The involvement of the Mid1/Cch1/Yvc1 calcium channels in Aspergillus fumigatus virulence

Abstract

Aspergillus fumigatus is a major opportunistic pathogen and allergen of mammals. Calcium homeostasis and signaling is essential for numerous biological processes and also influences A. fumigatus pathogenicity. The presented study characterized the function of the A. fumigatus homologues of three Saccharomyces cerevisiae calcium channels, voltage-gated Cch1, stretch-activated Mid1 and vacuolar Yvc1. The A. fumigatus calcium channels cchA, midA and yvcA were regulated at transcriptional level by increased calcium levels. The YvcA::GFP fusion protein localized to the vacuoles. Both ΔcchA and ΔmidA mutant strains showed reduced radial growth rate in nutrient-poor minimal media. Interestingly, this growth defect in the ΔcchA strain was rescued by the exogenous addition of CaCl2. The ΔcchA, ΔmidA, and ΔcchA ΔmidA strains were also sensitive to the oxidative stress inducer, paraquat. Restriction of external Ca(2+) through the addition of the Ca(2+)-chelator EGTA impacted upon the growth of the ΔcchA and ΔmidA strains. All the A. fumigatus ΔcchA, ΔmidA, and ΔyvcA strains demonstrated attenuated virulence in a neutropenic murine model of invasive pulmonary aspergillosis. Infection with the parental strain resulted in a 100% mortality rate at 15 days post-infection, while the mortality rate of the ΔcchA, ΔmidA, and ΔyvcA strains after 15 days post-infection was only 25%. Collectively, this investigation strongly indicates that CchA, MidA, and YvcA play a role in A. fumigatus calcium homeostasis and virulence.

Figure 1. The identification of CchA, MidA, and YvcA orthologues in A. fumigatus.

(A) CchA, (B) MidA, and (C) YvcA. Ion_trans 1 to 4 indicate four domains PF00520 (http://pfam.sanger.ac.uk/family/Ion_trans), each with six transmembrane-spanning regions. HR and SP indicate a hydrophobic regions and a signal peptide, respectively. The topologies were predicted using SMART database (http://smart.embl-heidelberg.de) and TMpred program (http://ch.embnet.org/software/TMPRED_form.html). The signal peptide was predicted by using SignalP 4.1 program (http://www.cbs.dtu.dk/services/SignalP/).

Figure 2. mRNA accumulation of the A. fumigatus cchA, midA, and yvcA genes.

The wild-type and mutant strains were grown for 16 hours in YG medium at 37°C and the mycelia was either transferred to YG without any calcium for 30 minutes (Control:C) or for YG supplemented with 200 mM of calcium. RT-qPCR was used to quantify calcium channel mRNA abundance, which was normalized using β-tubulin (Afu1g10910). The relative quantitation of cchA, midA, yvcA, and β-tubulin was determined by comparison with a standard curve (C_T –values plotted against logarithm of the DNA copy number). The results presented are the means (± standard deviation) of four sets of experiments.

Figure 3. YvcA::GFP localizes to the vacuoles.

Arrows indicate the vacuoles (insets). (DIC: Differential Interference Contrast; CMCA: CellTracker Blue). Bars, 5 µm.

Figure 4. Growth of A. fumigatus parental and calcium channel mutant strains in different media.

(A) The parental, ΔcchA, ΔmidA, ΔcchA ΔmidA, ΔyvcA and complementing strains in the presence or absence of calcium chloride or paraquat. (B) Dry weight (mg) of the parental, ΔcchA, ΔmidA, and ΔyvcA grown in liquid MM or YG cultures from 24 to 96 hours. The results are the means (± standard deviation) of three sets of experiments.

Figure 5. Growth of A. fumigatus parental, ΔcchA, ΔmidA, ΔcchA ΔmidA, ΔyvcA and complementing strains strains in MM and YAG supplemented with different cyclosporine concentrations.

Figure 6. Restriction of external Ca²⁺ supply by the addition of Ca²⁺-chelating agent EGTA affects the growth of the ΔcchA and ΔmidA mutant strains.

The parental, ΔcchA, ΔmidA, and ΔcchAΔmidA strains were grown in liquid MM supplemented with 0, 10 or 20 mM for 16 hours at 30°C. Bars, 5 µm.

Figure 7. Susceptibility of A. fumigatus parental and calcium channel mutant strains to antifungal agents.

YG agar plates were inoculated with 2×10⁷ conidia. Etest strips for voriconazole, amphotericin, Anidulafungin, ketoconazole, itraconazole, and posaconazole were overlaid on the media, and the plates were incubated at 37°C for 16–20 hours. Representative pictures showing the inhibition ellipses and the endpoint values obtained with the Etest strips are shown. Experiments with all antifungal drugs were performed in triplicate.

Figure 8. The ΔcchA, ΔmidA, and ΔyvcA strains have decreased accumulation of calcium in the cytoplasm.

The relative levels of intracellular calcium in the parental, ΔcchA, ΔmidA, and ΔyvcA mutant strains were determined using the calcium-sensitive dye Fluo-3. The relative Ca²⁺ concentration was determined based on the fluorescence ratio after dual-wavelength excitation (fluorescent intensity at 526 nm [FI506 nm]/[FI526 nm]. Data shown are means of three repetitions (± standard deviations). Statistical analysis was performed by using t-test (*, p<0.01) comparing the treatments at 20, 100, and 200 mM to the 0 mM CaCl₂.

Figure 9. A. fumigatus cchA contributes to virulence in neutropenic mice.

(A) Comparative analysis of parental, ΔcchA, and ΔcchA::cchA ⁺ strains in a neutropenic murine model of pulmonary aspergillosis. A group of 10 mice per strain was intranasally infected with a 20 µl suspension of conidiospores at a dose of 5.0×10⁴. (B) Histological analysis of infected murine lung was performed 72 hours after infection with the wild-type strain and it reveals invasion of the murine lung epithelium. (C) Fungal burden was determined 48 hours post-infection by real-time qPCR based on 18S rRNA gene of A. fumigatus and an intronic region of the mouse GAPDH gene. Fungal and mouse DNA quantities were obtained from the Ct values from an appropriate standard curve. Fungal burden was determined through the ratio between ng of fungal DNA and mg of mouse DNA. The results are the means (± standard deviation) of five lungs for each treatment. Statistical analysis was performed by using t-test (*, p<0.01).

Figure 10. A. fumigatus midA contributes to virulence in neutropenic mice.

(A) Comparative analysis of parental, ΔmidA, and ΔmidA::midA ⁺ strains in a neutropenic murine model of pulmonary aspergillosis. Experimental details are described in Figure 8A. (B) Histological analysis of infected murine lung was performed 72 hours after infection with the wild-type strain and it reveals invasion of the murine lung epithelium. (C) Fungal burden was determined 48 hours post-infection by real-time qPCR based on 18S rRNA gene of A. fumigatus and an intronic region of the mouse GAPDH gene. Experimental details are described in Figure 8C.

Figure 11. A. fumigatus yvcA contributes to virulence in neutropenic mice.

(A) Comparative analysis of parental, ΔyvcA, and ΔyvcA::yvcA ⁺ strains in a neutropenic murine model of pulmonary aspergillosis. Experimental details are described in Figure 8A. (B) Histological analysis of infected murine lung was performed 72 hours after infection with the wild-type strain and it reveals invasion of the murine lung epithelium. (C) Fungal burden was determined 48 hours post-infection by real-time qPCR based on 18S rRNA gene of A. fumigatus and an intronic region of the mouse GAPDH gene. Experimental details are described in Figure 8C.