0 is the initial scattering intensity before injection of any titrants.where E. coli RecO can bind both ss and dsDNA and anneal complementary ssDNA when complexed with SSB  monitoring RecO Trp fluorescence quenching and fluorescein fluorescence quenching or anisotropy using 5’-fluorescein labeled (dT)L (Fl-(dT)L). Finally, we used ssDNA composed of dT interspersed with the fluorescent analogue, etheno(dA) (3m(dT)3 with m = 3, 8, 17) and monitored the enhancement of 317(dT)3, referred to as (71), with RecO at two DNA concentrations (0.1 and 0.2 μM) in buffer BTP  at 25°C show different plateau levels for the normalized enhancement of 35 with RecO yielding similar inconsistencies . Reverse titrations of RecO with poly(dT) monitoring Trp fluorescence quenching mentclass2pt{minimg Figure  also shos Figure . These r71 with RecO under the same solution conditions  used to monitor To examine this further, we monitored light scattering during a titration of 0.2 μM (mentclass2pt{minimL (25 nM DNA molecules), of different lengths  with RecO (5 μM stock) monitoring light scattering in buffer BTP . Scattering intensities were normalized using Eq. 35, (dT)70 and (dT)140. Furthermore, the RecO to (dT)L ratio at which the onset of light scattering occurs increases with increasing DNA length at RecO to (dT)L molar ratios of 5.2 for (dT)35, 6 for (dT)70 and 7.1 for (dT)140 0 Figure –D. This 15, did not exhibit observable scattering. This indicates that RecO binding to dsDNA is more prone to aggregation compared to ssDNA of similar length.Similar scattering experiments were performed with dsDNA of 18 and 60 bp . Figure S2 shows increases in scattering intensities for both ds18 and ds60 with the onset of scattering occurring at higher RecO/DNA ratios for the longer DNA as in the case of ssDNA. However, a notable difference is that scattering is readily observed even for the short 18 bp DNA whereas ssDNA of similar length, (dT)L complexes formed in the presence of excess RecO that result in light scattering. Experiments were performed using a 20-fold molar excess of RecO (4 μM RecO) over ssDNA (200 nM) where light scattering intensities reached a plateau for all (dT)L with L = 35, 70 and 140 nts  and a small amount of Cy3-labeled (dT)L (1:100 molar ratio)  of the same length except for the experiments with (dT)70, which were performed by mixing with 3’-Cy3-(dT)68. As shown in Figure L. These structures did not merge or split over several minutes of imaging, suggesting that the structures are not dynamic, but are solid aggregates. These aggregates appeared larger and more elongated for the longer (dT)L, consistent with the higher light scattering intensity for these longer DNA lengths 15 ratio o Figure . It is pEscherichia coli single stranded binding (SSB) protein interacts with RecO via the last nine amino acids of SSB’s C-terminal intrinsically disordered tails (SSB-Ct) (K = (1.2 ± 0.3) × 107 M−1 in buffer BTP  L   ,33,36,57) . In the presence of SSB-Ct, however, the onset of scattering occurred at higher RecO/DNA ratios than in the absence of SSB-Ct . The maximum scattering intensities were similar for ds18 in the absence and in the presence of SSB-Ct, but the maximum intensity was reduced ∼3-fold for ds60 in the presence of SSB-Ct.L at a 20-fold molar excess of RecO (4 μM) over (dT)L  as before. Images showed mostly black background indicating that the binding of SSB-Ct to RecO significantly reduced the formation of the aggregated RecO–DNA structures 35 complexes, and only a few small fluorescent aggregates were observed for the RecO-(dT)68 and (dT)140 complexes, significantly reduced in size and number . This is consistent with the significantly reduced light scattering intensities observed in the presence of SSB-Ct L ternary complex. To clarify this, we performed sedimentation velocity experiments as described below.We next used confocal fluorescence microscopy to examine the effect of the SSB-Ct peptide 24 μM) on mixtures of RecO and(dT)s Figure . No aggr4 μM on mL). For these experiments, we used only a 4-fold molar excess of RecO over Cy3-(dT)L, a molar ratio such that no light scattering is observed for any of the (dT)L examined. Experiments were performed by monitoring absorbance at 546 nm, which only detects the Cy3-(dT)L and Cy3-(dT)L-bound to protein. Sedimentation velocity experiments were performed with 0.56 μM Cy3-(dT)L and 2.24 μM RecO in the absence and presence of SSB-Ct. Figure c(s) distributions for Cy3-(dT)L  in the absence of RecO. A single symmetric c(s) peak is observed for each DNA, indicating that each is a single homogeneous species. Weight average sedimentation coefficients of 0.5 S, 0.7 S, 0.9 S and 1.2 S were estimated for Cy3-(dT)15, Cy3-(dT)35, Cy3-(dT)68 and Cy3-(dT)140, respectively. Upon addition of a four-fold molar excess of RecO over ssDNA in the absence of SSB-Ct, the c(s) species distributions still showed only single peaks with only slight increases in sedimentation coefficients of 0.6 S, 0.7 S, 1.0 S and 1.2 S, indicating weak binding of RecO to ssDNA under these conditions s Figure .15, Cy3-(dT)35, Cy3-(dT)68 and Cy3-(dT)140, respectively L binding affinity. We also note that the c(s) species distributions for Cy3-(dT)68 and Cy3-(dT)140 in Figure L complexes form when SSB-Ct-RecO binds to these longer ssDNA molecules.However, when a 6-fold molar excess of SSB-Ct (13.4 μM) was included with RecO  over Cy3-(dT)15 or Cy3-(dT)35 (0.56 μM), and a six-fold molar excess of SSB-Ct (13.4 μM) over RecO at three rotor speeds . The sedimentation equilibrium profiles showed only a single exponential for the RecO complexes with both Cy3-(dT)15 and Cy3-(dT)35. This is consistent with the c(s) species distributions from sedimentation velocity that showed only a single species under these same conditions 35.The N-terminal domain of RecO contains the DNA binding domain , however RecO 2.2 μM over x Figure  and 70.1x Figure . These a4O and RecR4O2, but primarily RecR4O2 and some excess RecR dimer . This contrasts with significant aggregation in the absence of RecR, particularly for longer DNA L resulting in the inhibition of aggregation. To clarify this, we performed sedimentation velocity experiments with RecOR-ssDNA in the absence and presence of SSB-Ct.We have shown that RecR exists in a dimer-tetramer equilibrium under the conditions of our experiments and that RecO binding promotes RecR tetramer formation  . FurthercR dimer . Howeverwith DNA ,91, the A Figure . In the E. coli RecR does not bind to either SSB-Ct or DNA (4O/RecR4O2 equilibrium to favor a RecR4O complex (L (0.56 μM)  in the absence and presence of SSB-Ct (13.4 μM), monitoring Cy3 absorbance. This [RecO]:[RecR] molar ratio yields primarily RecR4O2 in the absence of SSB-Ct and RecR4O in the presence of SSB-Ct  alone are 0.5 S, 0.7 S, 0.9 S and 1.2 S  mixture results in decreases in the weight average sedimentation coefficients to 2.6 S, 3.8 S, 4.7 S and 4.8 S L , we observe wide, asymmetric c(s) distributions at >3 S indicating that multiple SSB-Ct-RecOR-(dT)L complex species are present. In addition to the RecOR-(dT)L species, experiments with (dT)140 140 complex. Since the presence of multiple species complicates the identification of the RecOR-ssDNA complexes for the longer (dT)L, we performed sedimentation equilibrium experiments on the mixture of RecO, RecR, SSB-Ct and Cy3-(dT)15, which displays a symmetric sedimentation velocity peak at 2.9 S, suggesting a homogeneous species  to the RecR 8.96 μM), RecO (2.24 μM) and (dT)S Figure . This cof SSB-Ct , a desta μM, RecOs Figure . Further15 (0.56 μM) and SSB-Ct (13.4 μM) was examined 15 (121.2 kDa). Even the upper limit of the MW estimate is 10.3 kDa less than the predicted MW (155.6 kDa) for two (dT)15 molecules bound to RecR4O2 along with two SSB-Ct molecules. In contrast to the symmetric peak observed in the presence of SSB-Ct, the asymmetric peak for (dT)15 in in the absence of SSB-Ct 15 and RecR4O2-(dT)15 complexes. Therefore, the transition from RecR4O2 to RecR4O upon binding of SSB-Ct is observed even when the RecOR complexes are bound to (dT)15.Sedimentation equilibrium experiments were performed to estimate the MW and thus identify the composition of the RecOR complexes. A mixture of RecO 2.24 μM) RecR (8.96 μM) and Cy3-(dT)d Figure . Each sey Figure , blue. T.24 μM Re4O and RecR4O2, and SSB-Ct binding to RecO preferentially stabilizes the RecR4O complex. In this study we report evidence for a second allosteric effect of the SSB-Ct on RecO binding to DNA. Aggregation of RecO–ssDNA complexes is inhibited when SSB-Ct is pre-bound to RecO. This is not due to the inability of SSB-Ct-bound RecO to interact with ssDNA, as we have shown that (dT)L from 15 to 140 nts interacts with SSB-Ct-bound RecO when RecO is in excess over ssDNA. We also observe that RecO–ssDNA aggregation is completely inhibited in the presence of RecR even in the absence of SSB-Ct, demonstrating an allosteric effect of RecR on binding of RecO to ssDNA.We showed previously that the SSB-Ct exerts an allosteric effect on RecOR complex formation . Two typ45 and (dT)70, Ryzhikov et al. (Using a pull-down assay with (dT)v et al.  showed tv et al. . These iv et al. .4O complex should be populated rather than RecR4O2 complexes ractions . The binractions . Such a  35 mode 35 mode ,56,95 th)35 mode ,77,81. F)35 mode ,69–73, asubtilis ,76. A chThe data underlying this article will be shared on reasonable request to the corresponding author.gkad084_Supplemental_FileClick here for additional data file.


Litomosoides sigmodontis with the aim of identifying novel vaccination strategies for filarial infections. Subcutaneous injection of irradiated L3 larvae in combination with poly(I:C) or 3pRNA resulted in neutrophil recruitment to the skin, accompanied by higher IP-10/CXCL10 and IFN-β RNA levels. To investigate the impact on parasite clearance, BALB/c mice received three subcutaneous injections in 2-week intervals with irradiated L3 larvae in combination with poly(I:C) or 3pRNA prior to the challenge infection. Vaccination with irradiated L3 larvae in combination with poly(I:C) or 3pRNA led to a markedly greater reduction in adult-worm counts by 73% and 57%, respectively, compared to the immunization with irradiated L3 larvae alone (45%). In conclusion, activation of nucleic acid-sensing immune receptors boosts the protective immune response against L. sigmodontis and nucleic acid-receptor agonists as vaccine adjuvants represent a promising novel strategy to improve the efficacy of vaccines against filariae and potentially other helminths.More than two-hundred-million people are infected with filariae worldwide. However, there is no vaccine available that confers long-lasting protection against filarial infections. Previous studies indicated that vaccination with irradiated infective L3 larvae reduces the worm load. This present study investigated whether the additional activation of cytosolic nucleic acid receptors as an adjuvant improves the efficacy of vaccination with irradiated L3 larvae of the rodent filaria  Schistosoma spp., four vaccine candidates, including the Schistosoma haematobium glutathione S-transferase, Schistosoma mansoni fatty acid-binding protein, S. mansoni tetraspanin, and S. mansoni calpain, are being evaluated in clinical trials with aluminum-based adjuvants [S. haematobium glutathione S-transferase) [Onchocerca volvulus, there has been progress using recombinant O. volvulus antigen, i.e., Ov-103 and Ov-RAL-2, which induced protections of up to 64% in combination with alum-based adjuvants in a murine model [Ov-103 and Ov-RAL-2 in the bovine model utilizing infections with Onchocerca ochengi led to a significant decrease in the rate of infection and development of microfilaridermia in cows that were infected via natural exposure [Brugia malayi (Bm-103 and Bm-RAL-2) coadministered with alum resulted in a significant worm reduction (61%) and reduced the embryogenesis of the remaining worms in a gerbil model [O. volvulus is currently in late preclinical development. For filarial infections in general, one of the best efficacies is obtained when irradiated L3 larvae are used for vaccinations, which can be used to study protective immune responses and explore novel vaccination approaches [Helminth infections affect more than 1 billion people and put a high socioeconomic burden on endemic countries . The elidjuvants . Initialsferase) . For thene model ,9. Furthil model . The putproaches ,13.Litomosoides sigmodontis murine model to identify new vaccination strategies. L. sigmodontis establishes chronic infections in susceptible BALB/c mice and triggers immune responses that resemble those of human filarial infections [L. sigmodontis L3 larvae every week resulted in the generation of parasite-specific IgG1 antibodies and a reduction in the adult-worm burden following a challenge infection of around 50% [In this project, we used the fections ,15,16. Pound 50% ,13,17,18One of the challenges to developing vaccines against helminth infections is the modulation of the host’s immune system by the parasite. During helminth infections, the host’s immune system is strongly regulated by the parasite in order to facilitate its survival in the host ,20. TypeSo far, a possible contribution of nucleic acid immunity in the context of helminth infection has not been addressed. Nucleic acid immunity is best studied in the context of viral infections where it triggers dominant type 1-associated immune responses . DiffereIn this study, we show that the use of nucleic acid receptor agonists as a vaccine adjuvant enhances local immune responses, including the production of type I IFN during immunization, which results in increased parasite-specific immune responses and enhanced worm clearance after infectious challenge. Consequently, we present the use of the cytosolic RNA receptors agonists as a novel strategy to improve filarial vaccination efficacy.Meriones unguiculatus were purchased from Janvier Labs, Saint-Berthevin, France. All animals were housed in individually ventilated cages within the animal facility at the IMMIP, University Hospital Bonn, with unlimited access to food and water and a 12 h day/night cycle.Six-week-old female BALB/c mice and eight-week-old Meriones unguiculatus were naturally infected with L. sigmodontis via exposure to Ornithonyssus bacoti mites containing the infective L3 larvae for 24 h as previously described [Mice and escribed . L. sigmodontis L3 larvae, M. unguiculatus were euthanized 5 days after the infection with an overdose of isoflurane , and the L3 larvae were isolated by pleural lavage with 25 mL warm Advanced-RPMI medium . To generate To quantify the adult-worm burden, mice were euthanized 63 days after the infection with an overdose of isoflurane , and the adult worms were isolated by pleural lavage with 8–10 mL cold PBS . The isolated worms were quantified, and the gender of the adult worms was determined. g, 5 min, RT) and the supernatant was discarded. Microfilariae in the pellet were then counted via light microscopy.For microfilariae quantification, 50 µL of peripheral blood were drawn from the facial vein via a puncture with a 4 mm animal lancet  to EDTA tubes and incubated with 950 μL 1× red blood cell lysis buffer  for 10 min at room temperature (RT). The blood was then centrifuged  and 20 µg/mouse 3pRNA [®  according to the manufacturer’s protocol at an N/P ratio of 8.Twenty µg/mouse poly(I:C) ,37,38,39se 3pRNA ,41 . The photon energy of the radiation source was 10 MeV with a dose rate of 24 Gy/min. L3s were irradiated with 450 Gy [Attenuation of L3 larvae by irradiation was performed at the Department of Radiation Oncology, University Hospital Bonn, Germany. Radiation was performed using a TrueBeam STxh 450 Gy  and applThen, 25 irradiated L3s  and agonFor the skin analysis , the rigIn the vaccination experiments, mice were subcutaneously injected in the neck with 25 L3 larvae attenuated by 450 Gy radiation (‘att.’) with or without an agonist three times in two-week intervals. Two weeks after the last immunization, blood was drawn, and the mice were naturally infected (day 0). Further, blood was drawn on days 50, 57, and 63 and ex vivo analysis was performed on day 63 after the infection.g, 5 min, RT) and the serum was stored at −20 °C until analysis.Blood for serum was collected from the facial vein of animals via a puncture with a 4 mm animal lancet  and stored at RT. The clotted samples were centrifuged (6000× 2) was excised post mortem. Half of the skin was stored in 700 µL Trizol  at −20 °C until RNA isolation. The other half was minced and incubated at 37 °C on a shaker (350 rpm) for 75 min in an RPMI medium , supplemented with 10% FCS , 1% Penicillin /Streptomycin (10 mg/mL) , 2 mM L-Glutamine , 0.25 mg/mL Liberase TL , and 0.5 mg/mL DNase I . The reaction was stopped with RPMI medium supplemented with 10 mM EDTA  and 2% FCS. Cells were passed through a 70 µm cell strainer , centrifuged at 400× g for 5 min at 4 °C and taken up in MACS buffer , 2 mM EDTA ). Cells were then counted with a CasyR TT Cell Counter + Analyser System , and 1 × 106 cells were analyzed by flow cytometry.Four hours after the injection of L3 larvae and/or agonists, the skin . Cells were washed with MACS buffer, counted with a CasyR TT Cell Counter + Analyser System , and 1 × 106 cells were analyzed by flow cytometry.The pleural lavage was performed with 8–10 mL cold PBS. The first mL was collected and centrifuged at 400× g for 5 min at 4 °C. RBC lysis was performed according to the manufacturer’s protocol . Afterward, the cells were washed with MACS buffer and taken up in RPMI medium  supplemented with 10% FCS , 1% Penicillin /Streptomycin (10 mg/mL)  and 2 mM L-Glutamine . Cells were then counted with a CasyR TT Cell Counter + Analyser System , and 1 × 106 cells were analyzed by flow cytometry. For ELISA analysis, 2 × 106 cells/mL were plated and stimulated with 2.5 µg/mL Concanavalin A  or 25 µg/mL crude L. sigmodontis adult-worm extract (LsAg) for 72 h in 48-well plates. The supernatant was stored at −20 °C until further analysis.Isolated spleens were perfused with 0.5 mg/mL collagenase VIII buffer , minced, and incubated at 37 °C for 30 min on a shaker with 350 rpm. MACS buffer was added, and a single-cell suspension was generated by passing the cells through a 70 µm metal strainer. The cells were centrifuged at 400× g for 10 min at 4 °C. The protein concentrations of crude extracts were determined using the Advanced Protein Assay .For LsAg preparation, freshly isolated adult worms were rinsed in sterile PBS before being mechanically homogenized under sterile conditions. Insoluble material was removed by centrifugation at 400× ® 20  and blocked for 2 h at RT with PBS containing 1% bovine serum albumin (BSA) . Sera from day 0, isolated from the blood that was drawn after the immunization and prior to the challenge infection, were diluted 1:50, sera from following time points after challenge infection were diluted 1:1000 in 1% BSA/PBS and incubated o/n at 4 °C. Plates were washed and biotinylated murine IgE, IgG1 or IgG2a/b antibodies , at a dilution of 1:400 in 1% BSA/PBS, were added and incubated for 2 h at RT on a shaker. After washing, streptavidin-HRP  was added for 30 min at RT on a shaker. After washing, TMB  was added. Upon coloration, the reaction was stopped with 2 M H2SO4 . Reading was performed at 450 nm and 570 nm wavelengths using a SpectraMax190  with Soft Max Pro 7 software .Plates were coated with 20 µg/mL LsAg diluted in PBS overnight (o/n) at 4 °C. Plates were washed with PBS containing 0.5% TweenM. unguiculatus 5 days after natural L. sigmodontis infection. Peritoneal cells were isolated from naïve BALB/c WT donor mice and 2 × 105 peritoneal cells were cocultured with 10–12 L3 larvae in RPMI-medium , supplemented with 25% pooled serum drawn from immunized animals immediately prior to the challenge infection . The motility of L3 larvae was scored under the microscope on a daily basis for a total of three days. The following scores were used to assess the motility: 4: fast and continuous movement, 3: slower but continuous movement, 2: slower and discontinuous movement, 1: movement discontinuous and limited to larval ends, 0: no movement observed within 30 s. The assay was adapted from Veerapathran et al. . L3 larvCytokines were quantified in the first mL of pleural wash and the 72 h splenocyte culture supernatant. IL-5 and IFN-γ were quantified using Invitrogen™ eBioscience™ ELISA Ready-SET-Go!™ . IP-10 (CXCL10), RANTES (CCL5), and Eotaxin 1 (CCL11) were quantified using DuoSet ELISA kits . The manufacturers’ protocol was followed. 2SO4  served as a stopping solution. Reading was performed at 450 nm and 570 nm wavelength using a SpectraMax190  with Soft Max Pro 7 software .The 2 M H6 cells were used per flow cytometry staining. For surface staining, cells were incubated for 20 min with a mastermix prepared in Fc-block ). Mastermixes were prepared as combinations of the following antibodies. If not stated otherwise, the antibodies were purchased from BioLegend, San Diego, CA, USA: CD3 , CD4 , CD8 , CD11b , CD11c , CD45 , CD86 , FOXP3 , GATA3 , I-Ab , Ly6C , Ly6G , purified RELM-α polyclonal, rabbit,  combined with goat anti-rabbit Al488 , RORγt , and T-bet , SiglecF . The 1 × 10® Software V10 . Fluorescence minus one (FMO) controls were used for evaluation.For intracellular stainings, cells were incubated in a fixation/permeabilization buffer  for 20 min at RT. Cells were washed and blocked overnight in Fc-block   in PBS with 1:1000 rat IgG ) at 4 °C. The next day, cells were permeabilized with a permeabilization buffer for 20 min at RT  and stained with mastermix containing antibodies for extracellular and intracellular targets for 45 min at 4 °C. After staining, the cells were washed. Data acquisition was performed on a CytoFLEX S  and analysis with FlowJo® 2 mL Soft Tissue Homogenizing Ceramic Beads Tube  using the Precellys® 24 machine, program “6000 − 3 × 60 − 120”. The homogenate was incubated at RT for 5 min in a fresh vial and 70 µL of 1-Bromo-3-chloropropane (BCP)  was added. The sample was then vortexed and incubated for 2–3 min at RT. After centrifugation for 15 min at 12,000× g at 4 °C, 350 µL of the aqueous phase was transferred into a 2 mL reaction tube and placed into QIAcube  for automated RNA isolation. The animal tissue and cell protocol, including an on-column DNase digest, was followed using the RNeasy® Mini kit . The 70% ethanol was exchanged with 100% ethanol. Skin samples stored in 700 µL Trizol were homogenized in a Precellys® Reverse Transcription Kit  using oligoDT12–18 primer  and RNaseOUTTM recombinant ribonuclease inhibitor . The mastermixes were prepared using the HotStarTaq® DNA Polymerase kit  and SYBRTM Green Nucleic Acid Stain . Samples were run on a Rotor-Gene Q  and analyzed with Rotor-Gene Q Series software .cDNA was generated from 1 µg RNA with the OmniscriptPrimer sequences: β-actin: forward: 5’ TGACAGGATGCAGAAGGAGA 3’, reverse: 5’ CGCTCAGGAGGAGCAATG 3’. IP-10/CXCL10: forward: 5’ GCCGTCATTTTCTGCCTCAT 3’, reverse: 5’ GCTTCCCTATGGCCCTCATT 3’. IFN-β: forward: 5’ CAGGCAACCTTTAAGCATCAG 3’, reverse: 5’ CCTTTGACCTTTCAAATGCAG 3’.p values < 0.05. For the comparison of MF+ mice, Fisher’s exact test was used to test for significant differences. For the comparison of antibody/motility levels over time (p < 0.1) were confirmed by repeated experiments, the trends were indicated in the figure.GraphPad Prism software version 8  was used for statistical analysis. The Kruskal–Wallis test followed by Dunn’s post hoc multiple comparisons was used to test for significant differences between multiple groups. The Mann–Whitney U-test was used to test for significant differences between two unpaired groups. Data are shown as median with interquartile ranges. Considered significant were ver time G–I, a twver time F or Tukever time G–I was u+ cells in the skin. Only the injection of poly(I:C) or 3pRNA, but not R848 or CpG-C, induced a strong local type I IFN response (p = 0.03)  .L. sigmodontis L3 larvae and local as well as systemic immune responses were analyzed four hours after the immunization (+ DCs in the skins of mice injected with att. L3 larvae and poly(I:C) or 3pRNA, compared to the 0.9% NaCl control or the injection of att. L3 larvae alone (p = 0.008) and IP-10 (p = 0.02) compared to the injection with att. L3 larvae alone  and 3pRNA were selected for further analysis. Poly(I:C) is a known agonist of the RNA sensors TLR3, MDA5, and RIG-I ,48, whilnization A. Flow cnization B. At theae alone C,D. The ae alone E. Levelsae alone F,G. In aae alone F,G. HoweL. sigmodontis immunization strategy. To that end, mice were subcutaneously immunized with att. L3 larvae in combination with poly(I:C) or 3pRNA every two weeks for a total of three times (L. sigmodontis-specific IgE compared to nonimmunized mice (L. sigmodontis-specific IgG1 (L. sigmodontis-specific IgG2a/b (Since the treatment with poly(I:C) or 3pRNA enhanced local immune responses, the agonists were included in the ee times A. This wzed mice B. All imfic IgG1 C. The im IgG2a/b D. Howeve IgG2a/b D. L. sigmodontis L3s, the medium was supplemented with the serum of immunized mice, and the motility of L3s was scored with a five-point scale over time  and d50 to d63 (p = 0.0433). IgG1 levels were lowest in unimmunized mice on day 37 after infection, but highest at the following time points  (p = 0.0129) and d57 (p = 0.0208) in comparison to d37. IgG2a/b levels were lowest in nonimmunized animals (p = 0.0156). Overall, the immunization with att. L3 larvae induced parasite-specific functional antibodies, which were enhanced by the use of nucleic acid-receptor agonists as adjuvants.Parasite-specific antibodies were measured in serum obtained 37, 50, 57, and 63 days after challenge infection. At all times, parasite-specific IgE was lowest in the unimmunized control group G. In all to d37) H. IgG1 l animals I. The imp = 0.0002) and a significant reduction when compared to the immunization with att. L3 larvae alone by direct comparison . The use of 3pRNA in combination with att. L3 larvae for immunization resulted in a significant reduction in worm burden by 57% compared to unimmunized animals . No differences were observed regarding the sex of the worms  by the use of poly(I:C) or 3pRNA as the agonist, respectively.Of note, none of the immunization regimens led to a complete absence of MF-positive mice , indicatp = 0.01) in the pleural cavity of mice immunized with a combination of att. L3 larvae and poly(I:C) and reduced by the trend in the pleural cavity of mice immunized with att. L3 larvae and 3pRNA (p = 0.08). The concentration of the chemokine CCL5/RANTES and the proinflammatory cytokine IFN-γ were significantly decreased in the pleural cavities of mice immunized with a combination immunization of att. L3 larvae with poly(I:C) or 3pRNA, compared to unimmunized mice  and 3pRNA in the spleen compared to control animals F,H. FreqTaken together, the use of the nucleic acid receptor agonists poly(I:C) or 3pRNA significantly reduced the adult-worm burden, increased parasite-specific immune responses, and enhanced immunization efficacy.L. sigmodontis L3 larvae was successful in enhancing parasite-directed immune responses during subsequent infection, resulting in a reduced adult-worm burden of 73% and 57%, respectively.In this study, we demonstrated that the subcutaneous injection of att. L3 larvae along with nucleic acid receptor agonists poly(I:C) and 3pRNA enhanced local immune responses. Furthermore, the implementation of poly(I:C) and 3pRNA as adjuvants during the immunization with irradiated L. sigmodontis and may present a novel strategy to boost the efficacy of helminth vaccines that are currently developed. The agonist poly(I:C) mainly targets the cytosolic RNA sensors MDA5 and RIG-I but is also known to activate the endosomal RNA sensor TLR3 [L. sigmodontis L3 larvae [L. sigmodontis infection, and DC frequencies decreased after agonist injection. Therefore, the local activation with poly(I:C) and 3pRNA might enhance the activation of innate but also adaptive immune responses targeting the invading L3 larvae. Our results demonstrated that nucleic acid receptor agonists such as poly(I:C) and 3pRNA improve the efficacy of a vaccine against the filarial nematode sor TLR3 ,48, whilsor TLR3 . Upon su3 larvae ,51. Ther3 larvae . In lineL. sigmodontis L3 larvae resulted in the generation of parasite-specific antibodies [Schistosoma, where the activation of the nucleic acid receptors TLR7/8 and TLR9 enhanced the immunization against Schistosoma japonicum [In the context of vaccination with irradiated L3 larvae, poly(I:C) as well as 3pRNA enhanced the efficacy of the immunization. Previous studies showed that the immunization with irradiated tibodies , a reductibodies ,17,53,54tibodies . In contaponicum . Similar.Overall, the presented results indicate that type I IFNs may be protective during filarial infection, and targeting the nucleic acids receptors TLR3, RIG-I, and MDA5 by their agonists poly(I:C) and 3pRNA can enhance vaccination efficacy by strengthening protective immune responses and present alternative adjuvants in the context of filarial vaccinations


Wax-less flowering Chinese cabbage wlm1 (wax-less mutation 1), which was fine-mapped on chromosome A09 by bulked segregant analysis sequencing (BSA-seq) of B.rapa genome V3.0. There was only one gene (BraA09g066480.3C) present in the mapping region. The homologous gene in Arabidopsis thaliana is AT1G02205 (CER1) that encodes an aldehyde decarboxylase in the epidermal wax metabolism pathway. Semi-quantitative reverse transcription PCR and transcriptome analysis indicated that BraA09g066480.3C was expressed in ‘FT’ but not in ‘CX001’. BraA09g066480.3C was lost in the CXA genome to which ‘CX001’ belonged.To clarify the mechanism of the wax deficiency, the wax-less flowering Chinese cabbage doubled-haploid (DH) line ‘CX001’ and Chinese cabbage DH line ‘FT’, obtained from isolated microspore culture, were used in the experiments. Genetic analysis showed that the wax-less phenotype of ‘CX001’ was controlled by a recessive nuclear gene, named BraA09g066480.3C was the causal gene for wax-less flowering Chinese cabbage ‘CX001’. This study will lay a foundation for further research on the molecular mechanism of epidermal wax synthesis in flowering Chinese cabbage.The work presented herein demonstrated that The online version contains supplementary material available at 10.1186/s12870-023-04424-3. Epidermal wax is the grayish-white fatty substance that coats the surfaces of the above-ground parts of terrestrial plants . Plant eThe synthesis of cuticular wax begins in the plastids of epidermal cells . Acylaceeceriferum (cer) mutant in Arabidopsis thaliana, and subsequently a great many genes involved in the regulation, biosynthesis, and transport of waxes were identified. Wax-related genes have been reported in many plant species such as maize (Zea mays) [Hordeum vulgare) [Cucumis sativus) [Solanum lycopersicum) [Triticum aestivum) [(Oryza sativa) [Sorghum bicolor) [BnCER1 was mapped to A09 chromosome in Brassica napus [BrWax1 was mapped to A01 chromosome [BoCER1 had a 2,722-bp insertion [BrCER1 gene of the wax less mutant of non-heading Chinese cabbage had a 39-bp deletion [Brcer1 gene of Chinese cabbage, there was a single-base C to T transition which resulted in the loss of epidermal wax [wlm1 mutation has not been fine mapped in flowering Chinese cabbage.Dellaert et al.  reportedea mays) , barley vulgare) , cucumbesativus) , tomato ersicum) , wheat (estivum) , rice (O sativa) , and sorbicolor) . The epica napus  In cabbaromosome ; BoCER1 nsertion  respectideletion . In the rmal wax . The genwlm1 in flowering Chinese Cabbage using BSA-Seq and found that the loss of BraA09g066480.3C on chromosome A09 resulted in plants with a wax-less, smooth-surface phenotype. Semi-quantitative RT-PCR analysis and transcriptome analysis showed that BraA09g066480.3C was not expressed in ‘CX001’. The genome sequence of CXA (the wax-less line) in the Brassicaceae Database indicated that the locus BraA09g066480.3C has been lost in flowering Chinese cabbage line ‘CX001’. This suggests that BraA09g066480.3C plays a key role in wax synthesis in flowering Chinese Cabbage.In this study, scanning electron microscopy (SEM) observation showed that the surfaces of ‘CX001’ plants are smooth and are without wax. We mapped the wax-less gene 1, F2, and BC1 populations for use in genetic analysis and gene mapping. F1 crossed with ‘FT’ to get BC1P1, crossed with ‘CX001’ to get BC1P2. The plants were cultivated in a greenhouse at Shenyang Agriculture University. The seeds were germinated on infiltrated filter paper and then vernalized for 15 days in a 4-degree refrigerator. After vernalization, the seeds were planted in seedling trays and grown in the greenhouse. After 25 days, the seedlings were transplanted into nutrition bowls within the greenhouse. During the bolting stage, the presence of epicuticular wax on stems and leaves was visually assessed to determine its presence. Wax-covered plants exhibited a grayish-white appearance, while wax less plants had a glossy surface.‘CX001’ is a doubled-haploid line of flowering Chinese cabbage, obtained from microspore culture isolated from ‘Super Grade 50 Sweet Flowering Chinese Cabbage’, with bright green leaves and stems. ‘FT’ is a DH line of Chinese cabbage, obtained from microspore culture isolated from ‘FT50’. The materials were planted in Shenyang Agricultural University. ‘CX001’ and ‘FT’ were used as parents to prepare FBuds, stems, leaves, and siliques of ‘CX001’ and ‘FT’ were harvested for SEM analysis. The leaves were cut into 0.5 cm squares and fixed overnight in pentanediol, dehydrated, frozen in liquid nitrogen, lyophilized, and plated with gold. The samples were observed with a Hitachi Regulus 8100 (Japan) scanning electron microscope.Fresh leaves were harvested at the bolting stage for DNA and RNA extraction. The cetyltrimethylammonium bromide (CTAB) method  was used2 plants to construct the WL-pool. Genomic DNA was isolated from the parental lines and WL-pool, fragmented into 400 bp fragments by sonication. These fragments underwent end repair, adapter ligation, purification, and PCR amplification for library preparation. Following library construction, preliminary quantification was performed using Qubit 2.0, and insert fragment lengths were determined using Agilent BioAnalyzer 2100. The average fragment length was appropriate. qPCR accurately quantified the library’s effective DNA concentration to ensure quality. The library was then sequenced using the Novaseq 6000 platform , and the sequencing mode was PE150. The off-machine data was quality-controlled with FastQC v0.11.7 [B.rapa genome V3.0 Database (http://www.brassicadb.cn/#/Download/) using BWA v0.7.12 [For BSA-seq, we selected 50 wax-less F v0.7.12  and the SNP and InDel detection was performed using GATK v3.8.1 UnifiedGenotyper . The SNPAfter SNPs and INDELs were called, the SNP-index was calculated to locate regions related to the wax-less trait. The SNP-index value was obtained by following the methods in ‘Genome sequencing reveals agronomically important loci in rice using MutMap’ .wlm1 gene by InDel molecular marker linkage analysis. The sequences within the candidate intervals were downloaded from the B.rapa genome V3.0 database. InDel primer design was accomplished via utilization of Primer Premier 5.0 software [2 population. Primer screening and population linkage analysis were performed using 10 μL system PCR reactions and agarose gel electrophoresis. The recombination values were converted to the distance of the genetic map (cM) according to Kosambi's mapping function [Validation and further shortening of the candidate intervals of the BSA-seq-identified software . Polymorfunction .http://ccb.jhu.edu/software/hisat2/index.shtml) to assemble the transcripts. Based on the Hisat2alignment results, the transcripts were assembled using StringTie v2.1.4 [Stems of ‘FT’ and ‘CX001’ were selected for RNA extraction, with three replicates per sample. The integrity and purity of the total RNA were determined using the Bioanalyzer 2100 and the RNA 6000 nano LabChip kit. After passing quality inspection, a sequencing library was constructed that was sequenced on an Illumina Hiseq4000 instrument using the PE150 mode. The raw read data was transformed into nucleotide sequences by base calling. The data was preprocessed by removing reads with joints and those that contained poly-A and poly-G tracts, reads containing > 5% Ns, and low-quality reads in which > 20% of the bases had Q scores < 10. The clean data reads were aligned to the reference genome using Hisat2 software  enrichment analysis, we used topGO v2.40.0  and founP-value < 0.05 using the hypergeometric distribution method [For Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, we used clusterprofiler  and founn method .wlm1-1 were designed to amplify cDNA fragment  assays were performed on a QuantStudio 6 PCR system using Ultra SYBR Mixture dye . The internal control for gene expression was the ACT gene. Three biological replicated and three technical replicated each. The 2Compared with the gray phenotype of ‘FT’ plants, the wax-less flowering Chinese cabbage plants exhibited a smooth green surface Fig. a, b. SEM1 and F1′ generation plants all showed the gray epidermal phenotype, indicating that the wax-less trait from ‘CX001’ was controlled by one or more recessive nuclear genes. In a population consisting of 4,390 F2 plants, 3,287 were waxy and 1,103 plants showed the wax-less phenotype, with a waxy/wax-less segregation ratio of 2.98:1. A Chi-squared test showed that the segregation ratio in the F2 population fitted the expected segregation ratio of 3:1 (χ2 = 0.038 < χ20.05 = 3.84) for a single recessive gene. The segregation ratio of the BC1P2 generation was 1:1 (χ2 = 0.186), and all of the BC1P1 progeny showed the waxy phenotype, indicating that the wax-less phenotype of ‘CX001’ was controlled by a recessive nuclear gene  from the WL-pool, ‘CX001’, and ‘FT’, respectively. We used BWA  to alignwlm1 gene in flowering Chinese cabbage, 20 pairs of Indel marker primers  were obtained from ‘CX001’, and 129,714,776 raw reads (19.59 Gb) were obtained from ‘FT’ , ‘lipid biosynthetic process’ (GO:0008610), ‘oxidoreductase activity’ (GO:0016491) and ‘oxidation–reduction process’ (GO:0055114). Among these, the term ‘iron ion binding’ (GO:0005506) consisted of 98 DEGs, with 44 upregulated and 54 downregulated genes; the term ‘lipid biosynthetic process’ (GO:0008610) consisted of 40 DEGs, with 26 upregulated and 14 downregulated genes; the term ‘oxidoreductase activity’ (GO:0016491) consisted of 408 DEGs, with 201 upregulated and 207 downregulated genes; the term ‘oxidation–reduction process’ (GO:0055114) consisted of 344 DEGs, with 168 upregulated and 176 downregulated genes.GO enrichment analysis was performed on 8,202 DEGs which were enriched in 1,700 GO terms Table Sc, d. BraBraA09g066480.3C was specifically enriched in the ‘cutin, suberine, and wax biosynthesis’pathway (brp00073), which contained a total of 19 differentially expressed genes. Among these genes, 7 were upregulated, namely BraA09g024730.3C, BraA09g000630.3C, BraA04g026730.3C, BraA06g018310.3C, BraA07g028970.3C, BraA05g007850.3C, and BraA01g017490.3C. Additionally, there were 12 downregulated genes in this pathway, specifically BraA09g024730.3C, BraA09g000630.3C, BraA04g026730.3C, BraA06g018310.3C, BraA07g028970.3C, BraA05g007850.3C, and BraA01g017490.3C.KEGG enrichment analysis showed that the DEGs were enriched to 127 pathways –34, and BraA09g066480.3C (CER1) in ‘FT’ was much higher than in ‘CX001’, with a maximum log2Fold-Change value of -10.2. Compared with ‘FT’, the expression of BraA05g036180.3C (CYP77A6), BraA09g066190.3C (CYP86A4), BraA07g030740.3C (CER6), and BraA03g004300.3C (KCD) was down-regulated in ‘CX001’; BraA07g028970.3C (HTH), BraA04g026730.3C (CER1-like2), BraA05g007850.3C (CER1-like2), BraA01g015290.3C (CER2), and BraA04g032210.3C (CYTB5-C) were up-regulated in ‘CX001’. BraA06g011410.3C (WIN1), BraA02g038430.3C (MYB30), and BraA09g007290.3C (MYB96) that belong to the transcriptional regulation pathway were differentially expressed in ‘FT’ and ‘CX001’. BraA02g038430.3C (MYB30) and BraA09g007290.3C (MYB96) were up-regulated and BraA06g011410.3C (WIN1) was down regulated in ‘CX001’. In the cuticular wax transport pathway, BraA05g026040.3C (ABCG12) was down regulated in ‘CX001’. qRT-PCR analysis showed that cuticular wax-related gene expression patterns were consistent with RNA-seq data, validating the accuracy and reliability of the transcriptome data  and the Arabidopsis Information Resource (https://www.arabidopsis.org/) were used to analyze the BraA09g066480.3C gene within the mapping region, and the homologous gene in Arabidopsis is AT1G02205 (CER1). In a study of Arabidopsis cer1-1 mutant, it was found that the mutation of the CER1 resulted in a smoother epidermis and greener color, and the composition of the waxy layer was also changed. The authors speculated that CER1 may be related to enzymes involved in alkane synthesis [BraA09g066480.3C, and the replaced sequence also contains only one gene, BraA09g003690.3C  database (https://www.ncbi.nlm.nih.gov)  [cer-1 locus, and the wax-less trait was accompanied by semi-sterility. Koornneef et al. (1989) [cer1, cer3, cer6, cer8, and cer10. These mutants exhibited partial sterility under conditions of low humidity and near-complete sterility under conditions of high humidity. McNevin et al. (1993) [Arabidopsis thaliana eceriferum mutants, including cer1. Wang et al. (2017) [Brcer1 from the epidermal wax mutant of non-heading Chinese cabbage. A mutation in Brcer1 was also reported in Chinese cabbage, and it resulted in the loss of epidermal wax [Epidermal wax represents a crucial barrier between the aerial parts of the plant and the environment for terrestrial plants. The first report of wax-related mutants was in . 1979)  found 53. (1989)  elucidat. (1993)  used T-D79  foundCER1, CER3, and MAH1 in siliques, flowers, stems, leaves, and seedlings suggests that they play a crucial role in the epidermal wax formation pathway [cer1 mutant plants, a significant reduction in the components of epidermal wax was observed, while a slight increase in the content of aldehydes was noted [cer1 mutation in flowering Chinese cabbage may lead to the failure of aldehydes to be converted to alkanes in the decarbonylation pathway, which further leads to the failure of alkanes to participate in the synthesis of secondary alcohols and ketones. SEM observation showed that there was a thick layer of epidermal wax on the surface of ‘FT’ plants, while the buds, stems, leaves, and siliques of ‘CX001’ were very smooth, with almost no wax powder, and the stomata were clearly visible.Aarts et al. (1995)  reported pathway . In the as noted , 39, 40.CER1 genes have been reported in many species. Hu et al. (2009) [TaCer1 from wheat for the first time. Alkio et al. (2012) [PaCER1 was involved in the formation of epidermal wax in sweet cherry. CER1-1 played a key role in citrus fruit wax formation [CsCER1 was involved in VLCFA biosynthesis in cucumber [MdCER1 from apple and found that it played an important role in the epidermal wax synthesis pathway. Similarly, CER1 alleles have also been studied in species of Brassica. In cabbage, Bol018504 had a 2,722-bp insertion [Bol018504 is homologous to CER1. The Brcer1 gene of the wax less mutant of non-heading Chinese cabbage had a 39-bp deletion [Brcer1 gene of Chinese cabbage, there was a single-base C to T transition, the mutation site is located on the fourth exon, causing the conformation of the 255th amino acid to change from hydroxyl to benzene ring, and the amino acid sequence to change from S to F, that is, from serine to phenylalanine, which resulted in the loss of epidermal wax [wlm1 was lost in the flowering Chinese cabbage line ‘CX001’. During species evolution, a loss of the wlm1 gene occurred in ‘CX001’, but it did not have any discernible impact on the growth and development of the plants. Furthermore, this genetic alteration resulted in the desirable trait of wax-less in the individuals. and this is the first report of gene loss in flowering Chinese cabbage.. 2009)  cloned t. (2012)  showed tormation . CsCER1 cucumber . Qi et acucumber  cloned M009  clondeletion . In the rmal wax . We founwlm1 was fine mapped to a 12.70 kb region of chromosome A09, and the only gene in this region, BraA09g066480.3C, was lost from the genome of the flowering Chinese cabbage line ‘CX001’. BraA09g066480.3C was predicted to be the candidate gene for wlm1. BraA09g066480.3C encodes an aldehyde decarboxylase, which is a key enzyme in the epidermal wax biosynthesis pathway. The results of this study will extablishy a foundation for further research on the molecular mechanism of epidermal wax synthesis in flowering Chinese cabbage and other species in the Brassicaceae.In summary, BraA09g066480.3C on chromosome A09 resulted in plants with a wax-less, smooth-surface phenotype. Transcriptome analysis and semi-quantitative RT-PCR showed that BraA09g066480.3C was not expressed in wax-less ‘CX001’. BraA09g066480.3C was lost from the genome of the flowering Chinese cabbage line ‘CX001’. This study will lay a foundation for further research on the molecular mechanism of epidermal wax synthesis in flowering Chinese cabbage.We mapped the wax-less gene wlm1 in flowering Chinese cabbage and found that the loss of Additional file 1:Table S1. PCR primer sequences used for semi-quantitative RT-PCR, qRT-PCR, Primer1,Primer2 and the amplification of BraA09g066480.3C. Table S2. Region of 90% confidence interval. Table S3. SNPof 90% confidence interval. Table S4. Region of 95% confidence interval. Table S5. SNPof 95% confidence interval. Table S6. PCR primers used to amplify InDel markers in the candidate region. Table S7. Statistical trancriptome data. Table S8. DEGs in diferent enriched GO terms. Table S9. KEGG enrichment analysis of the DEGs. Table S10. Epidermal wax-related differentially expressed genes from ‘FT’ and ‘CX001’. Table S11. The sequence information obtained from amplification in‘FT’ and ‘CX001’.Additional file 2:Fig. S1. Original picture of Fig. Additional file 3:Fig. S2. Original picture of Fig.


Influenza B virus (IBV) contributes to substantial influenza-mediated morbidity and mortality, particularly among children. Similar to influenza A viruses (IAV), the hemagglutinin (HA) and neuraminidase (NA) of IBV undergo antigenic drift, necessitating regular reformulation of seasonal influenza vaccines. NA inhibitors, such as oseltamivir, have reduced activity and clinical efficacy against IBV, while M2 channel inhibitors are only effective against IAV, highlighting the need for improved vaccine and therapeutics for the treatment of seasonal IBV infections. We have previously described a potent human monoclonal antibody (hMAb), 1092D4, that is specific for IBV NA and neutralizes a broad range of IBVs. The anti-viral activity of MAbs can include direct mechanisms such as through neutralization and/or Fc-mediated effector functions that are dependent on accessory cells expressing Fc receptors and that could be impacted by potential host-dependent variability. To discern if the in vivo efficacy of 1092D4 was dependent on Fc-effector function, 1092D4 hMAb with reduced ability to bind to Fc receptors (1092D4–LALAPG) was generated and tested. 1092D4–LALAPG had comparable in vitro binding, neutralization, and inhibition of NA activity to 1092D4. 1092D4–LALAPG was effective at protecting against a lethal challenge of IBV in mice. These results suggest that hMAb 1092D4 in vivo activity is minimally dependent on Fc-effector functions, a characteristic that may extend to other hMAbs that have potent NA inhibition activity.  Seasonal influenza vaccines significantly reduce influenza associated morbidity and mortality, but continuing antigenic drift of circulating influenza strains results in substantial variations in efficacy. In the United States, influenza B virus (IBV) infections comprised, on average, ~25% of influenza infections in the 2010–2011 through 2019–2020 seasons and ~1% of infections in the 2021–2022 through 2022–2023 seasons ,5 and ha50 values have been reported for IBV compared to influenza A viruses (IAV) [Available treatments for IBV infection include the neuraminidase (NA) inhibitor oseltamivir; however, much higher ICes (IAV) ,9. M2 ches (IAV) ,11. Althes (IAV) ,12. A loes (IAV) ,14,15. GSeveral hMAbs have been reported that have broad activity against a wide range of influenza viruses, including those that target the stem region of HA, which appear to be at least partially dependent on Fc-mediated effector functions for its in vivo activity ,17,18,19We have previously developed 1092D4, a fully human hMAb that is able to inhibit NA enzymatic activity and directly neutralize a wide range of IBV isolates, including both the Victoria and Yamagata lineages . To dete2. For IBV infection, MDCK cells were maintained in post-infection medium , 1% PSG, and 1 μg/mL tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin ) and incubated at 33 °C with 5% CO2. Transfected HEK293T cells were maintained in DMEM with 5% Fetal Clone II  and 1× antibiotic/antimycotic  at 37 °C in a 5% CO2 incubator. Influenza B/Malaysia/2506/04, B/Florida/04/06, and B/New York/PV00081/18 were kindly provided from Dr. Florian Krammer at Icahn School of Medicine at Mount Sinai. B/Brisbane/60/2008 was previously described [Cells and viruses: Madin-Darby canine kidney  and human embryonic kidney  cells were grown in Dulbecco’s modified Eagle’s medium  supplemented with 5% fetal bovine serum  and 1% PSG  at 37 °C with 5% COescribed .E. coli cells. Resulting colonies were again sequenced to ensure the correct insertion. Purified plasmid DNA of either the parental heavy or the LALAPG heavy chain and parental light chains were transfected into 60–80% confluent HEK293T cells in 10 cm tissue culture treated dishes using JetPrime  as previously described [2 atmosphere while culturing in DMEM with 5% Fetal Clone II (Cytiva) and 1× antibiotic/antimycotic (Corning/Mediatech). Media were harvested and replaced every two to three days. Harvested media were concentrated using 100,000 MWCO Amicon Ultra centrifugal filters  and then purified using Protein A magnetic beads  as described in the product literature.hMAb production. A new antibody vector was created by cutting out the IgG1 constant region of AbVec hIgG1 (GenBank: FJ475055.1) using SalI and HindIII restriction enzymes and replacing it with a newly synthesized oligonucleotide (IDT) containing the L234A, L235A, and P329G (LALAPG) mutations. Insertion was confirmed via sequencing. The previously cloned hMAb 1092D4  underwenescribed . TransfeEnzyme-linked immunosorbent assay (ELISA): ELISA plates  were coated with recombinant NA of B/Florida/04/2006 , B/Colorado/06/2017 , or B/Austria/1359417/2021  at 2 µg/mL. hMAbs were diluted in PBS, and binding was detected with HRP-conjugated anti-human IgG .FcγRI binding. Both 1092D4 and 1092D4–LALAPG were coated onto ELISA plates  at either 2, 1, 0.5, or 0.25 µg/mL. Plates were sealed and stored at 4 °C overnight before being washed 3× in PBS. Biotinylated Human Fc gamma RI/CD64  was diluted to 1, 0.1, and 0 µg/mL, and 50 µL was added to wells in triplicate for each antibody concentration. After 1 h of shaking at room temperature, plates were washed 5× in PBS with 0.05% tween, and 50 µL of 2 µg/mL Streptavidin–horseradish peroxidase  was added and incubated for 1 h, shaking at room temperature. Plates were again washed 5× in PBS with 0.05% tween, and 50 µL of TMB substrate  was added to wells and plates incubated for 5 min at room temperature. To stop the reaction, 50 µL of 1N HCl was added to wells and absorbance was read at 450 nm.6 yellow-green neutravidin-fluorescent beads (Life Technologies) per reaction in a 25 μL of final volume. Antigen-coated beads were subsequently washed twice in PBS–BSA (0.1%) and transferred to a 5 mL Falcon round bottom tube . hMAbs, diluted at 0.5 or 5 μg/mL, were added to each tube in a 20 μL of reaction volume and incubated for 2 h at 37 °C in order to allow Ag–Ab binding. Then, 250,000 THP-1 cells (human monocytic cell line obtained from NIH AIDS Reagent Program) were added to the cells and incubated for 3 h at 37 °C. At the end of incubation, 100 μL 4% paraformaldehyde was added to fix the samples. Cells were then assayed for fluorescent bead uptake by flow cytometry using a BD Biosciences Symphony. The phagocytic score of each sample was calculated by multiplying the percentage of bead positive cells (frequency) by the degree of phagocytosis measured as mean fluorescence intensity (MFI) and divided by 106. Values were normalized to background values (cells and beads without mAb) and an isotype control to ensure consistency in values obtained on different assays. Finally, the phagocytic score of the testing hMAb was expressed as the fold increase over BSA-coated beads.Antibody-dependent cellular phagocytosis (ADCP): ADCP activity of the hMAbs was measured as previously described ,36 with Arachis hypogaea (peanut) was added to plates (2 µg/mL) and incubated for 2 h in the dark. Plates were washed three times, and 50 µL of TMB substrate  was added to wells and plates incubated for 20 min at room temperature. To stop the reaction, 50 µL of 1N HCl was added to wells and absorbance was read at 450 nm. A concentration of rNA was selected that was approximately 90% of the peak of the linear range of the resulting curve (~0.03 µg/mL) for inhibition assays. Both the 1092D4 parental and LALAPG variant hMAbs were diluted 1:2 on a washed fetuin-coated plate to cover a range from 0.006 to 12 µg/mL. The rNA was then added at the predetermined concentration to all but the “background” wells. Plates were sealed and incubated for approximately 20 h at 37 °C before being washed and developed as described above. Optical densities were corrected using the mean of the “background” wells, and then percent inhibition was calculated from the mean of wells incubated with the rNA alone. The IC50 was determined using non-linear regression in GraphPad Prism (Version 9.5.1).Enzyme linked lectin assay (ELLA). ELLA were performed essentially as previously described by Bernard et al. . In shor4 cells/well, 96-well plate format, quadruplicates) were infected with 200 PFU/well of the indicated virus. After 1 h of viral adsorption, cells were maintained in a post-infection medium -treated trypsin (Sigma)) containing two-fold serial dilutions (starting concentration of 50 µg/mL) of the hMAbs and incubated at 33 °C with 5% CO2. At 72 h post-infection, virus neutralization titer (NT) was evaluated following crystal violet staining of the cells and expressed as the lowest concentration of the hMAb to prevent the virus-induced cytopathic effect (CPE). Microneutralization assay. The neutralizing activity of both 1092D4 and 1092D4–LALAPG hMAbs against B/Malaysia/2506/04, B/Florida/04/06, B/Brisbane/60/2008, and B/New York/PV00081/18 was performed using virus neutralization assay as previously described [4 plaque-forming units (PFU)/mouse of B/Malaysia/2506/04. At days 2 and 4 post-infection (p.i.), viral replication was determined in the lungs of the infected mice. Briefly, four mice from each experimental group were euthanized by administration of a lethal dose of avertin and exsanguination, and lungs were surgically extracted and homogenized in 1 mL of PBS using Precellys tissue homogenizer . Virus titers (PFU/mL) were determined by standard plaque assay as previously described [In vivo studies: The prophylactic activities of both 1092D4 and 1092D4–LALAPG hMAbs were tested against B/Malaysia/2506/04 in mice. Briefly, female C57BL/6 mice (8 weeks of age) were purchased from the Jackson Laboratory and maintained in the animal facility at Texas Biomedical Research Institute under specific pathogen-free conditions and ABSL2 containment. Mice were intraperitoneal treated with a single dose of 2 or 20 mg/kg of each hMAb, isotype control, or PBS (mock treated) 12 h prior to viral challenge. For virus infection, mice were anesthetized following gaseous sedation in an isoflurane chamber and inoculated intranasally with 10To minimize the ability of 1092D4 IgG1 to bind to FcγRs, the well-described L234A and L235A substitutions (LALA) to residues in the CH2 domain, which form part of the FcγR binding site, and the P329A substitution (PG) in the C1q binding site to minimize binding to C1q ,33,38,39The LALAPG mutation is expected to reduce the ability of 1092D4 to bind FcR and subsequently diminish Fc-mediated effector functions. As expected, 1092D4-LALPG exhibited minimal binding to the FcγRI as compared to 1092D4 A. Simila50 of 0.049 and 0.037 μg/mL, respectively. These results indicate that the ability to inhibit the enzymatic activity of NA is not Fc-dependent. The primary function of influenza NA is to cleave sialic acid residues from the cell surface, enabling the release of the mature virus which can then infect new cells. Consistent with our previous findings that 1092D4 can potently inhibit NA enzymatic activity , 1092D4–The ability of 1092D4 and 1092D4–LALAPG to inhibit viral infection was measured using a microneutralization assay. Both were able to similarly neutralize all IBVs tested at NT < 1.0 μg/mL, including the Victoria lineage strains B/Malaysia/2506/2004, B/Brisbane/60/2008, and the recent B/New York/PV00081/2018; and the Yamagata lineage strain B/Florida/04/2006 . NTs forp = 0.0135). All doses of 1092D4 and 1092D4–LALAPG resulted in significant reduction in lung virus titers at 2 dpi, with no significant difference between equal doses of 1092D4 and 1092D4–LALAPG  required euthanasia by 7 days p.i., while all 1092D4- and 1092D4–LALAPG-treated mice survived infection A. All IB4–LALAPG C. At 4 dImproved vaccines and therapeutics for influenza that are sufficiently robust to tolerate its continued viral mutations and antigenic drift variation are needed. The pre-clinical and clinical efficacy of NA-directed antibodies and antivirals highlight the therapeutic potential of targeting NA. Here, we demonstrate that a hMAb that has activity against a broad range of IBV isolates potently inhibits NA enzymatic activity and can mediate protection against lethal IBV challenge without the reliance on Fc-effector functions.The ability of Abs to mediate anti-viral activity through Fc-dependent activities such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-mediated processes, as well as Fc-independent activities such as neutralization and viral enzyme inhibition, provides multiple and often complementary mechanisms by which the humoral immunity can respond to viruses. Fc-dependent effector functions are particularly critical for the anti-viral activity of Abs that do not recognize epitopes that can result in direct neutralization or inhibition of virus activity. A concern for such Fc-dependent Abs is extensive neutrophil, eosinophil, and NK cell accumulation in the lungs along with alveolar macrophage activation, which could result in hyperinflammation and the associated cytokine storm and pathology that negates the antiviral activity of the Ab ,41,42,43A few highly conserved epitopes on the influenza virus have been identified which have garnered attention with respect to universal vaccine development, including the stem region of HA and the enzymatic active site of NA. Thus far, HA stem Abs predominantly require Fc-effector functions for in vivo activity ,18,47,48Reducing the Fc-effector function of the previously identified NA-specific hMAb 1092D4 did not impact its capacity to bind or inhibit NA activity, nor did it alter the ability to neutralize influenza B in vitro. This lack of difference also persisted when antibodies were administered to mice prior to infection with a lethal dose of B/Malaysia/2506/04. Although Fc-effector function of 1092D4 can likely contribute to some of the viral clearance, we can conclude from this work that direct inhibition of NA through binding is likely a major anti-viral mechanism of 1092D4. NA-inhibiting antibodies may be key prophylactic and/or therapeutic agents, especially in the young or immunocompromised individuals, where other therapies are not available or are ineffective as a result of emerging drug-resistant viral variants. Research on such antibodies deserves further consideration.