The Transcriptome Response to Azole Compounds in Aspergillus fumigatus Shows Differential Gene Expression across Pathways Essential for Azole Resistance and Cell Survival.

The opportunistic pathogen Aspergillus fumigatus is found on all continents and thrives in soil and agricultural environments. Its ability to readily adapt to novel environments and to produce billions of spores led to the spread of azole-resistant A. fumigatus across the globe, posing a threat to many immunocompromised patients, including critically ill patients with severe influenza or COVID-19. In our study, we sought to compare the adaptational response to azoles from A. fumigatus isolates that differ in azole susceptibility and genetic background. To gain more insight into how short-term adaptation to stressful azole compounds is managed through gene expression, we conducted an RNA-sequencing study on the response of A. fumigatus to itraconazole and the newest clinically approved azole, isavuconazole. We observed many similarities in ergosterol biosynthesis up-regulation across isolates, with the exception of the pan-azole-resistant isolate, which showed very little differential regulation in comparison to other isolates. Additionally, we found differential regulation of membrane efflux transporters, secondary metabolites, iron metabolism, and various stress response and cell signaling mechanisms.

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

Olorofim (F901318) is a new antifungal currently under clinical development that shows both in vitro and in vivo activity against a number of filamentous fungi including Aspergillus fumigatus. In this study, we screened A. fumigatus isolates for intrinsic olorofim-resistant A. fumigatus and evaluated the ability of A. fumigatus to acquire an olorofim-resistant phenotype. No intrinsic resistance was found in 975 clinical A. fumigatus isolates. However, we found that isolates with increased olorofim MICs (> 8 mg/L) could be selected using a high number of conidia and olorofim exposure under laboratory conditions. Assessment of the frequency of acquired olorofim resistance development of A. fumigatus was shown to be higher than for voriconazole but lower than for itraconazole. Sequencing the PyrE gene of isogenic isolates with olorofim MICs of >8 mg/L identified various amino acid substitutions with a hotspot at locus G119. Olorofim was shown to have reduced affinity to mutated target protein dihydroorotate dehydrogenase (DHODH) and the effect of these mutations was proven by introducing the mutations directly in A. fumigatus. We then investigated whether G119 mutations were associated with a fitness cost in A. fumigatus. These experiments showed a small but significant reduction in growth rate for strains with a G119V substitution, while strains with a G119C substitution did not exhibit a reduction in growth rate. These in vitro findings were confirmed in an in vivo pathogenicity model.

Hmg1 Gene Mutation Prevalence in Triazole-Resistant Aspergillus fumigatus Clinical Isolates.

Recently, mutations in the 3-hydroxy-3-methylglutaryl-coenzyme-A-reductase-encoding gene (hmg1), a gene involved in ergosterol production, were associated with triazole-resistance in Aspergillus fumigatus. In this study, we determined the prevalence and characteristics of hmg1 mutations in a collection of clinical triazole-resistant A. fumigatus isolates collected during 2001-2019 from two international mycology reference centers: the Belgian National Reference Center for Mycosis and the Center of Expertise in Mycology Radboudumc/CWZ. Clinical isolates with and without cyp51A gene mutations and randomly selected wild-type (WT) controls were included. Isolates were characterized by in vitro susceptibility testing, cyp51A and hmg1 sequencing, and short tandem repeat typing. Available clinical records were analyzed for previous triazole exposure. In 23 isolates (24%) of the 95 triazole-resistant A. fumigatus isolates, hmg1 gene mutations were observed; including 5/23 (22%) isolates without cyp51A gene mutations and 18/72 (25%) with cyp51A mutations. Four previously described hmg1 gene mutations (E105K, G307R/D, G466V, and S541G) and two novel mutations (W273S and L304P) were found; 4/23 (17%) in the sterol-sensing-domain region. No triazole-antifungal exposure was reported in 75% (9/12) of patients harboring an isolate with hmg1 gene mutations. Three of 39 WT isolates (8%) contained a hmg1 gene mutation; E105K (2-isolates) and S541G. Hmg1 gene mutations were predominantly found in A. fumigatus with cyp51A mutations with voriconazole MICs ≥ 8 mg/L.

Facilitators of adaptation and antifungal resistance mechanisms in clinically relevant fungi.

Opportunistic fungal pathogens can cause a diverse range of diseases in humans. The increasing rate of fungal infections caused by strains that are resistant to commonly used antifungals results in difficulty to treat diseases, with accompanying high mortality rates. Existing and newly emerging molecular resistance mechanisms rapidly spread in fungal populations and need to be monitored. Fungi exhibit a diversity of mechanisms to maintain physiological resilience and create genetic variation; processes which eventually lead to the selection and spread of resistant fungal pathogens. To prevent and anticipate this dispersion, the role of evolutionary factors that drive fungal adaptation should be investigated. In this review, we provide an overview of resistance mechanisms against commonly used antifungal compounds in the clinic and for which fungal resistance has been reported. Furthermore, we aim to summarize and elucidate potent generators of genetic variability across the fungal kingdom that aid adaptation to stressful environments. This knowledge can lead to recognizing potential niches that facilitate fast resistance development and can provide leads for new management strategies to battle the emerging resistant populations in the clinic and the environment.

Phenotypic plasticity and the evolution of azole resistance in Aspergillus fumigatus; an expression profile of clinical isolates upon exposure to itraconazole.

BACKGROUND: The prevalence of azole resistance in clinical and environmental Aspergillus fumigatus isolates is rising over the past decades, but the molecular basis of the development of antifungal drug resistance is not well understood. This study focuses on the role of phenotypic plasticity in the evolution of azole resistance in A. fumigatus. When A. fumigatus is challenged with a new stressful environment, phenotypic plasticity may allow A. fumigatus to adjust their physiology to still enable growth and reproduction, therefore allowing the establishment of genetic adaptations through natural selection on the available variation in the mutational and recombinational gene pool. To investigate these short-term physiological adaptations, we conducted time series transcriptome analyses on three clinical A. fumigatus isolates, during incubation with itraconazole. RESULTS: After analysis of expression patterns, we identified 3955, 3430, 1207, and 1101 differentially expressed genes (DEGs), after 30, 60, 120 and 240 min of incubation with itraconazole, respectively. We explored the general functions in these gene groups and we identified 186 genes that were differentially expressed during the whole time series. Additionally, we investigated expression patterns of potential novel drug-efflux transporters, genes involved in ergosterol and phospholipid biosynthesis, and the known MAPK proteins of A. fumigatus. CONCLUSIONS: Our data suggests that A. fumigatus adjusts its transcriptome quickly within 60 min of exposure to itraconazole. Further investigation of these short-term adaptive phenotypic plasticity mechanisms might enable us to understand how the direct response of A. fumigatus to itraconazole promotes survival of the fungus in the patient, before any "hard-wired" genetic mutations arise.