Insecticidal Toxicity of Yersinia frederiksenii Involves the Novel Enterotoxin YacT

Abstract

The genus Yersinia comprises 19 species of which three are known as human and animal pathogens. Some species display toxicity toward invertebrates using the so-called toxin complex (TC) and/or determinants that are not yet known. Recent studies showed a remarkable variability of insecticidal activities when representatives of different Yersinia species (spp.) were subcutaneously injected into the greater wax moth, Galleria mellonella. Here, we demonstrate that Y. intermedia and Y. frederiksenii are highly toxic to this insect. A member of Y. Enterocolitica phylogroup 1B killed G. mellonella larvae with injection doses of approximately 38 cells only, thus resembling the insecticidal activity of Photorhabdus luminescens. The pathogenicity Yersinia spp. displays toward the larvae was higher at 15°C than at 30°C and independent of the TC. However, upon subtraction of all genes of the low-pathogenic Y. enterocolitica strain W22703 from the genomes of Y. intermedia and Y. frederiksenii, we identified a set of genes that may be responsible for the toxicity of these two species. Indeed, a mutant of Y. frederiksenii lacking yacT, a gene that encodes a protein similar to the heat-stable cytotonic enterotoxin (Ast) of Aeromonas hydrophila, exhibited a reduced pathogenicity toward G. mellonella larvae and altered the morphology of hemocytes. The data suggests that the repertoire of virulence determinants present in environmental Yersinia species remains to be elucidated.

Keywords: Galleria mellonella; YacT; Yersinia; enterotoxin; insecticidal activity.

Figure 1

Time course of G. mellonella infection assays with Y. frederiksenii and Y. intermedia. The strains were pregrown overnight at 30°C, and the cultures were serially diluted. Aliquots of 5–7.5 μl from a 10⁴-fold or a 10⁵-fold dilution were used for infection, corresponding to (A) 1.44–5.50 × 10³ Y. intermedia CFU and 1.43 × 10³–10⁴ Y. frederiksenii CFU, or (B) 120 Y. intermedia CFU, 95 Y. frederiksenii CFU, and 145 Y. enterocolitica W22703 CFU. Three independent experiments per strain were performed, with three groups composed of (A) 25, 29, and 30 larvae (Y. intermedia) and 20, 22, and 23 larvae (Y. frederiksenii), or (B) 15 larvae each. The larvae were incubated at 20°C and monitored daily. Error bars represent the standard error of the mean of three experiments.

Figure 2

Dose-dependent toxicity of Yersinia strains. Survival assays were performed by infection of G. mellonella larvae with P. luminescens, Y. frederiksenii, Y. intermedia, Y. enterocolitica 8081, Y. enterocolitica W22703, and E. coli DH5α. Infected larvae were incubated at 15°C; few experiments (^*) were performed at 20°C. The CFU used for the injection, the number of larvae, and the error bars (mean of experiments with three groups of larvae) are indicated. Groups of 15 larvae each were independently infected.

Figure 3

Temperature-dependent pathogenicity of Yersinia strains against G. mellonella larvae. (A) Larvae were each infected with eight and 95 Y. frederiksenii CFU, respectively, and incubated at 15°C and 30°C; the experiment at 15°C with an infection dose of eight was performed with 30 ungrouped larvae. (B) Infection was done with 120 Y. intermedia CFU and the larvae incubated at 15°C and 30°C. Infection with E. coli DH5α was used here as a control for all experiments. (C) 57 Y. enterocolitica 8081 CFU were used to infect G. mellonella larvae, which were incubated at 15°C; in a further assay, larvae infected with 40 CFU were incubated at 30°C; the experiment with an infection dose of 57 CFU was done with 3 × 10 larvae. (D) Infection assays were performed with 9 × 10⁵ or with 145 Y. enterocolitica W22703 CFU at 15°C and 30°C. In all experiments, three groups of 15 larvae each were independently infected with the exceptions mentioned earlier. Error bars represent the standard deviations. The larvae survival rate was plotted against day's p. i.

Figure 4

Phenotypes of G. mellonella larvae after infection. (A) Larvae were infected with 95 Y. frederiksenii CFU, incubated at 15°C and 30°C, and monitored until day 6 p. i.; larvae injected with 5 μl of LB medium served as control. (B) Infection was done with Y. frederiksenii (5 μl of a 10³ dilution of an overnight culture), with Y. intermedia (5 μl of a 10⁴ dilution), and with P. luminescens (5 μl of a 10⁴ dilution). Photographs were taken 3 days p. i. The larvae were incubated at room temperature (R.T.).

Figure 5

Y. frederiksenii ΔyacT exhibits attenuated virulence. (A) Y. frederiksenii/pACYC184, Y. frederiksenii ΔyacT, Y. frederiksenii ΔyacT/pACYC184, and Y. frederiksenii ΔyacT /pACYC-yacT were used to infect G. mellonella larvae that were incubated for 10 days at 15°C. Infection doses and TD₅₀ values are indicated in Table 3. In all experiments depicted in the Kaplan–Meier plot, three independent infection experiments per strain were monitored, with groups composed of 15 larvae each. (B) Additionally, larvae infected in parallel were homogenized at the indicated time points and the number of viable Y. frederiksenii cells were enumerated. Gray boxes: Y. frederiksenii, black boxes: Y. frederiksenii ΔyacT. Standard deviations of three replicates are shown.

Figure 6

Effect of purified YacT of Y. frederiksenii on hemocytes. (A) Six Microliter of purified YacT or of PBS (control) were injected. After 1 day, hemolymph preparations of G. mellonella larvae were fixed with methanol (upper row) or with aceton (lower row) and then stained by Giemsa solution. The hemocytes derived from YacT-treated larvae began to form some aggregates in comparison with controls (lower row). (B) Hemolymph preparations of larvae 1 day after oral infection with 6 μl of an overnight culture of Y. frederiksenii and Y. frederiksenii ΔyacT. Arrows point to changes of hemocyte morphology and chromatin density, the latter one as visible by the weaker nuclear staining. Photos of representative preparations are shown; the scale is indicated. Microscope Olympus BX53 was used with 600 × magnification.