Effect of allyl-isothiocyanate on survival and antimicrobial peptide expression following oral bacterial infections in Drosophila melanogaster

Abstract

Since infections with antibiotic-resistant bacteria cause increasing problems worldwide, the identification of alternative therapies is of great importance. Plant-derived bioactives, including allyl-isothiocyanate (AITC), have received attention for their antimicrobial properties. The present study therefore investigates the impact of AITC on survival and antimicrobial peptide (AMP) levels in Drosophila melanogaster challenged with the fly pathogenic bacteria Pectobacterium carotovorum subsp. carotovorum and Leuconostoc pseudomesenteroides. AITC, a sulfur-containing compound derived from glucosinolates, exhibits antimicrobial properties and has been suggested to modulate AMP expression. By using D. melanogaster, we demonstrate that AITC treatment resulted in a concentration-dependent decrease of survival rates among female flies, particularly in the presence of the Gram-negative bacterium Pectobacterium carotovorum subsp. carotovorum, whereas AITC did not affect survival in male flies. Despite the ability of isothiocyanates to induce AMP expression in cell culture, we did not detect significant changes in AMP mRNA levels in infected flies exposed to AITC. Our findings suggest sex-specific differences in response to AITC treatment and bacterial infections, underlining the complexity of host-pathogen interactions and potential limitations of AITC as a preventive or therapeutic compound at least in D. melanogaster models of bacterial infections.

Keywords: Drosophila melanogaster; allyl-isothiocyanate; anti-microbial peptides; bioactive plant compound; gene expression; infection; survival.

Figure 1

Experimental procedure of survival analysis. (A) D. melanogaster were initially exposed to a 100-mM sucrose solution with or without L. pseudomesenteroides or P. carotovorum subsp. carotovorum for 18 h and were subsequently transferred on either a control or an AITC-supplemented diet (0.125 mM or 0.250 mM) for the rest of their lifetime. (B) D. melanogaster were initially kept on either a control or an AITC-supplemented diet (0.125 mM or 0.250 mM) for 10 days, followed by an exposure to a 100-mM sucrose solution with or without L. pseudomesenteroides or P. carotovorum subsp. carotovorum for 18 h and were subsequently transferred on a control diet for the rest of their lifetime.

Figure 2

Experimental procedure of generating fly samples for subsequent mRNA analysis. (A) D. melanogaster were initially exposed to a 100-mM sucrose solution with P. carotovorum subsp. carotovorum or L. pseudomesenteroides for 18 h. (B) D. melanogaster were fed either a control diet or an AITC-supplemented diet (0.125 mM or 0.250 mM) for 10 days. (C) D. melanogaster were initially kept on either a control or an AITC-supplemented diet (0.125 mM or 0.250 mM) for 10 days and were subsequently transferred to a 100-mM sucrose solution with or without L. pseudomesenteroides or P. carotovorum subsp. carotovorum for 18 h. .

Figure 3

Relative food intake of female (A) and male (B) D. melanogaster fed either a control diet or an AITC-supplemented diet (0.125 mM or 0.250 mM), respectively, for 10 days. Bars show the mean ± SD of three independent experiments. Significant differences between groups were tested by applying one-way ANOVA followed by Dunnett’s multiple-comparison test. Significance was accepted at p < 0.05. *p < 0.05.

Figure 4

Survival curves and median survival time of female (B, D) and male (C, E) D. melanogaster initially exposed to a control solution (W/O Infection) (B, C) or a solution with L. pseudomesenteroides (LP) or P. carotovorum subsp. carotovorum (ECC) (D, E) for 18 h and subsequently transferred on either a control or an AITC-supplemented diet (0.125 mM or 0.250 mM) for the rest of their lifetime. The experiment procedure is shown in (A). Results show the three independent experiments. Significant differences between treatments were tested by the Kaplan–Meier approach and a log-rank test. Significance was accepted at p < 0.05. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 5

Survival curves and median survival time of female (B, D) and male (C, E) D. melanogaster initially exposed to a solution with L. pseudomesenteroides (LP) (B, C) or P. carotovorum subsp. carotovorum (ECC) (D, E) for 18 h and subsequently transferred on either a control or an AITC-supplemented diet (0.125 mM or 0.250 mM) for the rest of their lifetime. The experiment procedure is shown in (A). Results show three independent experiments. Significant differences between treatments were tested by the Kaplan–Meier approach and a log-rank test. Significance was accepted at p < 0.05. *p < 0.05, ****p < 0.0001.

Figure 6

Survival curves and median survival time of female (B, D) and male (C, E) D. melanogaster initially kept on either a control or an AITC-supplemented diet (0.125 mM or 0.250 mM) for 10 days then exposed to a control solution (W/O infection) (B, C) or a solution with L. pseudomesenteroides (LP) or P. carotovorum subsp. carotovorum (ECC) (D, E) for 18 h and subsequently transferred on a control diet for the rest of their lifetime. The experiment procedure is shown in (A). Results show two independent experiments. Significant differences between treatments were tested by the Kaplan–Meier approach and a log-rank test. Significance was accepted at p < 0.05. **p < 0.01.

Figure 7

Survival curves and median survival time of female (B, D) and male (C, E) D. melanogaster initially kept either on a control or an AITC-supplemented diet (0.125 mM or 0.250 mM) for 10 days then exposed to a solution with L. pseudomesenteroides (LP) (B, C) or P. carotovorum subsp. carotovorum (ECC) (D, E) for 18 h and subsequently transferred on a control diet for the rest of their lifetime. The experiment procedure is shown in (A). Results show two independent experiments. Significant differences between treatments were tested by the Kaplan–Meier approach and a log-rank test. Significance was accepted at p < 0.05. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 8

Relative mRNA expression levels of dro in female (A, C) and male (B, D) D. melanogaster exposed to a 100-mM sucrose solution with P. carotovorum subsp. carotovorum (ECC) or L. pseudomesenteroides (LP) for 18 h (A, B) and D. melanogaster fed either a control diet or an AITC-supplemented diet (0.125 mM or 0.250 mM) for 10 days (C, D). mRNA levels were determined in fly samples from three independent experiments with three replicates containing 10 flies each. Bars show the mean ± SD. Significant differences between groups were tested by applying one-way ANOVA followed by Dunnett’s multiple comparison test. Significance was accepted at p < 0.05.

Figure 9

Relative mRNA expression levels of mtk in female (A, C) and male (B, D) D. melanogaster exposed to a 100-mM sucrose solution with P. carotovorum subsp. carotovorum (ECC) or L. pseudomesenteroides (LP) for 18 h (A, B) and D. melanogaster fed either a control diet or an AITC-supplemented diet (0.125 mM or 0.250 mM) for 10 days (C, D). mRNA levels were determined in fly samples from three independent experiments with three replicates containing 10 flies each. Bars show the mean ± SD. Significant differences between groups were tested by applying one-way ANOVA followed by Dunnett’s multiple-comparison test. Significance was accepted at p<0.05.

Figure 10

Relative mRNA expression levels of dro in female (A, C, E, G) and male (B, D, F, H) D. melanogaster initially kept on either a control or an AITC-supplemented diet (0.125 mM or 0.250 mM) for 10 days and subsequently exposed to a 100-mM sucrose solution with or without L. pseudomesenteroides (LP) or P. carotovorum subsp. carotovorum (ECC) for 18 h. mRNA levels were determined in fly samples from three independent experiments with three replicates containing 10 flies each. Bars show the mean ± SD. Significant differences between groups were tested by applying one-way ANOVA followed by Dunnett’s multiple-comparison test. Significance was accepted at p < 0.05.

Figure 11

Relative mRNA expression levels of mtk in female (A, C, E, G) and male (B, D, F, H) D. melanogaster initially kept on either a control or an AITC-supplemented diet (0.125 mM or 0.250 mM) for 10 days and subsequently exposed to a 100-mM sucrose solution with or without L. pseudomesenteroides (LP) or P. carotovorum subsp. carotovorum (ECC) for 18 h. mRNA levels were determined in fly samples from three independent experiments with three replicates containing 10 flies each. Bars show the mean ± SD. Significant differences between groups were tested by applying one-way ANOVA followed by Dunnett’s multiple comparison test. Significance was accepted at p < 0.05. *p < 0.05.