An unusual Toll/MyD88-mediated Drosophila host defence against Talaromyces marneffei

Abstract

Talaromycosis, caused by Talaromyces marneffei (T. marneffei, formerly known as Penicillium marneffei), is an opportunistic invasive mycosis endemic in tropical and subtropical areas of Asia with high mortality rate. Despite various infection models established to study the immunological interaction between T. marneffei and the host, the pathogenicity of this fungus is not yet fully understood. So far, Drosophila melanogaster, a well-established genetic model organism to study innate immunity, has not been used in related research on T. marneffei. In this study, we provide the initial characterization of a systemic infection model of T. marneffei in the D. melanogaster host. Survival curves and fungal loads were tested as well as Toll pathway activation was quantified by RT-qPCR of several antimicrobial peptide (AMP) genes including Drosomycin, Metchnikowin, and Bomanin Short 1. We discovered that whereas most wild-type flies were able to overcome the infection, MyD88 or Toll mutant flies failed to prevent fungal dissemination and proliferation and ultimately succumbed to this challenge. Unexpectedly, the induction of classical Toll pathway activation readouts, Drosomycin and Bomanin Short 1, by live or killed T. marneffei was quite limited in wild-type flies, suggesting that the fungus largely escapes detection by the systemic immune system. This unusual situation of a poor systemic activation of the Toll pathway and a strong susceptibility phenotype of MyD88/Toll might be accounted for by a requirement for this host defence in only specific tissues, a hypothesis that remains to be rigorously tested.

Keywords: Drosophila melanogaster infection model; Toll pathway activation; detection of fungal infections; resistance; talaromycosis.

Figure 1.

Susceptibility of MyD88 mutant flies to talaromyces marneffei infection.(a) injection model and natural infection model of talaromyces marneffei in w [A5001] flies and MyD88 mutant flies at the dose of 100 conidia/fly and 10⁶ conidia/mL (5 mL for each tube of flies), respectively. (b) Standard food, food without potassium sorbate and food with gentamicin were used in the injection model at the dose of 100 conidia/fly. (c) Dose–response curves of injected MyD88 mutant flies fed on the food without potassium sorbate. The quantity of flies in total is indicated to the right of the group.

Figure 2.

Susceptibility of Toll, key, and ΔAMP14 mutant flies to talaromyces marneffei infection.(a) survival of Toll mutant flies injected with T. marneffei at the dose of 100 conidia/fly. (b) Survival of key mutant flies injected with T. marneffei at the dose of 100 conidia/fly. (c) Survival of ΔAMP14 mutant flies injected with T. marneffei at the dose of 100 conidia/fly. The data correspond to pooled data from at least three independent experiments. N, times of independent experiments; Tm, talaromyces marneffei infection. ****, P<0.0001; ns, no significance, P>0.05.

Figure 3.

Proliferation and dissemination of Talaromyces marneffei in MyD88 flies.(a) fungal loads of w[A5001] flies and MyD88 mutant flies on 3-5 days post infection (dpi). (b) Fungal load of w [A5001] flies on 14 dpi and fungal load upon death (FLUD) of MyD88 mutant flies. (c) Different appearances of MyD88 mutant flies’ carcasses under the stereomicroscope at 80 × magnification. (d) Fungal loads of different tagmata (head, thorax, abdomen) of dying MyD88 mutant flies. (b, d) red data points correspond to dead flies that present a reddish color, as shown in (c). Flies were infected at the dose of 100 conidia/fly. The data correspond to pooled data from several independent experiments (results from each single experiment are represented by a specific symbol shape (circles, triangles, squares, and diamonds)) and described by Median with interquartile range for they were non-normally distributed data. N, times of independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, no significance, P > 0.05.

Figure 4.

Talaromyces marneffei is a poor elicitor of the toll pathway.(a) steady-state transcript levels of Drosomycin, BomS1 and Metchnikowin on 1-3 dpi, 7dpi, and 14dpi flies as measured by RTqPCR in wild-type w [A5001] flies. (b) Steady-state transcript levels of Drosomycin, BomS1 and Metchnikowin on 1-3 dpi, 7dpi, and 14dpi flies as measured by RTqPCR in MyD88 mutant flies. (c) Steady-state transcript levels of Drosomycin, BomS1 and Metchnikowin in w [A5001] flies infected with T. marneffei (live conidia, unless otherwise indicated: UV- or heat-killed) and/or Micrococcus luteus (OD₆₀₀ = 50, 4.6 nL). In the case of double infections, flies were first challenged with T. marneffei and then secondarily after 0 to 3 days (tm xdpi) as indicated with M. luteus (the time of analysis was one day after M. luteus challenge). (A-C) flies were infected at the dose of 100 conidia/fly.