Molecular Mechanism Of Host Tropism In Bovine Pasteurella Multocida Type A

Pasteurella multocida(P.multocida)is a Gram-negative coccobacillus that can infect a variety of livestocks and poulty,causing huge economic losses to the breeding industry.According to the difference of capsular polysaccharides,P.multocida can be divided into 5 capsular serotypes A,B,D,E,and F.Among them,type A strains have the most extensive host infectivity,such as causing fowl cholera in poulty and wild birds,pneumonia in cattle,sheep,and pigs,bovine respiratory syndrome,rabbit pneumonia,and atrophic rhinitis.Studies have showed that strains isolated from different host species have exhibited different host predilection,for example,non-avian P.multocida strains cannot infect poultry.In recent years,some articles based on comparative genomics analysis have tried to explain the host tropism mechanism of P.multocida from different aspects,but failed to draw a clear conclusion.The previous research of our laboratory have found that avian P.multocida of serotype A can both infect poultry(chickens,ducks)and mammals(mice,rabbits,piglets),while bovine P.multocida of serotype A can only infect mammals but not poultry.In order to explore the host tropism mechanism of serotype A bovine P.multocida,this study have selected bovine strain type A PmCQ2 and avian strain type A PmQ as the reprentative stains,mice and chickens as model animals,to dissect the underlying factors and molecular basis of host tropism,from both aspects of pathogen and host,repectively.1.Screening of host tropism-related genes based on the whole genome sequencesFrom the perspective of pathogens,two reasons may determine the difference in tropism between bovine and avian-derived strains,one is the presence or absence of genes,and the other is the mutations in homologous genes.To this end,this research have used the second-generation plus third-generation high-throughput sequencing platform to determine the whole genome sequence of PmCQ2 and PmQ.Four bovine(36950,HB01,P1062,and Pm-3)and avian(FDAARGOS＿216,FDAARGOS＿218,X73,and P1059)serotype A P.multocida strains complete genome sequences were selected to analye the core gene families with PmCQ2 and PmQ,respectively.It was found that there are 1740 and 1831 core gene families in bovine and avian strain groups respectively.And two gene families were uniquely existed in the avian strains,namely hypothetical protein coding gene family and the Ton B-dependent receptor coding gene family,suggesting that the genes of these two gene families may be the key that bovine strains cannot infect poultry.This study have attempted to construct PmQ mutants to verify their contribution in host tropism,but failed to obtain corresponding mutant strains.This study continues to analyze its homologous genes of avian and bovine strains.At early time,our laboratory have isolated a bovine type A strain PmCQ7,and whole genome evolution analysis showed that its original host may be poulty.Further study showed its outer membrane proteins Omp A and Omp H have adapted to bovine strains,but whole genome were significantly different from other bovine strains in evolutionary relationship,which suggested that these two protein maight participate in the host tropism change in P.multocida.Thus,this study have constructed an overexpression vector for the omp A gene from the avian strain PmQ,then transferred into the bovine strain PmCQ2.The expression analysis revealed that the avian source omp A gene were stable expressed in PmCQ2-OE,and the relative expression level was 25 times that of its own omp A,while the expression of omp A gene of PmCQ2 itself dropped to 1/12 of that of the wild strain.The survival rate and bacterial colonization of chickens infected with recombinant bacteria PmCQ2-OE were measured,and the results showed that there was no significant difference from PmCQ2,indicating that Omp A alone could not reverse the host tropism of PmCQ2.2.Screening of host tropism-related metabolic pathways based on bacterial transcriptome in vivo and in vitroIn order to further screen the genes that may be involved in the host tropism from the gene transcription level,this study uses mice and chickens as model animals to establish PmCQ2 and PmQ infection models,respectively.Because PmCQ2 does not infect chicks,we use high-dose(2×10~9 CFU/chicken)intraperitoneal injection to simulate the infection state of chickens,so that the bacterial load of PmCQ2 in the lung tissue of chickens can be maintained at about 10~5 CFU/mg at 4,8,and 16 h.In the same way,the colonization amount of the chickens lung tissue infected with 10~3CFU of PmQ could reached 10~4 CFU/mg at 16 h.For mice,PmCQ2 and PmQ were challenged via nasal drops with 10~6 CFU/mouse,and the bacterial loads in lung tissue of mice can reach 10~4-10~5CFU/mg at 16 h post infection.The lung tissues of mice and chickens at 16 h post infection were subjected to HE staining to observe the pathological changes.The results showed that the lung tissues of the mice infected with PmCQ2 and PmQ,and the PmQ infected chickens had a large number of inflammatory cell infiltration and obvious damage,while the PmCQ2 infected chickens had very slight symptoms.Therefore,it was determined that lung tissues were taken at 16 h post infection for transcriptome sequencing.Meantime,considering the difference in body temperature between mammals and poultry,the in vitro transcriptome assay samples were cultured at 37℃and41℃in simulating different body temperatures.Comparing the transcriptome between 41℃and 37℃,a total of 33 genes differentially expressed(DEGs)were found in PmCQ2,of which 16 genes were up-regulated and 17 genes were down-regulated,and GO enrichment analysis showed that the molecular functions of oxidoreductase activity and Antioxidant activity is significantly enriched.While,there are 120 DEGs in PmQ,of which 27 genes are up-regulated and 93 genes are down-regulated,and GO enrichment analysis indicates that the receptor activity and molecular transduction activity of molecular functions are significantly enriched.Afterwards,the PmCQ2-infected mice were compared with those in vitro at37℃,645 up-regulated genes and 747 down-regulated or undetected genes were found,while PmCQ2-infected chickens compare to those at 41℃in vitro revealed 661 up-regulated genes and1191 genes down-regulated or undetectable.Intersection analysis of these genes revealed that 227genes of PmCQ2 showed an up-regulation trend in the infected mice,while down-regulated or undetected in the infected chickens.Classification and verification of these genes showed that there are four gene clusters of PmCQ2 was up-regulated in infected mice but down-regulated in infected chickens.They are phosphate transport system(pst SCAB),arginine transport system(art PIQMJ),tryptophan synthesis(trp AB),and peptide transport system(sap ABCDF).Correspondingly,these genes were both showed increasing tendency in PmQ-infected mice and chickens,suggesting that these transporters and synthesis-related genes may be involved in the host tropism of PmCQ2.3.Differences analysis of mice and chickens immune response to different host-isolated strainsFirst,RNA seq sequencing was performed on the lung tissues of uninfected mice and PmCQ2-and PmQ-infected mice.The results showed that,compare to the control mice,1356 DEGs were identified in PmCQ2 infected mice lung,including 556 up-regulated genes and 800 down-regulated genes,whereas,PmQ infection revealed 1610 DEGs,with 642 up-regulated genes and 968down-regulated genes.KEGG enrichment analysis showed that in both PmCQ2 and PmQ infected mice,Cytokine-cytokine receptor interaction,Hematopoietic cell lineage,Osteoclast differentiation,and Calcium、TNF、PI3K-Akt and MAPK signaling pathway are all significantly enriched.Cell type enrichment analysis showed that macrophages are the most significantly enriched cell types in the lung tissue of mice infected with both PmCQ2 and PmQ,followed by granulocytes,lung cells,mast cells,etc.Furthermore,the expression levels of macrophage surface marker genes and M1/M2polarization-related genes in lung tissues were further determined by Q-PCR,and the results indicated that macrophages were significantly enriched in the lung tissues of PmCQ2 and PmQ infected mice,and M1 polarization macrophage was dominated during infection.Q-PCR verification of immune response-related genes showed that they were consistent with the results of transcriptome sequencing,meantime,apoptosis-related genes(Fas,Ctsd,Atf4,and Tnfsf1α)were significantly up-regulated in the lung tissues of PmCQ2 and PmQ-infected mice.The cytokine levels in lung tissues were also tested by ELISA,and the results showed that pro-inflammatory cytokines,such as,TNF-α,IL-6,IL-1β,etc.were increased significantly after PmCQ2 and PmQ infection,while anti-inflammatory cytokines IL-4 and IL-10 did not change.In addition,mouse peritoneal macrophages infected by PmCQ2 and PmQ analysis also showed that macrophages were polarized toward M1.The above results demonstrated that in the lung of mouse infected with PmCQ2 and PmQ,many immune cells mainly macrophages were significantly enriched,and the polarization of pro-inflammatory M1 macrophages was dominant,thus,induced a severe inflammatory response of host.Thereafter,transcriptome of lung tissue of uninfected chickens and PmCQ2-and PmQ-infected chickens were sequenced.The analysis showed that the PmCQ2 infected chickens were screened out1,427 DEGs,including 633 up-regulated genes and 794 down-regulated genes.In the PmQ-infected group,1649 DEGs were obtained,of which 831 were up-regulated and 818 were down-regulated.Comparing the PmQ infected and PmCQ2 infected chickens,318 DEGs(232 up-regulated and 86down-regulated)were identified.Some immune-related genes were selected for Q-PCR verification and the results showed that they were consistent with the transcriptome sequencing outcome,indicating the reliablity of transcriptome results.GO functional analysis of DEGs in PmCQ2infected chickens were enriched in leukocyte migration,granulocytes chemotaxis and migration,and bacterial defense response,etc.,while the DEGs of PmQ infected chickens are concentrated in immune response,inflammatory response,cytokine response,etc.KEGG enrichment analysis indicated that there are 30 significant enrichment pathways for PmCQ2 infection,and 33 pathways in PmQ infection.Among them,JAK-STAT and Apoptosis signaling pathways were only enriched after PmQ infection,while NOD-like Receptor signaling pathways were only enriched in PmCQ2infected chickens.Moreover,apopotosis related genes expression analysis revealed ap Cell type enrichment analysis showed that macrophages were the most enrichment cell type in chicken lung infected by PmCQ2 and PmQ.When compared PmQ infection with PmCQ2 infection,macrophages was also the most significant cell types.Further analysis of the macrophage-related gene expression pattern,suggesting that different defense strategies and apopotosis of macrophages in the PmCQ2and PmQ infected chickens,which might be the important reasons for the different results of PmCQ2 infection.4.Differences in macrophage regulation are the key to the host tropism of Pasteurella multocida type AIn order to further explore the mechanism of PmCQ2 host tropism at the cellular level,this study selected chicken macrophage cell line HD11 as the cell model to infect PmCQ2 and PmQ,respectively.Taking into account the results of transcriptome analysis of chicken infection,we first detected cell apoptosis of HD11.It was found that PmQ infected HD11 can induce a higher proportion of cell apoptosis,and the cytotoxicity caused by PmQ infection is significantly higher than that of PmCQ2 infection.Then,we found that PmCQ2 infection of HD11 induces cells to differentiate into a spindle shape,while PmQ infection of HD11 induces cells to turn into round,and the anti-phagocytosis ability of PmQ is significantly higher than that of PmCQ2.Next,the transcriptome of HD11 infected by PmCQ2 and PmQ was sequenced,and the results showed that in PmCQ2-infected HD11,Focal adhesion(FA)related genes were significantly up-regulated but not in PmQ infection.The expression of FA marker genes Zyxin and p-FAK was further determined by WB and found that they were increased significantly in PmCQ2 infection,while PmQ infection showed a downward trend.Meanwhile,immunofluorescence detection of F-actin expression showed significantly increased in the PmCQ2 infection group,indicating that PmCQ2 infection of HD11promoted the production of FA,and PmQ showed an inhibitory effect.So how does the increase of FA affect cell apoptosis?Further analysis of the FA downstream pathways of HD11 infected by PmCQ2 revealed that PmCQ2 activates AKT,which subsequently promotes NF-κB phosphorylation and mediates the expression of downstream anti-apoptotic genes(GADD45B,BCL2L1,BCL2A1,and BIRC2).Meantime,compared with PmQ infection,PmCQ2 infection activation of AKT could phosphorylate Fox O1,and results in transfers out of the nucleus of p-Fox O1,thus decreased the expression of downstream pro-apoptotic genes(BCL6,PKL2,PKL3,and KLF2).Otherwise,by inhibiting the formation of FA using Lat.A,the PmCQ2 infection of HD11 displayed a similar degree of apoptosis caused by PmQ infection.In addition,knock-dowan of Zyxin and Fox O1 by si RNA can both eliminate the anti-apoptotic effect of PmCQ2 infection of HD11.In summary,PmCQ2 infection of chicken macrophages HD11 can promote a Zyxin-dependent FA formation,which could increase the downstream AKT phosphorylation via FAK activation,thereby activating the IKBα/NF-κB pathway and Fox O1 phosphorylation,and mediating anti-apoptosis.Conversely,PmQ infection of HD11 will induce a large number of cell apoptosis,which might leading to immune deficiency of chicken and eventually cause the death of host.