Article Figures & Data
Figures
- FIG 1
(A) Paradoxical growth observed with caspofungin (CSP) in the AF293 strain but not in the echinocandin-resistant strain (EMFR-S678P). Caspofungin at 1 μg/ml is active against the AF293 strain, causing a significant growth defect, but higher concentrations (2 and 4 μg/ml) result in a paradoxical growth recovery. The EMFR-S678P strain demonstrated resistance to caspofungin at all concentrations. (B to D) The Ca2+ chelator BAPTA blocked caspofungin-induced paradoxical growth of AF293 and CEA10 and also effectively abrogated echinocandin resistance of the EMFR-S678P strain. In all cases, 1 × 104 spores of each strain were spotted onto GMM agar plates and cultured for 5 days at 37°C.
- FIG 2
(A) Inclusion of CaCl2 slightly rescued growth inhibition by caspofungin at 0.5 to 2 μg/ml. Spores (1 × 104) of the AF293 strain were cultured in the presence of various concentrations of caspofungin supplemented with 2.5 mM and 5 mM CaCl2. Growth was monitored for a period of 5 days at 37°C. (B) The Ca2+ channel blocker verapamil at 1 mM inhibited caspofungin (CSP) paradoxical growth, indicating the requirement of extracellular Ca2+ for inducing paradoxical growth. In all cases, 1 × 104 spores of each strain were spotted onto GMM agar in the absence or presence of verapamil in combination with 4 μg/ml caspofungin for 5 days at 37°C.
- FIG 3
Caspofungin (CSP) and micafungin (MFG) induced perturbation in cytosolic free calcium ([Ca2+]c) concentration with reproducible Ca2+ signatures. Four micrograms per milliliter of each echinocandin or GMM alone was added, and the [Ca2+]c was monitored over 60 min. (A to D) A. fumigatus CEA10 (AEQ) grown in the wells of multiwell plates was either treated directly with caspofungin or micafungin (A), pretreated for 30 min with 1 mM verapamil (B), pretreated with 20 μM trifluoperazine (TFP) (C), or pretreated with 1 mM BAPTA (D) before the addition of 4 μg/ml of the drugs. (E) Summary of the mean maximum [Ca2+]c amplitudes for each of the treatments ∼3 min after the drug treatments.
- FIG 4
(A) Quantification of the transcription of calmodulin (cmdA) and calcineurin A (cnaA) by real-time reverse transcription-PCR in the AF293 strain following exposure to increasing concentrations of caspofungin. Conidia were grown for 20 h in liquid GMM in the absence of any drug. Caspofungin was added at concentrations of 0, 1, and 4 μg/ml for an additional 4-h incubation. Results are presented as the mean fold change (2−ΔΔCT) ± standard deviation compared to the untreated condition (black columns). (B) The calmodulin inhibitor trifluoperazine inhibited caspofungin (CSP) paradoxical growth with 4 μg/ml caspofungin in a dose-dependent manner, consistent with the caspofungin-induced activation of calmodulin being necessary to activate downstream calcineurin signaling. Spores (1 × 104) of the AF293 strain were cultured in the presence of various concentrations of TFP alone or in combination with 4 μg/ml caspofungin. Growth was monitored for a period of 5 days at 37°C.
- FIG 5
Phosphorylation profile of the A. fumigatus CnaA SPRR in the presence of caspofungin. (A and B) Tandem mass spectra of EELEDETPT[pS]VSP[pS]APSPPLPMDVESSEFK (A) and EELEDETPTSVSPSAP[pS]PPLPMDVESSEFK (B) in the CnaA subunit reveal three unique phosphorylated serine residues (S406, S410, and S413) clustered within the SPRR following treatment with 4 μg/ml caspofungin. The presence of each identified C-terminal (y) and N-terminal (b) product ion is indicated within the peptide sequence. The y-series and b-series ions are C-terminal and N-terminal ions, respectively, and qualitatively describe the confidence in identification from the MS/MS spectrum. For additional verification of the unique localization of phosphorylation between the two peptides, corresponding full MS extracted ion chromatograms of m/z 1,131.14 (±10 ppm) and 1,104.48 (±10 ppm) are shown on the right of each mass spectrum and illustrate a clear chromatographic shift in retention time between the species. (C) Phosphorylation profile of the A. fumigatus CnaA SPRR in the presence of micafungin. Tandem mass spectra of the phosphorylated peptide EELEDETPTSVSPSAP[pS]PPLPMDVESSEFK in the CnaA subunit reveal a unique phosphorylated serine residue (S413) within the SPRR. The presence of each identified C-terminal (y) and N-terminal (b) product ion is indicated within the peptide sequence. For additional verification of the unique localization of phosphorylation, the corresponding full MS extracted ion chromatogram of m/z 1,104.48 (±10 ppm) is shown on the right of the mass spectrum and illustrates a clear chromatographic shift in retention time between the species.
Tables
- TABLE 1
Differential phosphorylation of CnaA and CnaB in response to caspofungin and micafungin treatment identified by TiO2 enrichment and LC-MS/MS analysise
Condition/proteina Peptide sequenceb Phosphorylated residue(s) m/z Charge Mass error (ppm) Mascot ion scorec Ascore localization probabilityd (%) Caspofungin 1 CnaA RI[pS]MSAGSGR S537 551.25 2 −0.1 56.8 98 CnaB A[pS]VGTSQLLDNIVSASNFDRDEVDR S21 930.10 3 −2.9 76.1 99 Caspofungin 4 CnaA EELEDETPT[pS]VSP[pS]APSPPLPMDVESSEFK S406, S410 1,131.14 3 −1.8 49.8 95, 99 CnaA EELEDETPTSVSPSAP[pS]PPLPMDVESSEFK S413 1,104.48 3 −1.2 64.9 95 CnaA RI[pS]MSAGSGR S537 551.25 2 1.2 50.5 97 CnaB A[pS]VGTSQLLDNIVSASNFDRDEVDR S21 930.1 3 −3.2 93.3 99 Micafungin 1 CnaA RI[pS]MSAGSGR S537 551.25 2 0.8 54 88 CnaB A[pS]VGTSQLLDNIVSASNFDRDEVDR S21 930.1 3 −2.6 87 99 Micafungin 4 CnaA EELEDETPTSVSPSAP[pS]PPLPMDVESSEFK S413 1,104.48 3 −3.9 65.2 99 CnaA RI[pS]MSAGSGR S537 551.25 2 0.2 69.7 94 CnaB A[pS]VGTSQLLDNIVSASNFDRDEVDR S21 930.10 3 −0.1 88.2 99 ↵a Caspofungin 1 and caspofungin 4 indicate caspofungin used at 1 μg/ml and 4 μg/ml; micafungin 1 and micafungin 4 indicate micafungin used at 1 μg/ml and 4 μg/ml.
↵b [pS] indicates phosphorylated residue.
↵c Mascot identity score of >41 indicates identity or extensive homology (P < 0.05).
↵d Probability of phosphorylated residue localization based on Ascore algorithm.
↵e CnaA and CnaB subunit peptides contained uniquely identified phosphorylation residues or combinations of phosphorylation residues following treatment with caspofungin (1 and 4 μg/ml) and micafungin (1 and 4 μg/ml). Due to the proximity of multiple phosphorylatable residues within these peptides, mass spectra of each identification were submitted to an independent algorithm (Ascore) which assigns confidences to the localization of each phosphorylation.
Supplemental material
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