| Aeromonas hydrophila is a major pathogen causing motile Aeromonas septicemia(MAS),and it has been recognised as a pathogen to human,livestock and fish.To date,none of the A.hydrophila strains isolated in China has been whole-genome sequenced,and the predominant epidemic strains which could cause the outbreak of MAS in China were not determined.In addition,the pathogenesis of A.hydrophila is very complex,and so far,there are still many virulence factors not found.In this study,epidemic A.hydrophila strains isolated in China were whole-genome sequenced and comparative genomics analysis was performed,deciphering the genetic information of epidemic strains.Also,three metabolic pathways were found to be specific to epidemic strains,and may be the key reason leading to the high virulence of epidemic strains.Based on the comprative genomics analysis,ST251 clonal group strains were determined as the main epidemic strains in China and USA along with the analysis of their isolated location and source.In addition,this study established a method to assess the virulence of Aeromonas strains with Tetrahymena as a model host,and constructed a mutant library consisting of 1030 mutant strains using transposon insertion mutagenesis.After using Tetrahymena as a model to screen the virulence-associated genes,12 genes were identified.Moreover,this study also constructed trkll and chiA gene deletion strains,and performed preliminary study on their function in bacterial pathogenicity.1.Complete genome sequencing and comprative genomic analysis of Aeromonas hydrophilaFor the first time,we provide two complete genomes of epidemic A.hydrophila strains NJ-35 and J-1 which isolated in China in different time,and we performed comparative genomic analyses of five epidemic strains,together with the environmental strain ATCC 7966T.It showed that the epidemic strains isolated in China and USA were highly similar to each other,and all of them belong to ST(sequence type)251 clonal group.We also found that the known virulence factors,including a typeⅢsecretion system,a typeⅥsecretion system and lateral flagella,are not required for the high virulence of the ST251 clonal group.Additionally,our work identifies 27 regions of genomic plasticity(RGPs)including prophage and O-antigen regions,and also identifies three utilization pathways for myo-inositol,sialic acid and L-fucose providing clues regarding the factors that underlie the epidemic and virulent nature of ST251 A.hydrophila.2.The geographila distribution and biological characteristics of ST251 Aeromonas hydrophilaIn this study,we determined that the ST251 clonal group strains are the predominant epidemic strains in China and USA based on their isolated location and source.The analysis of the virulence factors of 30 A.hydrophila strins showed that all of the strains do not have typeⅢsecretion system and lateral flagella system,suggesting they are not required for the high virulence of the ST251 clonal group.On the contrary,typeⅡsecretion system are conserved in all strains,indicating that it is necessary and sufficient for the secretion of extracellular products,however,it can not be used as the bacterial virulence marker.The pathogenic characteristics of ST251 strains showed that,there were no regular differences between ST251 strains and other ST strains in biofilm-forming ability,motility,hemolytic activity and protease activity of extracelluar products.However,the ST251 clonal group strains could take advantage of inositol,sialic acid and L-fucose for growth and survival in mice,suggesting these metabolic pathways presented in the bacteria may help them get nutrients in the host body,thereby enhancing their ability to survive and proliferate,and enabling them stronger pathogenicity.3.Tetrahymena:an alternative model host for evaluating virulence of Aeromonas strainsThe aim of this study was to assess virulence of Aeromonas spp.using two model hosts,a zebrafish assay and Tetrahymena-Aeromonas co-culture,and to examine whether data from the Tetrahymena thermophila model reflects infections in the well-established animal model.First,virulence of 39 Aeromonas strains was assessed by determining the 50%lethal dose(LD50)in zebrafish.LD50 values ranging from 1.3×102 to 3.0×107 indicated that these strains represent a high to moderate degree of virulence and could be useful to assess virulence in the Tetrahymena model.In Tetrahymena-Aeromonas co-culture,we evaluated the virulence of Aeromonas by detecting relative survival of Aeromonas and Tetrahymena.An Aeromonas isolate was considered virulent when its relative survival was greater than 60%,while the Aeromonas isolate was considered avirulent if its relative survival was below 40%.When relative survival of T.thermophila was lower than 40%after co-culture with an Aeromonas isolate,the bacterial strain was regarded as virulent.In contrast,the strain was classified as avirulent if relative survival of T.thermophila was greater than 50%.Encouragingly,data from the 39 Aeromonas strains showed good correlation in zebrafish and Tetrahymena-Aeromonas co-culture models.The results provide sufficient data to demonstrate that Tetrahymena can be a comparable alternative to zebrafish for determining the virulence of Aeromonas isolates.4.Identification of novel virulence-associated genes using Tetrahymena as a model host by transposon mutagenesis of Aeromonas hydrophilaIn this study,ST251 A.hydrophila strain NJ-35 was used as the parental strain to construct a mutant library consisting of 1030 mutant strains using transposon insertion mutagenesis.Firstly,94 virulence-attenuated mutant strains were identified using Tetrahymena as a model host,then 33 mutant strains which LD50 to zebrafish increased at least one magnitude were identified using zebrafish as a model host,eventually,19 transposon insertion sites were identified.Nine of the insertion sites are located in non-coding regions,the other 12 insertion sites are located in genes encoding the pilus gene cluster transcription regulator,glucose-1-phosphate thymidylyltransferase(RmlA),chitinase,mechanosensitive ion channel protein,potassium transporter(TrkH)and so on.All of the mutant strains showed decreased virulence to Tetrahymena,zebrafish and HEp-2 cells,however,their phenotypic characteristics in other abilities including growth and athletic ability are not consistent.This study identified novel virulence-associated genes in A.hydrophila and contributed to the further study of the bacterial pathogenesis.5.Construction and biological characteristics of trkH-deleted and chiA-deleted Aeromonas hydrophila strainsIn this study gene-deleted strains of chiA and trkH were constructed using homologous recombination technology with ST251 Aeromonas hydrophila NJ-35 as a parental strain and pYAK1 as a suicide plasmid.The corresponding gene-complemented strains were also constructed using pMMB207 as a shuttle plasmid.Once chiA was deleted,the mutant strain showed no difference in growth and motility compared to the wild type strains,however,its virulence to Tetrahymena,zebrafish and HEp2 cells was significantly decreased.Moreover,the ability of chiA-deleted strain to survive in mice was significantly decreased,and the hemolytic activity and protease activity of its extracellular products were slightly decreased.For trkH-deletied strain,its biological characteristics were similar to chiA-deleted strain except for that its virulence to to Tetrahymena,zebrafish and HEp-2 cells decreased more significantly.This study showes that chiA and trkH play important roles in the infection of A.hydrophila,and they may be required to the pathogenicity of the bacteria,however,it still needs further study to clarify their specific roles. |
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