Components of a new gene family of ferroxidases involved in virulence are functionally specialized in fungal dimorphism

Abstract

Mucormycosis is an emerging angio-invasive infection caused by Mucorales that presents unacceptable mortality rates. Iron uptake has been related to mucormycosis, since serum iron availability predisposes the host to suffer this infection. In addition, iron uptake has been described as a limiting factor that determines virulence in other fungal infections, becoming a promising field to study virulence in Mucorales. Here, we identified a gene family of three ferroxidases in Mucor circinelloides, fet3a, fet3b and fet3c, which are overexpressed during infection in a mouse model for mucormycosis, and their expression in vitro is regulated by the availability of iron in the culture media and the dimorphic state. Thus, only fet3a is specifically expressed during yeast growth under anaerobic conditions, whereas fet3b and fet3c are specifically expressed in mycelium during aerobic growth. A deep genetic analysis revealed partially redundant roles of the three genes, showing a predominant role of fet3c, which is required for virulence during in vivo infections, and shared functional roles with fet3b and fet3c during vegetative growth in media with low iron concentration. These results represent the first described functional specialization of an iron uptake system during fungal dimorphism.

Figure 1

Conservation analysis of the proteins Fet3a, Fet3b and Fet3c. (A) Schematic comparison of M. circinelloides Fet3 proteins (shown as McFet3a, McFet3b and McFet3c) with S. cerevisiae FET3 (ScFET3), depicting protein domain architecture, signal peptide and transmembrane helixes. (B) Phylogenetic analysis of well-characterized fungal ferroxidases (pink background) and laccases (green background) and their relationship with M. circinelloides putative ferroxidases, showing support values for each node. Protein names and ID numbers are listed on Supplementary Table S1.

Figure 2

Differential expression of fet3a, fet3b and fet3c genes in infected mice and mycelia. The bars indicate the relative gene expression (mean ± s.d.) of the target genes in infected mice (in vivo, black bars, n = 5) and mycelia (in vitro, white bars, n = 5) compared to mycelia, after normalization with the 18S rRNA expression. Data were analyzed using a two-tailed unpaired t test (****p < 0.0001, T-test).

Figure 3

Induction of the genes fet3a, fet3b and fet3c by low iron availability. Upper panels show levels of fet3a, fet3b and fet3c mRNAs (A,B and C, respectively). Total RNA was extracted from mycelia of the wild type strain R7B grown in liquid culture for 24 hours in YNB pH 3.2, then the mycelia was washed and grown in either L15 or L15 + FeCl₃ 350 μM. To analyze expression of the fet3 genes in response to iron limitation, the mycelia grown in L15 were washed and transferred to an iron depleted media (with FBS or 1,10-phenanthroline) and samples were taken at 30 min, 60 min and 120 min. Middle panels show mRNA loading controls, which were performed by re-probing the membranes with a rRNA 18S probe. Lower panels show relative accumulation of fet3a, fet3b and fet3c mRNAs (A, B and C, respectively) by normalization with the 18S rRNA signals. Significant differences respect to control L15 medium are indicated with an *(p < 0.01, unpaired t-test). The cropped blots are displayed in the main figure, the black lines surrounding blots indicate the cropping lines. The scanned full blots are presented in Supplementary Fig. 2.

Figure 4

Disruption of genes fet3a, fet3b and fet3c. (A) Schematic representation of wild-type and mutant loci after homologous recombination with the disruption fragments of genes fet3a (left), fet3b (middle) and fet3c (right). The position of the probes used (A, B and C) and the expected sizes of the restriction fragments are indicated. Dashed lines are genomic sequences not included in the disruption fragment. (B) Southern blot analysis of the wild-type recipient strain (WT) and transformants obtained with the disruption fragments after ten vegetative cycles in selective medium. Genomic DNA (1 μg) was digested with HindIII and hybridized with probes A, B and C, which recognized wild type and disrupted alleles, but could discriminate between them. The positions and sizes of the GeneRuler DNA ladder mixture (M) (Fermentas) are indicated. The cropped blots are displayed in the main figure, the black lines surrounding blots indicate the cropping lines. The scanned full blots are presented in Supplementary Fig. 3. Asterisk (*) indicated that marker M(Kb) was revealed after a second re-hibridization of the same membrane.

Figure 5

Generation of double deletion mutants Δfet3a/Δfet3b, Δfet3c/Δfet3b and Δfet3a/Δfet3c. (A) Schematic representation of wild type and mutant loci after homologous recombination with the disruption fragments of genes fet3b (left) and fet3c (right). The position of the probes used (B and C) and the expected sizes of the restriction fragments are indicated. Dashed lines, sequences not included in the disruption fragment. (B) Southern blot analysis of the wild-type recipient strain (WT) and transformants obtained with the disruption fragments after ten vegetative cycles in selective medium. Genomic DNA (1 μg) was digested with HindIII (transformants for fet3b, left and right) or XbaI (transformants for fet3c, middle) and hybridized with probes B and C, which recognized wild-type and disrupted alleles, but could discriminate between them. The positions and sizes of the GeneRuler DNA ladder mixture (M) (Fermentas) are indicated. The cropped blots are displayed in the main figure, the black lines surrounding blots indicate the cropping lines. The scanned full blots are presented in Supplementary Fig. 3.

Figure 6

Growth defects of single and double deletion mutants in the genes fet3a, fet3b and fet3c. The diameter of ten independent colonies was measured from single deletion mutants Δfet3a, Δfet3b and Δfet3c in (A), and double deletion mutants Δfet3a/Δfet3b, Δfet3c/Δfet3b and Δfet3a/Δfet3c in (B), and compared to their corresponding wild-type control strains. Cultures were grown in solid minimal media YNB pH 4.5 supplemented with 50 μM of 1,10-Phenanthroline for 48 hours. In (C) and (D) single and double deletion mutants (respectively) and wild-type strains were grown as described above, but using media without 1,10-Phenanthroline and supplemented with FeCl₃ 350 µM.

Figure 7

Differential expression of the genes fet3a, fet3b and fet3c during dimorphic development. Upper panels show levels of fet3a, fet3b and fet3c mRNAs (A,B and C, respectively) during yeast and mycelial growth. Two cultures of 50 ml of YPG 4.5 inoculated with 10⁶ spores/ml were grown in a tightly closed 50-ml conical tube for 15 hours to induce yeast growth. Next, one of the tubes was opened and transferred to an open 500 ml flask in which it was shaken for 2 hours to promote the mycelial growth. These two cultures were repeated adding either 1,10-phenanthroline 10 μM or FeCl₃ 350 μM. Middle panels show mRNA loading controls, which were performed by re-probing the membranes with a rRNA 18S probe. Lower panels show relative expression of the genes fet3a, fet3b and fet3c mRNAs (A,B and C, respectively) by normalization with the 18S rRNA signals. The cropped blots are displayed in the main figure, the black lines surrounding blots indicate the cropping lines. The scanned full blots are presented in Supplementary Fig. 4.

Figure 8

Virulence test of single and double deletion mutants in the ferroxidase genes in a murine host model. (A) Virulence assays using spores of the wild type strain and the single deletion mutants Δfet3a, Δfet3b and Δfet3c. (B) Virulence assays using spores of the wild type strain and the double deletion mutants Δfet3a/Δfet3b, Δfet3c/Δfet3b and Δfet3a/Δfet3c. Immunosuppressed mice were injected with 1 × 10⁶ sporangiospores of each strain.