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

CDR1, a multidrug resistance gene from Candida albicans, contains multiple regulatory domains in its promoter and the distal AP-1 element mediates its induction by miconazole

We previously demonstrated that the CDR1 gene, encoding a multidrug transporter in Candida albicans, is differentially upregulated by various drugs and steroids. In order to get an insight into the molecular basis of the induction of this gene we analyzed its promoter region. The transcription start site was mapped to 63 nucleotides upstream of the initiating ATG. Reporter assays revealed the presence of four upstream activating and four upstream repressing sequence domains along the entire promoter. Like the native gene, promoter-luciferase recombinants showed enhanced activity in response to various stresses like drugs, human steroid hormones and heavy metals. Mutational analysis demonstrated that while the proximal promoter (−345/+1) contains all the regulatory domains required for its induction by various other stresses, the miconazole response is mediated via the distal promoter (−857/−1147), harboring an AP-1 site. The involvement of the AP-1 element in mediating the latter effect was evident by an increase in AP-1 binding activity following miconazole treatment

Oxidative stress in cardiovascular diseases

Oxidative stress caused by various oxygen containing free radicals and reactive species (collectively called "Reactive Oxygen Species" or ROS) has long been attributed to cardiovascular diseases. In human body, major oxidizing species are super oxide, hydrogen peroxide, hydroxyl radical, peroxy nitrite etc. ROS are produced from distinct cellular sources, enzymatic and non-enzymatic; have specific physicochemical properties and often have specific cellular targets. Although early studies in nineteen sixties and seventies highlighted the deleterious effects of these species, later it was established that they also act as physiological modulators of cellular functions and diseases occur only when ROS production is deregulated. One of the major sources of cellular ROS is Nicotinamide adenine dinucleotide phosphate oxidases (Noxes) that are expressed in almost all cell types. Superoxide and hydrogen peroxide generated from them under various conditions act as signal transducers. Due to their immense importance in cellular physiology, various Nox inhibitors are now being developed as therapeutics. Another free radical of importance in cardiovascular system is nitric oxide (a reactive nitrogen species) generated from nitric oxide synthase(s). It plays a critical role in cardiac function and its dysregulated generation along with superoxide leads to the formation of peroxynitrite a highly deleterious agent. Despite overwhelming evidences of association between increased level of ROS and cardiovascular diseases, antioxidant therapies using vitamins and omega 3 fatty acids have largely been unsuccessful till date. Also, there are major discrepancies between studies with laboratory animals and human trials. It thus appears that the biology of ROS is far complex than anticipated before. A comprehensive understanding of the redox biology of diseases is thus needed for developing targeted therapeutics