Bacterial strains and growth conditions
The bacterial strains and plasmids used are listed in Additional file 1. We used B. suis bv2 CITA 198 (herein Bs2WT) because, although Bs2WT and the B. suis bv2 reference strain (B. suis bv2 Thomsen) have the same PCR-RFLP pattern [3], the former shows a virulence pattern in mice typical of B. suis bv2 field strains and the latter is attenuated (Additional file 2).
Bacteria were routinely grown either in standard tryptic soy broth (TSB; Scharlau, Barcelona, Spain) or TSA (TSB supplemented with agar [Pronadisa, Laboratorios Conda, Spain]) at 37 °C. For the studies in mice, vaccines and challenge strains were grown on Blood Agar Base (BAB; Oxoid, UK). When needed, media were supplemented with 5% sucrose, diaminopimelic acid (DAP [Sigma]; 1 mM), 0.2% activated charcoal, kanamycin (Km) at 50 µg/mL or at 35 µg/mL, ampicillin (Amp) at 100 µg/mL and/or chloramphenicol (Cm) at 20 µg/mL (all from Sigma). The lactate-glutamate-glycerol-vitamins synthetic medium of Gerhardt’s [35] was supplemented with 1 mM methionine (mGSM) (this amino acid is required for growth of some Brucella strains in synthetic media [32] including Bs2WT (Zúñiga-Ripa, unpublished observations). All strains were stored at − 80 °C in skimmed milk (Scharlau, Barcelona, Spain) or in TSB supplemented with 0.5% yeast extract (Pronadisa, Laboratorios Conda, Spain) (TYSB) and 7% dimethylsulfoxide.
Sequence analyses and DNA manipulation
Genomic sequences of B. suis bv2 Thomsen (ATCC 23445 or NCBI:txid470137), B. melitensis 16M (ATCC 23456 or NCBI:txid224914) and B. abortus 2308 (NCBI:txid359391) were obtained from databases at the National Center for Biotechnology Information (NCBI) or Kyoto Encyclopedia of Genes and Genomes (KEGG: https://www.genome.jp/kegg/). Since the genomic sequence of Bs2WT was not available, the ORFs of interest were PCR amplified and sequenced by “Servicio de Secuenciación del Centro de Investigación Médica Aplicada” (CIMA, Pamplona, Spain). Sequence alignments were performed with Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/).
Plasmids and chromosomal DNA were extracted with QIAprep® Spin Miniprep Kit and QIAamp® DNA Miniprep Kit (Qiagen GmbH, Hilden, Germany). When needed, DNA was purified from agarose gels using a QIAquick® Gel Extraction Kit (Qiagen GmbH, Hilden, Germany). Primers were designed using Primer 3 input (http://primer3.ut.ee/) and synthesized by Sigma-Genosys Ltd (Haverhill, United Kingdom).
Mutant construction
We obtained Bs2ΔwadB, Bs2ΔwadD, Bs2ΔppdK, Bs2ΔppdKΔpckA, Bs2ΔppdKΔwadB and Bs2ΔppdKΔwadD (all with O-chain) and Bs2ΔwbkF and Bs2Δwzm (R; O-chain defective) by in-frame deletion of the corresponding genes (Table 1 and Additional file 1). For the construction of Bs2ΔwadB, Bs2ΔwadD and Bs2ΔppdK, we used the suicide plasmids previously shown to generate in-frame deletions in B. abortus (the sequences where oligonucleotides hybridize are identical in Bs2WT) [29, 30, 33, 34]. To this end, the plasmids were extracted from E. coli TOP10F´ and transformed into E. coli β2150, a diaminopimelic acid (DAP) auxotrophic donor strain [36]. For Bs2ΔwadB construction, the suicide plasmid pJQKΔwadB [30] was introduced in Bs2WT by tri-parental mating with conjugative E. coli β2150-pJQKΔwadB and E. coli β2150-pRK2013 as helper strain [37]. The first recombination event was selected by Km resistance and DAP independence and confirmed by PCR. The allelic exchange by double recombination was selected for growth on sucrose and Km sensitivity. The same strategy was followed to construct Bs2ΔwadD and Bs2ΔppdK using suicide plasmids pJQKΔwadD [29] and pJQKΔppdK [34]. The double mutants Bs2ΔppdKΔwadB and Bs2ΔppdKΔwadD were constructed by deletion of wadB or wadD in Bs2ΔppdK as described above. The double mutant Bs2ΔppdKΔpckA was constructed by deleting pckA in Bs2ΔppdK using plasmid pJQKΔpckA [34]. The loss of the plasmid concomitant with the gene deletion in each of these mutants was confirmed by PCR with the corresponding oligonucleotides (see Additional file 3).
For the construction of Bs2Δwzm, we first generated two PCR fragments: oligonucleotides wzm-F1 and wzm-R2 (Additional file 4) were used to amplify a 484 bp fragment including codons 1 to 31 of BMEI1415, as well as 390 bp upstream of the start codon. Oligonucleotides wzm-F3 and wzm-R4 (Additional file 4) were used to amplify a 447 bp fragment including codons 247 to 265 of BMEI1415 and 385 bp downstream of the stop codon. These fragments were ligated by overlapping PCR using wzm-F1 and wzm-R4 for amplification, and the complementary regions between R2 and F3 for overlapping. The resulting sequence, containing the wzm deletion allele, was cloned into pCR2.1 (Invitrogen) to generate plasmid pCR2.1Δwzm, sequenced to ensure the maintenance of the reading frame, subcloned into the BamHI and XbaI sites of the suicide plasmid pJQK [38] and transformed into competent E. coli β2150. The resulting suicide pJQK-derived plasmid (pJQKΔwzm) was introduced in Bs2WT by conjugation following the procedure described above. Bs2ΔwbkF was constructed following a similar strategy using oligonucleotides wbkF-F1 and wbkF-R2 (Additional file 4) that amplified a 448 bp fragment (including codons 1 to 19 of BMEI1426 as well as 390 bp upstream of the BMEI1426 start codon) and oligonucleotides wbkF-F3 and wbkF-R4 (Additional file 4) that amplified a 505 bp fragment (including codons 301 to 335 of BMEI1426 and 398 bp downstream of the BMEI1426 stop codon). Both fragments were ligated, cloned into pCR2.1 to generate plasmid pCR2.1ΔwbkF, subcloned into pJQK (pJQKΔwbkF) and transformed into competent E. coli β2150. After conjugation with Bs2WT, we performed PCRs (primers are described in Additional file 3) to screen the resulting colonies for the wbkF or wzm deletion.
We constructed a kanamycin resistant challenge strain (Bs2::Tn7KmR) to discriminate the challenge and vaccine strains in the mouse protection experiments (see below). For this, we inserted the pUC18R6KT-miniTn7Km vector into the Bs2WT chromosome by tetra-parental conjugation and selected the conjugants harboring this modified miniTn7 by plating onto TSA-DAP-Km. The construct (Bs2::Tn7KmR) was examined by PCR [39] for the correct insertion and orientation of the transposon between the glmS and recG genes [40, 41].
Bacteriological typing
All strains were typed following established Brucella procedures [2]: i.e. colonial morphology, urease, agglutination with anti-A and anti-M monospecific sera recognizing the cognate epitopes in the O-chain of Brucella S-LPS, susceptibility to thionine, fuchsine and safranin dyes and sensitivity to Tb, Wb, Iz and R/C phages. S/R colony morphology was studied by the crystal violet dye exclusion test and by acriflavine agglutination [2].
Growth curves
To obtain inocula preconditioned for growth in each test medium (TSB or mGSM), bacteria were first grown in 10 mL of TSB in a 50 mL flask at 37 °C with orbital shaking for 18 h. In the case of TSB growth curves, these exponentially growing bacteria were harvested by centrifugation, resuspended at an O.D.600 nm of 0.1 in the same medium and 200 µL/well aliquots were dispensed by triplicate in Bioscreen multi-well plates. In the case of mGSM growth curves, the exponentially growing bacteria in TSB were harvested by centrifugation, resuspended in 10 mL of mGSM at an optical density at 600 nm (O.D.600 nm) of 0.1, and incubated at 37 °C with orbital shaking for 18 h. These preconditioned bacteria were harvested by centrifugation, resuspended at an O.D.600 nm of 0.1 in the same medium and 200 µL/well aliquots were dispensed by triplicate in Bioscreen multi-well plates. The plates were cultivated in a Bioscreen C (Lab Systems) apparatus with continuous shaking at 37 °C. Growth was monitored spectrophotometrically at 420–580 nm every 30 min over a 60 h-period. All experiments were performed in triplicate (biological replicates). Wells containing sterile medium were used as negative controls in all experiments.
LPS characterization
LPS from Bs2WT and mutants in LPS genes (Table 1) were extracted by the proteinase K sodium dodecyl sulfate (SDS) protocol [42], an extraction procedure valid for S and R Brucella [17, 43]. Briefly, phenol-inactivated bacteria (grown in TSB) were centrifuged (20 min at 6 500 × g) and washed with saline. The cells were suspended in 2% SDS-60 mM Tris–HCl buffer (pH 6.8), heated at 100 °C for 10 min and treated with proteinase K (60 μL of a 2.5 mg/mL stock in Tris–HCl per each mL of suspension) for 3 h at 55 °C and overnight at room temperature. Then, samples were centrifuged (30 min at 20 000 × g) and the supernatants precipitated with 3 volumes of methanol containing 1% sodium acetate-saturated methanol at -20 °C for 60 min. The precipitates were collected by centrifugation (15 min at 5 000 × g), and resuspended in 10 mL of distilled water, precipitated again with methanol and sedimented again by centrifugation. The pellets were resuspended in 3 mL of 60 mM Tris-HCl (pH 6.8), digested once with nucleases [20 µL/mL each of DNase and RNase stock solutions at 0.5 mg/mL in Tris–HCl (MP Biomedicals and Sigma-Aldrich, respectively)] at 37 °C for 30 min and treated again with proteinase K (60 µL/mL, 3 h at 55 °C). Finally, the LPS were harvested by centrifugation (15 min at 5 000 × g), suspended in 1 mL of distilled water and stored at − 20 °C.
For LPS characterization, LPS samples were analyzed in 15% polyacrylamide gels (37.5:1 acrylamide/methylene-bisacrylamide ratio) in Tris-HCl-glycine and revealed by the periodate-alkaline silver method [44]. For western blots, gels were electro-transferred onto nitrocellulose sheets (Amersham-GE Healthcare Life Scientific, Germany; 0.45 µm pore) using 25 mM Tris, 192 mM glycine (pH 8.3) and 20% (vol/vol) methanol in a Trans-Blot Semi-Dry Transfer Cell (Bio-Rad) at a constant voltage of 8 V and 200 mA for 30 min. Membranes were blocked with 5% skimmed milk in phosphate buffered saline (PBS; 10 mM phosphate 140 mM NaCl, 2.7 mM KCl [pH 7.4]; Medicago, Sweden) with 0.05% Tween 20 (PBS-T) overnight at 4 °C, and then washed with the same buffer. Membranes were then incubated with primary antibody diluted 1:500 in PBS-T overnight at room temperature, washed with PBS-T and bound immunoglobulins were detected with peroxidase labeled protein G and 4-chlorine-1-naphtol-H2O2. Monoclonal antibody A68-24G12/A08 from the ascitic fluid of an infected mouse, which recognizes core epitopes [45], and a serum of a rabbit infected with B. melitensis 16M and bled 45 days later were used.
Mouse experiments
Seven-week-old female BALB/c mice (ENVIGO, Harlan) were lodged in cages in a BSL-3 facility (ES/31-2010-000132) with water and food ad libitum for 2 weeks before and during the experiments in accordance with the current European (directive 86/609/EEC) and Spanish (RD 53/2013) legislations.
For virulence assessment, groups of 5 mice per time-point were inoculated with each mutant (Bs2ΔwadB, Bs2ΔwadD, Bs2ΔppdK, Bs2ΔppdKΔwadB, Bs2ΔppdKΔwadD, Bs2Δwzm and Bs2ΔwbkF) or Bs2WT. Inocula were prepared harvesting BAB grown bacteria in sterile buffered saline (BSS; 0.85% NaCl, 0.1% KH2PO4, 0.2% K2HPO4; pH 6.85) and adjusting the bacterial suspension to 1 × 109 CFU/mL (inoculum used for 1 × 108 CFU doses in the immunization with R vaccines). This suspension was then diluted to 1 × 106 CFU/mL to prepare the inocula used for S vaccines (1 × 105 CFU doses). Mice were inoculated intraperitoneally (IP) with 0.1 mL of each inoculum and the exact dose was determined retrospectively on triplicate BAB plates. At several intervals after inoculation, mice were euthanized, the spleens were aseptically removed, individually weighed, homogenized in 9 volumes of BSS and serial tenfold dilutions plated by triplicate on BAB plates. After 5 days at 37 °C, CFU were counted, colonies checked by the crystal violet exclusion test and the identity confirmed by PCR. The data (mean CFU/spleen) were normalized by logarithmic transformation, and mean log10 CFU/spleen values and standard deviations (SD) calculated for plotting and statistical comparison by one-way ANOVA and Fisher's Protected Least Significant Differences (PLSD) tests.
To determine the protective efficacy, groups of 5 BALB/c mice were inoculated subcutaneously (SC) with 105 CFU/mouse of Bs2ΔppdK, Bs2ΔwadB and Bs2ΔppdKΔwadB or 108 CFU/mouse of Bs2ΔwbkF and Bs2Δwzm. The Rev1 reference vaccine (105 CFU/mouse) or BSS inoculated mice were used as effective-vaccine and unvaccinated controls, respectively. Four weeks after vaccination, all animals were challenged IP with 105 CFU of the virulent Bs2::Tn7KmR strain. After 2 weeks, CFU/spleen numbers of the challenge strain were determined by plating on BAB Km. Values of residual vaccine were also calculated by subtracting CFU numbers on BAB Km to those obtained on BAB. Results were expressed as the mean log10 CFU/spleen ± SD (n = 5) and statistical comparisons between vaccines and controls for challenge strain values were made using ANOVA and PLSD tests. The virulence of the Bs2::Tn7KmR challenge strain used in this work was verified in a previous experiment in BALB/c mice (n = 5) by IP infection (105 CFU/mouse) and CFU/spleen counting 2 and 6 weeks later. This strain showed identical CFU in BAB-S and BAB-Km, and virulence pattern identical to that of Bs2WT (not shown).