The high affinity iron permease is a key virulence factor required for Rhizopus oryzae pathogenesis

Abstract

Rhizopus oryzae is the most common cause of mucormycosis, an angioinvasive fungal infection that causes more then 50% mortality rate despite first-line therapy. Clinical and animal model data clearly demonstrate that the presence of elevated available serum iron predisposes the host to mucormycosis. The high affinity iron permease gene (FTR1) is required for R. oryzae iron transport in iron-depleted environments. Here we demonstrate that FTR1 is required for full virulence of R. oryzae in mice. We show that FTR1 is expressed during infection in diabetic ketoacidosis (DKA) mice. In addition, we disrupted FTR1 by double cross-over homologous recombination, but multinucleated R. oryzae could not be forced to segregate to a homokaryotic null allele. Nevertheless, a reduction of the relative copy number of FTR1 and inhibition of FTR1 expression by RNAi compromised the ability of R. oryzae to acquire iron in vitro and reduced its virulence in DKA mice. Importantly, passive immunization with anti-Ftr1p immune sera protected DKA mice from infection with R. oryzae. Thus, FTR1 is a virulence factor for R. oryzae, and anti-Ftr1p passive immunotherapy deserves further evaluation as a strategy to improve outcomes of deadly mucormycosis.

Fig. 1

FTR1 is expressed in DKA mice infected intravenously with R. oryzae. (A) FACS analysis of R. oryzae transformed with plasmid containing the reporter gene GFP driven by either the FTR1 promoter or the constitutively expressed ACT1 promoter and grown in iron-rich or iron-depleted conditions. R. oryzae M16 transformed with an empty plasmid was used as a negative control. (B) FTR1 is expressed in the brains of DKA mice infected with R. oryzae expressing GFP under the control of FTR1p. For anti-GFP Ab stain, tissue section was stained with rabbit polyclonal antibody to GFP then counter stained with FITC conjugated anti-rabbit antibody. For DIC, confocal image showing non-fluorescent R. oryzae at the time of infection. Arrows denote fungal elements in infected brains. Magnification, X 400.

Fig. 2

Disruption cassette integrates in FTR1 locus but complete elimination of FTR1 could not be achieved. (A) A diagram summarizing the strategy used to achieve FTR1 disruption. PyrF (998 bp) was used as a selectable marker flanked by 606 and 710 bp fragments of FTR1-5′ UTR and FTR1-3′ UTR, respectively. (B) Gel electrophoresis showing integration of the disruption cassette in a representative putative ftr1 null mutant (KO) but not in the wild-type (WT) (see 5′UTR and 3′UTR). Primers FTR1 P11 and FTR1 P12 were used to amplify 503 bp from the FTR1 ORF only from the wild-type but not from the putative ftr1 null mutant (see FTR1). Primers PyrF P9 and PyrF P18 to test for possible reciculization of the transformed plasmid with expected band of 2094 bp were also used (see self ligation). (C) Comparison of growth rate of R. oryzae wild-type, R. oryzae PyrF-complemented, or putative ftr1 null mutants grown on different sources of iron on iron-limited or iron-rich media. Growth was measured after 48 h for media containing 10 or 1000 μM of FeCl₃ or FeSO₄ or 100 μM of ferrioxamine, while growth was measured after 72 h for medium supplemented with 100 μM heme. Values (n=12 from four independent transformants with their growth measured in three experiments with similar results) are expressed as increase in mycelial diameter growth on solid growth medium in cm/h. * P<0.05 compared to wild-type or R. oryzae PyrF-complemented strains. (D) Gel electrophoresis showing lack of amplification of FTR1 after one round of purification of the putative null mutants on iron-rich medium (1000 μM FeCl₃) and amplification of the FTR1 from the same isolate following growth on iron-depleted medium (i.e. 100μM ferrioxamine) for 96 h. Amplification of actin (600 bp) was used to control for DNA loading.

Fig. 3

Confirmation of the lack of complete disruption of FTR1 in the multinucleated R. oryzae. (A) DAPI stain of swollen R. oryzae spores showing the presence of multiple nuclei with a single spore. Arrows denote nuclei. Original magnification, x1000. (B) Gel electrophoresis showing lack of amplification of FTR1 after 14 passages of the putative null mutants on iron-rich medium (1000 μM FeCl₃) and amplification of the FTR1 from the same isolate following growth on iron-depleted medium (i.e. 100 μM ferrioxamine) for 96 h. Amplification of actin (600 bp) was used to confirm the integrity of DNA used as template and the absence of PCR inhibitors. (C) Southern blot confirming the integration of the disruption cassette in the putative ftr1 (7380 bp band is present only in DNA sample extracted from putative ftr1 grown in iron-rich medium) and almost complete elimination of the FTR1 copy (lack of 1960 bp in DNA sample extracted from putative ftr1 grown in iron-rich medium).

Fig. 4

Reduced copy number results in compromised ability of R. oryzae to take up iron. (A) qPCR demonstrating reduced copy number of the wild-type FTR1 in the putative ftr1 null mutant compared to R. oryzae PyrF-complemented strain or to the same mutant grown in iron-depleted medium. (B) Gel electrophoresis of samples taken from the qPCR tube showing the amplification specificity for the FTR1 product. (C) qPCR demonstrating reduced copy number of the disrupted ftr1 allele in the putative ftr1 null when grown in iron-limited medium following passing in iron-rich medium (n=9 samples from two independent transformants). (D) The putative ftr1 mutant demonstrated reduced ability to acquire ⁵⁹Fe compared to R. oryzae wild-type or R. oryzae PyrF-complemented strains. ⁵⁹Fe uptake by wild-type, R. oryzae PyrF-complemented, or putative ftr1 mutant. Germinated spores were incubated with 0.1 μM ⁵⁹FeCl₃ (a concentration in which high-affinity iron permeases are induced (Fu et al., 2004)). *P <0.05 when compared with R. oryzae wild-type, R. oryzae PyrF-complemented strains, or the same strain grown on different concentrations of iron. Data (n= 9 from three separate experiments using three independent transformants) are expressed as medians + interquartile ranges.

Fig. 5

Reduction of FTR1 copy number reduces R. oryzae virulence in the DKA mouse models. (A) Survival of DKA mice (n=6 for wild-type and 5 for pyrF null mutant) infected with R. oryzae wild-type (3.3 × 10³) or pyrF null mutant (2.6 × 10³). (B) A representative of the putative ftr1 null mutant demonstrated comparable growth to R. oryzae PyrF-complemented strain on YPD or CSM-URA media. (C) Survival of mice (n=13 per group from two experiments with similar results) infected i.v. with R. oryzae wild-type (average inoculum of 4.3 × 10³ spores), R. oryzae PyrF-complemented strain (average inoculum of 4.2 × 10³ spores) or with putative ftr1 null mutant (average inoculum of 3.2 × 10³ spores). *, P<0.0005 compared to wild-type or PyrF-complemented strains. (D) Survival of mice (n= 9) infected intranasally with R. oryzae wild-type (4.3 × 10³ spores), R. oryzae PyrF-complemented strain (5.1 × 10³ spores) or putative ftr1 null mutant (5.3 × 10³ spores). *, P=0.04 compared to wild-type or PyrF-complemented strains.

Fig. 6

Inhibition of FTR1 expression reduces R. oryzae ability to take up ⁵⁹Fe in vitro. (A) Plasmid pRNAi-pdc intron used to generate FTR1::RNAi strains. (B) RT-PCR showing lack of expression of FTR1 in R. oryzae transformed with RNAi plasmid (T₁ and T₃-T₅) compared to R. oryzae transformed with empty plasmid (C, control). Primers amplifying the 18s rDNA served as a control to demonstrate the integrity of starting sample and lack of PCR inhibitors. (C) A representative of the RNAi transformants demonstrated comparable growth to the R. oryzae M16 transformed with empty plasmid on CSM-URA media. (D) ⁵⁹Fe uptake by wild-type, R. oryzae M16 transformed with the empty plasmid, or one of the RNAi transformants. Germinated spores were incubated with 0.1 μM ⁵⁹FeCl₃ (a concentration in which high-affinity iron permeases are induced (Fu et al., 2004)). *P <0.05 when compared with R. oryzae wild-type or R. oryzae M16 transformed with empty plasmid. Data (n= 9 from three separate experiments) are expressed as medians ± interquartile ranges.

Fig. 7

Inhibition of FTR1 expression reduces virulence of R. oryzae in the DKA mouse models and passive immunization with anti-Ftr1p sera protects DKA mice from R. oryzae infection. (A) Survival of mice (n=8) infected i.v. with R. oryzae transformed with empty plasmid (control strain, 2.9 × 10³ spores) or with RNAi plasmid targeting expression of FTR1 (FTR1-i, 4.1 × 10³ spores). *, P<0.001. (B) Survival of mice (n=9) infected intranasally with R. oryzae transformed with empty plasmid (control strain, 2.8 × 10³ spores) or with RNAi plasmid targeting expression of FTR1 (FTR1-i, 7.6 × 10³ spores). *, P<0.02. (C) Kidney or brain Fungal burden of mice (n=8) infected i.v. with R. oryzae transformed with empty plasmid (control strain, 4.2 × 10³ spores) or with RNAi plasmid targeting expression of FTR1 (FTR1-i, 5.1 × 10³ spores). *, P<0.0006 and \, P<0.04 compared to control strain. Data are expressed as medians + interquartile ranges. The y-axes reflect lower limits of detection of the assay. (D) Survival of mice (n=8) infected intranasally with R. oryzae (intended inoculum of 2.5 × 10⁷ spores and actual inhaled inoculum of 9 × 10³ spores) and treated with serum collected from mice immunized with either Ftr1p or proteins collected form empty plasmid clone. *, P<0.007.