Bacterial strains and plasmids used in the present study are listed in Table I. S. suis 2 strains were cultured in brain-heart infusion (BHI) broth (Difco; BD Biosciences, Franklin Lakes, NJ, USA) supplemented with 5% (v/v) calf serum or BHI agar at 37°C. Escherichia coli strain DH5α was used for cloning purposes and was cultured in Luria broth (LB) or LB agar at 37°C (20). When recombinants were screened or cultured, plates or broth were supplemented with appropriate antibiotics at the following concentrations: i) Spectinomycin (Spc), 100 µg/ml for S. suis 2 with plasmid pSET4s and 50 µg/ml for DH5α with plasmid pSET4; ii) ampicillin (Amp), 100 µg/ml for DH5α with plasmid pMD18-T; iii) erythromycin (Ery), 8 µg/ml for S. suis 2 with plasmid pAT18 and 150 µg/ml for DH5α with plasmid pAT18 (all antibiotics from Sigma-Aldrich, St. Louis, MO, USA). DNA extraction, cloning, transformation, and other molecular techniques used in the present study were implemented following protocols described previously (20). All primer synthesis and DNA sequencing were outsourced to Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

The primers used in the current study are presented in Table II. The PCR was performed according to a standard protocol. Each reaction was conducted using a 50 µl mixture containing 5 µl 10X buffer, 50 pmol each primer, 2 mM each deoxynucleoside triphosphate, 5 units Ex Taq polymerase (all obtained from Takara Biotechnology Co., Ltd., Dalian, China) and 5 µl supernatant of denatured bacteria. The PCR was performed with a Techgene FTGENE2D thermocycler (Techne Ltd., Duxford, UK). In order to generate a vapE gene-deleted mutant, a primer set was designed with a 194 bp internal deletion in the vapE gene by overlap extension polymerase chain reaction (OE PCR), using denatured bacteria as the DNA template (20). Two pairs of primers (VapE-1/VapE-2 and VapE-3/VapE-4) were used to independently amplify the 674 and 743 bp fragments of vapE, including flanking sequences from genomic DNA of the wild-type SS2 strain, ZY458. 16S rDNA was used as the reference gene, the primers were as follows: Sense, 5′-AGAGTTTGATCCTGGCTCAG-3′ and antisense, 5′-ACGGCTACCTTGTTACGACTT-3′. Amplification was performed as follows: i) Initial denaturation at 94°C for 3 min; ii) 30 cycles of 94°C for 30 sec, 60°C for 30 sec and 72°C for 50 sec; and iii) a final elongation step at 72°C for 10 min. Primers VapE-2 and VapE-3 contained 15 nt stretches complementary to each other; thus, the PCR fragments were fused by OE PCR using primers VapE-1 and VapE-4. The following PCR protocol was used: 3 min at 94°C, followed by 30 cycles at 94°C for 30 sec, 59°C for 30 sec and 72°C for 1 min 40 sec, followed by 72°C for 10 min. The resultant 1,417 bp PCR product contained an internal in-frame deletion of 194 bp in the vapE gene (from 1 to 194 nt). The PCR product was purified by DNA Gel Extraction kit (Takara Biotechnology Co., Ltd.) Band subsequently cloned into a pMD18-T vector using a pMD18-T Vector Cloning kit (Takara Biotechnology Co., Ltd.) to generate the pMD18-T::ΔvapE plasmid. The pMD18-T::ΔvapE plasmid was digested with BamHI and EcoRI enzymes (Takara Biotechnology Co., Ltd., Dalian, China), and the DNA fragment containing the mutated vapE (ΔvapE) was then cloned into a pSET4s thermosensitive suicide plasmid (21) to generate the pSET4s::ΔvapE suicide plasmid. The resultant plasmid was confirmed by DNA sequencing and transfected into a ZY458 SS2 strain to screen for deletion mutants as described by Takamatsu et al (22). Subsequently, the SS2 strain ZY458 was electrotransfected with pSET4s::ΔvapE using the ECM 399 electroporation system (BTX Harvard Apparatus, Inc., Holliston, MA, USA) at 2,000 V, and cultured at 28°C in the presence of Spc to select the recombinant. The resultant ZY458 (pSET4s::ΔvapE) cells were cultured in BHI broth with Spc at 28°C until the early logarithmic growth phase, and then were shifted to 37°C and incubated for an additional 10 h. Subsequently, the cultures were diluted and spread onto BHI agar plates without antibiotic and incubated overnight at 37°C. The cultures were screened for mutants that had lost the vectors and had exchanged their wild-type allele for a genetic segment containing the ΔvapE gene as a consequence of homologous recombination via a double cross-over. A resultant 458ΔvapE mutant strain was verified by PCR amplification with the primers VapE-5/VapE-R and further confirmed by DNA sequencing. The PCR cycle protocol, performed on Techgene FTGENE2D thermocycler, was as follows: i) Initial denaturation at 94° for 3 min; ii) 30 cycles of 94°C for 30 sec, 50°C for 30 sec and 72°C for 40 sec; and iii) a final elongation step at 72°C for 10 min.

To generate a functionally complemented strain of 458ΔvapE, the structural gene of vapE with its promoter sequence (from 320 bp upstream of the start codon to 439 bp downstream of the stop codon) was amplified using primers VapE-F and VapE-R. PCR was performed on a Techgene FTGENE2D thermocycler under the following conditions: i) Initiation and elongation, 3 min at 94°; ii) 30 cycles of denaturation for 30 sec at 94°, annealing for 30 sec at 50°C; and iii) elongation for 2 min at 72°C; and a final elongation step at 72°C for 10 min. The PCR product (2,292 bp) was purified using the DNA Gel Extraction kit and subsequently cloned into pMD18-T to generate the plasmid, pMD18-T::vapE. The vapE fragment was then subcloned into shuttle vector pAT18. The resultant plasmid was verified by DNA sequencing, and subsequently used to electrotransfect the mutant strain 458ΔvapE using the ECM 399 electroporation system, as described above (BTX Harvard Apparatus, Inc.), which was plated onto BHI agar supplemented with Ery to screen for the complemented strain, 458ΔvapE (pvapE).

Wild-type strain S. suis 2 ZY458, mutant strain 458ΔvapE and complemented strain 458ΔvapE (pvapE) were separately inoculated into 100 ml BHI broth and incubated at 37°C. Samples of culture were monitored by spectrophotometry using a T6 UV spectrophotometer (Beijing Purkinje General Instrument Co., Ltd, Beijing, China). The absorbance was measured at 600 nm in a quartz cuvette (Beijing Purkinje General Instrument Co., Ltd.) at 1 h intervals. BHI broth minus the inoculation of bacteria served as a blank.

The present study was approved by the Review Board of the Academy of Military Medical Sciences (Changchun, China). All animal experiments were conducted in accordance with the accepted standards of the Animal Care and Use Committee of Academy of Military Medical Sciences. The protocol was approved by the Animal Care and Use Committee of Academy of Military Medical Sciences and all efforts were made to minimize suffering. All experiments involving mice were conducted in accordance with the Council for International Organizations of Medical Sciences: International Guiding Principles for Biomedical Research Involving Animals (23). The bacterial cultures were serially diluted in BHI broth and plated onto BHI agar plates in order to determine the colony forming unit (CFU)/ml. The working cultures for experimental infection were adjusted to a final concentration of 1×10⁹ CFU/ml.

Female BALB/c mice (age, 4 weeks; weight, 13–14 g) were housed at 24±1°C and 60% relative humidity in a 12-h light/dark cycle with access to food and water ad libitum. They were randomly divided into four groups (12 mice/group), and individual groups were injected intraperitoneally (i.p.) with 100 µl of the ZY458 wild-type strain, ZD89 carrier strain, 458ΔvapE mutant strain or 458ΔvapE (pvapE) complemented strain cultures. The animals were monitored daily for 1 week for mortality and clinical signs, including depression, swollen eyes, ruffled hair, lethargy and nervous symptoms. Tissues of the heart, liver, spleen, lung and kidney from infected mice (12 mice/group) were harvested for detection of the SS2 bacteria by plating onto BHI agar plates. Positive cultures were confirmed by PCR using VapE-F and VapE-R primers (Table II).

To evaluate the pathogenicity of SS2, the capacity of the bacteria to colonize the tissues of the heart, liver, spleen, lung and kidney of infected mice was analyzed using a colonization assay. Further BALB/c (13–14 g) mice were randomly divided into three groups (nine mice/group) and injected i.p. with 1×10⁸ CFU/mouse of one of the ZY458, ZD89 or 458ΔvapE strains. One mouse from each group was euthanized by cervical dislocation at 12, 24 and 36 h post-infection, and the tissues were collected and ground with an electric pestle (Tiangen Biotech Co., Ltd., Beijing, China) in 0.9% NaCl (0.03 g tissue/ml). The supernatants were diluted 10-fold and plated onto BHI agar plates. Following an overnight incubation, the bacterial colonies were counted and the data were expressed as CFU/g of tissue, as described previously (24).

The primers VapE-F and VapE-R were designed on the basis of the published sequence of the SS2 strain, 05ZYH33 (GenBank accession no. NC_009442), to detect the vapE gene from the genomic DNA of SS2 strains ZY458, SP3, SP6, 1330, ZD89, B22, B3 and SP8 by PCR. 16S rDNA served as the reference gene. Each reaction was conducted using a 25 µl mixture containing 2.5 µl 10X buffer, 25 pmol each primer, 2 mM each deoxynucleoside triphosphate, 2.5 units Taq polymerase and 2.5µl supernatant of denatured bacteria. The PCR was performed with Techgene FTGENE2D thermocycler, under the following conditions: i) Initiation and elongation for 3 min at 94°; ii) 30 cycles of denaturation for 30 sec at 94°, annealing for 30 sec at 50°C and elongation for 2 min at 72°C; and iii) a final elongation step at 72°C for 10 min. Amplicons were visualized by running at 100 V for 30 min on a 1% agarose gel containing ethidium bromide (Takara Biotechnology Co., Ltd.). A DL2000 DNA ladder (Takara Biotechnology Co., Ltd.) was used as a size marker.

A vapE deletion mutant strain, 458ΔvapE, was generated by a homologous replacement method using ZY458 as the parent strain. The vapE gene knock-out mutant strain was confirmed by PCR (Fig. 1). Comparing the nucleotide sequences using basic local alignment search tool (BLAST) searching revealed that a 194 bp segment of the vapE gene (from 1 to 194 nt of the vapE coding sequence) had been deleted without alteration of the remaining sequence.

The PCR-amplified structural gene of vapE was cloned into a pAT18 shuttle vector. The resultant plasmid was confirmed by PCR (Fig. 1) and designated as pAT18::vapE, which was then electrotransfected into 458ΔvapE cells to produce the complemented strain, 458ΔvapE (pvapE). A BLAST analysis of the nucleotide sequences in the National Center for Biotechnology Information database with the vapE gene confirmed that the amplified 2,292 bp fragment was identical with the other strains.

To determine whether the deletion of the vapE gene leads to a defect in bacterial growth, the growth characteristics of wild-type SS2 strain ZY458, the vapE deletion mutant, 458ΔvapE, and the complemented strain, 458ΔvapE (pvapE), were compared at 37°C in BHI broth. The optical density values of the bacterial cultures at 600 nm were measured. The growth curves indicated that the growth rates of the mutant strains 458ΔvapE and 458ΔvapE (pvapE) were similar to that of the wild-type strain, ZY458 (Fig. 2).

To further assess the effect of vapE deletion on the pathogenesis of SS2, four groups of mice were infected with one of the SS2 wild-type strain ZY458, the carrier strain, ZD89, or the mutant strains, 458ΔvapE or 458ΔvapE (pvapE). The results indicated that all mice infected with wild-type SS2 exhibited severe clinical symptoms, including depression, apathy, fever, anorexia, emaciation, swollen eyes and neural disorders, and died within 2 days of infection (Table III). Similarly, animals infected with the complemented strain 458ΔvapE(pvapE) developed severe clinical symptoms and 83.3% of the mice died 2 days post-infection. By contrast, 100% of the mice infected with mutant strain 458ΔvapE exhibited only mild clinical symptoms in the first 2 days post-infection, and recovered fully within a week. None of the mice injected with ZD89 developed any clinical symptoms. Furthermore, SS2 bacteria were recovered from the organs of mice infected with ZY458 and 458ΔvapE (pvapE). However, no bacteria were detected in the heart, liver, spleen, lung or kidney of any mice infected with 458ΔvapE or ZD89 over a test period of 7 days post-infection (data not shown).

To further evaluate the virulence attenuation of 458ΔvapE, a bacterial colonization assay was performed. As indicated in Table IV, a reduction in clone-forming efficiency was observed in the infected mice. Mice infected with ZY458 died within 36 h, in contrast, mice infected with 458ΔvapE remained alive with 10⁵–10⁶ CFU/g of tissue.

To determine whether the vapE gene exists only in virulent strains of SS2, PCR amplification of the vapE gene was performed using primers of VapE-F and VapE-R (Table II). A 2,292 bp portion of the target fragment was amplified from the virulent strains (ZY458, SP3, SP6), but not from any of the avirulent (1330) and carrier strains that were investigated (ZD89, B22, B3 and SP8) (Fig. 3).