Supplemental Materials for Colabardini et al 2021

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 of 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 ∆crzACEA17 mutant, the ∆crzAAf293
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.