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A High-Affinity Iron Permease Essential for Candida albicans Virulence

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Science  12 May 2000:
Vol. 288, Issue 5468, pp. 1062-1064
DOI: 10.1126/science.288.5468.1062

Abstract

Microbial pathogens must compete with the iron-withholding defense systems of their host to acquire this essential nutrient. Here, two high-affinity iron permease genes, CaFTR1 andCaFTR2, were isolated. CaFTR1 expression was induced under iron-limited conditions and repressed when iron supply was sufficient, whereas the expression of CaFTR2 was regulated in a reversed manner. Mutants lacking CaFTR1 but not CaFTR2 exhibited a severe growth defect in iron-deficient medium and were unable to establish systemic infection in mice. Thus, CaFTR1-mediated iron-uptake mechanism constitutes a virulence factor of Candida albicans and may be a target for the development of anti-Candida therapies.

Candida albicans is the cause of most fungal infections in immunocompromised patients, and systemic candidiasis may have fatal consequences (1). Pathogenicity of C. albicans has been attributed to several factors that enable the pathogen to damage and penetrate tissues, to escape host immune systems, and to establish systemic infection (2–4). However, little is known about how C. albicans acquires certain essential nutrients that are highly limiting in the host. One such nutrient is iron, which functions as an obligate cofactor of enzymes in many biological processes, such as DNA replication and electron transport (5). Mammals have developed an elaborate iron-withholding defense system against microbial infections, which collectively create an environment with virtually no free iron (6–8). Possession of specialized iron-uptake systems is intimately related to virulence of bacterial pathogens, such as Haemophilus, Neisseria, andYersinia (9–11).

To study iron transport in C. albicans and its relevance to pathogenesis, we screened a subtracted cDNA library (12) for genes differentially expressed in cells grown in an iron-limited medium and investigated their importance for virulence in mice. We isolated two related genes, CaFTR1 andCaFTR2, which are homologous to the high-affinity iron permease gene FTR1 of Saccharomyces cerevisiae(13). CaFTR1 and CaFTR2 encode two proteins consisting of 381 and 382 amino acids, respectively, sharing 83% identity with each other and ∼54% identity with FTR1. CaFTR1 and CaFTR2 contain multiple putative transmembrane domains and potential iron binding sites at positions similar to those in FTR1 (14).

We first used Northern blot analysis to examine CaFTR1and CaFTR2 expression in response to iron concentration change in the C. albicans strain SC5314 (15). ACaFTR1-specific probe detected a transcript of ∼1.66 kilobases (kb), and a CaFTR2-specific probe detected one of ∼1.4 kb (Fig. 1A). Both transcripts are sufficiently large to encode the proteins. Expression of the two genes was distinctly regulated. The highest amount of CaFTR1transcript was detected in cells grown in LIM1, and the amount decreased with the increase of iron concentration and was hardly detectable in cells grown in LIM1000 (12). The response ofCaFTR2 transcript level was the opposite of that ofCaFTR1 transcript, exhibiting the highest transcription level in cells grown in LIM1000 and a negligible level in cells grown in LIM1. From this differential regulation of the two genes, we would predict CaFTR1 to be a major iron permease used by C. albicans to grow in iron-deficient environments.

Figure 1

Iron-regulated expression and iron permease activity of CaFTR1 and CaFTR2. (A) Iron-regulated expression of CaFTR1 and CaFTR2 inC. albicans. (B) Correction of iron-dependent growth defect of S. cerevisiae ftr1 mutant byCaFTR1 and CaFTR2. S. cerevisiae ftr1mutant was transformed with a vector expressing eitherCaFTR1 or CaFTR2 under the control of Gal1-10 promoter or the vector alone. Multiple transformants were picked and patched onto low- (SD50) and high-iron (SD350) plates containing either glucose or galactose. The plates were incubated for 4 days at 30°C. (C) High-affinity iron uptake. Wild-type (WT) and ftr1 strains transformed with CaFTR1, orCaFTR2, or the vector (three clones each) were examined for high-affinity iron uptake.

To determine whether CaFTR1 and CaFTR2 were functionally equivalent to FTR1, we tested whether they could rescue the iron-dependent growth defect of a S. cerevisiae ftr1 strain (16). CaFTR1 and CaFTR2 were expressed from an S. cerevisiae centromeric vector under the control of a galactose-inducible promoter. The ftr1 cells transformed with either CaFTR1 or CaFTR2exhibited little growth on the low-iron plate containing glucose in contrast to the considerable growth on the plate containing galactose (Fig. 1B). Transformants carrying the vector alone could not grow on the low-iron plate even in the presence of galactose. All transformants grew equally well on the iron-sufficient plates containing either glucose or galactose, that is, under conditions in which the low-affinity iron transporter functions (17). We also determined 59Fe uptake by these transformants (18). The ftr1 cells transformed with the vector exhibited no intracellular iron accumulation when59FeCl3 was supplied at 2 μM, a concentration in which only high-affinity permeases are active [(17) and Fig. 1C]. Introduction of eitherCaFTR1 or CaFTR2 restored the iron uptake to ∼30 and 60% of the amount exhibited by the wild-type cells. Previous studies showed that FTR1 function requires interaction with a ferrous oxidase FET3 (13). Heterologous expression of theSchizosacchacromyces pombe iron permease gene FIP1alone in an S. cerevisiae ftr1 mutant could not correct the iron uptake defect (19). Our result suggests that the two C. albicans permeases may be able to interact with FET3 to constitute a partially functional iron transporter. This possibility is supported by the much higher identity in amino acid sequence between CaFTR and FTR1 (∼54%) than between FIP1 and FTR1 (∼43%). We also observed that the ftr1 mutants transformed with eitherCaFTR1 or CaFTR2 exhibited no increased iron uptake, when the culture medium contained 1 mM FeCl3(20), a concentration that would repress FET3expression (19).

To elucidate the respective roles of CaFTR1 andCaFTR2, we constructed gene-deletion mutants forCaFTR1 or CaFTR2 or both genes from the C. albicans strain CAI4 (14). In the process of deleting the CaFTR1 gene, we consistently found three copies of the gene in the genome, concluding that that is likely the natural status of the gene. We first evaluated the growth of all theCaftr strains in iron-limiting medium. The wild-type SC5314 and all the Ura3+ Caftr strains (containing oneCaURA3 copy at the gene disruption locus) were first grown in SD200 to saturation and then diluted to 1 × 104cells/ml in LIM0. Caftr1, Caftr2, and Caftr1 Caftr2 strains were able to grow at a rate similar to that of SC5314 to near saturation in this iron-deprived medium. However, when each culture, after reaching saturation, was diluted again to 1 × 104 cells/ml in LIM2 and LIM200, Caftr1 andCaftr1 Caftr2 strains failed to grow in LIM2 but grew as well as SC5314 in LIM200 (Fig. 2A). TheCaftr2 strain grew normally in both media. Other researchers have also demonstrated the necessity of multiple passages of yeast cells in iron-deprived medium for the manifestation of growth inhibition (21, 22), presumably because the cells must first exhaust the intracellular iron stores accumulated during growth in iron-sufficient media. Reintroduction ofCaFTR1 on a C. albicans autonomous-replicating plasmid pABSK1 (23) into Caftr1 andCaftr1 Caftr2 strains restored their growth in LIM2. Thus, strains lacking CaFTR1 but not CaFTR2 were unable to grow in the iron-limiting medium.

Figure 2

Phenotypes of Caftr strains. (A) Caftr1 strains fail to grow in iron-limited medium. All the strains were grown in uridine-dropout LIM2 and LIM200 media and monitored by measuring OD600 at 1.5-hour intervals. The strains are SC5314, Caftr1, Caftr2, Caftr1 Caftr2; the Caftr1 and Caftr1 Caftr2strains transformed with pABSK1 expressing CaFTR1(Caftr1::CaFTR1 and Caftr1 Caftr2::CaFTR1); and the same mutants transformed with the vector pABSK1. (B) Caftr1strain is defective in high-affinity iron uptake. The same strains used in the growth assay were subjected to the iron-uptake assay. The assay was also conducted on the Caftr1 and Caftr1 Caftr2 strains transformed with pABSK1 expressingCaFTR2 under the control of CaFTR1 promoter (Caftr1::pP1O2 and Caftr1 Caftr2::pP1O2) and on the Caftr1 strain transformed with pABSK1 expressing GFP-tagged CaFTR1 (Caftr1::CaFTR1-GFP). (C) Strains lacking CaFTR1 were avirulent. Twelve mice per group were injected with early stationary-phase yeasts of a C. albicans strain (1 × 106 viable cells per mouse in 0.2 ml of phosphate-buffered saline) grown in SD200-uridine and monitored for survival over a 42-day period. (D) Strains lacking CaFTR1 did not colonize the kidney of injected mice. The kidney sections were stained with periodic acid–Schiff's stain. The photomicrographs show corresponding cortical regions. (a) A representative appearance of tissues from mice infected with the strains lacking CaFTR1; (b) a representative appearance of tissues infected with strains carrying functionalCaFTR1.

To determine whether the Caftr strains were defective in high-affinity iron uptake, we assayed 59FeCl3accumulation in cells that had been grown in an iron-limited medium (Fig. 2B). Iron accumulations in the Caftr1 and Caftr1 Caftr2 strains were reduced to near background level. In contrast, the Caftr2 cells accumulated an amount of 59Fe similar to that of SC5314. Reintroducing CaFTR1 on pABSK1 into the Caftr1 and Caftr1 Caftr2 strains resulted in an accumulation of 59Fe nearly twice that in SC5314 cells, likely because of the ability of the plasmid to exist in multiple copies. Introducing the plasmid without the CaFTR1gene had no effect. Thus, CaFTR1 was responsible for nearly all of the high-affinity iron-uptake activity in the cells grown under iron-limiting conditions.

The CaFTR1 gene was essential for C. albicans to grow in an iron-deficient medium. Was it also required for C. albicans to grow in vivo? We inoculated mice with variousCaftr mutants to examine their lethality and colonization of kidneys. Again, the Ura3+ strains were used because Ura3 strains have reduced virulence (24). An inoculum of 1 × 106 cells of SC5314 or the Caftr2 strain resulted in 100% mortality within 4 days of injection (Fig. 2C). In contrast, all of the animals inoculated with Caftr1 or Caftr1 Caftr2 strains survived to the 42-day end point. Transformation of CaFTR1on pABSK1 into Caftr1 or Caftr1 Caftr2 cells fully restored the lethality. The kidneys from two mice of each group were removed on day 2. Microscopic examination revealed that the kidneys from mice inoculated with strains lacking CaFTR1appeared normal, whereas the tissues from mice injected with strains containing a functional CaFTR1 gene (or genes) showed lesions filled with both hyphal and yeast cells (Fig. 2D). The colony-forming units from animals of the former group were 1.4 ± 0.26 × 104 (n = 8) per gram of kidney in contrast to 3.8 ± 1.81 × 106(n = 6) from the animals of the latter group. Thus,CaFTR1 was indeed essential for C. albicans to grow and to cause systemic infection in the host. Other possible iron-uptake mechanisms, if there are any, are either not sufficient for the organism to acquire iron in vivo or they function in a CaFTR1-dependent manner.

The CaFTR2 gene was not required for C. albicansto grow in iron-deficient conditions. Could CaFTR2 function as a high-affinity iron permease in C. albicans when expressed?CaFTR1 gene promoter was used to control the expression ofCaFTR2 from pABSK1 (25). The Caftr1and Caftr1 Caftr2 strains transformed with this plasmid accumulated iron in amounts comparable to that in the same mutant cells transformed with the plasmid expressing CaFTR1 (Fig. 2B, plasmid pP1O2), suggesting that CaFTR2 may function as high-affinity iron permease in C. albicans. However, its function was not required for C. albicans to grow in highly iron-limiting environments, consistent with the lack of CaFTR2 expression under similar conditions. The presence of a pair of functionally similar but differentially regulated genes suggests that these genes evolved to function in different environments. C. albicansalso possesses a pair of pH-regulated genes, PHR1 andPHR2. They are maximally expressed at distinct pH and play different roles in morphogenesis (26).

The iron-transport function of CaFTR1 requires it to reside on the cell surface. To determine its cellular localization, CaFTR1 was tagged with the green fluorescent protein (GFP) at its COOH-terminus and expressed from the CaFTR1 promoter on pABSK1 (27).Caftr1 cells transformed with this plasmid exhibited high amounts of iron uptake, suggesting a functional fusion protein (Fig. 2B, Caftr1::CaFTR1-GFP). The transformants were then grown in either SD50 or SD1000 to exponential phase. Horse serum (10%) was added to a separate set of cultures to induce filamentous growth. Cells of both yeast and filamentous forms grown in SD50 were visualized to have a bright fluorescent periphery (Fig. 3), consistent with localization to the plasma membrane. The cells expressing the GFP alone showed intracellular fluorescence. Cells grown in SD1000 exhibited only faint disfused background fluorescence (28).

Figure 3

Cell surface localization of CaFTR1. The Ura3 Caftr1 was transformed with pABSK1 expressing either a CaFTR1-GFP fusion or GFP alone. The transformants were grown in SD50 to exponential phase.

Thus, CaFTR1 is required for C. albicans to acquire iron in iron-deficient environments both in vitro and in vivo. Deletion of the CaFTR1 gene abolished the pathogen's capacity to establish systemic infection in mice. Among all the strategies speculated to be important for C. albicans to acquire iron in vivo (29), CaFTR1 appears to play a central role and represents a potential target for the development of anti-Candida therapies.

  • * To whom correspondence should be addressed. E-mail: mcbwangy{at}imcb.nus.edu.sg

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