Pyoverdine Mediated Killing Of Caenorhabditis Elegans By Pseudomonas Syringae And Role Of Iron In Its Pathogenicity

Pseudomonas syringae is a Gram-negative bacterium which is well studied for its pathogenicity against plants,however,its pathogenic interaction with animal host such as Caenorhabditis elegans,has been recently confirmed.Iron is one of the most abundant elements on natural environment,but its bioavailability is comparatively low.A variety of microorganisms have developed various iron uptake systems with high affinity capacity.These include heme uptake system,siderophores and the corresponding membrane receptors.Primary siderophore of Pseudomonas species is pyoverdine.It is a low molecular weight,high affinity iron scavenging fluorescent molecule that is produced by fluorescent pseudomonads only under iron-limiting conditions.Besides iron scavenging property,pyoverdine also serves as bacterial toxin.In this dissertation,the interaction between P.syringae MB03 and C.elegans under iron limiting conditions in a liquid based pathogenicity model,and the role of pyoverdine were investigated.Pyoverdine genetic cluster of MB03 was initially predicted using Anti-SMASH.Pseudomonas aeruginosa PAO1 pyoverdine genetic locus was used as a blue print for annotation of pyoverdine genetic locus of MB03,since almost all of the genes required for the biosynthesis and secretion of pyoverdine has been identified in PAO1.In comparisons with the pyoverdine genetic cluster of PAO1,it showed that P.syringae MB03 lack five orthologs(pvd X,pvd Y,pvd A,pvd F and fpv I).Genes pvd X and pvd Y are predicted to encode the regulatory corresponding proteins,whereas pvd A and pvd F are predicted to encode the enzymes required for the synthesis of L-ornithine hydroxylase and N5-formyl-N5 hydroxyornithine residues respectively,which are present in the peptide chain of pyoverdine produced by P.aeruginosa PAO1.Instead of these enzymes,a gene(namely syr P)is present in the pyoverdine cluster of P.syringae MB03 which is hydroxyasparate enzyme.It is proposed that this enzyme is involved in the ?-hydroxylation of hydroxyasparate residues that are present in the peptide chain of MB03-PVD.Additionally,in PAO1,two sigma factors pvd S and fpv I are involved in the activation of pyoverdine biosynthetic genes and pyoverdine receptors fpv A,respectively,however,fpv I is absent in the genome of P.syringae MB03,instead a pvd S IS box is found in the front of first fpv A gene,suggesting that pvd S could be involved in the transcription of fpv A genes.Pyoverdine consists of three structural domains,i.e.chromophore(most conserved part),peptide chain(most variable part),and side chain(carboxylic acid derivatives).Non-ribosomal peptide synthetases(NRPS)are big enzymes which are present in the pyoverdine genetic cluster of MB03 and are involved in the biosynthesis of peptide chain and chromophore part of pyoverdine.In silico characterization of five putative pyoverdine NRPS genes of P.syringae MB03 by NRPS predictor revealed that peptide chain of MB03-PVD is in linear form and consists of L-lys,D-Asp,L-Thr,L-Thr,L-Ser,D-Asp,and L-Ser.The production of pyoverdine by the MB03 is inversely related to the Fe3+ concentration in the medium.The spectrofluorophotometric data support this hypothesis and revealed that critical inhibitory concentration of iron for pyoverdine production is about 20 μmol/L.Certain genetic,biochemical and spectrofluorophotometric analyses of pyoverdine biosynthetic genes were performed to confirm their role in pyoverdine production.Two pyoverdine biosynthetic genes were disrupted by using Rec TE recombination system.As pyoverdine is a secreted non-ribosomal polypeptide,so the cell free filtrates from MB03,?pvd D and ?pvd J were analyzed by LC-MS and spectrofluorophotometer.Both the mutants lack the characteristic yellowish-greenish color,indicated that the mutants failed to produce pyoverdine.In contrast to the lacking of the characteristics peaks corresponding to pyoverdine in mass spectra of both mutants,the wild-type MB03 produced two types of pyoverdine with 1123.412 m/z and 1142.4165 m/z.To explore the role of iron in the pathogenicity of MB03 against C.elegans,MB03 and Escherichia coli OP50(C.elegans)were grown in M9 medium(that enhances the production of pyoverdine)with or without iron supplementation.Pyoverdine also endorses the production of several virulence factors in P.syringae,such as AHL,EPS and Tabtoxin.Therefore,it is hypothesized that iron supplementation during overnight growth reduce the production of these virulent determinants in that way decreasing killing.To test this hypothesis,we split a filtrate produced from an overnight MB03 culture and incubated half with 100 μmol/L of iron overnight.MB03-filtrate showed significant killing(p=0)that was attenuated by addition of exogenous iron(100 μmol/L)during growth of MB03 in M9 or by overnight incubation of MB03 filtrate with exogenous iron.We have also found that killing of worms starts after 30 h,by these results we conclude that time is necessary for lethal interaction to be established.For liquid killing(LK)assay,MB03,?pvd D and ?pvd J were grown in iron-poor M9 medium.It is hypothesized that pyoverdine is the active factor in the filtrate of MB03 causing worms death.To support this hypothesis,liquid killing assays of purified pyoverdine were performed in parallel.The concentration of purified pyoverdine used in LK assay was comparable with the concentration of pyoverdine produced by MB03 in M9 medium in 48 h.Relative death rate of worms exposed to various filtrates was measured.As expected,MB03 filtrate showed significant killing(p=0)as compared to M9 and OP50 filtrate.The role of pyoverdine in LK assay was confirmed by attenuated virulence of both mutants,as both mutants showed little death of worms.To confirm the attenuated virulence of mutants was due to deficient in production of pyoverdine,the purified pyoverdine was added in the mutants to measure the relative death rate which was comparable with wild type.Liquid killing Assay implies that pyoverdine is the active factor in the filtrates causing worms death.By these results we conclude that pyoverdine is very important in MB03-mediated LK,which is confirmed by the attenuated virulence of mutants defective in pyoverdine biosynthesis and comparable killing of both mutants by addition of purified pyoverdine.The role of pyoverdine in the normal growth of P.syringae MB03 was investigated by measuring the growth curve of MB03 and both the mutants.The spectrometric data disclosed that both mutants showed retarted growth in iron-poor M9 medium.Thereby,it is suggesting that pyoverdine is vital for P.syringae MB03,as iron acquisition is typically required for normal growth of bacteria.In conclusion,the results of this study suggested that P.syringae MB03 has genetic potential to produce pyoverdine.Gene knock out by Rec TE recombination system showed the importance of pyoverdine biosynthetic genes in pyoverdine production,normal bacterial growth and pathogenicity against C.elegans.P.syringae requires prolonged interaction between the host and pathogen in liquid based pathogenicity model.To the best of our knowledge,this was the first study which revealed the role of pyoverdine in Pseudomonas syringae–C.elegans liquid based pathogenicity model.