The Pathogenicity of Pseudomonas syringae MB03 against Caenorhabditis elegans and the Transcriptional Response of Nematicidal Genes upon Different Nutritional Conditions

Abstract

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.

Keywords: Caenorhabditis elegans; Pseudomonas syringae MB03; gut colonization; pathogenicity; transcriptional response.

FIGURE 1

Bioassays of Pseudomonas syringae MB03 against Caenorhabditis elegans. (A) Killing of C. elegans by P. syringae MB03. Assay was conducted on four different media (PG, BHI, King’s B, and NGM). Fastest killing was observed on PG medium. (B) Shows lawn leaving behavior of C. elegans. P. syringae MB03 was grown on the PG, BHI, king’s B, and NGM media as bacterial lawn. L4 synchronized worms (40–50) were placed approximately at the center of bacterial lawn. Fraction of worms which was out of lawn was determined after regular time intervals. (C) Food preference assay shows choice index of C. elegans for P. syringae MB03 (-1 represents complete avoidance, 0 represents equal preference, and +1 represent complete preference). On PG medium, worms strictly avoided P. syringae MB03 whereas on NGM, worms did not avoid P. syringae MB03. (D) Growth of L4 worms on different media compared to control. L4 synchronized worms were placed on bacterial lawns and area of worms was determined. Worms grown on E. coli OP50 on same medium were used as control and normalized size of treated worm was determined by dividing treated worm area to worm grown on E. coli OP50. Statistical analysis was done by applying one-way ANOVA where * represents significant difference at p-value <0.05.

FIGURE 2

Response of C. elegans to the secretion of P. syringae MB03, ΔkdpD, and ΔkdpE. Newly developed secretion assay was performed to observe toxicity avoidance behavior of worms. Assay was performed on PG medium. (A) Shows choice index of worms for ‘test lawn’ (E. coli OP50 bacterial lawn made on secretion spot of test strains). P. aeruginosa PAF was used as control. (B) Schematic representation of secretion assay.

FIGURE 3

Colonization of gut of C. elegans by P. syringae MB03, ΔkdpD, and ΔkdpE. Worms were fed on bacterial strain harboring GFP expressing vector. At regular intervals worms were repeatedly washed with M9 buffer and examined under fluorescence microscope. Upper panel represents differential interference contrast image and lower panel represents florescence image of worm grown on P. syringae MB519 and P. syringae DK519 (ΔkdpD), P. syringae EK519 (ΔkdpE) on PG medium.

FIGURE 4

Transcriptional variation of candidate virulence genes of P. syringae MB03 during host–pathogen interaction. P. syringae MB03 was grown on PG, BHI, King’s B, and NGM media and then, L4 worms were added on bacterial lawns. After 24 h, total RNA was extracted for qRT-PCR analysis. P. syringae MB03 grown in the absence of worms was used as control sample for relative quantification of gene expression. Genes for 16S rRNA and recA were used as indigenous control. Statistical analysis was done by applying one-way ANOVA.

FIGURE 5

Evaluation of pathogenicity of P. syringae MB03 and its mutants (ΔkdpD and ΔkdpE). (A) For killing assay, all the test strains were grown over PG medium for 24 h and (40–50) L4 worms were added on bacterial lawn. Dead and live worms were determined after every 24 h. (B) Lawn leaving assay was performed on PG medium. Fraction of L4 worms out of the lawn was determined after every 5 h. (C) Food preference assay was conducted on PG medium. Fraction of worms on OP50 lawn and test strain lawn was determined after 15 h. (D) Growth assay was performed to determine effect of pathogenicity of test strain on the size of worms. Worms fed on E. coli OP50 were used as control to normalize size of treated worms. Statistical analysis was done by applying one-way ANOVA where * represents significant difference at p-value <0.05.

FIGURE 6

Percent worms colonized by P. syringae MB519, P. syringae DK519, and P. syringae EK519. GFP was expressed in P. syringae and its mutants. Strains were grown on PG medium and 80–100 L4 synchronized worms were fed on bacterial strains. Number of GFP expressing worms was determined under fluorescence microscope at regular intervals. Statistical analysis was done by applying one-way ANOVA where * represents significant difference at p-value <0.05 and ‘n.s.’ represent not significant results.