| Haemophilus parasuis is defined as a conditional pathogenic bacteria which colonized on the upper respiratory tract of porcine. It caused Gl?sser’s disease characterized by fibrinous polyserositis, polyarthritis and meningitis. Since serotypes of its strains lack of immunological cross-resistance caused by different serotypes and various virulence, so far there have not been effective methods of prevention and control of H. parasuis disease. Increasingly studies have focused on the virulence factors of the H. parasuis and identified lipooligosaccharide(LOS) is the potential one. LOS can mediate adhesion of H. parasuis and induce the release of inflammatory mediators. However the function of genes related to LOS synthesis remains to be further studied.Lgt B/lex-1 are two genes wait to be studied in H. parasuis while researches of Lgt B/lex-1 have been reported from other bacteria. β-1,4-galactosyltransferase encoded by lgt B/lex-1 is a member of glycosyltransferase 25 family,which is involved in LOS synthesis. This study aims to analyze gene function of lgt B/lex-1 and investigate their roles in the pathogenic process.First, deletion vector p ZQ001 of lgt B and deletion vector p ZQ002 of lex-1 were constructed according to the genetic techniques established by our lab. The two deletion vectors were transformed into wild type parent strain HPS SC096 by natural transformation to obtain the mutant Δlgt B or Δlex-1; Moreover the complementary vector p ZQ003 or p ZY004 were constructed to yield complementary strain Δlgt B-c or Δlex-1-c by transforming the vector into corresponding gene-deleted strain. Those strains were verified by PCR and sequencing.To investigate the variation of the LOS profile, the extracted LOSs were analysed by SDS-PAGE gels, then observed by silver stain. It showed that the LOS from Δlgt B or Δlex-1 mutant migrated significantly faster than that of the wild-type SC096 strain, which indicated reduced molecular masses and truncated structures of LOS, while the LOSs from Δlgt B-c and Δlex-1-c strains migrated equally with the wild-type LOS.The abilities of resistance to the serum in the mutant strains were investigated. The results demonstrated that the Δlgt B mutant or Δlex-1 mutant was significantly more sensitive in 50% porcine sera compared with the wild-type SC096 strain, displaying 15.0% and 54.46% survival rates, respectively, and complementation of the lex-1 mutant restored the serum-resistant phenotype. However, the survival rate of complementation of the lgt B mutant was 8.51%, with approximately 10 fold reduction in 50% porcine sera. To explain the reasons for increased serum sensitivity of Δlgt B-c strain, q RT-PCR analysis of m RNA levels of lgt B in the wild-type SC096 strain and Δlgt B-c strain was performed. The results showed that, relative to SC096 strain, Δlgt B-c strain had 27.05-fold increased transcriptional levels of lgt B, which indicated that lgt B should be overexpressed in the complemented strain.The adherence and invasion have been identified as important virulence mechanism in H. parasuis. In this study, adhesion and invasion to the PK-15 cells were assessed in constructed strains. The results demonstrated that compared to the wild-type SC096 strain, there were significantly less adhesion and invasion by Δlgt B or Δlex-1 mutants, indicating lack of lgt B/lex-1 could decreased the ability of mutants to adhere to and invave on host cells.Finally, 250 g healthy guinea pigs were infected with the Δlgt B mutant and wild-type SC096 strain in comparison with virulence by intraperitoneal inoculation. The results showed that guinea pigs infected with the wild-type SC096 strain appeared decreased food and water intake, lethargy, rough hair disorder and difficulty breathing, while the guinea pigs infected with the Δlgt B mutant had no obvious clinical symptoms. Abdominal cavity of guinea pigs infected with the wild-type SC096 strain appeared much fibrinous exudate, while the guinea pigs infected with the Δlgt B mutant had no pathological changes, which showed that compared to the wild-type strain, the Δlgt B mutant had significantly reduced virulence. |
PDF Full Text Request