| Haemophilus parasuis as an important bacterial pathogen which has a harmful effect on the global pig industry, it has caused great economic losses. Lipopolysaccharide(LPS) is the major constituent of the outer membranes of Gram-negative bacteria, and it is also an important virulence factor. The LPS generally contains Lipid A, core oligosaccharide and O-specific antigen. Core oligosaccharide plays an important role in the pathogenesis of LPS. The rfaE gene is generally considered to be involved in the formation of ADP-heptose, a substrate of heptosyltransferase which is speculated in the synthesis of LPS core oligosaccharide structure. However, the role of rfaE gene in the pathogenesis of LPS has not been known. The aim of this study is to construct the rfaE deficient mutant(ΔrfaE) and complementary strain of H. parasuis serovar 4 clinical strain SC096, and we investigated the serial of biological characteristics of ΔrfaE mutant. The LPS was extracted, and the effect and mechanism of LPS on the inflammatory response of cells and organism were studied. The results obtained are as follows: 1. The construction of H. Parasuis SC096 ΔrfaE mutant and complementary strainThe aim of this study was to study the role of rfaE gene in the pathogenic mechanism of H. Parasuis SC096 strain. Using the modified natural transformation method, we constructed the plasmid pZZ2 containing erythromycin resistant fragment. After transforming into SC096 strain, ΔrfaE mutant strain were obtained. The PCR detection and gene sequence analysis showed that the ΔrfaE mutant strain were successfully constructed in this study. In addition, a single-copy, chromosome-based complementation plasmid pZZ3 was constructed and transformed into the ΔrfaE mutant, cΔrfaE mutant strain were obtained. The PCR detection and gene sequence analysis showed that the complemented strain were successfully constructed in this study. It laid the foundation for studying the effect and mechanism of rfaE gene in the pathogenesis of HPS. 2. Study the biological characteristics of ΔrfaE mutantIn order to study the effect of rfaE gene on the variation of the LPS glycofoms, growth characteristic, sensibility in the serum and adherence to and invasion to host cells in wild-type SC096 strain. The LPS of H. parasuis SC096 strain, rfaE mutant and its complementation was extracted. The extracted LPS was loaded onto the SDS-PAGE gels, and observed by silver stain. It showed that the LPS from ΔrfaE mutant(ΔrfaE-LPS) migrated significantly faster than that of the wild-type LPS(WT-LPS). the LPS from cΔrfaE mutant migrated equally with the WT-LPS. Compared to the wild-type SC096 strain, loss the rfaE gene led to an obvious growth defect. The generation time of the ΔrfaE mutant(~69 min) was significantly longer than that of the wild-type strain(~59 min)(p<0.05), while the complemented strain restored the growth phenotype(~60 min). We investigated the ΔrfaE mutant in adherence to and invasion to porcine kidney epithelial cells(PK-15) and porcine umbilical vein endothelial cells(PUVEC). Compared to wild-type SC096 strain, the ΔrfaE mutant exhibited 10-fold less efficient adherence(p<0.001). Likewise, it also showed 12-fold reductions invasion capacity(p<0.001) a significantly reduced ability to adhere to and invade PK-15 and PUVEC cells. The abilities of the resistance to the serum in the ΔrfaE mutant strain were investigated. The wild-type SC096 strain exhibited a highly resistant to the bactericidal activity in serum resistance. Compared to the wild-type SC096 strain, deletion the rfaE gene resulted in approximately 30-fold reductions in survival percentage in 50% sera and 36-fold reductions in survival percentage in 90% sera(p<0.001). The complemented strain restored the serum-resistant phenotype. The above results suggested that the loss of rfaE gene expressed truncated LPS structures, slower growth, significantly greater sensitivity to complement-mediated serum and reduced ability to adhere to and invade PK-15 and PUVEC cells in wild-type SC096 strain. 3. Study the expression of inflammatory cytokines induced by ΔrfaE mutant LPSIn order to study the effect of rfaE gene on the expression of inflammatory cytokines induced by H. parasuis LPS. The LPS of H. parasuis SC096 strain, rfaE mutant and its complementation was extracted. The PAMs were stimulated with LPS(5 μg/mL and 10 μg/mL) from H. parasuis SC096, ΔrfaE mutant(ΔrfaE) and its complementation(cΔrfaE) for 2 h, 4 h, 6 h, 12 h and 24 h. The RNA was extracted and reversed into cDNA. The mRNA transcription of IL-1α, IL-1β, IL-6, IL-8 and TNF-α were then detected by Real-time PCR. The ribosomal protein L4(RPL4) as reference gene. The data were analyzed using the 2-△△CT method. The result showed that the mRNA transcription of IL-1α in PAMs induced by 5 μg/mL LPS from H. parasuis for 2 h, 4 h, 6 h,12 h and 24 h is significantly higher than the mRNA transcription of IL-1α in PAMs induced by 5 μg/mL LPS from ΔrfaE(P<0.05), the mRNA transcription of IL-1β, IL-6, IL-8 in PAMs induced by 5 μg/mL LPS from H. parasuis for 12 h and 24 h are remarkably higher than the mRNA transcription of IL-1α in PAMs induced by 5 μg/mL LPS from ΔrfaE(P<0.05), the mRNA transcription of TNF-α in PAMs induced by 5 μg/mL LPS from H. parasuis for 24 h is notably higher than the mRNA transcription of IL-1α in PAMs induced by 5 μg/mL LPS from ΔrfaE(P<0.05), compared to the WT-LPS group, the expression of IL-1α and IL-8 in PAMs induced by 5 μg/mL ΔrfaE-LPS showed 2-fold reductions, respectively; the mRNA transcription of IL-1α, IL-8 and TNF-α in PAMs induced by 10 μg/mL LPS from H. Parasuis always significantly higher than that 10 μg/mL LPS from ΔrfaE mutant(P<0.05), compared to the WT-LPS group, the expression of IL-1α and IL-8 in PAMs induced by 10 μg/mL ΔrfaE-LPS showed 7-fold and 4-fold reductions, respectively. At the same time, the cytokine mRNA transcription level in PAMs induced by 5 and 10 μg/mL LPS from cΔrfaE can restore to the level in PAMs induced by LPS from H. parasuis.The BALB/c mice were intraperitoneal injected with 80 μg WT-LPS, ΔrfaE-LPS and cΔrfaE-LPS. The expression of IL-1α, IL-6, IL-8 and TNF-α in serum were detected by commercial ELISA kits. The result showed that the expression of IL-1α, IL-6, IL-8 and TNF-α in BALB/c mice induced by WT-LPS are up-regulated. The expression of pro-inflammatory cytokines showed time dependent. Compared to the WT-LPS group, the expression of IL-1α, IL-6, IL-8 and TNF-α in BALB/c mice induced by ΔrfaE-LPS showed 4-fold, 5-fold, 3-fold and 8-fold reductions, respectively. The inflammatory cytokines expression level in BALB/c mice induced by cΔrfaE-LPS can restore to the level in BALB/c mice induced by WT-LPS. The above results confirmed that the HPS-LPS induced the inflammation response in both PAMs and BALB/c mice. The ΔrfaE-LPS induced the inflammation response in both PAMs and BALB/c mice were significantly decreased, suggesting that the rfaE gene regulated the inflammation response induced by HPS-LPS. 4. Study the NF-κB and MAPK signaling pathway induced by ΔrfaE mutant LPSDue to the ΔrfaE-LPS induced the inflammation response in both PAMs and BALB/c mice were significantly decreased. In this study, Real-time PCR and Western blot methods were used to study the effect of rfaE gene on the NF-κB and MAPK signaling pathway induced by HPS-LPS. Real-time PCR results showed that the mRNA transcription of TLR4, MD2, MAP2K2, ERK, P38 and JNK in PAMs induced by WT-LPS is up-regulated, and significantly higher than the mRNA transcription of TLR4, MD2, MAP2K2, ERK, P38 and JNK in PAMs induced by ΔrfaE-LPS. The signal molecules mRNA transcription level in PAMs induced by cΔrfaE-LPS can restore to the level in PAMs induced by WT-LPS. The mRNA transcription of NF-κB in PAMs no significant difference was observed. Western blot results showed that the protein of IκBα in PAMs induced by WT-LPS is significantly lower than the protein of IκBα in PAMs induced by ΔrfaE-LPS, which decreased about 1-flod. The NF-κB p65 phosphorylation and MAPK p38 phosphorylation in PAMs induced by WT-LPS is significantly higher than the NF-κB p65 phosphorylation, MAPK p38 phosphorylation in PAMs induced by ΔrfaE-LPS, which respectively decreased about 2-flod and 1-flod. The protein level in PAMs induced by cΔrfaE-LPS can restore to the level in PAMs induced by WT-LPS. The date confirmed that the loss of rfaE could effectively block the NF-κB and MAPK p38 signaling pathways and reduce the inflammatory process in PAMs induced by H. parasuis SC096 LPS.In this study, ΔrfaE mutant was successfully constructed. The study found that the rfaE gene was involved in the synthesis of LPS, serum resistance and adherence to and invasion of host cells in HPS SC096 strain. In addition, we also confirmed that the rfaE gene mediated LPS induction of pro-inflammatory cytokines(IL-1α, IL-1β, IL-6, IL-8 and TNF-α) expression in PAMs, which was dependent on both the NF-κB and MAPK p38 signaling pathways. |
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