The siderophore iron transporter of Candida albicans (Sit1p/Arn1p) mediates uptake of ferrichrome-type siderophores and is required for epithelial invasion

Abstract

The human fungal pathogen Candida albicans contains a close homologue of yeast siderophore transporters, designated Sit1p/Arn1p. We have characterized the function of SIT1 in C. albicans by constructing sit1 deletion strains and testing their virulence and ability to utilize a range of siderophores and other iron complexes. sit1 mutant strains are defective in the uptake of ferrichrome-type siderophores including ferricrocin, ferrichrysin, ferrirubin, coprogen, and triacetylfusarinine C. A mutation of FTR1 did not impair the use of these siderophores but did affect the uptake of ferrioxamines E and B, as well as of ferric citrate, indicating that their utilization was independent of Sit1p. Hemin was a source of iron for both sit1 and ftr1 mutants, suggesting a pathway of hemin uptake distinct from that of siderophores and iron salts. Heterologous expression of SIT1 in the yeast Saccharomyces cerevisiae confirmed the function of Sit1p as a transporter for ferrichrome-type siderophores. The sit1 mutant was defective in infection of a reconstituted human epithelium as a model for human oral mucosa, while the SIT1 strain was invasive. In contrast, both sit1 and SIT1 strains were equally virulent in the mouse model of systemic infection. These results suggest that siderophore uptake by Sit1p/Arn1p is required in a specific process of C. albicans infection, namely epithelial invasion and penetration, while in the blood or within organs other sources of iron, including heme, may be used.

FIG. 1.

Disruption of SIT1. (A) The wild-type genomic structure of SIT1 and the disruption fragment containing the URA blaster module (hisG-URA3-hisG) are shown (top). The relevant diagnostic restriction sites at the examination of the genomic configuration of SIT1 are HindIII (H), KpnI (K), and PvuII (P); the BglII (Bg*) and BamHI (B*) sites at the ends of the disruption fragment were generated by PCR. Asterisks indicate the region used as a probe in the Southern blot (bottom). Genomic DNAs of the following strains were digested by KpnI and PvuII and analyzed by Southern blotting: C4-SB1 (lane 1)and C4-SB4 (lane 2) (SIT1/sit1Δ::hisG-URA3-hisG); C4-SH1.1 (lane 3) and C4-SH4.1 (lane 4) (SIT1/sit1Δ::hisG); C4-SHB1.1 (lane 5) (sit1Δ::hisG-URA3-hisG/sit1Δ::hisG); C4-SHB4.21 (lane 6) (SIT1/sit1Δ::hisG-URA3-hisG/sit1Δ::hisG); C4-SHH4/21-VII (lane 7) (SIT1/sit1Δ::hisG/sit1Δ::hisG); C4-SHHB4/21-VII (lane 8) (sit1Δ::hisG-URA3-hisG/sit1Δ::hisG/sit1Δ::hisG); C4-SHHH4/21-VII (lane 9) (sit1Δ::hisG/sit1Δ::hisG/sit1Δ::hisG); and CAI4 (lane 10) (SIT1/SIT1). (B) Strategy to reconstitute the SIT1 gene in the sit1 mutant C4-SHH1.1. Plasmid p1367-CaSITpORF was cut with ClaI (C) and integrated into the SIT1 promoter region by homologous recombination. As a control, plasmid p1367-CaSITp was integrated similarly. The diagnostic restriction sites used in Southern blots were EheI (E) and SspI (S); the respective probes for the plasmids are indicated above and below the sit1 region. Examples of Southern blots verifying correct integrations are shown next to the respective plasmids (see Materials and Methods).

FIG. 2.

Utilization of siderophores by C. albicans strains. The indicated strains were embedded in solid Lee's medium containing 550 μM BPDA, and sterile filter paper disks containing siderophores (10 μl) were placed on the agar surface. Plates were incubated at 37°C for 2 to 3 days and photographed. The concentrations of siderophores and other compounds are listed in Table 2.

FIG. 3.

Utilization of siderophores by CaSIT1 transformant of S. cerevisiae. Strain PH14(YCp22Gal/SIT1) was embedded in solid SGal medium containing 200 μM BPDA, and sterile filter paper disks containing siderophores (10 μl) were placed on the agar surface. Plates were incubated at 30°C for 2 to 3 days and photographed. See Table 2 for concentrations. FUS, fusigen.

FIG. 4.

Infection of an RHE model by 2 × 10⁶ C. albicans cells. RHE infected by strains CAF2-1 and C4-SHB1.1 are shown at 6 and 22 h of incubation; RHE infected by strains SM1813c and SM1814c are shown at 12 h of incubation. Note the deep penetration and formation of vacuoles by SIT1⁺ strains (arrows).

FIG. 5.

Virulence of C. albicans in mice. C. albicans cells (10⁶; yeast form) were injected into the tail veins of BALB/c mice (n = 10), and the survival of mice was monitored daily. The strains used were CAF2-1 (SIT1/SIT1) (Δ), C4-SHB1.1 (sit1/sit1) (⋄), C4-SH1.1 (SIT1/sit1) (○), and C4-SHHB4/21-VII (sit1/sit1/sit1) (□).