Non-heat shock responsive roles of HSF1 in Candida albicans are essential under iron deprivation and drug defense.

Recently, we have reported that the conditional mutant of the heat shock factor-1 (HSF1) in Candida albicans displays enhanced susceptibility not only towards a plant alkaloid, berberine, but also to diverse antifungal drugs. The present study attempts to identify additional phenotypes highlighting the non-heat shock responsive roles of HSF1 that could be correlated with the enhanced drug susceptibility. We uncover an intricate relationship between cellular iron and HSF1 mediated drug susceptibility of C. albicans. Interestingly, at 30°C, the conditional deletion of HSF1 while presented no growth defect, exhibited low intracellular iron. Notably, exogenous supplementation of iron reversed growth defects of HSF1 mutant when grown at 37°C. We provide evidence that the HSF1 mutant presents interesting phenotypes at basal conditions and are implicated in enhanced drug susceptibilities, dysfunctional mitochondria, decreased resistance towards oxidative stress and compromised cell wall integrity, all of which could be fully reversed upon iron supplementation. The HSF1 mutant also displayed defective filamentation at basal conditions under various solid hypha inducing media. Further, chelation of iron of HSF1 mutant cells led to severe growth defects and apparently triggers an iron starvation signal in the cell thus, demonstrating that HSF1 is essential for C. albicans cells to tolerate the iron deprivation stress. Together, apart from the well-established roles of HSF1 in reciprocation of thermal stress, this study extends its role under basal conditions and provides molecular insights into the role of HSF1 in iron deprivation and drug defense of C. albicans.

Molecular mechanisms of action of herbal antifungal alkaloid berberine, in Candida albicans.

Candida albicans causes superficial to systemic infections in immuno-compromised individuals. The concomitant use of fungistatic drugs and the lack of cidal drugs frequently result in strains that could withstand commonly used antifungals, and display multidrug resistance (MDR). In search of novel fungicidals, in this study, we have explored a plant alkaloid berberine (BER) for its antifungal potential. For this, we screened an in-house transcription factor (TF) mutant library of C. albicans strains towards their susceptibility to BER. Our screen of TF mutant strains identified a heat shock factor (HSF1), which has a central role in thermal adaptation, to be most responsive to BER treatment. Interestingly, HSF1 mutant was not only highly susceptible to BER but also displayed collateral susceptibility towards drugs targeting cell wall (CW) and ergosterol biosynthesis. Notably, BER treatment alone could affect the CW integrity as was evident from the growth retardation of MAP kinase and calcineurin pathway null mutant strains and transmission electron microscopy. However, unlike BER, HSF1 effect on CW appeared to be independent of MAP kinase and Calcineurin pathway genes. Additionally, unlike hsf1 null strain, BER treatment of Candida cells resulted in dysfunctional mitochondria, which was evident from its slow growth in non-fermentative carbon source and poor labeling with mitochondrial membrane potential sensitive probe. This phenotype was reinforced with an enhanced ROS levels coinciding with the up-regulated oxidative stress genes in BER-treated cells. Together, our study not only describes the molecular mechanism of BER fungicidal activity but also unravels a new role of evolutionary conserved HSF1, in MDR of Candida.

Calcineurin signaling and membrane lipid homeostasis regulates iron mediated multidrug resistance mechanisms in Candida albicans.

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.