Table 1.
Bacterial strains and plasmids used in this study.
Table 2.
Primers used for PCR amplification.
Figure 1.
Identification of Spx regulators in S. suis.
(A) Schematic representation of the spx locus in S. suis. The genes and the ORF number in S. suis SC84 genome are indicated. Arrows indicate the direction of transcription and do not represent the exact length. (B) Multiple sequence alignment of S. suis Spx proteins with related homologous proteins at the amino acid level. The multiple alignment was computed using ClustalW (http://www.genome.jp/tools/clustalw/), and the final image was generated using ESPript 3.0 (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). Identical residues are in white letters with red background, and similar residues are in red letters with white background. The secondary structure of Spx is shown on top. α: α-helix; β: β-sheet; T: β-turns/coils. The known crystal structure of B. subtilis Spx (Protein Data Bank entry 1Z3E) was used as a reference (33). The conserved CXXC and RPI motif discussed in the text are boxed, and the conserved Gly52 are labeled with an asterisk (*). The GenBank accession numbers are the following: B. subtilis Spx, NP_389032.1; S. aureus Spx, NP_374119.1; E. faecalis Spx, NP_816313.1; S. pneumoniae SpxA1, NP_358855.1; S. sanguinis SpxA1, YP_001034909.1; S. suis SpxA1, YP_003025001.1; S. mutans SpxA, NP_721528.1; S. pneumoniae SpxA2, NP_357767.1; S. sanguinis SpxA2, YP_001036156.1; S. suis SpxA2, YP_003024122.1; S. mutans SpxB, NP_722373.1.
Figure 2.
Construction and confirmation of the knockout mutant strains.
(A) Strategy for deletion of spxA1 in S. suis SC19 by homologous recombination. The plasmid pSET4s::spxA1 is used for the spxA1 gene knockout. LA and RA indicate the left and right arms of spxA1. (B) PCR confirmation of the mutant strains. The primer pairs used in the PCR analysis are indicated above the lanes. Genomic DNAs from the WT (lanes 2, 4, 6, and 8), ΔspxA1 (lanes 1 and 3) and ΔspxA2 strains (lanes 5 and 7), were used as templates. (C) RT-PCR identification of the mutant strains. Total RNAs were extracted from the WT, ΔspxA1 and ΔspxA2 strains. cDNAs generated from these RNA samples were subjected to RT-PCR analysis with primer pairs A1in1/A1in2 (for detection of spxA1 gene transcripts) or A2in1/A2in2 (for detection of spxA2 gene transcripts). The RT-PCR products were analyzed by electrophoresis on a 1% agarose gel (lanes 1 and 3, the WT strain; lanes 2, the ΔspxA1 strain; lanes 4, the ΔspxA2 strain).
Figure 3.
Growth curves of the WT, ΔspxA1 and ΔspxA2 strains.
The WT (circles), ΔspxA1 (squares) and ΔspxA2 (triangles) strains were grown in TSB with 10% newborn bovine serum at 37°C under static conditions (A) or in a shaking incubator set to 180 rpm (B). For stress conditions, strains were inoculated in the presence of 0.5 mM H2O2 (C), 0.01% SDS (D), 1.5% NaCl (E) or in the absence of newborn bovine serum (F) and incubated at 37°C under static conditions. Growth was evaluated by measuring OD600. The curves shown are representative of a typical experiment performed three times.
Figure 4.
Survival curves of mice infected with S. suis strains.
Groups of ten female BALB/c mice were inoculated intraperitoneally with the WT (circles), ΔspxA1 (squares) and ΔspxA2 (triangles) strains at a dose of 7.0×108 CFU (A), or 3.5×108 CFU (B). Survival was monitored over a 14 day period.
Figure 5.
Pathological examination of brain tissues of mice infected with indicated S. suis strains.
BALB/c mice were inoculated intraperitoneally with 3.5×108 CFU of the WT, ΔspxA1 and ΔspxA2 strains. Brain samples were collected from surviving mice on day 14 post-infection and prepared for pathological examination. (A) The meninges of mice infected with the WT strain were severely thickened, infiltrated by macrophages and neutrophils. (B) No obvious change was displayed in the meninges of the mice infected with ΔspxA1. (C) The meninges of ΔspxA2-infected mice were mildly thickened.
Figure 6.
Colonization of the WT, ΔspxA1 and ΔspxA2 strains in various tissues of mice.
Groups of five female BALB/c mice were inoculated intraperitoneally with 1.0×108 CFU of the WT (circles), ΔspxA1 (squares) and ΔspxA2 (triangles) strains. Blood, brain and spleen were collected at 24 h post-infection. Bacterial burdens from blood (A), brain (B) and spleen (C) were examined. Statistical analyses were performed using the two-tailed Mann-Whitney test.
Figure 7.
In vivo competitive index of ΔspxA1 and ΔspxA2 against the WT strain.
Groups of six female BALB/c mice were inoculated intraperitoneally with a mixture of ΔspxA1 and WT or ΔspxA2 and WT at a ratio of 1∶1. At 18 h post-infection, blood samples were collected and plated. The ΔspxA1/WT and ΔspxA2/WT ratios were determined by analyzing 70 colonies of each sample with colony PCR. The competitive index was determined as the mutant:WT ratio in blood samples divided by the ratio in the inoculum. A CI value of 1 indicates equal competitiveness. Mean CI values from six mice were compared to 1 using the two-tailed paired t test to determine whether the difference in competitiveness is significant. ***P<0.0001.
Figure 8.
Production of inflammatory cytokines in mice.
Serum levels of TNF-α (A) and IL-6 (B) in BLAB/c mice 6 h after infection with indicated S. suis strains at a dose of 2×108 CFU. Time course of production of TNF-α (C) and IL-6 (D) in BALB/c mice infected with the WT (circles), ΔspxA1 (squares) and ΔspxA2 (triangles) strains. Data are expressed as mean levels ± standard deviation from three mice for each strain at each time point. Statistical analyses were performed using the two-tailed unpaired t test. ***P<0.0001.
Figure 9.
Growth factors of the WT, ΔspxA1 and ΔspxA2 strains in pig blood.
Approximate 105 CFU of the WT, ΔspxA1 and ΔspxA2 strains were incubated in heparinized pig blood and incubated for 3 h at 37°C with end-to-end rotation. Growth factor was defined as the ratio of CFU in each sample after 1 or 3 h incubation over the CFU in the corresponding inoculum. The results shown are the means ± standard deviations of three independent experiments. The P values were obtained using the two-tailed unpaired t test.
Figure 10.
Correlation between DNA microarray data and qRT-PCR results.
The relative transcriptional level of 10 selected genes determined by DNA microarray and qRT-PCR analyses were log2 transformed, and the values were plotted against each other to evaluate their correlation. The genes analysed by qRT-PCR are listed in Table S1.
Table 3.
Expression ratios of genes involved in oxidative stress response and virulence in the mutant strains relative to the WT strain by microarray analysisa.