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

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.

Keywords: ABC transporter; CRISPR-Cas9; Mucor; azole; pleiotropic drug resistance.

Figure 1

Phylogenetic tree of PDR proteins inferred from 853 amino acid sequences using Maximum Likelihood method. The eight M. circinelloides Pdr proteins, C. neoformand CnAfr1 and the C. albicans Cdr1 and Cdr2 are indicated with bold characters. Numbers above or below branches are ultrafast bootstrap supports. Only values greater than 95% are shown.

Figure 2

Relative transcript levels of the pdr genes of M. circinelloides after azole treatment. MS12 was grown in RPMI-1640 liquid media at 25°C; transcript level of each gene measured in the untreated control was taken as 1. The presented values are averages of three independent experiments; error bars indicate standard deviation. Relative transcript values followed by * and ** significantly differed from the untreated control according to the paired t-test (*p < 0.05 and **p < 0.01).

Figure 3

Heat-map of transcript level of pdr genes in response to exposure to azoles. Red and green colors indicate down- and upregulation, respectively, while yellow corresponds to the transcription activity of the control.

Figure 4

Relative transcript levels of pdr genes under anaerobic condition. Transcript level of each gene was measured under aerobic condition was taken as 1. The presented values are averages of three independent experiments; error bars indicate standard deviation. For RNA extraction, fungal strains were cultivated on YNB for two days at 25°C. Relative transcript values followed by * and ** significantly differed from the value taken as 1 according to the paired t-test (*p < 0.05 and **p < 0,01).

Figure 5

Relative transcript levels of pdr genes in the MS12-Δpdr1/1 (A), MS12-Δpdr2 (B), MS12-Δpdr6 (C) MS12-Δpdr1-Δpdr2 (D) and MS12-Δpdr1-Δpdr6 (E) mutant compared to that in the original strain. Transcript level of each gene measured in the strain MS12 (black column) was taken as 1. The presented values are averages of three independent experiments (error bars indicate standard deviation). For RNA extraction, fungal strains were cultivated on YNB for two days at 25°C. Relative transcript levels significantly different from the value taken as 1 according to the paired t-test are indicated with * or ** (^* p < 0.05, ^** p < 0.01).

Figure 6

Colony diameters of the pdr deletion mutants and the original MS12 strain of Mucor circinelloides at 25°C on YNB medium. (A) shows the colony diameter of pdr1 deleted and complemented strains, (B, C) show the colony diameter of the single pdr2 and pdr6 knock out mutants, while (D, E) show the colony diameter of the double knock out strains. The presented values are averages; colony diameters were measured during three independent cultivation (error bars indicate standard deviation). Values followed by * and ** significantly differed from the corresponding value of the MS12 strain according to the two-way Anova (*p < 0.05; **p < 0.01).

Figure 7

Measurement of the R6G efflux activity. After uptake of rhodamine 6G, fungal cells were resuspended in PBS supplemented with 2 nM of D-glucose. Values followed by * and ** significantly differed from the corresponding value of the MS12 strain according to the two-way Anova (*p < 0.05; **p < 0.01), n.s. is non-significant.