Alteration of Fermentative Metabolism Enhances Mucor circinelloides Virulence

Abstract

The fungus Mucor circinelloides undergoes yeast-mold dimorphism, a developmental process associated with its capability as a human opportunistic pathogen. Dimorphism is strongly influenced by carbon metabolism, and hence the type of metabolism likely affects fungus virulence. We investigated the role of ethanol metabolism in M. circinelloides virulence. A mutant in the adh1 gene (M5 strain) exhibited higher virulence than the wild-type (R7B) and the complemented (M5/pEUKA-adh1⁺) strains, which were nonvirulent when tested in a mouse infection model. Cell-free culture supernatant (SS) from the M5 mutant showed increased toxic effect on nematodes compared to that from R7B and M5/pEUKA-adh1⁺ strains. The concentration of acetaldehyde excreted by strain M5 in the SS was higher than that from R7B, which correlated with the acute toxic effect on nematodes. Remarkably, strain M5 showed higher resistance to H₂O₂, resistance to phagocytosis, and invasiveness in mouse tissues and induced an enhanced systemic inflammatory response compared with R7B. The mice infected with strain M5 under disulfiram treatment exhibited only half the life expectancy of those infected with M5 alone, suggesting that acetaldehyde produced by M. circinelloides contributes to the toxic effect in mice. These results demonstrate that the failure in fermentative metabolism, in the step of the production of ethanol in M. circinelloides, contributes to its virulence, inducing a more severe tissue burden and inflammatory response in mice as a consequence of acetaldehyde overproduction.

Keywords: Mucor circinelloides; acetaldehyde; alcohol; alcohol dehydrogenase; dimorphism; ethanol; fermentative metabolism; mucoral; mucormycosis; oxidoreductases; virulence.

FIG 1

The adh1⁻ mutation increases virulence in M. circinelloides. (A) A total of 5 × 10⁶ spores from each of the different strains (M5, adh1⁻ mutant; R7B, adh1 wild type; M5/pEUKA-adh1⁺, adh1 mutant complemented) were inoculated using intraperitoneal administration in normal male BALB/c mice. Groups of 8 animals were treated with the different strains. The animals were observed daily until their death. (B) For C. elegans infection assays, 24-well flat-bottom plates were incubated with 10,000 spores and 20 nematodes per well in 1 ml of Lee medium; living nematodes were monitored every 12 h. Three independent experiments were registered in each host model of infection. The data were statistically analyzed using the Kaplan-Meier test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. When results were not considered significant, we did not provide an additional indication (P < 0.1).

FIG 2

The aerobic development of M. circinelloides is affected by mutation of the adh1 gene. A total of 5 × 10⁵ spores per ml were inoculated into minimal (Lee) liquid medium supplemented with leucine at 28°C with constant shaking. The germination rate (A) and growth (B) were registered at the indicated times for the three strains (M5, adh1⁻ mutant; R7B, adh1 wild type; M5/pEUKA-adh1⁺, adh1 mutant complemented). Four independent experiments were performed under the same conditions. Significance testing was performed using the unpaired Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. When results were not considered significant, we did not provide an additional indication (P < 0.1).

FIG 3

Mutation in the adh1 gene increases the invasiveness of M. circinelloides. A total of 5 × 10⁶ spores from each of the different strains of M. circinelloides (M5, adh1⁻ mutant; R7B, adh1 wild type; M5/pEUKA-adh1⁺, adh1 mutant complemented with adh1 wild type [WT]) were inoculated independently per mouse. Following 48 h of incubation, animals were sacrificed and the organs were removed to be observed (A). Asterisks indicate the presence of M. circinelloides hyphae in tissues subjected to Grocott staining. Images were taken with ×100 magnification under light microscopy. Bar = 200 μm. (B) Quantification of fungal load in the different mouse tissues by qPCR using tfc-1 from M. circinelloides. The values shown in the ordinate of the graph correspond to the ratio of the tfc-1 gene signal detected in the tissues by treatment with strain M5 or R7B. (C) We also analyzed the ratio of the tfc-1 gene from the tissues infected with M5/pEUKA-adh1 or R7B. Mouse β-actin gene was used to validate the use of the same amount of DNA from the different mouse tissues. Four independent experiments were performed under the same conditions. Significance testing was performed using the unpaired Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. When results were not considered significant, we did not provide an additional indication (P < 0.1).

FIG 4

Role of the adh1 mutation in the survival of M. circinelloides spores after H₂O₂ damage or macrophage phagocytosis. (A) Spores produced on YPG from the different strains were incubated with or without 4 mM H₂O₂ and incubated at 28°C. The survival rate was obtained after 24 h from the quotient of colonies from treatment versus no-treatment groups. (B) Germination of spores in the interaction with macrophages was seen under direct observation by light microscopy (×40). Scale bar = 20 μm. Arrows indicate germinating hyphae, and arrowheads indicate swelling spores. (C) The pkaR1 mRNA levels were quantified using qRT-PCR 1, 3, and 6 h after the spores and macrophages interacted. (D) Quantitation of killed spores by qPCR using tfc-1 after 6 h of incubation with mouse macrophages. A ΔCT analysis was performed to compare the mRNA and gene levels between the samples. (E) Quantitation of CFU from spores after 6 h of incubation with mouse macrophages. Figures show the average of three independent experiments. Significance testing was performed using the unpaired Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. When results were not considered significant, we did not provide an additional indication (P < 0.1).

FIG 5

The adh1⁻ mutant strain M5 of M. circinelloides triggers an enhanced inflammation response in mice. Spores from the different strains of M. circinelloides were inoculated in mice, and 48 h postinfection, the animals were sacrificed and the organs were removed for histological analysis using hematoxylin and eosin (A). The arrows show the glial brain cells. Alveolar structures are enclosed in brackets. The stars show the erythrocytes in the spleen, and the triangles show the hepatocyte morphology in the liver. Images were taken with ×100 magnification under light microscopy. Bar = 200 μm. mRNA quantification was performed in the organs, and levels of (B) Mip2, (C), Il-1β, and (D) Il-6 were determined using qRT-PCR. Expression is relative to Mus musculus β-actin. Significance testing was performed using the unpaired Student’s t test. *, P < 0.05; ** P < 0.01; ***, P < 0.001. When results were not considered significant, we did not provide an additional indication (P < 0.1).

FIG 6

The adh1⁻ mutant strain M5 of M. circinelloides increased the protein expression of cytokines in mouse tissue. The cytokine expression of (A) IL-1β and (B) IL-6 was determined using ELISA in liver and lung from mice previously infected for 48 h with the different strains. Significance testing was performed using the unpaired Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. When results were not considered significant, we did not provide an additional indication (P < 0.1).

FIG 7

Cell-free medium from cultures of the adh1⁻ mutant strain M5 of M. circinelloides is toxic to Caenorhabditis elegans. (A) Spores from the indicated strains were inoculated in Lee medium and grown under aerobic conditions at 28°C with constant shaking for 12 h, and then the cultures were filtered using 2-μm Millipore filters to obtain the cell-free medium culture (SS), which then was incubated in the presence of C. elegans. (B) The SS from all the strains were treated for 2 h at 37°C with pronase E or (C) at 37°C prior to incubation with the nematode. (D) The SS from all the strains were treated for 12 h at 25°C. All the SS were incubated with the nematodes. A total of 20 nematodes were used per well and incubated at 20°C for 2 h. The results presented are the average of four independent experiments. The data were statistically analyzed using the Kaplan-Meier test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. When results were not considered significant, we did not provide an additional indication (P < 0.1).

FIG 8

Acetaldehyde accumulation and its toxic effect during spore germination in M. circinelloides adh1^– and adh1⁺ strains. (A) The acetaldehyde from cell-free medium culture (from 12 h of fungal growth) was quantified using a mass spectrometry/flame ionization detector (MS-FID) method from different strains of M. circinelloides (M5, adh1⁻ mutant; R7B, adh1 wild type; M5/pEUKA-adh1⁺, adh1 mutant complemented). M5 cell-free medium culture was untreated (M5) or treated for 12 h at 25°C (M5 25°C) or at 37°C (M5 37°C). (B) Spore germination in the presence of exogenous addition of acetaldehyde in the various culture media. Four independent experiments were performed employing the same conditions. Significance testing was performed using the unpaired Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. When results were not considered significant, we did not provide an additional indication (P < 0.1).

FIG 9

The adh1⁻ mutant M5 from M. circinelloides detoxifies acetaldehyde through acetaldehyde dehydrogenase (ald2) overexpression. (A) Quantitation of the transcript levels of the acetaldehyde dehydrogenase gene (ald2) from wild-type (R7B) and adh1^– mutant (M5) at 12 h of fungal growth. (B) Acetic acid levels were measured in the cell-free culture medium at 12 h of growth. Four independent experiments were performed under the same conditions. Significance testing was performed using the unpaired Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. When results were not considered significant, we did not provide an additional indication (P < 0.1).

FIG 10

Mouse ALD activity inhibition increases the virulence of the adh1⁻ mutant M5 of M. circinelloides. Spores from the different strains of M. circinelloides were inoculated independently per mouse. Mice were separated into two groups, which were not treated (A) or treated (B) with disulfiram and inoculated with spores from the M. circinelloides strains (M5, adh1⁻ mutant; R7B, adh1 wild type; M5/pEUKA-adh1⁺, adh1 mutant complemented). A positive control was used (mice with disulfiram treatment under the presence of ethanol at 10% ad libitum). Two independent experiments were performed employing the same conditions using 8 mice under each condition tested. The data were statistically analyzed using the Kaplan-Meier test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. When results were not considered significant, we did not provide an additional indication (P < 0.1).