Study On The Regulatory Function Of Two-component System QseB/QseC Of Haemophilus Parasuis

Haemophilus parasuis is normally a benign swine commensal,but may cause Gl?sser's disease in swine,which is characterized by fibrinous polyserositis,arthritis and meningitis.Qse B/Qse C is global regulator of virulence in Enterobacteriaceae and Pasteurellaceae.Study the functions of two component system Qse B/Qse C in Haemophilus parasuis would play crucial roles in efforts to prevent and control this disease.In this study,we created ?qse B??qse C and ?qse BC mutant strains and investigated the role of Qse B/Qse C in the Haemophilus parasuis infection process.The biological characteristics of wild strains and their mutant strains were investigated via growth curve,biofilm formation,pressure tolerance,antibiotic resistance and mouse viru Lence.Moerover,comparative transcriptomics analysis was performed between the wild-type strain SC1401 and mutant strain ?qse B to explore the role and regulation mechanism of Qse B in the pathogenesis of Haemophilus parasuis.The results obtained in this study are presented below:1.Constructions of ?qse B??qse C and ?qse BC mutant strains in Haemophilus parasuisThe fusion fragments were cloned into p K18 mobsac B to construct plasmid p LQ1,p LQ2 and p LQ4,respectively.The recombinant plasmids were introduced into Haemophilus parasuis wild strains by natural transformation to obtain ?qse B,?qse C and ?qse BC mutant strains,respectively.The colonies growing on the kan-containing TSA plates represented the mutant strains.The target genes,which contained the entire open reading frame of qse B,qse C and qse BC double genes were cloned into plasmid PSF116 respectively.The recombinant plasmids were transferred to the corresponding deletion strains by natural transformation to obtain the complementary strains C-?qse B,C-?qse C and C-?qse BC,respectively.The complementary strains were selected on TSA supplemented with 20 ?g/m L gentamicin.The results of PCR and Western blotting showed that ?qse B,?qse C and ?qse BC double mutant,their complemented strains C-?qse B,C-?qse C and C-?qse BC were constructed successfully.2.Biological characteristics of ?qse B,?qse C and ?qse BC mutant strains and virulence in miceTo determine the biological functions of Qse B/Qse C,we investigated the biological characteristics of ?qse B??qse C and ?qse BC.Compared to the wild strain SC1401,the growth rate of ?qse B and ?qse BC mutant strains were significantly slower than that of the wild strain,and the complementary strain recovered the growth ability,while ?qse C didn't exhibit obvious growth defects compared with the wild strain.The biofilm formation assays showed that the amount of biofilm in ?qse B,?qse C and ?qse BC mutant strains showed significantly reduced compared to that in the wild-type strains.We also investigated the morphological changes of H.parasuis after the qse BC deletion using a scanning electronic microscope.The wild-type strain SC1401 formed a dense biofilm,while ?qse BC mutant strains had weakened biofilm formation,inducing bacterial swelling,or deformities,and even cell autolysis.The antibiotic susceptibility of ?qse BC was increased significantly in planktonic bacteria and in biofilms when exposed to ceftiofur,ampicillin,polymyxin B and doxycycline and so on.In the serum killing assays,the ?qse B and ?qse BC mutant strains showed significantly lower percent survival compared to the wild strain SC1401.The stress resistance assays showed that ?qse C and ?qse BC obviously weakened the ability of stress tolerance such as osmotic pressure,oxidative stress,heat shock.Mouse virulence experiment indicated that the lethal rates of the ?qse B,?qse C,and ?qse BC mutant strains were significantly lower than that of the wild strains.These results showed that two-component system Qse B/Qse C is involved in the regulation of various biological functions and required for virulence of Haemophilus parasuis.3.Transcriptomic analysis of both the wild strain SC1401 and ?qse B mutant strainsIn the two-component system Qse B/Qse C,Qse C senses external signal molecules and undergoes autophosphorylation,and transfer phosphate groups to Qse B,a transcription factor that regulates relevant motor and virulence gene expression in bacteria.In view of the obvious biological characteristics of the ?qse B mutant strain,RNA-seq technology were used to compare the transcriptome sequencing of wild strain SC1401 and ?qse B mutant strain in Haemophilus parasuis.The results showed that there were 624 different genes in ?qse B compared with the wild strain,351 up-regulated genes and 273 down-regulated genes.SC1401 and ?qse B co-expressed 2058 genes,SC1401 expressed 5 genes alone,and ?qse B expressed 31 genes alone.GO database analysis showed that the different genes between the two samples were enriched in biological processes(transport,response to stress,biosynthesis of organic cyclic compounds,transcriptional regulation)and molecular functions(ATPase activity,sequence-specific DNA binding,anti-Oxidation activity).KEGG database annotated the metabolic pathways of the two sample transcriptional sequencing results,and the different genes are enriched in ABC transport,oxidative phosphorylation,glycolysis/gluconeogenesis,TCA cycle,Pentose phosphate pathway and Phosphotransferase system.Based on the results of RNA-Seq,we mapped the regulatory network of the Qse B in Haemophilus parasuis.