Study On The Regulation Of ArcA Of Haemophilus Parasuis

Haemophilus parasuis,a commensal organism of the upper respiratory tract of pigs,can invade the host and cause severe systemic disease characterized by fibrinous polyserositis,arthritis,and meningitis,known as Glasser's disease.The disease has existed in many pig-raising countries in the world and become one of the most important bacterial diseases of pigs worldwide.So far,the virulence factors and disease-causing mechanisms associated with H.parasuis are largely unknown.As the global response regulator in many bacteria,the ArcA not only play a major role in maintaining the s anaerobic redox balance of bacteria,but are also known to afford energy metabolism,serum resistance and virulence mechanisms of bacteria.In this study,the arcA deficient mutant(?arcA)of a H.parasuis serovar 13 clinical strain EP3 were constructed by a modified natural transformation system,and we investigated the serial of biological characteristics of AarcA mutants.Furthermore,the comparative transcriptomics and comparative proteomics analysis was performed between the EP3 and AarcA mutant strains in order to explain the novel phenotypes.The results obtained in this study are as follow:1.Constructions of ?arcA mutant strains in H.parasuisThe PCR product arcA gene was inserted into the expression vector pET28a and then transformed into E.coli BL21 for expreesion.The expression conditions were optimized.The expressed fusion protein was mainly in the form of inclusion bodies.After purification,the protein was immunized mice to produce polyclonal antibody.To obtain the H.parasuis mutant,we used the pK18mobsacB suicide plasmid to construct mutant.Using natural transformation method,we constructed the plasmid pAD.After transforming into EP3 strain,AarcA mutant strains were obtained.In addition,a single-copy plasmid pCA was constructed and transformed into theAarcA mutant to obtain complemented strains.Many kanamycin-resistant transformants were obtained and checked for specified homologous recombination by PCR and western-blotting.The above results showed that AarcA mutant,its complemented and C-AarcA strains were successfully constructed in this study.2.Study on the biological characteristics of EP3 strain,?arcA mutant and C-AarcA strainCompared to the wild-type EP3 strain,the ?arcA mutant showed a pronounced growth defect under anaerobic conditions,whereas the complemented strain could restore the growth.The results suggested that ArcA a played an important role in growth of the H.parasuis EP3 strain under anaerobic conditions.In serum resistance,wild-type EP3 strain exhibited a highly resistant to the bactericidal activity.However,loss of ArcA expression in wild-type strain resulted in significantly increased sensitivity to serum killing.Complementation of the?arcA mutant restored the serum-resistant phenotype.The results suggested that an important role of H.parasuis ArcA appeared to be its ability to protect against the bactericidal activity of complement.However,there were no obvious differences in self-agglutination.In this study,the BALB/C mouse model was introduced to evaluate the virulence.A significant difference was observed,the virulence for the wild-type strain was increased to for the AarcA mutant strain,whereas the complemented strain restored to that of the wild-type-like phenotype.The biofilm formation assay indicated that the ?arcA mutant strain significantly weakened the ability to form biofilm compared with the wild-type EP3 strain.The results revealed that arcA was involved inbacterial biofilm formation.3.Transcriptomic analysis of both the wild-type and ?arcA strainsGiven the unexpected results in phenotype changes of the ?arcA mutant,RNA-seq technology were preformed of total bacterial transcriptome to seek to the differentially genes which were involved in the unexpected phenomenon in ?arcA mutant.The 73 different genes were determined that showed altered expression in ?arcA mutant under the culture conditions employed(1.5-fold or greater).Of these,57 genes displayed higher expression in the mutant than in wild-type,whereas 23 genes displayed lower expression.When the differentially expressed genes were organized by the cluster of orthologous groups(COG)entry present for each gene,the majority of the differentially expressed genes were involved in the sugar metabolism,transcription and translation factor,lipid metabolism,amino acid metabolism,nucleotide metabolism,chaperons and other.The results suggested that the differentially expressed genes were mainly involved in energy metabolism and membrane biogenesis.4.Proteomic analysis of both the wild-type and ?arcA strainsThe iTRAQ technololgy were preformed of total bacterial proteins to seek to the differentially expressed proteins which were involved in the unexpected phenomenon in AarcA mutant.The 119 different proteins were determined that showed altered expression in AarcA mutant under the culture conditions employed(1.5-fold or greater).Of these,54 proteins displayed higher expression in the mutant than in wild-type,whereas 65 proteins displayed lower expression.When the differentially expressed proteins were organized by the cluster of orthologous groups(COG)entry present for each protein,the majority of the differentially expressed proteins were involved in tricarboxylic acid cycle,phosphoinositol metabolism,dicarboxylic acid metabolism,sphingolipid metabolism,?-alanine metabolism,pentose phosphate pathway,propionic acid metabolism,glutathione metabolism,NAD metabolism,catalase,Purine metabolism,synthesis of secondary metabolites,nitrogen metabolism,glycolysis,fructose and mannose metabolism,galactose metabolism,starch and sucrose metabolism,pantothenic acid biosynthesis,other polysaccharide degradation.The results suggested that the differentially expressed proteins were mainly involved in energy metabolism and membrane biogenesis.Therefore,loss of Arc A expression resulted in the global protein expression changes could be responsible for novel phenotypes occurred in AarcA mutant.