The strains of S. suis and Escherichia (E.) coli used for cloning and mutant construction are listed in Table 1. The SS2 strain ZJ081101 was isolated from the lungs of a diseased pig. Brain heart infusion (BHI; Oxoid, UK) was used for culturing S. suis at 37℃, while Luria-Bertani broth or Luria-Bertani agar was used for culturing E. coli. For cloning and the selection of mutants, the following antibiotics (Sigma-Aldrich, USA) were mixed with the culture media: spectinomycin at 100 mg/mL for SS2 and 50 mg/mL for E. coli; chloramphenicol at 4 mg/mL for SS2 and 8 mg/mL for E. coli; and ampicillin at 100 mg/mL for E. coli.

To construct the stp1 deletion mutant from SS2 strain ZJ081101, the 5- and 3-flanking regions of stp1 were PCR-amplified with primer pairs L1/L2 and R1/R2 carrying SphI/SalI and BamHI/EcoRI restriction sites, respectively (Table 2). The cat gene (from pSET6s) was inserted at the SalI/BamHI sites to generate a gene cassette that was then transferred to the temperature-sensitive shuttle vector pSET4s after digestion by corresponding restriction enzymes to generate the stp1-knockout vector pSET4s-Δstp1 (panel A in Fig. 1). Procedures for selection of mutants by allelic exchange via double crossover have been described previously [3031]. The resulting Δstp1 mutant strain was verified by polymerase chain reation (PCR) amplification with primers In1/In2, Cat1/Cat2, Out1/Cat2, and Cat1/Out2 and confirmed by DNA sequencing [37].

This was carried out essentially as described by Neiss et al. [22]. Briefly, the bacterial cells were subjected to fixation in 2.5% glutaraldehyde in phosphate buffer (pH 7.0) and treatment with 1% OsO₄ in phosphate buffer, each followed by washing. Specimens were then dehydrated by a graded series of ethanol [29] from 30% to 100% and transferred to absolute acetone for 20 min. Specimens were then placed at room temperature in a mixture of absolute acetone and resin at a 1:1 ratio for 1 h, transferred to a 1:3 ratio of absolute acetone to resin mixture for 3 h, and to a Spurr's resin mixture for overnight. Before thin sectioning, specimens were embedded in capsules containing the embedding medium and heated at 70℃ for approximately 9 h. The bacterial sections were stained by uranyl acetate and alkaline lead citrate, each for 15 min, and inspected via transmission electron microscopy (Model H-7650; Hitachi, Japan).

The ORF of stp1 was amplified from the genome DNA of SS2 strain ZJ081101 by PCR with the primer pairs ES1-Stp1/ES2-Stp1 (Table 2). The verified complete gene was cloned, using the EcoR I and Bgl II sites, into pET-32a (Invitrogen, USA) which contains a 22 kDa His-tag-encoding sequence. The recombinant plasmid pET-Stp1 was transformed into E. coli Rosetta (DE3) for expression of the recombinant Stp1 (rStp1). E. coli pET-Stp1 cells in their log phase of growth were induced with 1 mmol/L of isopropyl-β-D–thiogalactopyranoside at 37℃ for 4 h. The rStp1 protein was purified by Ni²⁺-nitrilotriacetic acid affinity chromatography and concentrated by ultrafiltration (Millipore, USA). The filtrates were passed through a 0.22-µm membrane (Millipore) and held at −80℃ until use.

p-Nitrophenyl phosphate (pNPP) was used as a substrate as described [16] in 500 µL of phosphatase buffer [50 mM Tris HCl (pH 8.0), and 2 mM MnCl₂] containing 20 mM pNPP and 2 µg of Stp1. Hydrolysis of pNPP in the samples was measured after incubation for 10 min by determining the increase in absorbance of p-nitrophenol (pNP) at 405 nm (SpectraMax M2; Molecular Devices, USA).

The SS2 strains, both wild-type and mutant, were incubated at 37℃ for 6 to 7 h and used to prepare cytoplasmic and cell-wall proteins from whole bacterial cells for SDS-PAGE and western blotting. Whole bacterial lysates were prepared by treating the bacterial suspensions in a Precellys 24 homogenizer (Bertin Instruments, France) in the presence of 0.1 mm ceramic beads at 3,360 × g repeated 5 times for 30 sec each. The supernatant samples were collected as whole cell cytoplasmic proteins (CPP) after centrifugation and saved at −80℃. The precipitates were the cell-wall proteins (CWP) remaining after treatment with 1% Triton-100 in 50 mM phosphate-buffered saline (PBS).

The Bradford method was used to quantify the concentrations of the protein samples. Thirty micrograms of each protein sample were subjected to SDS-PAGE on 12% gels and transferred onto PVDF membranes (Millipore). The membranes were blocked with Tris-buffered saline containing 0.1% Tween 20 and 5% non-fat milk and subjected to probing with the antiserum to rStp1 or rGAPDH and to HRP-conjugated anti-rabbit IgG [Multisciences (Lianke) Biotech, China]. Chemiluminescence substrate (SuperSignal West Femto Maximum Sensitivity Substrate; Thermo Fisher Scientific, USA) was used to visualize the protein bands.

The mutant strain Δstp1 and its parent strain were grown in BHI containing gradient concentrations of H₂O₂ or paraquat (a chemical generator of superoxide anions) [6] and incubated at 37℃. The cultures were examined at 620 nm after 4 and 8 h of incubation on SpectraMax M2 microplate reader (Molecular Devices).

Adhesion of the Δstp1 mutant and its parent strain to murine endothelial cell line bEnd.3 and human epithelial cell line HEp-2 was determined as previously described [17]. Bacterial cultures at the mid-log phase (10⁷ CFU/well; CFU, colony-forming unit) were added to the cells in 24-well culture plates at 100 multiplicity of infection (MOI). The plates were spun at 800 × g for 10 min and subjected to incubation for 1 h incubation at 37℃ and 5% CO₂. The infected cells were washed with PBS and disrupted by repeated pipetting with sterile distilled water. The cell lysates were transferred to Eppendorf tubes and vortexed to release the bacteria. The samples were then 10-fold diluted in PBS and plated on BHI agar plates for bacterial counting. Adhesion (%) was defined as (CFU_Adh/CFU_Total) × 100 [33].

The murine macrophage cell line RAW264.7 was used to examine the effect of stp1 deletion on phagocytosis and intracellular survival according to a previously reported protocol [8]. Briefly, the cells were incubated in RPMI 1640 medium containing 10% fetal calf serum (Gibco, USA) in 24-well plates. Wild-type and mutant SS2 strains were incubated in BHI broth for 6 h at 37℃, pelleted by centrifugation, and resuspended in sterile PBS. The cells were infected for 30 min at 37℃ at 100 MOI, washed, and incubated for 1 h in fresh RPMI 1640 medium containing gentamicin (100 mg/mL) and penicillin G (5 mg/mL) to inactivate extracellular bacteria. Cells were then washed and incubated in the fresh medium containing 20 mg/mL of gentamicin. The cells were subjected to further incubation at 37℃ and 5% CO₂ for 1 or 2 h. After incubation, the cells were trypsinized and disrupted by repeated pipetting for bacterial enumeration on BHI agar plates. Survival (%) was calculated as CFU_2h/CFU_1h × 100.

BALB/c mice were used in experimental infections to evaluate the role of stp1 in SS2 virulence [36]. For determination of lethal dose 50 (LD₅₀), eleven groups of 4-week-old female BALB/c mice (10 per group) were randomly allocated. Five groups were assigned to each strain (wild-type or its mutant Δstp1), and the last group of each strain received PBS as a sham control treatment. Five gradient inoculum sizes were used for intraperitoneal infection. Mortality was recorded twice a day for 7 days post-infection. The improved Karber's method was used to calculate LD₅₀. The bacterial burdens in blood and organs were examined in mice receiving intraperitoneal inoculation of the wild-type strain or its mutant (10 mice per strain) at 6.0–8.0 × 10⁸ CFU/mouse. Mice were euthanized at 24 and 48 h post-infection (hpi), and their brain, kidney, liver, and spleen were removed and homogenized individually in 1 mL PBS at pH 7.4. The homogenates were 10-fold diluted in PBS and appropriate dilutions were plated onto BHI agar plates. Anti-coagulant blood samples (100 µL) were collected at 24 and 48 hpi. Animal experiments were conducted following the guidelines and approved protocols of the Laboratory Animal Management Committee of Zhejiang University (approval No.2015029).

All data are expressed as mean ± SD values of three independent experiments, each performed in triplicate in wells or tubes. The two-tailed Student's t-test was used to determine the statistical significance of differences between the stp1 deletion mutant and its parent strain.

A stp1 knockout mutant was produced by electrotransforming suicide vector pSET4s containing a CatR cassette (panel A in Fig. 1) into wild-type competent SS2 cells and was used to evaluate the role of stp1 in pathogenesis. The SS2 Δstp1 mutant was generated by allelic change of stp1 with cat, which was verified by sequencing of the PCR product. Null expression of Stp1 in the mutant was confirmed by absence of its band on the western blot probed with anti-Stp1 polyclonal antibody. The results confirmed successful inactivation of stp1 in the SS2 strain (Fig. 1).

The Δstp1 mutant had longer chains than its parent strains (panel A in Fig. 2). The two strains showed difference on their surfaces as revealed by transmission electron microscope: the Δstp1 mutant had much fewer ‘pilus-like’ surface structures than its parent strain (panels B and C in Fig. 2).

To determine whether Stp1 of SS2 is a functional phosphatase, we expressed Stp1 in E. coli. The results showed that rStp1 was successfully expressed as a 41 kDa His-tagged fusion protein. Hydrolysis of pNPP was measured by assessing an increase in absorbance of pNP at 405 nm over 10 min. We observed that rStp1 hydrolyzed pNPP in the presence of Mn²⁺ (Fig. 3).

The bEnd.3 and HEp-2 cell lines were used to determine the effects of stp1 deletion on adhesion of SS2 to host cells. Fig. 4 shows that the Δstp1 mutant exhibited better adhesion to both cell lines than that of its parent strain: 1.92 ± 0.07 vs. 0.30 ± 0.014 (p < 0.01) in bEnd.3 cells and 1.92 ± 0.86 vs. 0.40 ± 0.047 (p < 0.05) in HEp-2 cells.

Fig. 5 shows that there was no marked difference in phagocytosis between Δstp1 mutant and its parent strain. However, the mutant was more resistant than the wild-type strain to killing by macrophages (0.79 ± 0.08 vs. 0.16 ± 0.037, p < 0.05). By using the chemical peroxides H₂O₂ or paraquat in BHI broth, we observed that the Δstp1 mutant was more resistant than its parent strain to paraquat treatment at 5 to 10 mM (p < 0.01), but not to hydrogen peroxide (Fig. 6). These results suggest that deletion of stp1 enhanced resistance to phagocytic killing, probably by increasing the resistance to reactive oxygen species (ROS).

The role of SS2-stp1 in virulence to BALB/c mice was examined by assessing responses to inoculation of 2.44–2.47 × 10⁹ CFU of the relevant strains per mouse. Death rate was 80% for mice challenged with the wild-type strain and 20% when Δstp1 mutant-challenged. At day 5 post-infection, doubling the inoculum level led to 100% death (10/10) in mice receiving the wild-type strain, but only 60% (6/10) death in mice inoculated with the Δstp1 mutant. Compared to its parent strain, deletion of stp1 decreased the virulence of SS2 (LD₅₀: 2.7 × 10⁹ CFU for Δstp1 strain vs. 1.51 × 10⁹ CFU for parent strain; Table 3). Bacterial burdens in blood and internal organs were lower in the Δstp1 mutant than in its parent strain. At 48 hpi, bacterial load in organs of mice receiving the parent strain remained high, while the load at 48 hpi in mice inoculated with Δstp1 mutant was below the detection limit (2 log, i.e., 100 CFU) of the method used (Fig. 7). These findings indicate that inactivation of stp1 reduces virulence of SS2 in mice.