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

Gábor Nagy,Sándor Kiss,Csaba Vágvölgyi,Sándor Kocsubé,Kitti Bauer,Tamás Papp,Mónika Homa,Nóra Zsindely,Rakesh Varghese,Csilla Szebenyi,László Bodai

doi:10.3389/fcimb.2021.660347

Gábor Nagy, Sándor Kiss + Show 9 more

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https://doi.org/10.3389/fcimb.2021.660347

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Abstract

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.

Highlights

Mucorales species are ubiquitous saprophytes (Hoffmann et al, 2013), but several species are known as opportunistic human pathogens, which can cause a life-threatening infection commonly called as mucormycosis
After cultivating Mucor in liquid RPMI-1640 for 24 h at 25°C, it was treated with the corresponding azole, and incubated for another 16 h before RNA extraction
In the M. circinelloides genome database (DoE Joint Genome Institute; M. circinelloides CBS277.49v2.0; http://genome.jgi-psf. org/Mucci2/Mucci2.home.html), eight potential pleiotropic drug resistance (PDR) proteins were found by amino acid similarity search using the amino acid sequence of Cryptococcus neoformans Afr1 (UniProtKB: Q8X0Z3) and Candida albicans Cdr1p (UniProtKB: P43071)

Summary

Introduction

Mucorales species are ubiquitous saprophytes (Hoffmann et al, 2013), but several species are known as opportunistic human pathogens, which can cause a life-threatening infection commonly called as mucormycosis. Clinical manifestations of mucormycosis include rhino-cerebral, pulmonary, cutaneous, and disseminated forms in immunocompromised patients (Skiada et al, 2018; Jeong et al, 2019) These infections are generally associated with rapid progression and high mortality rates (i.e. from 20 to 100% depending on the underlying condition, the clinical presentation, and the treatment), their diagnosis and treatment are often difficult (Riley et al, 2016; Skiada et al, 2018). The spectrum of applicable drugs is extremely narrow as these fungi are intrinsically resistant to most of the routinely used antifungal agents including most azoles (Almyroudis et al, 2007; Drogari-Apiranthitou et al, 2012; Riley et al, 2016)

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