The in vitro Production Potentialities of Secondary Toxic Metabolites by the Fungal Factory Fusarium verticillioides Is, Fortunately, Largely Underestimated in Fields: Pioneering Study on Fumonisins

Abstract

This study presents fungi infrequently viewed as fungal factories for secondary metabolite production resources such as mycotoxins in Ascomycota. Additionally, we demonstrated that biochemical warfare of Fusarium verticillioides factory against animal cells is not only due to mycotoxins such as fumonisins, but acute cytotoxic firing is based on different excreted secondary metabolite series, potentially leading to animal and human diseases. In this study, fumonisins, which can be followed by in situ localization, quantification, or expression of the key gene implicated in their synthesis, are used to understand secondary metabolite production by this fungus. It is known that F. verticillioides produces mycotoxins such as fumonisins on cereals, but until now, there is no evidence demonstrating a method to totally block fumonisin production on feed and food. In this paper, we explained, what was never clearly established before, that fumonisin production depends on two bottlenecks. The fumonisin synthesis and secretion in fungal articles of the mycelium are medium-independent and follow the fungal cell cycle developmental program (ontogenesis). Conversely, the fumonisin excretion into the medium depends on its composition, which also impacts fumonisin biosynthesis level. Using a high-pressure freezing method, we showed that, in non-permissive fumonisin excretion (NPFE) medium, FB1 is sequestered inside extra-vesicles and in the first third of the cell wall next to the plasmalemma, leading to the hypothesis that the fungus develops mechanisms to protect its cytosolic homeostasis against this cytotoxic. In permissive fumonisin excretion (PFE) medium, leading to very high quantities of excreted fumonisins, FB1 localized inside extra-vesicles, crosses the entire cell wall thickness, and then releases into the medium. Our results demonstrated a delayed and lower expression of Fvpks gene in mycelium developed on NPFE medium as compared to PFE medium. Conversely, higher amounts of fumonisins were accumulated in NPFE-grown mycelium than in PFE-grown mycelium. Thus, our results demonstrated for the first time that we have to take into account that the synthesis and secretion inside the article of secondary metabolites depend on the occurrence of cryptic biochemical specialized articles, differentiated in the mycelium. However, those are not morphologically different from other colonial hyphae.

Keywords: Fusarium verticillioides; acute cytotoxic firing; biochemical warfare; fumonisin; fungal factory; mycotoxin; ontological genetic program; secondary metabolite production.

Figure 1

Growth kinetics of Fusarium verticillioides cultured in liquid PFE or NPFE media (1.5–10 d.a.i.). Fungal growth rate was estimated by mycelium dry weight obtained after F. verticillioides culture in liquid PFE or NPFE media (1.5–10 d.a.i.). Means ± standard deviation (SD) are calculated with n = 9, with CV% under 7.39 using GraphPad Prism (V 6.05) software.

Figure 2

Trevue diagram of relative Fvpks/α-Fvtef-subunit expression in liquid (PFE, NPFE at 3, 7, and 10 d.a.i.) or solid (PFE, NPFE at 10 d.a.i.) media during F. verticillioides culture. Tests were carried out on mycelia grown in PFE or NPFE liquid media (3, 7, and 10 d.a.i.) or on corresponding solid media (10 d.a.i.). Fvpks expression was calibrated to this of the housekeeping α-Fvtef subunit gene. All values represent means ± SD of three mRNA purifications in triplicates from three independent culture flasks (n = 27) with CV% comprised between 0.52 and 3.94, using GraphPad Prism (V 6.05) software.

Figure 3

Kinetics of FB1, FB2, FB3, and HFB1 accumulation in PFE liquid media (A) and kinetics of FB1, FB2, and FB3 accumulation in mycelium (B) during F. verticillioides culture from 1.5 to 10 d.a.i. Tests were carried out using PFE culture supernatants (A) and PFE-produced mycelia. (B) Fumonisins were quantified using standard curves (n = 3) for each HPLC quantification. All values represent means ± SD, with CV% determined between 0.53 and 13.99 (n = 27) for culture supernatants and between 1.22 and 8.4 for mycelia. In mycelia, quantity is expressed in mg/g of mycelium dry weight (MDW).

Figure 4

FB1, FB2, and FB3 accumulation in solid NPFE or PFE media and in mycelia developed on the respective media 10 d.a.i. Fumonisin were quantified in semi-solid PFE and NPFE media and in the corresponding dried mycelia, using standard curves. Results are presented in histograms. All values represent means ± SD with CV% comprised between 3.75 and 11.15 (n = 27). In mycelia, quantity is expressed in mg/g of mycelium dry weight (MDW).

Figure 5

(A-J) Electron microscopic structure of F. verticillioides articles grown on semi-solid NPFE and PFE media after high-pressure-freezing fixation. Sections of 80 nm were observed using Hitachi H7650 TEM (scale bar = 1 μm indicated on each figure). CW, cell wall; Ex Ve, external vesicle; MVB, multi-vesicular body; mi, mitochondria; N, nucleus; va, vacuole; ve, vesicle; WC, Woronin corpus.

Figure 6

FB1-IDEM localization on 80-nm sections of F. verticillioides articles cultured on semi-solid PFE (A) or NPFE (B) media, after high-pressure-freezing fixation using TEM. Sections are observed using anti-FBI rabbit polyclonal antibodies (1/200) and 5-nm gold labeled goat anti-rabbit IgG polyclonal antibodies (1/30) in TBST. (Aa) represents the negative control in which anti-FB1 antibodies have been omitted (no labeling can be detected). Scale bar is indicated on each figure; a–c = 500 μm, d,e = 100 μm. CW, cell wall; Ex Me, external medium; mi, mitochondria; MVB, multi-vesicular body; va, vacuole; ve, vesicle.

Figure 7

Effect-directed assays of acute cytotoxic biochemical warfare firing of F. verticillioides. Supernatants (A,B) and water (C,D) or ethyl acetate (E,F) mycelium extractions from F verticillioides cultured in PFE (A,C,E) or NPFE (B,D,F) liquid media. Acute cytotoxicity was tested during 24 h against Neuro 2A mouse cell line. Results are given as normalization between plates, using three independent experiments (n = 8 × 3). IC₅₀ is indicated and CV% was calculated under 10.