The putative flavin carrier family FlcA-C is important for Aspergillus fumigatus virulence

Abstract

Aspergillus fumigatus is an opportunistic fungal pathogen and the most important species causing pulmonary fungal infections. The signaling by calcium is very important for A. fumigatus pathogenicity and it is regulated by the transcription factor CrzA. We have previously used used ChIP-seq (Chromatin Immunoprecipitation DNA sequencing) aiming to identify gene targets regulated by CrzA. We have identified among several genes regulated by calcium stress, the putative flavin transporter, flcA. This transporter belongs to a small protein family composed of FlcA, B, and C. The ΔflcA null mutant showed several phenotypes, such as morphological defects, increased sensitivity to calcium chelating-agent ethylene glycol tetraacetic acid (EGTA), cell wall or oxidative damaging agents and metals, repre-sentative of deficiencies in calcium signaling and iron homeostasis. Increasing calcium concentrations improved significantly the ΔflcA growth and conidiation, indicating that ΔflcA mutant has calcium insufficiency. Finally, ΔflcA-C mutants showed reduced flavin adenine dinucleotide (FAD) and were avirulent in a low dose murine infection model.

Keywords: Aspergillus fumigatus; CrzA; FAD metabolism; calcium; putative flavin flcA-C transporter family.

Figure 1.

Phylogeny of A. fumigatus FlcA-C. The optimal tree for the A. fumigatus is represented. The tree was inferred using the Neighbor-Joining Method. Sequences were aligned with ClustalW and the tree was constructed by using MEGA6.

Figure 2.

The A. fumigatus flcA expression is dependent on CrzA. The qRT-PCR for the A. fumigatus (A) flcA, (B) flcB, and (C) flcC genes. The strains were grown for 16 hours at 37 °C and transferred to 200 mM CaCl₂ for 10 and 30 min. The results are expressed as fold increase of the control (in the absence of CaCl₂) and the results were normalized with the βtub expression (*, p < 0.001).

Figure 3.

The A. fumigatus ΔflcA has morphogenetic defects. The wild-type, ΔflcA-C, and their corresponding complementing strains were grown for 48 h at 37 °C on solid (A) or liquid MM (B). A. fumigatus wild-type and ΔflcA germlings were grown in liquid MM for 12 h and stained or not with calcofluor white (C, top panels, bars 5 µM) or for 20 h at 30 °C (C, lower panels, bars, 10 µM). (D) The edge of the colonies represented in the plates of (A). Bars, 50 µM. (E) The qRT-PCR for the A. fumigatus flcA-C genes in the wild-type, ΔflcA, ΔflcB, and ΔflcC strain. The strains were grown for 16 hours at 37 °C (time 0) and transferred to 200 mM CaCl₂ for 10 and 30 min. The results are expressed as the number of cDNA copies of a specific flc gene divided by the number of copies of the cDNA of the normalizer βtub (p < 0.001).

Figure 4.

The ΔflcA mutant strain is more sensitive to calcium stress, cell wall damaging and oxidative stressing agents. (A) The wild type, the mutant, and the complemented strains were grown on YAG medium with increasing concentrations of EGTA, cyclosporin, calcofluor white (CFW), congo red (CR), Sodium Dodecyl Sulfate (SDS), t-butyl hydroxyperoxide, and paraquat for 48 h at 37 °C. (B) Radial growth of the wild-type and ΔflcA mutant strains grown in YAG medium for 120 hours at 37 °C in the absence and presence of increasing CaCl₂ concentrations. The results are the average ± standard deviation of 3 repetitions. Statistical analysis was performed by using One-way Anova with post test Dunnett (*** p < 0.05).

Figure 5.

The A. fumigatus ΔflcA is more sensitive to metals. (A) The wild type, the mutant, and the complemented strains were grown on YAG medium with increasing concentrations of LiCl and MnCl₂. (B) The wild type and the mutant strains were grown for 48 h at 37 °C in MM+200 µM FeSO₄ or AMM+ferrozine+BPS. The data were normalized by dividing the dry weight of the treatments by the dry weight of the corresponding strains grown for 48 h at 37 °C in MM.

Figure 6.

FlcA::GFP accumulates in the apical tip, cytoplasm and in vesicles. The FlcA::GFP strain was grown for 12 h at 30 °C in MM and transferred to either MM+FeSO₄ or AMM+ferrozine+BPS for 1or 2 h. Bars, 5 μm.

Figure 7.

There is a decreased FAD transport in the ΔflcA-C mutants. FAD transport was observed in A. fumigatus wild-type and ΔflcA-C protoplasts.

Figure 8.

A. fumigatus ΔflcA-C mutants are avirulent. (A) Comparative analysis of wild type, mutant, and complemented strains in a neutropenic murine model of pulmonary aspergillosis. Mice in groups of 10 per strain were infected intranasally with a 20 μl suspension of conidia at a dose of 10⁵. Fungal burden was determined 48 h post-infection by real-time qPCR based on 18 S 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 5 lungs for each treatment. Statistical analysis was performed by using t-test. (A) The ΔflcA mutant compared to the wild-type and ΔflcA::flcA⁺ strains. (B) Fungal burden for ΔflcA mutant, wild-type and ΔflcA::flcA⁺ strains. (C) The ΔflcB mutant compared to the wild type and ΔflcB::flcB⁺ strains. (D) Fungal burden for ΔflcB mutant, wild-type and ΔflcB::flcB⁺ strains. (E) The ΔflcC mutant compared to the wild type and ΔflcC::flcC⁺ strains. (F) Fungal burden for ΔflcC mutant, wild-type and ΔflcC::flcC⁺ strains. PBS = phosphate Buffer Saline.