Functional characterization of an Aspergillus fumigatus calcium transporter (PmcA) that is essential for fungal infection

Abstract

Aspergillus fumigatus is a primary and opportunistic pathogen, as well as a major allergen, of mammals. The Ca(+2)-calcineurin pathway affects virulence, morphogenesis and antifungal drug action in A. fumigatus. Here, we investigated three components of the A. fumigatus Ca(+2)-calcineurin pathway, pmcA,-B, and -C, which encode calcium transporters. We demonstrated that CrzA can directly control the mRNA accumulation of the pmcA-C genes by binding to their promoter regions. CrzA-binding experiments suggested that the 5'-CACAGCCAC-3' and 5'-CCCTGCCCC-3' sequences upstream of pmcA and pmcC genes, respectively, are possible calcineurin-dependent response elements (CDREs)-like consensus motifs. Null mutants were constructed for pmcA and -B and a conditional mutant for pmcC demonstrating pmcC is an essential gene. The ΔpmcA and ΔpmcB mutants were more sensitive to calcium and resistant to manganese and cyclosporin was able to modulate the sensitivity or resistance of these mutants to these salts, supporting the interaction between calcineurin and the function of these transporters. The pmcA-C genes have decreased mRNA abundance into the alveoli in the ΔcalA and ΔcrzA mutant strains. However, only the A. fumigatus ΔpmcA was avirulent in the murine model of invasive pulmonary aspergillosis.

Figure 1. A. fumigatus has three S. cerevisiae PMC1 homologues.

Phylogram tree and multiple sequence alignment of calcium transporter orthologues were made in CLUSTAL W2 (http://www.ebi.ac.uk/Tools/clustalw2/index.html) using the default parameters. The followings proteins were used for the analysis: Afu3g10690 (pmcB; XP_754550); Afu7g01030 (pmcC; XP_746828); Afu1g10880 (pmcA; XP_752453); Afu2g05860 (calcium/mangenese P-type ATPase: XP_749715); Afu6g06740 (endoplasmic reticulum calcium ATPase: XP_750567); Afu5g05840 (calcium channel subunit Mid1: XP_754048); Afu2g05330 (vacuolar H+/Ca2+ exchanger: XP_749663); Afu1g11110 (calcium channel subunit Cch1: XP_752476); Afu4g04670 (calcium permease family membrane transporter: XP_746653); Afu2g07630 (vacuolar H+/Ca2+ exchanger: XP_755098); Afu2g05320 (calcium-proton exchanger: XP_749662); Afu1g04270 (calcium ion transporter Vcx1: XP_750174); and Afu4g03320 (similar to vacuolar H⁺/Ca²⁺ exchanger: XP_001481534).

Figure 2. Binding of GST::CrzA recombinant protein to pmcA-C promoters.

Gel shift analysis was performed using three DNA fragments of pmcA, pmcB and pmcC promoters as probes and 1.0 µg to 2.0 µg of the CrzA::GST recombinant protein. Lanes 1 to 7, pmcA probe; lane 1, no protein added. Lanes 8 and 9, mutated pmcA probe. Lanes 10 to 14, pmcB probe; lane 10, no protein added. Lanes 11 to 14, fragments from pmcB probe. Lanes 15 to 23, pmcC probe; lane 15, no protein added. Lanes 22 and 23, mutated pmcC probe. O, gel origin; SC, specific competitor; FP, free probe. The arrow indicates the CrzA::GST-DNA complexes.

Figure 3. The pmcC gene is an essential A. fumigatus gene.

(A) The alcA::pmcC strain was grown for 16 hours in MM+ 2% glycerol at 37°C and transferred into either MM+4% glucose or MM+2% glycerol +threonine 100 mM and grown for further 6 hours. The relative quantitation of pmcC and tubulin gene expression was determined by a standard curve (i.e., C_T –values plotted against a logarithm of the DNA copy number). The results are the means (± standard deviation) of four biological replicates. (B) Growth phenotypes of the alcA::pmcC mutant strain. The A. fumigatus wild-type and alcA::pmcC mutant strains were grown for 72 hours at 37°C in MM+4% glucose, MM+2% glycerol and MM+2% glycerol +threonine 100 mM.

Figure 4. The pmcA-C genes have increased mRNA abundance when exposed to calcium.

The absolute quantitation of pmcA, pmcB, and pmcC and tubulin gene expression was determined by a standard curve (i.e., C_T –values plotted against a logarithm of the DNA copy number). The results are the means (± standard deviation) of four biological replicates. (A–C) The mRNA abundance of pmcA-C in the wild-type, ΔpmcA, and ΔpmcB.

Figure 5. Growth phenotypes of the ΔpmcA mutant strain.

The A. fumigatus wild-type, ΔpmcA::pmcA ⁺ and ΔpmcA mutant strains were grown for 72 hours at 37°C in (A)YAG, YAG+25 ng/ml cyclosporin (Cs), MM, or MM+25 ng/ml Cs; (B) YAG+500 mM CaCl₂, YAG+25 ng/ml Cs+500 mM CaCl₂, MM+500 mM CaCl₂, or MM+25 ng/ml Cs+500 mM CaCl₂; (C) YAG+25 mM MnCl₂, YAG+25 ng/ml Cs+25 mM MnCl₂, MM+25 mM MnCl₂, or MM+25 ng/ml Cs+25 mM MnCl₂.

Figure 6. Growth phenotypes of the ΔpmcB mutant strain.

The A. fumigatus wild-type, ΔpmcB::pmcB and ΔpmcB mutant strains were grown for 72 hours at 37°C in (A)YAG, YAG+25 ng/ml cyclosporin (Cs), MM, or MM+25 ng/ml Cs; (B) YAG+500 mM CaCl₂, YAG+25 ng/ml Cs+500 mM CaCl₂, MM+500 mM CaCl₂, or MM+25 ng/ml Cs+500 mM CaCl₂; (C) YAG+25 mM MnCl₂, YAG+25 ng/ml Cs+25 mM MnCl₂, MM+25 mM MnCl₂, or MM+25 ng/ml Cs+25 mM MnCl₂.

Figure 7. The pmcA-C genes have increased mRNA abundance when exposed to manganese.

The absolute quantitation of pmcA, pmcB, and pmcC and tubulin gene expression was determined by a standard curve (i.e., C_T –values plotted against a logarithm of the DNA copy number). The results are the means (± standard deviation) of four biological replicates. (A–C) The mRNA abundance of pmcA-C in the wild-type, ΔpmcA, and ΔpmcB.

Figure 8. The ΔpmcA mutant strain has increased accumulation of calcium in the cytoplasm.

The relative levels of intracellular calcium in the wild-type, ΔpmcA and ΔpmcB mutant strains were determined using the calcium-sensitive dye Fura-2-AM. The relative Ca⁺² concentration was determined based on the fluorescence ratio after dual-wavelength excitation (fluorescent intensity at 340 nm [FI340 nm]/[FI380 nm]. Data shown are means of three repetitions ± standard deviations. Statistical analysis was performed by using either One-Way Anova with Newman-Keuls post-tests or Tukey's multiple comparison tests. *p<0.005.

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

(A) Comparative analysis of wild-type, ΔpmcA and ΔpmcA::pmcA ⁺ strains in a neutropenic murine model of pulmonary aspergillosis. A group of 10 mice per strain was infected intranasally with a 20 µl suspension of conidiospores at a dose of 5.0×10⁴. (B) Histological analysis of infected murine lung were performed 72 hours after infection with the wild-type strain reveals invasion of the murine lung epithelium (C) Fungal burden was determined 48 hours post-infection by real-time RT-PCR 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 µg of mouse DNA quantities. The results are the means (± standard deviation) of five lungs for each treatment.