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

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.

Characterization of Three Pleiotropic Drug Resistance Transporter Genes and Their Participation in the Azole Resistance of Mucor circinelloides.

Mucormycosis is a life-threatening opportunistic infection caused by certain members of the fungal order Mucorales. This infection is associated with high mortality rate, which can reach nearly 100% depending on the underlying condition of the patient. Treatment of mucormycosis is challenging because these fungi are intrinsically resistant to most of the routinely used antifungal agents, such as most of the azoles. One possible mechanism of azole resistance is the drug efflux catalyzed by members of the ATP binding cassette (ABC) transporter superfamily. The pleiotropic drug resistance (PDR) transporter subfamily of ABC transporters is the most closely associated to drug resistance. The genome of Mucor circinelloides encodes eight putative PDR-type transporters. In this study, transcription of the eight pdr genes has been analyzed after azole treatment. Only the pdr1 showed increased transcript level in response to all tested azoles. Deletion of this gene caused increased susceptibility to posaconazole, ravuconazole and isavuconazole and altered growth ability of the mutant. In the pdr1 deletion mutant, transcript level of pdr2 and pdr6 significantly increased. Deletion of pdr2 and pdr6 was also done to create single and double knock out mutants for the three genes. After deletion of pdr2 and pdr6, growth ability of the mutant strains decreased, while deletion of pdr2 resulted in increased sensitivity against posaconazole, ravuconazole and isavuconazole. Our result suggests that the regulation of the eight pdr genes is interconnected and pdr1 and pdr2 participates in the resistance of the fungus to posaconazole, ravuconazole and isavuconazole.