Comparative Genomics Of Pseudomonas Syringae MB03 And Insights Into Its Pathogenicity Mechanism Against Caenorhabditis Elegans

Pseudomonas syringae is a Gram-negative bacterium which belongs to the Phylum Gammaproteobacteria.Although it is well studied for its infection against plants however,its pathogenic interaction with animal host such as Caenorhabditis elegans has not been yet investigated such far,and it was conventionally considered as a harmless microorganism to animal.Previously,a P.syringae strain MB03 was isolated and identified from a frost-plant-tissue sample in this laboratory,which showed prominently high ice-nucleating activity over other isolates and the ability to cause frost damage and infection against host plants.Current study was designed to evaluate pathogenic potential of this strain against the model organism C.elegans.For this purpose,the genome of P.syringae MB03 was sequenced twice,and the comparative genomics was performed.The size of its current version of genome was identified to be 5.77 Mb which encoded 5029 genes.On the basis of multiple locus sequence analysis and mean amino acid identity matrix,P.syringae MB03 was found to be closest to P.syringae subsp.syringae B64,P.syringae subsp.syringae SM and P.syringae subsp.syringae HS191.In terms of gene content,comparison of P.syringae MB03 with P.syringae pv.syringae HS191,P.syringae pv.syringae B64 and P.syringae pv.syringae SM resulted in 377,386 and 397 strain specific genes of P.syringae MB03,respectively.Comparison with six P.syringae strains used as reference strains,revealed 159 strain specific genes which could be considered as P.syringae MB03 unique genes.In contrast to reference strains,comparative genomics revealed important virulence mechanisms including 10 type ?secretion system(T3SS)effector proteins,two clusters of type six secretion system(T6SS)and mango toxin cluster.Moreover,some unique genomic islands were also observed in the genome of P.syringae MB03 which contained some potential virulence factors.For further investigation of potential nematicidal genes,the genome of P.syringae MB03 was compared with P.aeruginosa PAO1 and PA14.Homo logs of 115 nematicidal genes of P.aeruginosa strains PA14 and PAO1 were found in P.syringae MB03.Different bioassays such as killing assay,lawn leaving assay,food preference assay and L4 growth assay were performed to evaluate the pathogenic potential of P.syringae on different growth media.The results of the killing assay showed that P.syringae MB03 was able to kill C.elegans under specific conditions,as the interaction between the host and the pathogen varied from non-pathogenic(assay on NGM medium)to pathogenic(assay on PG medium).Red fluorescent protein was used as a marker to study gut colonization and it was found that P.syringae MB03 had the ability to colonize gut of C.elegans when assay was performed on PG medium.To investigate the response of candidate nematicidal genes of P.syringae MB03,real time quantitative polymerase chain reaction(RT-qPCR)was performed on 41 selected genes.Results showed that genes for pyoverdine,phoQ/phoP,phoR/phoB and flagella were up regulated during the interaction of P.syringae MB03 and C.elegans on PG medium.Other than these,the genes for some proteases,such as pepP,clpA and clpS,were also up regulated while kdpD and kdpB were down regulated more than 3-fold on the NGM-C.elegans interaction model.To further elucidate the role of selected genes,phoB,phoP clpA,kdpD and kdpE were knocked out and the results showed attenuated virulence in the case of ?AclpA,?kdpD and ?phoP.These mutants were also retarded in their ability to colonize gut of worms.On contrary,?phoB showed defect in the metabolite secretion and showed a deceitful behavior to worms as worms failed to determine this mutant as pathogenic strain.Overall,our results suggested that the killing of C.elegans by P.syringae requires a prolonged interaction between the host and pathogen on an agar-based assay.Moreover,some toxic metabolites were secreted by the bacterial strain that were sensed by C.elegans.Pathogenicity of P.syringae MB03 appeared as a combinatorial action of secreted metabolite and gut colonization.Previously,it was believed that P.syringae could not damage animal cells.However,for the very first time,this study provided evidence of the pathogenic behavior of P.syringae against C.elegans.Our study would change the general perception about P.syringae as harmless species to animal models.