Candida albicans, unlike Saccharomyces cerevisiae, was able to use extracellular haemin as an iron source. Haemin uptake kinetics by C. albicans cells showed two phases: a rapid phase of haemin binding (with a K d of about 0?2 mM) followed by a slower uptake phase. Both phases were strongly induced in iron-deficient cells compared to iron-rich cells. Haemin uptake did not depend on the previously characterized reductive iron uptake system and siderophore uptake system. CaHMX1, encoding a putative haem oxygenase, was shown to be required for iron assimilation from haemin. A double DCahmx1 mutant was constructed. This mutant could not grow with haemin as the sole iron source, although haemin uptake was not affected. The three different iron uptake systems (reductive, siderophore and haemin) were regulated independently and in a complex manner. CaHMX1 expression was induced by iron deprivation, by haemin and by a shift of temperature from 30 to 37˚C. CaHMX1 expression was strongly deregulated in a Defg1 mutant but not in a Dtup1 mutant. C. albicans colonies forming on agar plates with haemin as the sole iron source showed a very unusual morphology. Colonies were made up of tubular structures that were organized into a complex network. The effect of haemin on filamentation was increased in the double DCahmx1 mutant. This study provides the first experimental evidence that haem oxygenase is required for iron assimilation from haem by a pathogenic fungus. INTRODUCTIONIron is required by most living systems. Iron availability plays a critical role in the host-pathogen relationship and in the virulence of bacteria and fungi [reviewed by Howard (1999) and Payne (1993)]. In the host, iron is predominantly intracellular, occurring as haem, iron-sulphur proteins or ferritin, with smaller quantities in other iron proteins. The limited amounts of extracellular iron are tightly held by proteins such as transferrin in the serum and lactoferrin on body surfaces. The withholding of iron by the host has been shown to block virulent infections by many pathogens, and high-affinity iron acquisition by pathogens often promotes virulent infections (Ratledge & Dover, 2000). Candida albicans is a saprophytic organism for humans; it can be found inhabiting external surfaces of the body, including the skin and the mucosae of the gut and mouth in more than half of the normal population. In some settings C. albicans invades tissue and disseminates systemically. Prolonged neutropenia, following cancer chemotherapy or bone marrow transplantation, is a major risk factor for invasive fungal infections, and Candida species are the most frequently implicated organisms (Rex et al., 1995). Recurrent oropharyngeal and oesophageal candidiasis is encountered in most AIDS patients (Laine & Bonacini, 1994). There has been a rapid rise in the frequency of drug resistance in clinical C. albicans infections (Rex et al., 1995); hence, there is a pressing need to identify new drug targets.The first studies about iron uptake by C. albicans emphasized the possi...

†These authors contributed equally to this study.We have studied the intracellular trafficking of Sit1 [ferrioxamine B (FOB) transporter] and Enb1 (enterobactin transporter) in Saccharomyces cerevisiae using green fluorescent protein (GFP) fusion proteins. Enb1 was constitutively targeted to the plasma membrane. Sit1 was essentially targeted to the vacuolar degradation pathway when synthesized in the absence of substrate. Massive plasma membrane sorting of Sit1 was induced by various siderophore substrates of Sit1, and by coprogen, which is not a substrate of Sit1. Thus, different siderophore transporters use different regulated trafficking processes. We also studied the fate of Sit1-mediated internalized siderophores. Ferrioxamine B was recovered in isolated vacuolar fractions, where it could be detected spectrophotometrically. Ferrioxamine B coupled to an inhibitor of mitochondrial protoporphyrinogen oxidase (acifluorfen) could not reach its target unless the cells were disrupted, confirming the tight compartmentalization of siderophores within cells. Ferrioxamine B coupled to a fluorescent moiety, FOB-nitrobenz-2-oxa-1,3-diazole, used as a Sit1-dependent iron source, accumulated in the vacuolar lumen even in mutants displaying a steady-state accumulation of Sit1 at the plasma membrane or in endosomal compartments. Thus, the fates of siderophore transporters and siderophores diverge early in the trafficking process.