Abstract
Aspergillus fumigatus is an opportunistic and allergenic pathogenic fungus, responsible for fungal infections in humans. A. fumigatus infections are usually treated with polyenes, azoles, or echinocandins. Echinocandins, such as caspofungin, can inhibit the biosynthesis of the β-1,3-glucan polysaccharide, affecting the integrity of the cell wall and leading to fungal death. In some A. fumigatus strains, caspofungin treatment at high concentrations induces an increase of fungal growth, a phenomenon called the caspofungin paradoxical effect (CPE). Here, we analyze the proteome and phosphoproteome of the A. fumigatus wild-type strain and of mitogen-activated protein kinase (MAPK) mpkA and sakA null mutant strains during CPE (2 μg/ml caspofungin for 1 h). The wild-type proteome showed 75 proteins and 814 phosphopeptides (corresponding to 520 proteins) altered in abundance in response to caspofungin treatment. The ΔmpkA (ΔmpkA caspofungin/wild-type caspofungin) and ΔsakA (ΔsakA caspofungin/wild-type caspofungin) strains displayed 626 proteins and 1,236 phosphopeptides (corresponding to 703 proteins) and 101 proteins and 1,217 phosphopeptides (corresponding to 645 proteins), respectively, altered in abundance. Functional characterization of the phosphopeptides from the wild-type strain exposed to caspofungin showed enrichment for transcription factors, protein kinases, and cytoskeleton proteins. Proteomic analysis of the ΔmpkA and ΔsakA mutants indicated that control of proteins involved in metabolism, such as in production of secondary metabolites, was highly represented in both mutants. Results of functional categorization of phosphopeptides from both mutants were very similar and showed a high number of proteins with decreased phosphorylation of proteins involved in transcriptional control, DNA/RNA binding, cell cycle control, and DNA processing. This report reveals novel transcription factors involved in caspofungin tolerance.IMPORTANCEAspergillus fumigatus is an opportunistic human-pathogenic fungus causing allergic reactions or systemic infections, such as invasive pulmonary aspergillosis in immunocompromised patients. Caspofungin is an echinocandin that impacts the construction of the fungal cell wall by inhibiting the biosynthesis of the β-1,3-glucan polysaccharide. Caspofungin is a fungistatic drug and is recommended as a second-line therapy for treatment of aspergillosis. Treatment at high concentrations induces an increase of fungal growth, a phenomenon called the caspofungin paradoxical effect (CPE). Collaboration between the mitogen-activated protein kinases (MAPK) of the cell wall integrity (MapkA) and high-osmolarity glycerol (SakA) pathways is essential for CPE. Here, we investigate the global proteome and phosphoproteome of A. fumigatus wild-type, ΔmpkA, and ΔsakA strains upon CPE. This study showed intense cross talk between the two MAPKs for the CPE and identified novel protein kinases and transcription factors possibly important for CPE. Increased understanding of how the modulation of protein phosphorylation may affect the fungal growth in the presence of caspofungin represents an important step in the development of new strategies and methods to combat the fungus inside the host.
Highlights
Aspergillus fumigatus is an opportunistic and allergenic pathogenic fungus, responsible for fungal infections in humans
We had observed that fungal mycelia grown in liquid cultures are more susceptible to high concentrations of caspofungin, and in agreement, the caspofungin paradoxical effect (CPE) of A. fumigatus strain CEA17 was already observed at 2 g/ml caspofungin in liquid minimal medium (MM) for 1 h, whereas the onset of the CPE was observed at 8 g/ml on solid medium [9]
To understand how A. fumigatus reacts to CPE concentrations and about the involvement of mitogen-activated protein kinase (MAPK) in that response, we perform proteomic and phosphoproteomic analysis of the wild-type, ΔsakA, and ΔmpkA strains left untreated or treated with 2 g/ml caspofungin for 1 h
Summary
Aspergillus fumigatus is an opportunistic and allergenic pathogenic fungus, responsible for fungal infections in humans. Aspergillus fumigatus is an opportunistic and allergenic pathogenic fungus that is responsible for a high incidence of fungal infections in humans and several pathologies in immunocompromised individuals Fungal infections, such as invasive aspergillosis (IA), are usually treated with polyenes, azoles, or echinocandins [1]. In some A. fumigatus strains, tolerance to caspofungin at high concentrations induces an increase in fungal growth, a phenomenon called the caspofungin paradoxical effect (CPE) [5, 6] Compensatory reactions such as the transcriptional stimulation of genes encoding chitin synthases and the subsequent increase of chitin content in the cell wall are due to the activation of stress-activated signaling pathways [4, 6]. A cross talk interaction between SakA and MpkA has already been observed for the adaptation to caspofungin [13, 16, 17]
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