Construction And Biological Characterization Of DppA1DppA2 Mutation In Actinobacillus Pleuropneumoniae

Actinobacillus pleuropneumoniae(APP)is a Gram-negative bacterium belonging to the genus Pasteurella.Porcine pleuropneumonia caused by A.pleuropneumoniae that is one of the most important respiratory diseases of pigs.The disease is mainly characterized by necrotic pneumonia and fibrinous pleuritis.Due to high mortality and mortality rate,the outbreak of porcine pleuropneumonia causes huge economic losses to the pig industry all over the world.Porcine pleuropneumonia is a highly contagious disease which is easily co-infected some other porcine respiratory pathogens.The control of porcine pleuropneumonia is not easy since the complex and un-clarified pathogenesis of A.pleuropneumoniae infection.Sulfur is an important nutrient that is essential for pathogenic bacteria survival in vitro and in vivo.At present,the mechanisms of sulfur nutrients transport in A.pleuropneumoniae,especially glutathione transport and its relationship with bacterial pathogenicity is still unclear.In this study,roles of possible dipeptide transporters DppAl and DppA2 in the process of sulfur transport were investigated.The main contents and results are as follows:1.Construction and identification of mutant strainsIn this study,single-gene deletion mutants AdppAl and ΔdppA2,and double-g ene deletion mutants ΔdppA1ΔdppA2-1 and ΔdppA1ΔdppA2-2 were constructed by homologous recombination and negative screening,separately,by using A.pleurop neumoniae standard strain WF83(serovar 7)as the wild type(WT)strain.Then c omplementation strains were obtained,by introduction of dppAl,dppA2,dppA1dpp A2 genes into the ΔdppA1ΔdppA2-1 strain,respectively,and named as ΔdppA1Δdp pA2-1-CΔA1,ΔdppA1ΔdppA2-1-CΔA2 and ΔdppA1ΔdppA2-1-CΔA1A22.Analysis of the growth ability of mutant strains in vitroThe growth ability of A.pleuropneumoniae strains WF83,ΔdppAl,ΔdppA2,ΔdppA1ΔdppA2-1 were evaluated in TSB medium and chemically defined medium(CDM)with various sulfur sources.The results showed that there was no significant difference between these three mutant strains and WT when cultured in TSB.When use glutathione as the sole sulfur source,the growth levels of ΔdppA1 and ΔdppA2 were nearly the same as WT,however,the growth ability of the double gene deletion mutationΔdppA1ΔdppA2-1 was significantly lower than that of the WT after lag phase.Trans-complementation of DppAl,DppA2 or DppA1DppA2 was unable to recover the growth disability of ΔdppA1ΔdppA2-1 in CDM supplemented with glutathione.In addition,the growth curves indicated that the growth ability of ΔdppA1ΔdppA2-2 was not significantly different from WT when use glutathione as the only sulfur source.3.Pathogenicity of ΔdppA1ΔdppA2-1As we known that the ability of WF83 and double gene deletion mutantΔdppA1ΔdppA2-1 is disabled to use glutathione.Therefore,the relationship between the ability of glutathione utilization and virulence were determined,by using mouse and piglet infection models.The results showed that the competitive infection index ofΔdppA1ΔdppA2-1 and WT in mouse lung tissues was 1.35,which was higher than the attenuation cutoff value(0.20).And the competitive indices of AdppA1ΔdppA2-1 and WT in pig lung tissues were determined.The results showed that average CI value was 0.91,which was higher than the weakening threshold(0.20),indicating that the deletion of dppAl and dppA2 genes did not decrease the pathogenicity of A.pleuropneumoniae,and the utilization of glutathione is probably not regarded to the virulence of A.pleuropneumoniae.In conclusion,this study preliminarily shows that deletion of dppAldppA2 as well as the sequence between these two genes affects the growth ability of A.pleuropneumoniae in glutathione CDM medium,indicating that this deleted sequence is related to A.pleuropneumoniae glutathione utilization.However,DppAl and DppA2 probably not contribute to this process.Although the deletion of this sequence affected the utilization of glutathione by A.pleuropneumoniae,the pathogenicity in mice and piglets was not significantly decreased.Although our present study was unable to provide direct evidence between DppAl DppA2 and glutathione utilization in A.pleuropneumoniae,it offers some materials and clues for further investigation of the roles of DppAlDppA2,and the relationship between sulfur nutrients transport and bacterial pathogenesis of A.pleuropneumoniae.