Dual RNA-Seq Analysis Of Glaesserella Parasuis And NPTr Cells And Exploration Of The Function Of VtaA2 Protein

Glaesserella parasuis(G.parasuis)belongs to the family Pasteurellaceae and is an extracellular symbiotic bacterium that parasitizes the upper respiratory tract of pigs and is also part of the upper respiratory microbiota of pigs.In certain cases,G.parasuis can induce Gl(?)sser disease in the swine,with features that can cause pneumonia,arthritis and meningitis in pigs.Gl(?)sser disease in pigs caused by G.parasuis infection can cause significant economic losses to the swine industry,but its prevention and control remains a challenge because the pathogenicity of G.parasuis is not fully understood.During bacterial infection,the interaction between pathogen and host leads to large-scale changes in gene expression in both organisms.To facilitate our ability to better understand the mechanisms of interaction,this study used an in vitro model,using G.parasuis to infect Newborn pig tracheal(NPTr)cells to detect the transcriptional response of both G.parasuis and NPTr cells by using Dual RNA-seq,which in turn helped to reveal the interaction mechanism between GPS and NPTr cells;and the membrane protein gene VtaA2,which is elevated in GPS after infection with NPTr cells,was also selected for investigation,and the effect of VtaA2 protein on GPS in the pathogenic process was studied.The specific results is as follows:1.Dual RNA-seq analysis of G.parasuis-infected NPTr cells:In this study,the NPTr cells were infected with G.parasuis in vitro,the bacterial control and cellular control groups were transcriptome sequenced with the infected group,and four cDNA libraries were constructed: cDNA library of GPS in infected NPTr cells,cDNA library of cultured organisms within TSB/V/S,cDNA library of infected NPTr cells and cDNA library of uninfected NPTr cells.726 DEGs of G.parasuis were screened,of which 383 genes increased,343 genes were decreased;NPTr cells had 3,420 DEGs,of which 2,295 genes were increased and 1,125 genes were decreased.The results of the analysis of G.parasuis differential genes showed that during infection,phosphate metabolism(phn D,E,C),arginine metabolism(Art M,I,Q,P),molybdate metabolism(Mod A,B,C),iron ion transport(Afu A,B,C,Sit A,B,C,D and Fhu D,B,C),heme uptake(Ccm A,B,C,D),lipoprotein metabolism(Lol C,E,D),spermine/putrescine metabolism(Pot A,B,C,D),and other nutrient uptake related genes were upregulated and expressed,and virulence factors such as Cap D,Gal U,CDT and other virulence factors were up-regulated,but most of the previously reported virulence factors were downregulated.Differential gene analysis of NPTr cells revealed that "Toll-like receptor signaling pathway","Focal adhesion","Oxidative phosphorylation","PI3K-AKT signaling pathway","Adhesions junction","TNF signaling pathway" and "ECM-receptor interaction",etc.,have undergone significant changes,especially in immune signaling and extracellular matrixrelated pathways,such as Cxcl 8,Cxcl 10,CCL 5,IL-6,collagen,laminin,integrin,etc.,were increased,indicating that these genes have a great role in the anti-G.parasuis infection during the infection process.2.Study on the function of VtaA2 protein in G.parasuis:This research used the method of homologous recombination to construct the G.parasuis deletion mutant of VtaA2 gene,and then used plasmid to clone the VtaA2 gene into the deletion mutant to obtain the complementary strain.We used PCR and qPCR methods to demonstrate the success of VtaA2 gene deletion.Growth curves indicate that the VtaA2 gene does not interfere with the strain’s growth after deletion and complementation.;the resistance of the three strains to complement in the serum was significantly higher in the deletion strain than in the wild strain and the complementary strain using healthy pig serum,indicated that VtaA2 may be involved in the process of complement killing G.parasuis.The analysis of biofilm and agglutination assay showed that VtaA2 protein could affect the agglutination process of G.parasuis and further affect the formation of G.parasuis biofilm.In terms of anti-phagocytic ability,the absence of VtaA2 gene did not effect its phagocytosis by PAM cells..The mouse virulence test and the mouse bacterial load test proved that the lethality of the deletion strain to mice was lower than that of the wild strain and the complementary strain,and the bacterial load in the liver and lung of the mice was significantly lower than that of the wild strain and complementary strain.Finally,it was proved that VtaA2 was involved in the adhesion of G.parasuis to NPTr cells by adhesion invasion assay and laser confocal.The above results were combined to identify genes and related pathways that play an important role in GPS pathogenesis and NPTr cell resistance through Dual RNA-Seq analysis of GPS interactions with NPTr cells,and to identify that VtaA2 protein in GPS can regulate the pathogenic ability of Glaserella parasuis.It provides new ideas for subsequent insight into the pathogenic mechanism of Glaserella parasuis.