In this study, we compared the effects of altered membrane lipid composition on the localization of two membrane drug transporters from different superfamilies of the pathogenic yeast Candida albicans. We demonstrated that in comparison to the major facilitator superfamily multidrug transporter CaMdr1p, ATP-binding cassette transporter CaCdr1p of C. albicans is preferentially localized within detergent-resistant membrane (DRM) microdomains called 'rafts.' Both CaCdr1p and CaMdr1p were overexpressed as green fluorescent protein (GFP)-tagged proteins in a heterologous host Saccharomyces cerevisiae, wherein either sphingolipid (⌬sur4 or ⌬fen1 or ⌬ipt1) or ergosterol (⌬erg24 or ⌬erg6 or ⌬erg4) biosynthesis was compromised. CaCdr1p-GFP, when expressed in the above mutant backgrounds, was not correctly targeted to plasma membranes (PM), which also resulted in severely impaired drug resistance. In contrast, CaMdr1p-GFP displayed no sorting defect in the mutant background and remained properly surface localized and displayed no change in drug resistance. Our data clearly show that CaCdr1p is selectively recruited, over CaMdr1p, to the DRM microdomains of the yeast PM and that any imbalance in the raft lipid constituents results in missorting of CaCdr1p.

We have cloned and overexpressed multidrug transporter CaMdr1p as a green fluorescent proteintagged protein to show its capability to extrude drug substrates. The drug extrusion was sensitive to pH and energy inhibitors and displayed selective substrate specificity. CaMdr1p has a unique and conserved antiporter motif, also called motif C [G(X 6 )G(X 3 )GP(X 2 )GP(X 2 )G], in its transmembrane segment 5 (TMS 5). Alanine scanning of all the amino acids of the TMS 5 by site-directed mutagenesis highlighted the importance of the motif, as well as that of other residues of TMS 5, in drug transport. The mutant variants of TMS 5 were placed in four different categories. The first category had four residues, G244, G251, G255, and G259, which are part of the conserved motif C, and their substitution with alanine resulted in increased sensitivity to drugs and displayed impaired efflux of drugs. Interestingly, first category mutants, when replaced with leucine, resulted in more dramatic loss of drug resistance and efflux. Notwithstanding the location in the core motif, the second category included residues which are part of the motif, such as P260, and those which were not part of the motif, such as L245, W248, P256, and F262, whose substitution with alanine resulted in a severe loss of drug resistance and efflux. The third category included G263, which is a part of motif C, but unlike other conserved glycines, its replacement with alanine or leucine showed no change in the phenotype. The replacement of the remaining 11 residues of the fourth category did not result in any change. The putative helical wheel projection showed clustering of functionally critical residues to one side and thus suggests an asymmetric nature of TMS 5.

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Taken together, our results demonstrate that the absence of ergosterol, which is one of the constituents of membrane microdomains (rafts), has a direct effect on drug susceptibility and morphogenesis of C. albicans.

CaMdr1p is a multidrug MFS transporter of pathogenic Candida albicans. An over-expression of the gene encoding this protein is linked to clinically encountered azole resistance. In-depth knowledge of the structure and function of CaMdr1p is necessary for an effective design of modulators or inhibitors of this efflux transporter. Towards this goal, in this study, we have employed a membrane environment based computational approach to predict the functionally critical residues of CaMdr1p. For this, information theoretic scores which are variants of Relative Entropy (Modified Relative Entropy REM) were calculated from Multiple Sequence Alignment (MSA) by separately considering distinct physico-chemical properties of transmembrane (TM) and inter-TM regions. The residues of CaMdr1p with high REM which were predicted to be significantly important were subjected to site-directed mutational analysis. Interestingly, heterologous host Saccharomyces cerevisiae, over-expressing these mutant variants of CaMdr1p wherein these high REM residues were replaced by either alanine or leucine, demonstrated increased susceptibility to tested drugs. The hypersensitivity to drugs was supported by abrogated substrate efflux mediated by mutant variant proteins and was not attributed to their poor expression or surface localization. Additionally, by employing a distance plot from a 3D deduced model of CaMdr1p, we could also predict the role of these functionally critical residues in maintaining apparent inter-helical interactions to provide the desired fold for the proper functioning of CaMdr1p. Residues predicted to be critical for function across the family were also found to be vital from other previously published studies, implying its wider application to other membrane protein families.

The COVID-19 patients, both infected and recovered are rapidly contracting mucormycetes infections due to the ‘Mucorales’ order, under Zygomycetes class of fungi. The mucorales fungi commonly known to exist in our natural surroundings including soil, but the frequency of incidences was never rampant. This sudden spike in infections, is locally known as ‘black fungus,’ and is affecting various organs, including- eyes, sinuses, nose, brain, skin, intestine, lungs, etc. The severity of situation is ascertainable from the fact that, in certain cases surgical eye/jaws removal persists as the only viable option to avert mortality, as therapeutic interventions are limited. This epidemic situation intrigued experts to investigate the probable reason behind this unpredicted escalation in reported cases, including in recuperated COVID-19 patients, as person-to-person spread of infection is not common. The comparison of physiological parameters in healthy and COVID-19 afflicted patients highlights that the underlying conditions including diabetes mellitus, steroidal therapy, lymphopenia (decreased CD4+ and CD8+ lymphocytes), deregulated cytokine release storm, elevated free iron levels (hemosiderosis) in blood and insulin insensitivity are playing major roles in deteriorating conditions in rarely pathogenic fungal infections. This review is an attempt to explain the rationalities that makes people vulnerable to mucormycetes infection.

The development of MDR (multidrug resistance) in yeast is due to a number of mechanisms. The most documented mechanism is enhanced extrusion of drugs mediated by efflux pump proteins belonging to either the ABC (ATP-binding cassette) superfamily or MFS (major facilitator superfamily). These drug-efflux pump proteins are localized on the plasma membrane, and the milieu therein affects their proper functioning. Several recent studies demonstrate that fluctuations in membrane lipid composition affect the localization and proper functioning of the MDR efflux pump proteins. Interestingly, the efflux pumps of the ABC superfamily are particularly susceptible to imbalances in membrane-raft lipid constituents. This review focuses on the importance of the membrane environment in functioning of the drug-efflux pumps and explores a correlation between MDR and membrane lipid homoeostasis.

By exploiting the biosynthetic pathways of raft lipid constituents, in this study we demonstrate that fluctuations in either sphingolipid or ergosterol levels result in increased drug sensitivity and morphological defects in Candida albicans cells. We show that any change in either ergosterol composition by conditionally disrupting ERG1 or in sphingolipid composition by homozygously disrupting its biosynthetic gene IPT1 leads to improper surface localization of a major ABC (ATP-binding cassette) drug efflux protein, Cdr1p. Results suggest that sterol/sphingolipid-rich membrane microdomains play an important role in positioning and functional maintenance of the integral efflux protein. The impaired ability of erg1/ipt1 mutant cells to efflux drugs mediated through Cdr1p appears to be the main cause of increased drug sensitivity of Candida cells.