| Streptococcus suis type 2 (S.suis 2) is an important zoonotic pathogen associated with a wide range of diseases in pigs, including meningitis, septicemia, pneumonia, endocarditis, arthritis, acute death, it causes serious economic effect. According to the capsular polysaccharide (CPS), Streptococcus suis can be divided into 33 serotypes (1-31,33 and 1/2). S. suis 2 can also be transmitted to human beings through respiratory tract or wound contamination.The disease was distributed worldwide, not only to the swine industry, but also raised considerable international concerns among the public health professionals.The mod gene of S. suis 2 type III restriction and modification system (R-M) is a regulatory gene, can regulate the expression of many proteins, it were screened through the in vivo induced antigen technology (IVIAT), and play an important role in bacteria infection process. This study constructed a mod gene deletion mutant and plasmids complementary strains, and study the impact of mod gene deletion on the biological characteristics, Using gene chip technology to study the regulatory mechanism of mod gene at the transcriptional level.The following experiments are conducted:1. Construction and identification of SS2 mod gene mutant strain and complementary strainThe flanking DNA sequences to mod were amplified from the chromosomal DNA of S.suis SC-19 and were ligated to the thermosensive suicide vector pSET4s.The resulting plasmid pSET4s-modU-ermb-modD was electroporated into the wild strain SC-19. The suspected mutant was further confirmed by Southern blotting analysis to ensure that an isogenic knockout mutant of mod (namelyΔmod) was successfully constructed. For complementation analysis, a DNA fragment containing mod gene and its upstream promoter was amplified and cloned into the E. coli-S.suis shuttle vector pSET2. Then the resulting plasmid pET2s-mod was electrotransformed into the Amod mutant to screen the complemented strain Cmod with selection pressure of Spc and PCR confirmation.2.Effect of mod deletion on the general biological characteristics of S.suis SC-19The general biological characteristics of the S.suis SC-19 and the Amod mutant were compared under the same conditions. S.suis SC-19 and the Amod mutant have no difference in the growth and Gram stain, but in the observation under scanning electron microscopy and transmission electron microscopy, the mutant strain have a thick outer surface, The complementary strain have no significant difference in the surface compared with the wild strain.3.1mpact of Amod mutant on the virulence of S.suis SC-19To evaluate the impact of mod deletion on the virulence of SC-19, two sets of experimental infections were carried out in parallel:①Five weeks old piglets (4/group) were challenged nasal drip with SC-19 and Amod at a dose of 2×106CFU. Results of experimental infections showed that knockout of mod eliminated the lethality of S.suis SC-19.②Competitive colonization experiment was adopted to analyze the capability of each strain to colonize the various tissues of piglet. Groups of 3 piglets were inoculated nasal with the wild strain SC-19, Amod mutant and a 1:1 mixture of SC-19/Amod, respectively. Co-colonization experiment results showed that the Amod mutant infected the specific tissues less effectively than the wild strain.4. Analysis of mod Gene Regulation S.suis SC-19 gene expressionTo gain further insights into the network mediated by mod, whole-genome gene chip was applied to reveal the differential transcription profiles between the wild strain SC-19 with the Amod mutant. In total,83 genes compared to the wild strain SC-19 were up regulated, mainly in glycolysis, ATP binding, iron ion binding, oxidative phosphorylation,TCA cycle, fatty acid biosynthesis and purine metabolism; 108 genes were down regulated, mainly related to pyrimidine, ABC transport system, PTS. |
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