Analysis Of Comparative Proteomics And Genomics Of Different Virulent Strains Of Aeromonas Veronii And Preliminary Study On Functions Of Related Genes

Recently,the increasing cases indicate that A.veronii is one of the serious pathogens which can infect mammals and aquatic organisms and it is of great importance to food safety.A.veronii and its congeneric species have been treated as the quarantine objects of water quality and food safety in several countries.At present,the cases reported are increasing year by year,and its prevalence shows a clear upward trend at home and abroad.Related research shows that virulence of A.veronii has gradually strengthened,but relatively less research has been carried out on pathogenic mechanism.There are less studies on virulence factor at home and abroad and most of them are focused on common virulence factor.Studies on new virulence factor of A.veronii will be helpful to further investigate its pathogenic mechanism.In the present study,we analyzed and compared the proteome in different virulent strains of A.veronii by TMT firstly.The differentially expressed proteins between different virulent strains were screened and their possible relationships were predicted.And the whole genome of A.veronii TH0426 strain which infects fish was sequenced and analyzed for the first time.At the same time,the determination of the genome framework of the attenuated strain AV161 and the non-virulent strain CL8155 was completed.The comparative analysis of the genomes revealed a significant difference in the genome level of A.veronii TH0426,AV161 and CL8155 strains.In addition,the genetic evolution of 39 A.veronii strains isolated from different sources was analyzed by comparative genomics,and the genetic evolution of A.veronii was preliminarily understood.Finally,the function of aha and lamB genes encoding Porin and LamB proteins expressed highly in TH0426 were analyzed by gene knockout technique.The main results were as follows:（1）The comparative proteome analysis showed that the expression of proteins in TH0426,AV161 and CL8155 was significantly different,and there had a variety of up-regulated and down-regulated proteins between each other.And 78 differentially expressed proteins were identified between TH0426 and AV161,90 differentially expressed proteins between TH0426 and CL8155,and 92 differentially expressed proteins were identified between AV161 and CL8155.Among them,a total of 16 proteins in TH0426 were identified and their expression was significantly higher than that of AV161 and CL8155,after GO and KEGG enrichment analysis,these proteins were mainly involved in two metabolic pathways: bacterial chemotaxis process and microbial metabolic process in different environments.In addition,there was a positive correlation between the expression level of lysine decarboxylase CadA,endonuclease L-PSP,maltoporin protein LamB,pullulanase and aerolysin and the virulence of the strains.It is speculated that these five proteins may be closely related to the virulence of A.veronii.The results of real-time quantitative PCR and MRM were consistent with the results of TMT identification,and it indicated the results of differentially expressed proteins were accurate and reliable.（2）The analysis of the whole genome of A.veronii TH0426 showed that there were many factors of virulence gene clusters in the genome such as fimbriae,flagella,toxins,iron ion uptake system and various types of secretion system（T2SS,T3 SS and T6SS）.And two genes（AMS64₁9745 and AMS64₂0505）encoding chitinase and two genes（AMS64₀0725 and AMS64₀5940）encoding pullulanase were also found in the TH0426 genome.Similarly,the results of identifying differentially expressed proteins（first chapter）also showed that the expression of these two enzymes in TH0426 was significantly higher than that in AV161 and CL8155.The comparative analysis of the virulence factors of TH0426,AV161 and CL8155 suggested that only TH0426 contained two sets of complete type III secretion system,and the API2 type III secretion system and iron ion transport system were unique for TH0426.In addition,compared with AV161 and CL8155,TH0426 also had some other unique functional gene clusters such as TA systems,pre-phage sequences,CRISPR-Cas systems and recombinant systems.These functional gene clusters may play an important role in resistance to infection,adaptation to different environments,genetic evolution,and pathogenicity of TH0426.（3）The comparative genome analysis of 39 A.veronii strains isolated from different sources suggested that there were 1993 shared core genes which might be necessary to maintain the essential characteristics of A.veronii.The phylogenetic tree based on these core genes showed that strain AMC34 was distant from other 38 A.veronii strains,while the 38 A.veronii strains were closely related.The results of average nucleotide identity（ANI）analysis demonstrated the genetic relationship between strain AMC34 and other 38 strains was distant,which indicates that strain AMC34 probably does not belong to A.veronii.The results also showed that the evolution of A.veronii had no obvious correlation with the location of the isolates,but might be related to their sources,to a certain extent.The strains isolated from the same or similar sources had a closer relationship,which indicates that the host environment may have a more obvious impact on the evolution of A.veronii.（4）The aha and lamB gene deletion mutants of A.veronii TH0426 were constructed successfully,and represented by △ aha and △ lamB,respectively.Compared with the wild-type strain,the biofilm forming ability of △aha decreased by 3.7 times significantly and the adhesion and invasive ability to EPC cells also decreased by 2.3 times.And the pathogenicity of △aha to zebrafish and mice was significantly reduced,the LD50 to zebrafish increased by 80.4 times and the LD50 to mice increased by 10.1 times,compared with the wild-type strain.Similarly,the biofilm forming ability of △lamB decreased by 5.6 times significantly and the adhesion and invasive ability to EPC cells also decreased by 1.8 times.And the LD50 of △lamB to zebrafish increased by 13.7 times and the LD50 to mice increased by 5.6 times,compared with the wild-type strain.The results suggested that the virulence of △aha and △lamB decreased significantly.The stability of flagella was destroyed and the swimming ability of the deletion strains was completely lost after deletion of aha and lamB genes.In summary,the differences between virulent,attenuated and non-virulent strains of A.veronii were studied preliminarily.The results showed that the highly expressed proteins and the specifically functional gene clusters in the virulent strain,such as specific type III secretion system API2 and iron ion uptake system,might be the reasons why A.veronii TH0426 has higher virulence than AV161 and CL8155.The results of genetic evolution analysis suggested that the evolution of A.veronii had no obvious correlation with the location of the isolates,but the influence of host environment on A.veronii evolution was more obvious.The adhesion ability and virulence of TH0426 decreased significantly after deletion of aha and lamB genes encoding Porin and LamB proteins expressed highly in TH0426,which indicates that these highly expressed proteins may be closely related to the pathogenicity of TH0426.These findings will provide a basis for further elucidating the pathogenesis of A.veronii TH0426.