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Caspofungin Treatment of Aspergillus fumigatus Results in ChsG-Dependent Upregulation of Chitin Synthesis and the Formation of Chitin-Rich Microcolonies.

ABSTRACT: Treatment of Aspergillus fumigatus with echinocandins such as caspofungin inhibits the synthesis of cell wall ?-1,3-glucan, which triggers a compensatory stimulation of chitin synthesis. Activation of chitin synthesis can occur in response to sub-MICs of caspofungin and to CaCl2 and calcofluor white (CFW), agonists of the protein kinase C (PKC), and Ca(2+)-calcineurin signaling pathways. A. fumigatus mutants with the chs gene (encoding chitin synthase) deleted (?Afchs) were tested for their response to these agonists to determine the chitin synthase enzymes that were required for the compensatory upregulation of chitin synthesis. Only the ?AfchsG mutant was hypersensitive to caspofungin, and all other ?Afchs mutants tested remained capable of increasing their chitin content in response to treatment with CaCl2 and CFW and caspofungin. The resulting increase in cell wall chitin content correlated with reduced susceptibility to caspofungin in the wild type and all ?Afchs mutants tested, with the exception of the ?AfchsG mutant, which remained sensitive to caspofungin. In vitro exposure to the chitin synthase inhibitor, nikkomycin Z, along with caspofungin demonstrated synergistic efficacy that was again AfChsG dependent. Dynamic imaging using microfluidic perfusion chambers demonstrated that treatment with sub-MIC caspofungin resulted initially in hyphal tip lysis. However, thickened hyphae emerged that formed aberrant microcolonies in the continued presence of caspofungin. In addition, intrahyphal hyphae were formed in response to echinocandin treatment. These in vitro data demonstrate that A. fumigatus has the potential to survive echinocandin treatment in vivo by AfChsG-dependent upregulation of chitin synthesis. Chitin-rich cells may, therefore, persist in human tissues and act as the focus for breakthrough infections.

SUBMITTER: Walker LA

PROVIDER: S-EPMC4576100 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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Caspofungin Treatment of Aspergillus fumigatus Results in ChsG-Dependent Upregulation of Chitin Synthesis and the Formation of Chitin-Rich Microcolonies.

Walker Louise A LA Lee Keunsook K KK Munro Carol A CA Gow Neil A R NA

Antimicrobial agents and chemotherapy 20150713 10

Treatment of Aspergillus fumigatus with echinocandins such as caspofungin inhibits the synthesis of cell wall β-1,3-glucan, which triggers a compensatory stimulation of chitin synthesis. Activation of chitin synthesis can occur in response to sub-MICs of caspofungin and to CaCl2 and calcofluor white (CFW), agonists of the protein kinase C (PKC), and Ca(2+)-calcineurin signaling pathways. A. fumigatus mutants with the chs gene (encoding chitin synthase) deleted (ΔAfchs) were tested for their resp ...[more]

PMID: 26169407

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Project description: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.

Project description:Aspergillus fumigatus is the leading cause of pulmonary fungal diseases. Azoles have been used for many years as the main antifungal agents to treat and prevent invasive aspergillosis. However, in the last 10 years there have been several reports of azole resistance in A. fumigatus and new strategies are needed to combat invasive aspergillosis. Caspofungin is effective against other human-pathogenic fungal species, but it is fungistatic only against A. fumigatus Resistance to caspofungin in A. fumigatus has been linked to mutations in the fksA gene that encodes the target enzyme of the drug β-1,3-glucan synthase. However, tolerance of high caspofungin concentrations, a phenomenon known as the caspofungin paradoxical effect (CPE), is also important for subsequent adaptation and drug resistance evolution. Here, we identified and characterized the transcription factors involved in the response to CPE by screening an A. fumigatus library of 484 null transcription factors (TFs) in CPE drug concentrations. We identified 11 TFs that had reduced CPE and that encoded proteins involved in the basal modulation of the RNA polymerase II initiation sites, calcium metabolism, and cell wall remodeling. One of these TFs, FhdA, was important for mitochondrial respiratory function and iron metabolism. The ΔfhdA mutant showed decreased growth when exposed to Congo red or to high temperature. Transcriptome sequencing (RNA-seq) analysis and further experimental validation indicated that the ΔfhdA mutant showed diminished respiratory capacity, probably affecting several pathways related to the caspofungin tolerance and resistance. Our results provide the foundation to understand signaling pathways that are important for caspofungin tolerance and resistance.IMPORTANCEAspergillus fumigatus, one of the most important human-pathogenic fungal species, is able to cause aspergillosis, a heterogeneous group of diseases that presents a wide range of clinical manifestations. Invasive pulmonary aspergillosis is the most serious pathology in terms of patient outcome and treatment, with a high mortality rate ranging from 50% to 95% primarily affecting immunocompromised patients. Azoles have been used for many years as the main antifungal agents to treat and prevent invasive aspergillosis. However, there were several reports of evolution of clinical azole resistance in the last decade. Caspofungin, a noncompetitive β-1,3-glucan synthase inhibitor, has been used against A. fumigatus, but it is fungistatic and is recommended as second-line therapy for invasive aspergillosis. More information about caspofungin tolerance and resistance is necessary in order to refine antifungal strategies that target the fungal cell wall. Here, we screened a transcription factor (TF) deletion library for TFs that can mediate caspofungin tolerance and resistance. We have identified 11 TFs that are important for caspofungin sensitivity and/or for the caspofungin paradoxical effect (CPE). These TFs encode proteins involved in the basal modulation of the RNA polymerase II initiation sites, calcium metabolism or cell wall remodeling, and mitochondrial respiratory function. The study of those genes regulated by TFs identified in this work will provide a better understanding of the signaling pathways that are important for caspofungin tolerance and resistance.

Project description:Aspergillus fumigatus is the main causative agent of invasive pulmonary aspergillosis (IPA), a severe disease that affects immunosuppressed patients worldwide. The fungistatic drug caspofungin (CSP) 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, CSP induces a tolerance phenotype with partial reestablishment of fungal growth called CSP 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 CSP 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 CSP tolerance and that crzA deletion differentially impacts the SM production and growth of Af293 and CEA17. As opposed to the ΔcrzACEA17 mutant, the ΔcrzAAf293 mutant fails to activate cell wall remodeling genes upon CSP exposure, which most likely severely affects its macrostructure and extinguishes its CPE. This study 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.

Dataset Information

Caspofungin Treatment of Aspergillus fumigatus Results in ChsG-Dependent Upregulation of Chitin Synthesis and the Formation of Chitin-Rich Microcolonies.

Publications

Caspofungin Treatment of Aspergillus fumigatus Results in ChsG-Dependent Upregulation of Chitin Synthesis and the Formation of Chitin-Rich Microcolonies.

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