Publications by authors named "Nitesh Kumar Khandelwal"

17 Publications

  • Page 1 of 1

A Conserved Motif in Intracellular Loop 1 Stabilizes the Outward-Facing Conformation of TmrAB.

J Mol Biol 2021 08 29;433(16):166834. Epub 2021 Jan 29.

Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721, USA. Electronic address:

The ATP binding cassette (ABC) family of transporters moves small molecules (lipids, sugars, peptides, drugs, nutrients) across membranes in nearly all organisms. Transport activity requires conformational switching between inward-facing and outward-facing states driven by ATP-dependent dimerization of two nucleotide binding domains (NBDs). The mechanism that connects ATP binding and hydrolysis in the NBDs to conformational changes in a substrate binding site in the transmembrane domains (TMDs) is currently an outstanding question. Here we use sequence coevolution analyses together with biochemical characterization to investigate the role of a highly conserved region in intracellular loop 1 we define as the GRD motif in coordinating domain rearrangements in the heterodimeric peptide exporter from Thermus thermophilus, TmrAB. Mutations in the GRD motif alter ATPase activity as well as transport. Disulfide crosslinking, evolutionary trace, and evolutionary coupling analysis reveal that these effects are likely due to the destabilization of a network in which the GRD motif in TmrA bridges residues of the Q-loop, X-loop, and ABC motif in the NBDs to residues in the TmrAB peptide substrate binding site, thus providing an avenue for conformational coupling. We further find that disruption of this network in TmrA versus TmrB has different functional consequences, hinting at an intrinsic asymmetry in heterodimeric ABC transporters extending beyond that of the NBDs. These results support a mechanism in which the GRD motifs help coordinate a transition to an outward open conformation, and each half of the transporter likely plays a different role in the conformational cycle of TmrAB.
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http://dx.doi.org/10.1016/j.jmb.2021.166834DOI Listing
August 2021

A homologous overexpression system to study roles of drug transporters in Candida glabrata.

FEMS Yeast Res 2020 06;20(4)

Yeast Biofuel Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, 110067, India.

Considering the relevance of drug transporters belonging to ABC and MFS superfamilies in pathogenic Candida species, there has always been a need to have an overexpression system where these membrane proteins for functional analysis could be expressed in a homologous background. We could address this unmet need by constructing a highly drug-susceptible Candida glabrata strain deleted in seven dominant ABC transporters genes such as CgSNQ2, CgAUS1, CgCDR1, CgPDH1, CgYCF1, CgYBT1 and CgYOR1 and introduced a GOF mutation in transcription factor (TF) CgPDR1 leading to a hyper-activation of CgCDR1 locus. The expression system was validated by overexpressing four GFP tagged ABC (CgCDR1, CgPDH1, CaCDR1 and ScPDR5) and an MFS (CgFLR1) transporters genes facilitated by an engineered expression plasmid to integrate at the CgCDR1 locus. The properly expressed and localized transporters were fully functional, as was revealed by their several-fold increased drug resistance, growth kinetics, localization studies and efflux activities. The present homologous system will facilitate in determining the role of an individual transporter for its substrate specificity, drug efflux, pathogenicity and virulence traits without the interference of other major transporters.
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http://dx.doi.org/10.1093/femsyr/foaa032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7611192PMC
June 2020

In vitro characterization, ADME analysis, and histological and toxicological evaluation of BM1, a macrocyclic amidinourea active against azole-resistant Candida strains.

Int J Antimicrob Agents 2020 Mar 20;55(3):105865. Epub 2019 Dec 20.

Department of Biotechnology, Chemistry and Pharmacy, University of Siena, I-53100 Siena, Italy; Lead Discovery Siena s.r.l., Via Vittorio Alfieri 31, I-53019 Castelnuovo Berardenga, Italy; Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, BioLife Science Building, Philadelphia, PA 19122, USA.

Background: Candida species are one of the most common causes of nosocomial bloodstream infections among the opportunistic fungi. Extensive use of antifungal agents, most of which were launched on the market more than 20 years ago, led to the selection of drug-resistant or even multidrug-resistant fungi. We recently described a novel class of antifungal macrocyclic compounds with an amidinourea moiety that is highly active against azole-resistant Candida strains.

Objective: A compound from this family, BM1, was investigated in terms of in vitro activity against various Candida species, including C. auris isolates, interaction with the ABC transporter, CDR6, and in vivo distribution and safety.

Methods: In vitro assays (CYP inhibition, microsomal stability, permeability, spot assays) were used to collect chemical and biological data; animal models (rat) paired with LC-MS analysis were utilised to evaluate in vivo toxicology, pharmacokinetics, and distribution.

Results: The current research shows BM1 has a low in vivo toxicity profile, affinity for the renal system in rats, and good absorption, distribution, metabolism, and excretion (ADME). BM1 also has potent activity against azole-resistant fungal strains, including C. auris isolates and CDR6-overexpressing strains.

Conclusions: The results confirmed low minimum inhibitory concentrations (MICs) against several Candida species, including preliminary data vs. C. auris. BM1 has good ADME and biochemical characteristics, is suitable and safe for daily administration and is particularly indicated for renal infections. These data indicate BM1 and its derivatives form a novel, promising antifungal class.
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http://dx.doi.org/10.1016/j.ijantimicag.2019.105865DOI Listing
March 2020

ABC Transporter Genes Show Upregulated Expression in Drug-Resistant Clinical Isolates of : A Genome-Wide Characterization of ATP-Binding Cassette (ABC) Transporter Genes.

Front Microbiol 2019 16;10:1445. Epub 2019 Jul 16.

Amity Institute of Biotechnology and Integrative Sciences and Health, Amity University Gurugram, Gurgaon, India.

ATP-binding cassette (ABC) superfamily members have a key role as nutrient importers and exporters in bacteria. However, their role as drug exporters in eukaryotes brought this superfamily member to even greater prominence. The capacity of ABC transporters to efflux a broad spectrum of xenobiotics represents one of the major mechanisms of clinical multidrug resistance in pathogenic fungi including species. , a newly emerged multidrug-resistant fungal pathogen of humans, has been responsible for multiple outbreaks of drug-resistant infections in hospitals around the globe. Our study has analyzed the entire complement of ABC superfamily transporters to assess whether these play a major role in drug resistance mechanisms of . Our bioinformatics analyses identified 28 putative ABC proteins encoded in the genome of the type-strain CBS 10913T; 20 of which contain transmembrane domains (TMDs). Quantitative real-time PCR confirmed the expression of all 20 TMD transporters, underlining their potential in contributing to the drug-resistant phenotype. Changes in transcript levels after short-term exposure of drugs and in drug-resistant isolates suggested their importance in the drug resistance phenotype of this pathogen. orthologous to , a major multidrug exporter in other yeasts, showed consistently higher expression in multidrug-resistant strains of . Homologs of other ABC transporter genes, such as , , and , also displayed raised expression in a sub-set of clinical isolates. Together, our analysis supports the involvement of these transporters in multidrug resistance in .
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http://dx.doi.org/10.3389/fmicb.2019.01445DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6647914PMC
July 2019

Lipidomics Approaches: Applied to the Study of Pathogenesis in Candida Species.

Prog Mol Subcell Biol 2019 ;58:195-215

Amity Institute of Integrative Sciences and Health, Amity University Haryana, Gurgaon, Haryana, India.

High rate of reported cases of infections in humans caused by fungal pathogens pose serious concern. Potentially these commensal fungi remain harmless to the healthy individuals but can cause severe systemic infection in patients with compromised immune system. Effective drug remedies against these infections are rather limited. Moreover, frequently encountered multidrug resistance poses an additional challenge to search for alternate and novel targets. Notably, imbalances in lipid homeostasis which impact drug susceptibility of Candida albicans cells do provide clues of novel therapeutic strategies. Sphingolipids (SPHs) are unique components of Candida cells, hence are actively exploited as potential drug targets. In addition, recent research has uncovered that several SPH intermediates and of other lipids as well, govern cell signaling and virulence of C. albicans. In this chapter, we highlight the role of lipids in the physiology of Candida, particularly focusing on their roles in the development of drug resistance. Considering the importance of lipids, the article also highlights recent high-throughput analytical tools and methodologies, which are being employed in our understanding of structures, biosynthesis, and roles of lipids in fungal pathogens.
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http://dx.doi.org/10.1007/978-3-030-13035-0_8DOI Listing
August 2019

Vacuolar Sequestration of Azoles, a Novel Strategy of Azole Antifungal Resistance Conserved across Pathogenic and Nonpathogenic Yeast.

Antimicrob Agents Chemother 2019 03 26;63(3). Epub 2019 Feb 26.

Amity Institute of Biotechnology and Integrative Sciences and Health, Amity University Haryana, Gurgaon, India

Target alteration and overproduction and drug efflux through overexpression of multidrug transporters localized in the plasma membrane represent the conventional mechanisms of azole antifungal resistance. Here, we identify a novel conserved mechanism of azole resistance not only in the budding yeast but also in the pathogenic yeast We observed that the vacuolar-membrane-localized, multidrug resistance protein (MRP) subfamily, ATP-binding cassette (ABC) transporter of , Ybt1, could import azoles into vacuoles. Interestingly, the Ybt1 homologue in , Mlt1p, could also fulfill this function. Evidence that the process is energy dependent comes from the finding that a Mlt1p mutant version made by converting a critical lysine residue in the Walker A motif of nucleotide-binding domain 1 (required for ATP hydrolysis) to alanine (K710A) was not able to transport azoles. Additionally, we have shown that, as for other eukaryotic MRP subfamily members, deletion of the conserved phenylalanine amino acid at position 765 (F765Δ) results in mislocalization of the Mlt1 protein; this mislocalized protein was devoid of the azole-resistant attribute. This finding suggests that the presence of this protein on vacuolar membranes is an important factor in azole resistance. Further, we report the importance of conserved residues, because conversion of two serines (positions 973 and 976, in the regulatory domain and in the casein kinase I [CKI] consensus sequence, respectively) to alanine severely affected the drug resistance. Hence, the present study reveals vacuolar sequestration of azoles by the ABC transporter Ybt1 and its homologue Mlt1 as an alternative strategy to circumvent drug toxicity among pathogenic and nonpathogenic yeasts.
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http://dx.doi.org/10.1128/AAC.01347-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6395933PMC
March 2019

All about CDR transporters: Past, present, and future.

Yeast 2019 04 29;36(4):223-233. Epub 2018 Oct 29.

School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.

Drug resistance mechanisms in human pathogenic Candida species are continually evolving. Over the time, Candida species have acquired diverse strategies to vanquish the effects of various classes of drugs thereby, emanating as a serious life threat. Apart from the repertoire of well-established strategies, which predominantly comprise alteration, overexpression of drug targets, and chromosome duplication, Candida species have evolved a number of permeability constraints for antifungal drugs, via compromised drug import or increased drug efflux. For the latter, genome of Candida species harbour battery of exporters designated as Candida drug resistance genes. These genes predominantly encode membrane efflux transporters, which expel the incoming drugs and thus prevent toxic intracellular accumulation of drugs to manifest multidrug resistance. Such a phenomenon is restricted not only to Candida species but has been observed among many other pathogenic fungal species as well. Notably, the existence of large number of drug exporters in genomes of Candida species posits other pivotal roles for these efflux transporter proteins. The brief review discusses as to how the whole gamut of antifungal research has since been changed to include these new observations wherein reduced permeability of azoles across cell membrane of Candida cells is being implicated as one of the major determinants of antifungal susceptibilities, which all began with the identification of the first multidrug resistance gene CDR1, in Andre Goffeau's laboratory back in 1995.
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http://dx.doi.org/10.1002/yea.3356DOI Listing
April 2019

ABC transportome inventory of human pathogenic yeast Candida glabrata: Phylogenetic and expression analysis.

PLoS One 2018 28;13(8):e0202993. Epub 2018 Aug 28.

Yeast Biofuel Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India.

ATP-binding cassette (ABC) is one of the two major superfamilies of transporters present across the evolutionary scale. ABC superfamily members came to prominence due to their ability to extrude broad spectrum of substrates and to confer multi drug resistance (MDR). Overexpression of some ABC transporters in clinical isolates of Candida species was attributed to the development of MDR phenotypes. Among Candida species, Candida glabrata is an emerging drug resistant species in human fungal infections. A comprehensive analysis of such proteins in C. glabrata is required to untangle their role not only in MDR but also in other biological processes. Bioinformatic analysis of proteins encoded by genome of human pathogenic yeast C. glabrata identified 25 putative ABC protein coding genes. On the basis of phylogenetic analysis, domain organization and nomenclature adopted by the Human Genome Organization (HUGO) scheme, these proteins were categorized into six subfamilies such as Pleiotropic Drug Resistance (PDR)/ABCG, Multi Drug Resistance (MDR)/ABCB, Multi Drug Resistance associated Protein (MRP)/ABCC, Adrenoleukodystrophy protein (ALDp)/ABCD, RNase L Inhibitor (RLI)/ABCE and Elongation Factor 3 (EF3)/ABCF. Among these, only 18 ABC proteins contained transmembrane domains (TMDs) and were grouped as membrane proteins, predominantly belonging to PDR, MDR, MRP, and ALDp subfamilies. A comparative phylogenetic analysis of these ABC proteins with other yeast species revealed their orthologous relationship and pointed towards their conserved functions. Quantitative real time PCR (qRT-PCR) analysis of putative membrane localized ABC protein encoding genes of C. glabrata confirmed their basal expression and showed variable transcriptional response towards antimycotic drugs. This study presents first comprehensive overview of ABC superfamily proteins of a human fungal pathogen C. glabrata, which is expected to provide an important platform for in depth analysis of their physiological relevance in cellular processes and drug resistance.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0202993PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6112666PMC
February 2019

Inventory of ABC proteins and their putative role in salt and drug tolerance in Debaryomyces hansenii.

Gene 2018 Nov 17;676:227-242. Epub 2018 Jul 17.

Amity Institute of Biotechnology and Integrative Sciences and Health, Amity University Haryana, Amity Education Valley Gurgaon, 122413, India. Electronic address:

ATP-binding cassette (ABC) is one of the largest superfamily of proteins, which are ubiquitously present, performing variety of cellular functions. These proteins as drug transporters have been enticing substantial consideration because of their clinical importance. The present study focuses on genome wide identification of ABC proteins of an important halotolerant yeast Debaryomyces hansenii and explores their role in salt and drug tolerance. Our bioinformatics analysis identified a total of 30 putative ABC protein-coding genes whose expression at transcript level was confirmed by qRT-PCR. Our comparative phylogenetic analysis of nucleotide binding domains of D. hansenii and topology prediction categorized these proteins into six subfamilies; ABCB/MDR, ABCC/MRP, ABCD/ALDP, ABCF/YEF3, ABCE/RLI, and ABCG/PDR based on the nomenclature adopted by the Human Genome Organization (HUGO). Further, our transmembrane domain (TMD) predictions suggest that out of 30 ABC proteins, only 22 proteins possess either two or one TMD and hence are considered as membrane localized ABC proteins. Notably, our transcriptional dynamics of ABC proteins encoding genes following D. hansenii cells treatment with different salts and drugs concentrations illustrated variable transcriptional response of some of the genes, pointing to their role in salt and drug tolerance. This study first time provides a comprehensive inventory of the ABC proteins of a haploid D. hansenii which will be helpful for exploring their functional relevance.
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http://dx.doi.org/10.1016/j.gene.2018.07.029DOI Listing
November 2018

Phosphatidylserine decarboxylase governs plasma membrane fluidity and impacts drug susceptibilities of Candida albicans cells.

Biochim Biophys Acta Biomembr 2018 11 29;1860(11):2308-2319. Epub 2018 May 29.

Amity Institute of Integrative Sciences and Health, Amity University Haryana, Amity Education Valley, Gurgaon 122413, India. Electronic address:

Plasma membrane (PM) lipid composition imbalances affect drug susceptibilities of the human pathogen Candida albicans. The PM fundamental structure is made up of phospholipid bilayer where phosphatidylethanolamine (PE) contributes as second major phospholipid moieties, which is asymmetrically distributed between the two leaflets of the bilayer. PSD1 and PSD2 genes encode phosphatidylserine decarboxylase which converts phosphatidylserine (PS) into PE in C. albicans cells. Genetic manipulation of PSD1 and PSD2 genes is known to impact virulence, cell wall thickness and mitochondrial function in C. albicans. In the present study, we have examined the impact of PSD1 and PSD2 deletion on physiochemical properties of PM. Our fluorescence recovery after photobleaching (FRAP) experiments point that the PM of psd1Δ/Δ psd2Δ/Δ mutant strain displays increased membrane fluidity and reduced PM dipole potential. Further, the result of PSD1 and PSD2 deletion on the thermotropic phase behavior monitored by differential scanning calorimetry (DSC) showed that in comparison to WT, the apparent phase transition temperature is reduced by ~3 °C in the mutant strain. The functional consequence of altered physical state of PM of psd1Δ/Δ psd2Δ/Δ mutant strain was evident from observed high diffusion of fluorescent dye rhodamine 6G and radiolabelled fluconazole (FLC). The higher diffusion of FLC resulted in an increased drug accumulation in psd1Δ/Δ psd2Δ/Δ mutant cells, which was manifested in an increased susceptibility to azoles. To the best of our knowledge, these results constitute the first report on the effect of the levels of phospholipid biosynthesis enzyme on physiochemical properties of membranes and drug susceptibilities of Candida cells.
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http://dx.doi.org/10.1016/j.bbamem.2018.05.016DOI Listing
November 2018

Azole resistance in a mutant lacking the ABC transporter depends on TOR signaling.

J Biol Chem 2018 01 20;293(2):412-432. Epub 2017 Nov 20.

From the School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India,

ATP-binding cassette (ABC) transporters help export various substrates across the cell membrane and significantly contribute to drug resistance. However, a recent study reported an unusual case in which the loss of an ABC transporter in , orf19.4531 (previously named ROA1), increases resistance against antifungal azoles, which was attributed to an altered membrane potential in the mutant strain. To obtain further mechanistic insights into this phenomenon, here we confirmed that the plasma membrane-localized transporter (renamed for consistency with nomenclature) could efflux xenobiotics such as berberine, rhodamine 123, and paraquat. Moreover, a null mutant, NKKY101, displayed increased susceptibility to these xenobiotics. Interestingly, fluorescence recovery after photobleaching (FRAP) results indicated that NKKY101 mutant cells exhibited increased plasma membrane rigidity, resulting in reduced azole accumulation and contributing to azole resistance. Transcriptional profiling revealed that ribosome biogenesis genes were significantly up-regulated in the NKKY101 mutant. As ribosome biogenesis is a well-known downstream phenomenon of target of rapamycin (TOR1) signaling, we suspected a link between ribosome biogenesis and TOR1 signaling in NKKY101. Therefore, we grew NKKY101 cells on rapamycin and observed TOR1 hyperactivation, which leads to Hsp90-dependent calcineurin stabilization and thereby increased azole resistance. This finding was supported by data from a mouse model of systemic infection in which NKKY101 cells led to higher fungal load after fluconazole challenge than wild-type cells. Taken together, our study uncovers a mechanism of azole resistance in , involving increased membrane rigidity and TOR signaling.
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http://dx.doi.org/10.1074/jbc.M117.807032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5767851PMC
January 2018

pHluorin enables insights into the transport mechanism of antiporter Mdr1: R215 is critical for drug/H+ antiport.

Biochem J 2016 10 18;473(19):3127-45. Epub 2016 Jul 18.

School of Life Sciences, Jawaharlal Nehru University, New Delhi, India Amity Institute of Integrative Sciences and Health, Amity Institute of Biotechnology, Amity University Haryana, Amity Education Valley, Gurgaon 122413, India.

Multidrug resistance 1 (MDR1) is a member of the major facilitator superfamily that contributes to MDR of Candida albicans This antiporter belongs to the drug/H(+) antiporter 1 family, pairing the downhill gradient of protons to drug extrusion. Hence, drug efflux from cytosol to extracellular space and the parallel import of H(+) towards cytosol are inextricably linked processes. For monitoring the drug/H(+) antiporter activity of Mdr1p, we developed a new system, exploiting a GFP variant pHluorin, which changes its fluorescence properties with pH. This enabled us to measure the cytosolic pH correlated to drug efflux. Since protonation of charged residues is a key step in proton movement, we explored the role of all charged residues of the 12 transmembrane segments (TMSs) of Mdr1p in drug/H(+) transport by mutational analysis. This revealed that the conserved residue R(215), positioned close to the C-terminal end of TMS-4, is critical for drug/H(+) antiport, allowing protonation over a range of pH, in contrast with its H(215) or K(215) variants that failed to transport drugs at basic pH. Mutation of other residues of TMS-4 highlights the role of this TMS in drug transport, as confirmed by in silico modelling of Mdr1p and docking of drugs. The model points to the importance of R(215) in proton transport, suggesting that it may adopt two main conformations, one oriented towards the extracellular face and the other towards the centre of Mdr1p. Together, our results not only establish a new system for monitoring drug/H(+) transport, but also unveil a positively charged residue critical to Mdr1p function.
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http://dx.doi.org/10.1042/BCJ20160407DOI Listing
October 2016

Yeast ABC transporters in lipid trafficking.

Fungal Genet Biol 2016 08 1;93:25-34. Epub 2016 Jun 1.

Membrane Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.

Throughout its evolution, the ATP-binding cassette (ABC) transporter superfamily has experienced a rapid expansion in its substrate repertoire and functions. Of the diverse functions that these pumps offer, their drug transport properties have attracted considerable attention primarily owing to their clinical significance. Despite this fact, emerging evidence suggests that physiological substrates of transporters also affect the overall functioning of an organism. Lipids, as substrates of ABC transporters, constitute one feature found in all representative groups of the living kingdom. Due to the importance of lipid species in the cellular physiology of an organism, their proper distribution within cells is crucial. This fact is well exemplified by the vast number of medical conditions that have been caused as a result of perturbations in ABC transporter-mediated lipid transport in higher organisms. In yeasts, apart from providing transport functions, ABC transporters also coordinate regulatory networks with lipids. This review focuses on yeast ABC transporters involved in the transport of lipids and briefly discusses the integration of their regulatory network with that of the lipid species.
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http://dx.doi.org/10.1016/j.fgb.2016.05.008DOI Listing
August 2016

Pleiotropic effects of the vacuolar ABC transporter MLT1 of Candida albicans on cell function and virulence.

Biochem J 2016 06 29;473(11):1537-52. Epub 2016 Mar 29.

Amity Institute of Integrative Sciences and Health, Amity University Haryana, Amity Education Valley, Gurgaon 122413, India

Among the several mechanisms that contribute to MDR (multidrug resistance), the overexpression of drug-efflux pumps belonging to the ABC (ATP-binding cassette) superfamily is the most frequent cause of resistance to antifungal agents. The multidrug transporter proteins Cdr1p and Cdr2p of the ABCG subfamily are major players in the development of MDR in Candida albicans Because several genes coding for ABC proteins exist in the genome of C. albicans, but only Cdr1p and Cdr2p have established roles in MDR, it is implicit that the other members of the ABC family also have alternative physiological roles. The present study focuses on an ABC transporter of C. albicans, Mlt1p, which is localized in the vacuolar membrane and specifically transports PC (phosphatidylcholine) into the vacuolar lumen. Transcriptional profiling of the mlt1∆/∆ mutant revealed a down-regulation of the genes involved in endocytosis, oxidoreductase activity, virulence and hyphal development. High-throughput MS-based lipidome analysis revealed that the Mlt1p levels affect lipid homoeostasis and thus lead to a plethora of physiological perturbations. These include a delay in endocytosis, inefficient sequestering of reactive oxygen species (ROS), defects in hyphal development and attenuated virulence. The present study is an emerging example where new and unconventional roles of an ABC transporter are being identified.
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http://dx.doi.org/10.1042/BCJ20160024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4888455PMC
June 2016

The ABCs of Candida albicans Multidrug Transporter Cdr1.

Eukaryot Cell 2015 Dec 25;14(12):1154-64. Epub 2015 Sep 25.

Membrane Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.

In the light of multidrug resistance (MDR) among pathogenic microbes and cancer cells, membrane transporters have gained profound clinical significance. Chemotherapeutic failure, by far, has been attributed mainly to the robust and diverse array of these proteins, which are omnipresent in every stratum of the living world. Candida albicans, one of the major fungal pathogens affecting immunocompromised patients, also develops MDR during the course of chemotherapy. The pivotal membrane transporters that C. albicans has exploited as one of the strategies to develop MDR belongs to either the ATP binding cassette (ABC) or the major facilitator superfamily (MFS) class of proteins. The ABC transporter Candida drug resistance 1 protein (Cdr1p) is a major player among these transporters that enables the pathogen to outplay the battery of antifungals encountered by it. The promiscuous Cdr1 protein fulfills the quintessential need of a model to study molecular mechanisms of multidrug transporter regulation and structure-function analyses of asymmetric ABC transporters. In this review, we cover the highlights of two decades of research on Cdr1p that has provided a platform to study its structure-function relationships and regulatory circuitry for a better understanding of MDR not only in yeast but also in other organisms.
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http://dx.doi.org/10.1128/EC.00137-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4664872PMC
December 2015

An assessment of growth media enrichment on lipid metabolome and the concurrent phenotypic properties of Candida albicans.

PLoS One 2014 25;9(11):e113664. Epub 2014 Nov 25.

Membrane Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.

A critical question among the researchers working on fungal lipid biology is whether the use of an enriched growth medium can affect the lipid composition of a cell and, therefore, contribute to the observed phenotypes. One presumption is that enriched medias, such as YPD (yeast extract, peptone and dextrose), are likely to contain lipids, which may homogenize with the yeast lipids and play a role in masking the actual differences in the observed phenotypes or lead to an altered phenotype altogether. To address this issue, we compared the lipids of Candida albicans, our fungus of interest, grown in YPD or in a defined media such as YNB (yeast nitrogen base). Mass spectrometry-based lipid analyses showed differences in the levels of phospholipids, including phosphatidylinositol, phosphatidylglycerol, lyso-phospholipids; sphingolipids, such as mannosyldiinositolphosphorylceramide; and sterols, such as ergostatetraenol. Significant differences were observed in 70 lipid species between the cells grown in the two media, but the two growth conditions did not affect the morphological characteristics of C. albicans. The lipid profiles of the YNB- and YPD-grown C. albicans cells did vary, but these differences did not influence their response to the majority of the tested agents. Rather, the observed differences could be attributed to the slow growth rate of the Candida cells in YNB compared to YPD. Notably, the altered lipid changes between the two media did impact the susceptibility to some drugs. This data provided evidence that changes in media can lead to certain lipid alterations, which may affect specific pathways but, in general, do not affect the majority of the phenotypic properties of C. albicans. It was determined that either YNB or YPD may be suitable for the growth and lipid analysis of C. albicans, depending upon the experimental requirements, but additional precautions are necessary when correlating the phenotypes with the lipids.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0113664PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4244132PMC
January 2016

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

PLoS One 2014 8;9(8):e104554. Epub 2014 Aug 8.

School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0104554PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4126717PMC
December 2015
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