Resistance profiling of Aspergillus fumigatus to olorofim indicates absence of intrinsic resistance and unveils the molecular mechanisms of acquired olorofim resistance

Figures & data

Figure 1. Olorofim resistance frequency. Frequency of resistance observed of six A. fumigatus isolates when 10⁶–10⁹ spores were incubated on RPMI agar plates containing either 0.5 mg/L olorofim (OLO), 4 mg/L voriconazole (VOR) or 8 mg/L itraconazole (ITC). A. fumigatus ATCC 204305 b. A. fumigatus AZN 8196 c. A. fumigatus V052-35 (TR₃₄/L98H, azole resistant) d. A. fumigatus V139-36 e. A. fumigatus V180-37 and f. A. fumigatus V254-51. *P ≤ 0.05 **P ≤ 0.01, ns Not significant.

Table 1. Strains used in this study for resistance frequency analysis.

Strain	Cyp51A genotype	Source	Olorofim MIC (mg/L)	Voriconazole MIC (mg/L)	Itraconazole MIC (mg/L)
ATCC 204305	wildtype	Reference strain	0.016	0.25	0.125
AZN8196	wildtype	Radboudumc fungal database	0.031	0.25	0.125
V052-35	TR34/L98H	Radboudumc fungal database	0.031	8	>16
V139-36	wildtype	Radboudumc fungal database	0.063	0.25	0.25
V180-37	wildtype	Radboudumc fungal database	<0.016	0.5	0.5
V254-51	wildtype	Radboudumc fungal database	0.031	0.25	0.5
AF293	wildtype	Reference strain	0.016	0.5	0.5

Table 2. Mutations in the PyrE gene in isolates selected for olorofim resistance.

Parent Strain	Cyp51A genotype	Number of olorofim-resistant progeny sequenced strains	PyrE amino acid substitutions
			G119A	G119C	G119F	G119Y	G119S	G119V	H116P
ATCC 204305	wildtype	8	1	5			2
AZN8196	wildtype	6	2	2			1	1
V052-35	TR34/L98H	4		4
V139-36	wildtype	9	1	4		1	1	2
V180-37	wildtype	4	1	1	1				1
V254-51	wildtype	8	2	2			4
Af293	wildtype	10		3			3	4

Figure 2. IC50s of wildtype and mutant DHODH. The inhibition of DHODH activity by a range of olorofim concentrations was measured for the recombinant wild type Af293 enzyme and the Gly119 mutants indicated. Lines were fitted using log (inhibitor) vs response – Variable slope (four parameters) in Graphpad Prism. R squares were 0.998 for Af_DHODH (WT), 0.556 for Af_DHODH (G119V), 0.924 for Af_DHODH (G119A), 1.000 for Af_DHODH (G119S) and 0.9680 for Af_DHODH (G119C).

Figure 3. Radial growth rate of isolate AZN8196 and Af293 and olorofim-resistant progeny. Colony diameters are displayed for (a) isolate AZN8196 and 2 olorofim-resistant progeny isolates AZN8196_OLR1 (G119V) and AZN8196_OLR2 (G119C) with and (b) Af293, Af293_OLR5 (G119S), Af293_OLR7 (G119C) Af293 OLR9 (G119V).

Figure 4. In vivo virulence model. Survival of mice inoculated with (a) olorofim wildtype strain AZN8196 and olorofim-resistant progeny AZN8196_OLR1 and AZN8196_OLR2, and (b) olorofim wildtype strain Af293 and olorofim-resistant progeny Af293_OLR5, Af293_OLR7 and Af293_OLR9. Eight mice were inoculated with each strain.

Data availability statement

All data can be requested by the corresponding author upon request.