Heterogeneity in the transcriptional response of the human pathogen Aspergillus fumigatus to the antifungal agent caspofungin

Abstract

Aspergillus fumigatus is the main causative agent of invasive pulmonary aspergillosis (IPA), a severe disease that affects immunosuppressed patients worldwide. The fungistatic drug caspofungin is the second line therapy against IPA but has increasingly been used against clinical strains that are resistant to azoles, the first line antifungal therapy. In high concentrations, caspofungin induces a tolerance phenotype with partial reestablishment of fungal growth called caspofungin paradoxical effect (CPE), resulting from a change in the composition of the cell wall. An increasing number of studies has shown that different isolates of A. fumigatus exhibit phenotypic heterogeneity, including heterogeneity in their CPE response. To gain insights into the underlying molecular mechanisms of CPE response heterogeneity, we analyzed the transcriptomes of two A. fumigatus reference strains, Af293 and CEA17, exposed to low and high caspofungin concentrations. We found that there is a core transcriptional response that involves genes related to cell wall remodeling processes, mitochondrial function, transmembrane transport, and amino acid and ergosterol metabolism, and a variable response related to secondary metabolite (SM) biosynthesis and iron homeostasis. Specifically, we show here that the overexpression of a SM pathway that works as an iron chelator extinguishes the CPE in both backgrounds, whereas iron depletion is detrimental for the CPE in Af293 but not in CEA17. We next investigated the function of the transcription factor CrzA, whose deletion was previously shown to result in heterogeneity in the CPE response of the Af293 and CEA17 strains. We found that CrzA constitutively binds to and modulates the expression of several genes related to processes involved in caspofungin tolerance, and that crzA deletion differentially impacts the SM production and growth of Af293 and CEA17. As opposed to the ? crzA CEA17 mutant, the ? crzA Af293 mutant fails to activate cell wall remodeling genes upon caspofungin exposure, which most likely severely affects its macrostructure and extinguishes its CPE. This work describes how heterogeneity in the response to an antifungal agent between A. fumigatus strains stems from heterogeneity in the function of a transcription factor and its downstream target genes.

Every single conidium in Aspergillus fumigatus caspofungin tolerant strains are intrinsically caspofungin tolerant

“…CrzA regulates the activation of several stress responses and cell wall modifications (21, 19). Interestingly, crzA deletion in the clinical strain Af293 results in CPE loss (22, Figure 2C ), while crzA deletion in the CEA17 background is CPE + (23, 19, Figure 2C ), demonstrating intra-species differences or CPE heterogeneity. Differently from Δ crzA CEA17 mutant, the Δ crzA Af293 mutant cannot activate cell wall remodeling genes upon CAS exposure, affecting its CPE (23).…”

“…Interestingly, crzA deletion in the clinical strain Af293 results in CPE loss (22, Figure 2C ), while crzA deletion in the CEA17 background is CPE + (23, 19, Figure 2C ), demonstrating intra-species differences or CPE heterogeneity. Differently from Δ crzA CEA17 mutant, the Δ crzA Af293 mutant cannot activate cell wall remodeling genes upon CAS exposure, affecting its CPE (23). The CGIs for Δ crzA CEA17 and Δ crzA Af293 mutant strains are 100 and 0 %, respectively, when grown on MM+8 µg/ml independently if the conidia are derived from MM or MM+8 µg/ml ( Figure 2D, Supplementary Table S3 at 10.6084/m9.figshare.19178888 ).…”

Genome-wide patterns of non-coding sequence variation in the major fungal pathogenAspergillus fumigatus

“…Variation in non-coding regions can also contribute to phenotypic variation, including gene expression variation between and within species (Caroll, 2005;Hill et al, 2020). We have previously demonstrated that non-coding regions between two A. fumigatus reference strains, Af293 and A1163 (Brown et al, 2022;Colabardini et al, 2022), as well as between A. fumigatus and its non-pathogenic close relatives are highly variable (Brown et al, 2022). For example, we found that 418 A. fumigatus genes exhibit a different rate of evolution in their non-coding regions (relative to non-pathogenic close relatives), including the non-coding regions of 25 genes that are known genetic determinants of A. fumigatus virulence.…”

Aspergillus fumigatustranscription factor ZfpA regulates hyphal development and alters susceptibility to antifungals and neutrophil killing during infection

“…Conversely, ZfpA null hyphae have reduced branching, septation, and cell wall chitin. In addition to its role in hyphal development, ZfpA has been implicated in A. fumigatus response to stressors such as high calcium and the antifungals voriconazole and caspofungin [15–18]. However, whether ZfpA affects fungal growth and susceptibility to host immunity and antifungal treatment during infection remains unclear.…”