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Pyoverdine-Mediated Killing of Caenorhabditis elegans by Pseudomonas syringae MB03 and the Role of Iron in Its Pathogenicity.

ABSTRACT: The pathogenicity of the common phytopathogenic bacterium Pseudomonas syringae toward Caenorhabditis elegans has been recently demonstrated. However, the major virulence factors involved in this interaction remain unknown. In this study, we investigated the nematocidal activity of P. syringae against C. elegans under iron-sufficient/limited conditions, primarily focusing on the role of the ferric chelator pyoverdine in a P. syringae-C. elegans liquid-based pathogenicity model. Prediction-based analysis of pyoverdine-encoding genes in the genome of the wild-type P. syringae strain MB03 revealed that the genes are located in one large cluster. Two non-ribosomal peptide synthetase genes (pvdD and pvdJ) were disrupted via a Rec/TE recombination system, resulting in mutant strains with abrogated pyoverdine production and attenuated virulence against C. elegans. When used alone, pure pyoverdine also showed nematocidal activity. The role of iron used alone or with pyoverdine was further investigated in mutant and MB03-based bioassays. The results indicated that pyoverdine in P. syringae MB03 is a robust virulence factor that promotes the killing of C. elegans. We speculate that pyoverdine functions as a virulence determinant by capturing environmentally available iron for host bacterial cells, by limiting its availability for C. elegans worms, and by regulating and/or activating other intracellular virulence factors that ultimately kills C. elegans worms.

SUBMITTER: Bashir A

PROVIDER: S-EPMC7139650 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Pyoverdine-Mediated Killing of Caenorhabditis elegans by Pseudomonas syringae MB03 and the Role of Iron in Its Pathogenicity.

Bashir Anum A Tian Tian T Yu Xun X Meng Cui C Ali Muhammad M Li Lin L

International journal of molecular sciences 20200322 6

The pathogenicity of the common phytopathogenic bacterium Pseudomonas syringae toward Caenorhabditis elegans has been recently demonstrated. However, the major virulence factors involved in this interaction remain unknown. In this study, we investigated the nematocidal activity of P. syringae against C. elegans under iron-sufficient/limited conditions, primarily focusing on the role of the ferric chelator pyoverdine in a P. syringae-C. elegans liquid-bas ...[more]

PMID: 32235814

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Project description:Different species of the Pseudomonas genus have been reported for their pathogenic potential against animal cells. However, the pathogenicity of Pseudomonas syringae against Caenorhabditis elegans has never been reported. In this study, the interaction of P. syringae MB03 with C. elegans was studied. Different bioassays such as killing assay, lawn leaving assay, food preference assay, L4 growth assay and newly developed "secretion 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). The lawn leaving assay and the food preference assay illustrated that C. elegans identified P. syringae MB03 as a pathogen when assays were performed on PG medium. Green fluorescent protein was used as the reporter protein to study gut colonization by P. syringae MB03. Our results suggested that MB03 has the ability to colonize the gut of C. elegans. Furthermore, to probe the role of selected virulence determinants, qRT-PCR was used. The 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. On the other hand, kdpD and kdpB were down regulated more than threefold in the NGM - C. elegans interaction model. The deletion of the kdpD and kdpE genes altered the pathogenicity of the bacterial strain against C. elegans. Overall, our results suggested that the killing of C. elegans by P. syringae requires a prolonged interaction between the host and pathogen in an agar-based assay. Moreover, it seemed that some toxic metabolites were secreted by the bacterial strain that were sensed by C. elegans. Previously, it was believed that P. syringae could not damage animal cells. However, this study provides evidence of the pathogenic behavior of P. syringae against C. elegans.

Project description:BackgroundPseudomonas syringae pv. phaseolicola 1448a (P. syringae 1448a), the causative agent of bean halo blight, is a bacterium capable of occupying diverse biological niches. Under conditions of iron starvation P. syringae 1448a secretes siderophores for active uptake of iron. The primary siderophore of P. syringae 1448a is pyoverdine, a fluorescent molecule that is assembled from amino acid precursors by non-ribosomal peptide synthetase (NRPS) enzymes. Whereas other species of Pseudomonas often exhibit structural variations in the pyoverdine produced by different strains, all P. syringae pathovars previously tested have been found to make an identical pyoverdine molecule. P. syringae 1448a also appears to have the genetic potential to make two secondary siderophores, achromobactin and yersiniabactin, each of which has previously been detected in different P. syringae pathovars.ResultsFive putative pyoverdine NRPS genes in P. syringae 1448a were characterized in-silico and their role in pyoverdine biosynthesis was confirmed by gene knockout. Pyoverdine was purified from P. syringae 1448a and analyzed by MALDI-TOF and MS/MS spectroscopy. Peaks were detected corresponding to the expected sizes for the pyoverdine structure previously found in other P. syringae pathovars, but surprisingly P. syringae 1448a appears to also produce a variant pyoverdine species that has an additional 71 Da monomer incorporated into the peptide side chain. Creation of pyoverdine null mutants of P. syringae 1448a revealed that this strain also produces achromobactin as a temperature-regulated secondary siderophore, but does not appear to make yersiniabactin. Pyoverdine and achromobactin null mutants were characterized in regard to siderophore production, iron uptake, virulence and growth in iron limited conditions.ConclusionsThis study provides the first evidence of a P. syringae pathovar producing a side chain variant form of pyoverdine. We also describe novel IC₅₀ and liquid CAS assays to quantify the contribution of different siderophores across a range of iron starvation conditions, and show that although achromobactin has potential to contribute to fitness its contribution is masked by the presence of pyoverdine, which is a significantly more effective siderophore. Neither pyoverdine nor achromobactin appear to be required for P. syringae 1448a to cause bean halo blight, indicating that these siderophores are not promising targets for crop protection strategies.

Project description:Pseudomonas donghuensis HYS is more virulent than P. aeruginosa toward Caenorhabditis elegans but the mechanism underlying virulence is unclear. This study is the first to report that the specific gene cluster gtrA/B/II in P. donghuensis HYS is involved in the virulence of this strain toward C. elegans, and there are no reports of GtrA, GtrB and GtrII in any Pseudomonas species. The pathogenicity of P. donghuensis HYS was evaluated using C. elegans as a host. Based on the prediction of virulence factors and comparative genomic analysis of P. donghuensis HYS, we identified 42 specific virulence genes in P. donghuensis HYS. Slow-killing assays of these genes showed that the gtrAB mutation had the greatest effect on the virulence of P. donghuensis HYS, and GtrA, GtrB and GtrII all positively affected P. donghuensis HYS virulence. Two critical GtrII residues (Glu47 and Lys480) were identified in P. donghuensis HYS. Transmission electron microscopy (TEM) showed that GtrA, GtrB and GtrII were involved in the glucosylation of lipopolysaccharide (LPS) O-antigen in P. donghuensis HYS. Furthermore, colony-forming unit (CFU) assays showed that GtrA, GtrB and GtrII significantly enhanced P. donghuensis HYS colonization in the gut of C. elegans, and glucosylation of LPS O-antigen and colonization in the host intestine contributed to the pathogenicity of P. donghuensis HYS. In addition, experiments using the worm mutants ZD101, KU4 and KU25 revealed a correlation between P. donghuensis HYS virulence and the TIR-1/SEK-1/PMK-1 pathways of the innate immune p38 MAPK pathway in C. elegans. In conclusion, these results reveal that the specific virulence gene cluster gtrA/B/II contributes to the unique pathogenicity of HYS compared with other pathogenic Pseudomonas, and that this process also involves C. elegans innate immunity. These findings significantly increase the available information about GtrA/GtrB/GtrII-based virulence mechanisms in the genus Pseudomonas.

Dataset Information

Pyoverdine-Mediated Killing of Caenorhabditis elegans by Pseudomonas syringae MB03 and the Role of Iron in Its Pathogenicity.

Publications

Pyoverdine-Mediated Killing of <i>Caenorhabditis elegans</i> by <i>Pseudomonas syringae</i> MB03 and the Role of Iron in Its Pathogenicity.

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