Characterization of the Sterol 24-C-Methyltransferase Genes Reveals a Network of Alternative Sterol Biosynthetic Pathways in Mucor lusitanicus

Abstract

Certain members of the order Mucorales can cause a life-threatening, often-fatal systemic infection called mucormycosis. Mucormycosis has a high mortality rate, which can reach 96 to 100% depending on the underlying condition of the patient. Mucorales species are intrinsically resistant to most antifungal agents, such as most of the azoles, which makes mucormycosis treatment challenging. The main target of azoles is the lanosterol 14α-demethylase (Erg11), which is responsible for an essential step in the biosynthesis of ergosterol, the main sterol component of the fungal membrane. Mutations in the erg11 gene can be associated with azole resistance; however, resistance can also be mediated by loss of function or mutation of other ergosterol biosynthetic enzymes, such as the sterol 24-C-methyltransferase (Erg6). The genome of Mucor lusitanicus encodes three putative erg6 genes (i.e., erg6a, erg6b, and erg6c). In this study, the role of erg6 genes in azole resistance of Mucor was analyzed by generating and analyzing knockout mutants constructed using the CRISPR-Cas9 technique. Susceptibility testing of the mutants suggested that one of the three genes, erg6b, plays a crucial role in the azole resistance of Mucor. The sterol composition of erg6b knockout mutants was significantly altered compared to that of the original strain, and it revealed the presence of at least four alternative sterol biosynthesis pathways leading to formation of ergosterol and other alternative, nontoxic sterol products. Dynamic operation of these pathways and the switching of biosynthesis from one to the other in response to azole treatment could significantly contribute to avoiding the effects of azoles by these fungi. IMPORTANCE The fungal membrane contains ergosterol instead of cholesterol, which offers a specific point of attack for the defense against pathogenic fungi. Indeed, most antifungal agents target ergosterol or its biosynthesis. Mucormycoses-causing fungi are resistant to most antifungal agents, including most of the azoles. For this reason, the drugs of choice to treat such infections are limited. The exploration of ergosterol biosynthesis is therefore of fundamental importance to understand the azole resistance of mucormycosis-causing fungi and to develop possible new control strategies. Characterization of sterol 24-C-methyltransferase demonstrated its role in the azole resistance and virulence of M. lusitanicus. Moreover, our experiments suggest that there are at least four alternative pathways for the biosynthesis of sterols in Mucor. Switching between pathways may contribute to the maintenance of azole resistance.

Keywords: CRISPR-Cas9; azole resistance; eburicol; erg6 gene; ergosterol; mucormycosis; zymosterol.

FIG 1

Alternative pathways from lanosterol to fecosterol. The described pathway for Saccharomyces cerevisiae (13) is indicated with black arrows, that for Aspergillus fumigatus (20) is indicated with white arrows, and that for Cryptococcus neoformans (37) is indicated with gray arrows.

FIG 2

Relative transcript levels of the ergosterol biosynthesis genes in the erg6 knockout mutant strains. Strains were grown on YNB medium at 25°C; the transcript level of each gene measured in the MS12+pyrG control strain was taken as 1. The presented values are averages of three independent experiments of two independent isolates per mutant; error bars indicate standard deviations. Relative transcript values followed by asterisks were significantly differed from the untreated control according to the unpaired t test (*, P < 0.05; **, P < 0.01).

FIG 3

Colony diameters of the erg6 deletion mutants and the MS12+pyrG strain of Mucor lusitanicus at 20°C (A), 25°C (B), 30°C (C), and 35°C (D) on YNB medium. The presented values are averages; colony diameters were measured during three independent cultivations of two isolates per mutant (error bars indicate standard deviations). Values followed by asterisks were significantly different from the corresponding value of the MS12 strain according to two-way ANOVA; (*, P < 0.05; **, P < 0.01).

FIG 4

Effects of different stressors on growth abilities of erg6 mutants and MS12+pyrG strain. (A) Effect of Congo red (CR) on the growth of strains. (B) Effect of calcofluor white (CFW) on the growth of strains. (C) Effect of SDS on the growth of strains. The presented values are averages; colony diameters were measured during three independent cultivations of two independent isolates per mutant (error bars indicate standard deviations). Values followed by asterisks significantly differed from the corresponding value of the MS12 strain according to the two-way ANOVA (*, P < 0.05; **, P < 0.01).

FIG 5

Spore production after 7 days on malt extract agar. The presented values are averages; spores were counted from three independent cultivations of two independent isolates per mutant (error bars indicate standard deviations). Values followed by asterisks significantly differed from the corresponding value of the MS12+pyrG strain according to the unpaired t test (**, P < 0.01).

FIG 6

Survival of Galleria mellonella (n = 20) infected with the erg6 mutants or the control M. lusitanicus MS12+pyrG and MS12 strains. The presented values are averages; survival curves were determined from three independent cultivations of two independent isolates per mutant. Survival curves followed by asterisks significantly differed from the control strain according to the log-rank (Mantel-Cox) test (**, P ≤ 0.01). The results summarize the results of 3 independent experiments.

FIG 7

Heat map of sterol composition of different Mucor strains. Scale represents the ratio of peak area and internal standard (IS) peak area values. The baseline was set to 1.

FIG 8

Sterol composition of erg6 and MS12+pyrG strains. The presented values are averages; amounts of sterols were measured from three independent cultivations of two independent isolates per mutant (error bars indicate standard deviations). Values followed by asterisks significantly differed from the corresponding value of the MS12+pyrG strain according to the unpaired t test (*, P < 0.05; **, P < 0.01).

FIG 9

Sterol composition of MS12+pyrG and cyp51 knockout mutant strains. The presented values are averages; amounts of sterols were measured from three independent cultivations of two independent isolates per mutant (error bars indicate standard deviations). Values followed by asterisks significantly differed from the corresponding value of the MS12+pyrG strain according to the unpaired t test (*, P < 0.05; **, P < 0.01).

FIG 10

Relative transcript levels of DHCR and CDI genes in the erg6 knockout mutant strains. Strains were grown on YNB medium at 25°C; the transcript level of each gene measured in the MS12+pyrG control strain was taken as 1. The presented values are averages of three independent experiments of two independent isolates per mutant; error bars indicate standard deviations. Relative transcript values followed by asterisks significantly differed from the untreated control according to the unpaired t test (*, P < 0.05; **, P < 0.01).

FIG 11

Possible ergosterol biosynthetic pathways of M. lusitanicus. Black arrows indicate the main ergosterol biosynthetic pathway, while dashed arrows indicate the alternative sterol biosynthetic pathways.