DnaJ protein sequence retrieval
The complete (Uniprot Accession No. B5ZBQ4) amino acid sequence of U. urealyticum DnaJ was obtained from UniProt (http://www.uni-prot.org) in the FASTA format.DnaJ sequence conservation across different serovars of U. urealyticum was analysed using BLASTp, with a sequence identity of >95% and coverage percentage of >95%.
Analysis of physicochemical properties
Three physiochemical properties of the U. urealyticum DnaJ protein were analysed, including the theoretical number of transmembrane helices, MW and pI. The MW and pI were determined using Expasy ProtParam (https://web.expasy.org/protparam/). Transmembrane helices were studied using TMHMM (https://services.healthtech.dtu.dk/service.php?TMHMM-2.0), with a cut-off <1.
Structure prediction and assessment
The Phyre2 server (http://www.sbg.bio.ic.ac.uk/phyre2/) was used to predict the DnaJ 3D structure. Validation of the predicted 3D model through the generation of a Ramachandran plot was performed using PdbSum (http://www.ebi.ac.uk/thornton-srv/databases/pdbsum/Generate.html).
Antigenic epitope mapping
To determine the B-cell candidate linear epitopes, the full-length DnaJ protein sequence was analysed using BepiPred 2.0, with a 0.55 cut-off and a 15-me epitope length, and using ABCpred, with a 0.80 threshold value and a 16-me epitope length. Next, ElliPro (http://tools.iedb.org/ellipro/), available in IEDB, was used to perform structural B-cell epitope prediction, with a threshold value of >0.5, using the 3D model of DnaJ. Finally, the antigenicity of the linear and structural B-cell epitopes was further assessed using Vaxijen V2.0 (http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html).
To predict CTL epitopes, we followed the method reported by Paul et al. , scoring all peptides with the frequent HLA allele models, selecting the top 1% scoring peptides in the DnaJ sequence. For the helper T-lymphocyte epitope prediction, we applied a previously reported method with modifications , using the Tepitool resource in IEDB and a median consensus percentile of <20, which corresponds to a target-specificity threshold of 50%.
U. urealyticum and culture conditions
The standard strain of U. urealyticum serovar 8 (ATCC 27618) was cultivated in a pleuropneumonia-like organism with 16% foetal bovine serum (FBS, Gibco), 0.3% urea, 0.002% phenol red and 1,000 U/mL penicillin G. As described previously, U. urealyticum culture dilutions were inoculated onto a solid medium and incubated at 37 °C for 5 days to determine CFUs.
Recombinant DnaJ (rDnaJ) expression and purification
The genomic DNA (gDNA) of U. urealyticum strain 8 was used as a template for PCR to amplify the full-length DnaJ gene sequence. The following primers were used: forward primer, 5′-CGGGATCCATGGCGAAACGTGACTACTACG-3′ (the BamHI site is underlined), and reverse primer, 5′-CCGCTCGAGTTACTGCATCAAGCGGAGTTATTTGTTCACTTCTTTCAGCAGTTT-3′
(the XhoI site is underlined). The target DnaJ fragment (1,128 bp) was cloned into the expression vector pET28a using the BamHI and XhoI restriction sites, which harbour the His6 tag at the N-terminus. Recombinant pET28a-DnaJ was transformed into E. coli BL21 (DE3) cells. Before expression, the target DnaJ fragment integrity in the vector was confirmed by double digestion and sequencing.
rDnaJ expression was induced by 100 μg/mL kanamycin and 0.5 mM IPTG at 37 °C for 4 h. Bacterial cells were harvested and re-suspended in lysis buffer (10 mM imidazole, 20 mM NaH2PO4, 50 mM Tris-HCl (pH 7.8), 300 mM NaCl, 20% glycerol and 1% Triton X-100). For easy purification, rDnaJ with a His6 tag was expressed as a soluble protein, purified by Ni-NTA affinity chromatography, and step-eluted with different concentrations of imidazole (100–300 mM). The purified recombinant protein was investigated by 12.5% SDS- PAGE. Once purified, DnaJ was treated with ToxinEraser ™ Endotoxin Removal Kit (GenScript, Piscataway, NJ).
Studies were performed on 4–6-week-old female BALB/c mice and C57BL/6 mice from the Hunan SJA Laboratory Animal Co., China (Approval No. SCXK-Hunan-2019-0004). Specific-pathogen-free mice were housed in a barrier facility and handled by authorised personnel. All protocols were approved by the Institutional Animal Use Committee of the University of South China.
BMDC generation and stimulation
BMDCs were generated from C57BL/6 bone marrow precursors according to a previously described procedure [21, 22]. Briefly, the bone marrow precursors were differentiated for 6 d in Rosewell Park Memorial Institute (RPMI)-1640 with 15% FBS, 10 ng/mL granulocyte-macrophage colony-stimulating factor (R&D Systems), 10 ng/mL IL-4 (PeproTech) and 100 U/mL penicillin/streptomycin at 37 °C with 5% CO2. Flow cytometric analysis confirmed that the isolated cell purity was approximately 90% (Supplementary Figure 1). Immature BMDCs (5 × 106 cells/well) were plated onto 24-well plates and pulsed with DnaJ.
The BMDCs in the 24-well plates were incubated with DnaJ (5–80 μg/mL) for 48 h. The culture supernatant was collected and LDH activity was measured using a microplate reader at 570 nm.
Purified recombinant protein Uu-DnaJ (50 μg), emulsified with 100 μL of Freund’s complete adjuvant (Sigma-Aldrich, St. Louis, MO, USA) for the first inoculation (injected intramuscularly, 0 weeks), followed by two booster inoculations at 2-week intervals with Freund’s incomplete adjuvant (2 and 4 weeks). Negative control mice were injected with equal amounts of PBS or FA (PBS was emulsified in FA) at the immunising site. Serum samples were collected from the tail vein of each mouse weekly (0, 1, 2, 3, 4, 5 and 6 weeks). Two weeks after the last immunisation, the mice were euthanised and splenocytes were isolated for the immunoassays.
Genital tract infection challenge
Groups of mice were challenged with 1 × 107 CFU U. urealyticum (re-suspended in 30 μL of PBS) intravaginally 2 weeks after the last immunisation. Prior to the challenge, the oestrus cycle was synchronised by neck subcutaneous treatment with oestradiol benzoate (0.5 mg/mouse), increasing susceptibility to U. urealyticum infection. At weeks 0, 1, 2 and 3 after the genital challenge, vaginal or cervical samples were collected using swabs. Then, U. urealyticum was cultured in the samples, as previously described.
Western blot analysis
Three microliters of DnaJ lysate was separated by SDS-PAGE in a 12.5% gel and blotted onto a previously rehydrated polyvinylidene fluoride membrane. After 3 h of blocking with 5% skimmed milk and washing twice with Tris-buffered saline with Tween-20 (TBST), the membrane with the protein was incubated with DnaJ-immunised mouse serum or (Uu-serum) as the primary antibody and then kept overnight at 4 °C on a shaker. The following day, after four washes with TBST, the bands were incubated with anti-mouse IgG antibodies conjugated with horseradish peroxidase (HRP, 1:5,000) (Cell Signaling Technology, MA, USA) for 1.5 h at 37 °C. After the blots were rewashed with TBST, they were exposed to a chemiluminescence instrument (CLINX-6300, China). Finally, images were obtained using a film developer (EPSON-V370, Japan).
The antigen-specific antibody responses were assayed using ELISA in 96-well plates. For the antibody titres, the plates were coated with purified rDnaJ (10 μg/mL, 100 μL/well) overnight at 4 °C. The microplates were then blocked with 5% skim milk at 37 °C for 2 h. After blocking, the microplates were washed four times with PBS containing Tween 20 (PBST). The serum samples were serially diluted from 1:100 to 1:204,800 in dilution buffer, and 100 μL of the diluted serum was added to each well. After 2 h of incubation at 37 °C, the microplates were washed six times with PBST and HRP-conjugated goat anti-mouse IgG (1:10,000) was distributed into each well for 1 h at 37 °C. Then, 3,3’,5,5’-tetramethylbenzidine (substrate (100 μL/well) was added, the microplates were incubated at 37 °C for 20 min and the reaction was stopped using 2N H2SO4. The absorbance of the plates was measured at 450 nm using a microplate reader (Multiskan Mk-3, Thermo Fisher Scientific). The antibody titres were determined as the highest dilution of serum giving a detectable absorbance reading 2.1 times above the background average.
To further measure the serum IgG, IgA, IgM, IgG1, IgG2a, IgG2b and IgG3 responses, the microplates were coated with DnaJ. On the following day, the plates were incubated with the serum samples from the mice (1: 100 diluted in 5% non-fat milk in PBST). Horseradish peroxidase-conjugated goat anti-mouse IgA, IgM, IgG1, IgG2a, IgG2b or IgG3 was added at a 1:1,000 dilution to the designated well, following the same procedure as above.
Cytokine levels were determined in mouse splenocytes and BMDCs. The splenocytes were cultured at 2 × 106 cells/well in 24-well plates and stimulated with DnaJ (10 μg/mL) for 48 h. Alternatively, BMDCs (5 × 106 cells/well) were treated with 5–20 μg/mL DnaJ for 24 h. Then, supernatants were collected to measure the levels of secreted cytokines such as IFN-γ, TNF-α, IL-4, IL-10, IL-1β, IL-6 and IL-12p70 using the corresponding mouse ELISA kits, according to the manufacturer’s instructions (eBioscience, San Diego, USA).
Flow cytometry analysis
Mouse BMDCs or splenocytes were washed with BD Pharmingen Stain Buffer. For surface marker extracellular staining, the cells were incubated with anti-mouse CD11c (557400, BD Biosciences), CD80 (560526, BD Biosciences), CD86 (552692, BD Biosciences), MHC-II (562367, BD Biosciences), CD4 (553046, BD Biosciences) or CD8 antibodies (551162, BD Biosciences) at 4 °C for 30 min. For intracellular cytokine staining, the splenocytes were seeded at 37 °C for 8 h in Uu-DnaJ (10 μg/mL) of Golgi plugTM (51-2301KZ, BD Bioscience). The splenocytes were then treated for surface markers (CD4 or CD8), fixed/permeabilised with a Cytofix/Cytoperm solution (BD Bioscience) and then stained with anti-IFN-γ (557735, BD Biosciences) and anti-IL-4 (554435, BD Biosciences) antibodies at 20–25 °C for 30 min. All events were acquired on a FACSverse flow cytometer and analysed using the FlowJoV software (Tree Star).
To determine cytokine (IFN-γ, TNF-α, IL-1β, IL-10, IL-17a, (MCP-1), IL-1α and IL-6) concentrations in the cervical tissue, multi-analyte flow assay kits (Biolegend, San Diego, CA, USA) were used as indicated by the manufacturer.
Quantitative real-time polymerase chain reaction(qPCR)
U. urealyticum loads in the cervical tissue were determined as previously described . In short, 14 days after infection, mice were anesthetised and euthanised and the whole cervix was rapidly harvested and stored at −80 °C. gDNA from the cervix was extracted and qPCR was performed using gene-specific primers (U. urealyticum urease gene) on a Light Cycle 96 apparatus (Roche, Basel, Switzerland).
The mice were immunised with DnaJ and challenged with U. urealyticum, as described above. The reproductive tract was removed from immunised mice 14 days after infection, fixed with 4% paraformaldehyde and embedded in paraffin, and the paraffin-embedded reproductive tract samples (including the uterine horn) were cut into sections. For routine histology, the cervical tissue sections were stained with haematoxylin and eosin. In addition, to visualise the pathogen load in the mouse cervical tissues, immunohistochemical detection of U. urealyticum antigen was performed as described previously , and mouse anti-U. urealyticum was provided by the Pathogenic Biology Institute (University of South China, Hunan, China). All cervical tissue sections were blindly evaluated by two pathologists.
Detection of endotoxin removal effect in DnaJ
The endotoxin removal effect in the rDnaJ detection method was modified from a previous study. DnaJ was incubated in PBS containing 10 μg/mL PMB for 3 h at 4 °C. Moreover, for heat-inactivation (Boiling), DnaJ was incubated at 100 °C for 2 h. After 24 h of antigen pulsing, the surface molecules (CD80, CD86 and MHC-II) expression levels in CD11c+ BMDCs were measured by flow cytometry analysis.
BMDC and naïve T-cell co-culture
The allogeneicT cells were isolated from BALB/c mouse splenocytes using MojoSort™ Mouse Naïve CD4T cell Isolation Kit (480040, BioLegend) and co-cultured with BMDCs at a ratio of 1:10 for 3 days. The levels of IFN-γ, IL-4, IL-5 and IL-10 were quantified on the culture supernatant using commercially available ELISA kits (eBioscience, San Diego, USA).
Adoptive immunisation with DnaJ-pulsed BMDCs
The BMDCs were cultured and stimulated as described above. Six-week-old female C57BL/6 mice (four per group) were immunised with intravenous injection of 2 × 106 mature DnaJ-pulsed BMDCs. Booster immunisation was performed with the same number of DnaJ-pulsed BMDCs at 7-day intervals. Control groups were injected with equal amounts of PBS or untreated BMDCs. Serum was collected from all mice and these were sacrificed to isolate splenocytes for subsequent experiments (ELISA or flow cytometry analysis).
Statistical analysis was performed using one-way ANOVA followed by the Duncan test. All results are expressed as mean ± standard error of the mean. Prism version 8 software (GraphPad Inc, San Diego, CA, USA) was used for analysis. P-values lower than 0.05 were considered significant.