关键词: 猪链球菌4型, GalR, 生物学特性, 毒力

Abstract:

【Objective】 The study was carried out to investigate the effect of the deletion of the transcriptional regulator GalR gene of Streptococcus suis type 4 virulent strain SH1510 on the biological characteristics of bacteria in order to further study GalR regulation of galactose metabolism pathway and its pathogenesis, so as to provide a theoretical basis.【Method】 The SH1510ΔGalR, an SS4 virulent strain SH1510 transcriptional regulator GalR gene deletion strain, was constructed by homologous recombination double-crossover. Then, a preliminary screening of the deletion strain was performed by the internal amplification primers I1/I2 of GalR gene, and further identified the deletion strain SH1510ΔGalR by PCR and Western blotting. We performed Gram staining on the parental strain SH1510 and the deletion strain SH1510ΔGalR to compare morphological differences. We configured the basal medium, added glucose, sucrose and D-galactose to culture the bacteria, and plotted the bacterial growth curve to compare the bacteria’s utilization of different sugars. The bacterial concentration of the parent strain and the deletion strain were adjusted to 2.5×10 ⁹ CFU/mL, 5×10 ⁸ CFU/mL, 1×10 ⁸ CFU/mL and 2×10 ⁷ CFU/mL, respectively, and BALB/c mice were intraperitoneally injected. Then, the lethality of the mice was observed, and the strain LD₅₀ was calculated by using the Reed-Muench method. The parental strain and the deletion strain with a concentration of 2×10 ⁷CFU/mL were mixed in an equal volume of 1﹕1 and intraperitoneally injected into BALB/c mice. After 24 h, brain, spleen and blood were counted on chloramphenicol-resistant and non-resistant THB plates to compare the colonization ability of bacteria in mice. The internal environment with the whole blood of healthy pigs was simulated, and then the parent strain and the deletion strain were separately and added to whole blood of healthy pigs containing SS4 antiserum, and incubated at 37℃ for 2 h for plate counting to compare the viability of bacteria in whole blood.【Result】 PCR was performed by using the internal detection primer I1/I2, and the result showed that the deletion strain SH1510ΔGalR was negative. Further, primers C1/C2, O1/C2, O2/C1, and O1/O2 were used to identify the deletion strain SH1510ΔGalR, and 1 056, 2 121, 2 094, and 3 147 bp fragments were amplified, respectively, and the results were in agreement with expectations. The results of Western blotting showed that the parental strain SH1510 could specifically bind to the crude rabbit anti-GalR polyclonal serum, and a single band appeared at 37 kD, but the deletion strain SH1510ΔGalR showed no band. The results of PCR and Western blotting indicated that the deletion strain SH1510ΔGalR was successfully constructed. The parent strain and the deletion strain were stained by Gram. The optical microscopic observation showed that the parent strain and the deletion strain were arranged in a chain, the length was similar, and the morphology was not significantly different. Under the growth conditions of glucose and sucrose as the sole glycogen, the growth of the parent strain and the deletion strain was similar; when the glycogen was D-galactose, the growth rate and OD₆₀₀ value of the deletion strain were significantly lower than that of the parent strain. In addition, it could be seen from the highest OD₆₀₀ value that the utilization rate of sugar by Streptococcus suis SH1510 was glucose>sucrose>D-galactose. In the mouse pathogenicity test, the LD₅₀ of the parent strain and the deletion strain were 1×10 ⁸ CFU and 1.62×10 ⁸ CFU, respectively, and the pathogenicity of the deletion strain to the mouse was decreased by 1.62 times. In vivo competitive infection test results showed that the number of bacteria isolated from deleted strains in the brain, spleen and blood of the mice was much lower than that of the parent strains, and the difference was extremely significant (P＜0.01). The whole blood survival test showed that the survival rate of the parent strain was 35.2%, the survival rate of the deletion strain was 27.3%, and the survival rate of the deletion strain SH1510ΔGalR in the whole blood was significantly lower than that of the parent strain SH1510 (P＜0.05).【Conclusion】 In summary, the GalR gene could promote the utilization of galactose by Streptococcus suis serotype 4, and had direct or indirect regulation of the virulence of SH1510.

Key words: Streptococcus suis serotype 4, GalR, biological characteristics, virulence