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Using genetically encoded heme sensors to probe the mechanisms of heme uptake and homeostasis in Candida albicans.

ABSTRACT: Candida albicans is a major fungal pathogen that can utilise hemin and haemoglobin as iron sources in the iron-scarce host environment. While C. albicans is a heme prototroph, we show here that it can also efficiently utilise external heme as a cellular heme source. Using genetically encoded ratiometric fluorescent heme sensors, we show that heme extracted from haemoglobin and free hemin enter the cells with different kinetics. Heme supplied as haemoglobin is taken up via the Common in Fungal Extracellular Membrane (CFEM) hemophore cascade, and reaches the cytoplasm over several hours, whereas entry of free hemin via CFEM-dependent and independent pathways is much faster, less than an hour. To prevent an influx of extracellular heme from reaching toxic levels in the cytoplasm, the cells deploy Hmx1, a heme oxygenase. Hmx1 was previously suggested to be involved in utilisation of haemoglobin and hemin as iron sources, but we find that it is primarily required to prevent heme toxicity. Taken together, the combination of novel heme sensors with genetic analysis revealed new details of the fungal mechanisms of heme import and homeostasis, necessary to balance the uses of heme as essential cofactor and potential iron source against its toxicity.

SUBMITTER: Weissman Z

PROVIDER: S-EPMC7855654 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Candida albicans accumulates large amounts of the polyols glycerol and d-arabitol when the cells are exposed to physiological conditions relevant to stress and virulence in animals. Intracellular concentrations of glycerol are determined by rates of glycerol production and catabolism and of glycerol uptake and efflux through the plasma membrane. We and others have studied glycerol production in C. albicans, but glycerol uptake by C. albicans has not been studied. In the present study, we found that [(14)C]glycerol uptake by C. albicans SC5314 was (i) accumulative; (ii) dependent on proton-motive force; (iii) unaffected by carbon source; and (iv) unaffected by large molar excesses of d-arabitol or other polyols. The respective K(m) and V(max) values were 2.1 mM and 460 micromol h(-1) (g dry wt)(-1) in glucose medium and 2.6 mM and 268 micromol h(-1) (g dry wt)(-1) in glycerol medium. To identify the C. albicans glycerol uptake protein(s), we cloned the C. albicans homologues of the Saccharomyces cerevisiae genes GUP1 and STL1, both of which are known to be involved in glycerol transport. When multicopy plasmids encoding C. albicans STL1, C. albicans STL2 and C. albicans GUP1 were introduced into the corresponding S. cerevisiae null mutants, the transformants all acquired the ability to grow on minimal glycerol medium; however, only S. cerevisiae stl1 null mutants transformed with C. albicans STL1 actively took up extracellular [(14)C]glycerol. When both chromosomal alleles of C. albicans STL1 were deleted from C. albicans BWP17, the resulting stl1 null mutants grew poorly on minimal glycerol medium, and their ability to transport [(14)C]glycerol into the cell was markedly reduced. In contrast, deletion of both chromosomal alleles of C. albicans STL2 or of C. albicans GUP1 had no significant effects on [(14)C]glycerol uptake or the ability to grow on minimal glycerol medium. Northern blot analysis indicated that C. albicans STL1 was expressed in both glucose and glycerol media, conditions under which we detected wild-type active glycerol uptake. Furthermore, STL1 was highly expressed in salt-stressed cells; however, the stl1 null mutant was no more sensitive to salt stress than wild-type controls. We also detected high levels of STL2 expression in glycerol-grown cells, even though deletion of this gene did not influence glycerol uptake activity in glycerol-grown cells. We conclude from the results above that a plasma-membrane H(+) symporter encoded by C. albicans STL1 actively transports glycerol into C. albicans cells.

Project description:We previously demonstrated that iron deprivation enhances drug susceptibility of Candida albicans by increasing membrane fluidity which correlated with the lower expression of ERG11 transcript and ergosterol levels. The iron restriction dependent membrane perturbations led to an increase in passive diffusion and drug susceptibility. The mechanisms underlying iron homeostasis and multidrug resistance (MDR), however, are not yet resolved. To evaluate the potential mechanisms, we used whole genome transcriptome and electrospray ionization tandem mass spectrometry (ESI-MS/MS) based lipidome analyses of iron deprived Candida cells to examine the new cellular circuitry of the MDR of this pathogen. Our transcriptome data revealed a link between calcineurin signaling and iron homeostasis. Among the several categories of iron deprivation responsive genes, the down regulation of calcineurin signaling genes including HSP90, CMP1 and CRZ1 was noteworthy. Interestingly, iron deprived Candida cells as well as iron acquisition defective mutants phenocopied molecular chaperone HSP90 and calcineurin mutants and thus were sensitive to alkaline pH, salinity and membrane perturbations. In contrast, sensitivity to above stresses did not change in iron deprived DSY2146 strain with a hyperactive allele of calcineurin. Although, iron deprivation phenocopied compromised HSP90 and calcineurin, it was independent of protein kinase C signaling cascade. Notably, the phenotypes associated with iron deprivation in genetically impaired calcineurin and HSP90 could be reversed with iron supplementation. The observed down regulation of ergosterol (ERG1, ERG2, ERG11 and ERG25) and sphingolipid biosynthesis (AUR1 and SCS7) genes followed by lipidome analysis confirmed that iron deprivation not only disrupted ergosterol biosynthesis, but it also affected sphingolipid homeostasis in Candida cells. These lipid compositional changes suggested extensive remodeling of the membranes in iron deprived Candida cells. Taken together, our data provide the first novel insight into the intricate relationship between cellular iron, calcineurin signaling, membrane lipid homeostasis and drug susceptibility of Candida cells.

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Using genetically encoded heme sensors to probe the mechanisms of heme uptake and homeostasis in Candida albicans.

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