Immunotherapies against human bacterial and fungal infectious diseases: A review.

Nations' ongoing struggles with a number of novel and reemerging infectious diseases, including the ongoing global health issue, the SARS-Co-V2 (severe acute respiratory syndrome coronavirus 2) outbreak, serve as proof that infectious diseases constitute a serious threat to the global public health. Moreover, the fatality rate in humans is rising as a result of the development of severe infectious diseases brought about by multiple drug-tolerant pathogenic microorganisms. The widespread use of traditional antimicrobial drugs, immunosuppressive medications, and other related factors led to the establishment of such drug resistant pathogenic microbial species. To overcome the difficulties commonly encountered by current infectious disease management and control processes, like inadequate effectiveness, toxicities, and the evolution of drug tolerance, new treatment solutions are required. Fortunately, immunotherapies already hold great potential for reducing these restrictions while simultaneously expanding the boundaries of healthcare and medicine, as shown by the latest discoveries and the success of drugs including monoclonal antibodies (MAbs), vaccinations, etc. Immunotherapies comprise methods for treating diseases that specifically target or affect the body's immune system and such immunological procedures/therapies strengthen the host's defenses to fight those infections. The immunotherapy-based treatments control the host's innate and adaptive immune responses, which are effective in treating different pathogenic microbial infections. As a result, diverse immunotherapeutic strategies are being researched more and more as alternative treatments for infectious diseases, leading to substantial improvements in our comprehension of the associations between pathogens and host immune system. In this review we will explore different immunotherapies and their usage for the assistance of a broad spectrum of infectious ailments caused by various human bacterial and fungal pathogenic microbes. We will discuss about the recent developments in the therapeutics against the growing human pathogenic microbial diseases and focus on the present and future of using immunotherapies to overcome these diseases. Graphical AbstractThe graphical abstract shows the therapeutic potential of different types of immunotherapies like vaccines, monoclonal antibodies-based therapies, etc., against different kinds of human Bacterial and Fungal microbial infections.

Physicochemical and Anti-fungal Studies of the Pharmaceutical Co-crystal/Salt of Fluconazole.

Crystal engineering is one green alternative to organic synthesis that can be used to manipulate molecular behavior promptly and economically. We report the preparation and characterization of the pharmaceutical organic salt (FLC-C) of fluconazole (FLC) and organosulfonate (NDSA-2H), based on the sulfonate-pyridinium supramolecular synthon. Structural studies validate the crystallization of the two-component stoichiometric crystal with two molecules of water in the triclinic P1̅ space group. The anticipated proton transfer between the crystal forms leads to ionic interactions, augmenting the organic salt's thermal stability. Hirshfeld studies of FLC-C help to understand the role and significance of different types of intermolecular interactions responsible for crystal packing. The structural and theoretical studies indicate the absence of π-π interactions in FLC-C, which account for the incipience of solid-state emission in the product. The solubility studies establish augmented aqueous solubility of FLC-C over pristine FLC at physiological pH values of 2 and 7. Interestingly, in in vitro studies, FLC-C appears to serve as a potential alternative to FLC, displaying a wide spectrum of antifungal activity. FLC-C is active against several human pathogenic yeast strains, including the leading and emerging Candida strains (Candida albicans and Candida auris, respectively), at comparable and/or lower drug concentrations without showing any enhanced host cell toxicity. Interestingly, the pharmaceutical co-crystal also displays fluorescence properties inside the Candida cells.

Glucose metabolic reprogramming and modulation in glycerol biosynthesis regulates drug resistance in clinical isolates of Candida.

AIMS: The study is aimed at understanding the novel molecular mechanisms governing drug resistance in the opportunistic fungi belonging to the genus Candida. METHODS AND RESULTS: This is a multipronged study wherein different assays like drug susceptibility and whole cell proteome analysis, stress tolerance assay, measurement of total internal glycerol content, western blot analysis, reactive oxygen species (ROS) measurement, glucose uptake, lactate production, ATP generation, and NADPH measurements were made.The study reveals an incidence of different species of Candida in the northern most part of India (Kashmir valley). Resistant isolates, mostly resistant to azoles were reported across all the species. The study revealed a difference in resistance mechanisms between Candida albicans and C. glabrata clinical isolates. Further, such resistance mechanism (in the case of C. albicans) was mostly mediated by Hexokinase 2 (Hxk2) and Glucose-6-phosphate dehydrogenase (G6pd). Increased expression of Hxk2 was associated with increased glucose uptake, more lactate production, and more ATP generation in drug-resistant C. albicans. At the same time, increased G6pd expression was responsible for the increased production of NADPH, which imparts a better ROS scavenging potential. While in C. glabrata the resistance was linked with glycerol metabolism, where the drug-resistant isolate tends to accumulate more glycerol as an osmolyte in response to external stresses. This glycerol accumulation was found to be triggered by the HOG1-MAPK pathway. CONCLUSION: The study concludes that, like various human malignant tumors, there is a strong correlation between drug resistance and aberrant cellular metabolism in the opportunistic fungi belonging to the genus Candida.

ß-Nitrostyrene derivatives as broad range potential antifungal agents targeting fungal cell wall.

The prevalence of multidrug resistance has been increasingly witnessed during the past few decades. Resistance of human pathogenic fungi against the currently available antifungal agents has increased the frequency of fungal infections and associated mortality rates. The discovery of novel lead antifungal agents is important to challenge multidrug resistance. The present study examined the antifungal potential of chemically synthesized ß-Nitrostyrene derivatives. Among the eight ß-Nitrostyrene derivatives used in this study, SS45, SS46 and SS47 showed strong antifungal potential. The results show that ß-Nitrostyrene derivatives inhibited the growth of different species of human pathogenic Candida, particularly the highly prevalent C. albicans, C. glabrata and the emerging pathogenic C. auris species. Moreover, ß-Nitrostyrene derivatives also show strong antifungal activities against drug-resistant clinical isolates and drug transporter overexpressing fungal species. The drug susceptibility assays revealed that ß-Nitrostyrene derivatives are fungicidal and show the synergy of action when combined with antifungal drugs caspofungin and fluconazole. The transcriptomic study performed on C. albicans in the presence of ß-Nitrostyrene derivatives revealed the differential expression of genes related to cell wall metabolism. Mechanistically, ß-Nitrostyrene derivatives impact cell wall morphology, enhance ROS generation and modulate drug efflux. Collectively this study reveals that ß-Nitrostyrene derivatives have strong antifungal potential with a particular mode of activity similar to known cell wall perturbing antifungal agents and thus can be exploited as promising potential antifungal agents for further studies.

Metabolic flexibility and extensive adaptability governing multiple drug resistance and enhanced virulence in Candida albicans.

Commensal fungus-Candida albicans turn pathogenic during the compromised immunity of the host, causing infections ranging from superficial mucosal to dreadful systemic ones. C. albicans has evolved various adaptive measures which collectively contribute towards its enhanced virulence. Among fitness attributes, metabolic flexibility and vigorous stress response are essential for its pathogenicity and virulence. Metabolic flexibility provides a means for nutrient assimilation and growth in diverse host microenvironments and reduces the vulnerability of the pathogen to various antifungals besides evading host immune response(s). Inside the host micro-environments, C. albicans efficiently utilizes the multiple fermentable and non-fermentable carbon sources to sustain and proliferate in glucose deficit conditions. The utilization of alternative carbon sources further highlights the importance of understanding these pathways as the attractive and potential therapeutic target. A thorough understanding of metabolic flexibility and adaptation to environmental stresses is warranted to decipher in-depth insights into virulence and molecular mechanisms of fungal pathogenicity. In this review, we have attempted to provide a detailed and recent understanding of some key aspects of fungal biology. Particular focus will be placed on processes like nutrient assimilation and utilization, metabolic adaptability, virulence factors, and host immune response in C. albicans leading to its enhanced pathogenicity.

Glucose - The X factor for the survival of human fungal pathogens and disease progression in the host.

The incidence of human fungal infections is increasing due to the expansion of the immunocompromised patient population. The continuous use of different antifungal agents has eventually resulted in the establishment of resistant fungal species. The fungal pathogens unfold multiple resistance strategies to successfully tackle the effect of different antifungal agents. For the successful colonization and establishment of infection inside the host, the pathogenic fungi switch to the process of metabolic flexibility to regulate distinct nutrient uptake systems as well as to modulate their metabolism accordingly. Glucose the most favourable carbon source helps carry out the important survival and niche colonization processes. Adopting glucose as the center, this review has been put forward to provide an outline of the important processes like growth, the progression of infection, and the metabolism regulated by glucose, affecting the pathogenicity and virulence traits in the human pathogenic fungi. This could help in the identification of better treatment options and appropriate target-oriented antifungal drugs based on the glucose-regulated pathways and processes. In the article, we have also presented a summary of the novel studies and findings pointing to glucose-based potential therapeutic avenues to be explored to tackle the problem of globally increasing multidrug-resistant human fungal infections.

Identification of Genomewide Alternative Splicing Events in Sequential, Isogenic Clinical Isolates of Candida albicans Reveals a Novel Mechanism of Drug Resistance and Tolerance to Cellular Stresses.

Alternative splicing (AS)-a process by which a single gene gives rise to different protein isoforms in eukaryotes-has been implicated in many basic cellular processes, but little is known about its role in drug resistance and fungal pathogenesis. The most common human fungal pathogen, Candida albicans, has introns in 4 to 6% of its genes, the functions of which remain largely unknown. Here, we report AS regulating drug resistance in C. albicans Comparative RNA-sequencing of two different sets of sequential, isogenic azole-sensitive and -resistant isolates of C. albicans revealed differential expression of splice isoforms of 14 genes. One of these was the superoxide dismutase gene SOD3, which contains a single intron. The sod3Δ/Δ mutant was susceptible to the antifungals amphotericin B (AMB) and menadione (MND). While AMB susceptibility was rescued by overexpression of both the spliced and unspliced SOD3 isoforms, only the spliced isoform could overcome MND susceptibility, demonstrating the functional relevance of this splicing in developing drug resistance. Furthermore, unlike AMB, MND inhibits SOD3 splicing and acts as a splicing inhibitor. Consistent with these observations, MND exposure resulted in increased levels of unspliced SOD3 isoform that are unable to scavenge reactive oxygen species (ROS), resulting in increased drug susceptibility. Collectively, these observations suggest that AS is a novel mechanism for stress adaptation and overcoming drug susceptibility in C. albicansIMPORTANCE The emergence of resistance in Candida albicans, an opportunistic pathogen, against the commonly used antifungals is becoming a major obstacle in its treatment. The necessity to identify new drug targets demands fundamental insights into the mechanisms used by this organism to develop drug resistance. C. albicans has introns in 4 to 6% of its genes, the functions of which remain largely unknown. Using the RNA-sequencing data from isogenic pairs of azole-sensitive and -resistant isolates of C. albicans, here, we show how C. albicans uses modulations in mRNA splicing to overcome antifungal drug stress.

Quorum sensing: A less known mode of communication among fungi.

Quorum sensing (QS), a density-dependent signaling mechanism of microbial cells, involves an exchange and sense of low molecular weight signaling compounds called autoinducers. With the increase in population density, the autoinducers accumulate in the extracellular environment and once their concentration reaches a threshold, many genes are either expressed or repressed. This cell density-dependent signaling mechanism enables single cells to behave as multicellular organisms and regulates different microbial behaviors like morphogenesis, pathogenesis, competence, biofilm formation, bioluminescence, etc guided by environmental cues. Initially, QS was regarded to be a specialized system of certain bacteria. The discovery of filamentation control in pathogenic polymorphic fungus Candida albicans by farnesol revealed the phenomenon of QS in fungi as well. Pathogenic microorganisms primarily regulate the expression of virulence genes using QS systems. The indirect role of QS in the emergence of multiple drug resistance (MDR) in microbial pathogens necessitates the finding of alternative antimicrobial therapies that target QS and inhibit the same. A related phenomenon of quorum sensing inhibition (QSI) performed by small inhibitor molecules called quorum sensing inhibitors (QSIs) has an ability for efficient reduction of gene expression regulated by quorum sensing. In the present review, recent advancements in the study of different fungal quorum sensing molecules (QSMs) and quorum sensing inhibitors (QSIs) of fungal origin along with their mechanism of action and/or role/s are discussed.

W1038 near D-loop of NBD2 is a focal point for inter-domain communication in multidrug transporter Cdr1 of Candida albicans.

Candida drug resistance 1 (Cdr1), a PDR subfamily ABC transporter mediates efflux of xenobiotics in Candida albicans. It is one of the prime factors contributing to multidrug resistance in the fungal pathogen. One hallmark of this transporter is its asymmetric nature, characterized by peculiar alterations in its nucleotide binding domains. As a consequence, there exists only one canonical ATP-binding site while the other is atypical. Here, we report suppressor analysis on the drug-susceptible transmembrane domain mutant V532D that identified the suppressor mutation W1038S, close to the D-loop of the non-catalytic ATP-binding site. Introduction of the W1038S mutation in the background of V532D mutant conferred resistance for most of the substrates to the latter. Such restoration is accompanied by a severe reduction of ATPase activity, of about 85%, while that of the V532D mutant is half-reduced. Conversely, alanine substitution of the highly conserved aspartate D1033A in that D-loop rendered cells selectively hyper-susceptible to miconazole without an impact on steady-state ATPase activity, suggesting altogether that ATP hydrolysis may not hold the key to restoration mechanism. Analysis of the ABCG5/ABCG8-based 3D-model of Cdr1p suggested that the W1038S substitution leads to the loss of hydrophobic interactions and H-bond with residues of the neighbor NBD1, in the non-catalytic ATP-binding site area. The compensatory effect within TMDs accounting for transport restoration in the V532D-W1038S variant may, therefore, be mainly due to an increase in NBDs mobility at the non-catalytic interface.

Molecular Basis of Substrate Polyspecificity of the Candida albicans Mdr1p Multidrug/H+ Antiporter.

The molecular basis of polyspecificity of Mdr1p, a major drug/H+ antiporter of Candida albicans, is not elucidated. We have probed the nature of the drug-binding pocket by performing systematic mutagenesis of the 12 transmembrane segments. Replacement of the 252 amino acid residues with alanine or glycine yielded 2/3 neutral mutations while 1/3 led to the complete or selective loss of resistance to drugs or substrates transported by the pump. Using the GlpT-based 3D-model of Mdr1p, we roughly categorized these critical residues depending on their type and localization, 1°/ main structural impact ("S" group), 2°/ exposure to the lipid interface ("L" group), 3°/ buried but not facing the main central pocket, inferred as critical for the overall H+/drug antiport mechanism ("M" group) and finally 4°/ buried and facing the main central pocket ("B" group). Among "B" category, 13 residues were essential for the large majority of drugs/substrates, while 5 residues were much substrate-specific, suggesting a role in governing polyspecificity (P group). 3D superposition of the substrate-specific MFS Glut1 and XylE with the MDR substrate-polyspecific MdfA and Mdr1p revealed that the B group forms a common substrate interaction core while the P group is only found in the 2 MDR MFS transporters, distributed into 3 areas around the B core. This specific pattern has let us to propose that the structural basis for polyspecificity of MDR MFS transporters is the extended capacity brought by residues located at the periphery of a binding core to accomodate compounds differing in size and type.

Resistance to antifungal therapies.

The evolution of antifungal resistance among fungal pathogens has rendered the limited arsenal of antifungal drugs futile. Considering the recent rise in the number of nosocomial fungal infections in immunocompromised patients, the emerging clinical multidrug resistance (MDR) has become a matter of grave concern for medical professionals. Despite advances in therapeutic interventions, it has not yet been possible to devise convincing strategies to combat antifungal resistance. Comprehensive understanding of the molecular mechanisms of antifungal resistance is essential for identification of novel targets that do not promote or delay emergence of drug resistance. The present study discusses features and limitations of the currently available antifungals, mechanisms of antifungal resistance and highlights the emerging therapeutic strategies that could be deployed to combat MDR.

Newly identified motifs in Candida albicans Cdr1 protein nucleotide binding domains are pleiotropic drug resistance subfamily-specific and functionally asymmetric.

An analysis of Candida albicans ABC transporters identified conserved related α-helical sequence motifs immediately C-terminal of each Walker A sequence. Despite the occurrence of these motifs in ABC subfamilies of other yeasts and higher eukaryotes, their roles in protein function remained unexplored. In this study we have examined the functional significance of these motifs in the C. albicans PDR transporter Cdr1p. The motifs present in NBD1 and NBD2 were subjected to alanine scanning mutagenesis, deletion, or replacement of an entire motif. Systematic replacement of individual motif residues with alanine did not affect the function of Cdr1p but deletion of the M1-motif in NBD1 (M1-Del) resulted in Cdr1p being trapped within the endoplasmic reticulum. In contrast, deletion of the M2-motif in NBD2 (M2-Del) yielded a non-functional protein with normal plasma membrane localization. Replacement of the motif in M1-Del with six alanines (M1-Ala) significantly improved localization of the protein and partially restored function. Conversely, replacement of the motif in M2-Del with six alanines (M2-Ala) did not reverse the phenotype and susceptibility to antifungal substrates of Cdr1p was unchanged. Together, the M1 and M2 motifs contribute to the functional asymmetry of NBDs and are important for maturation of Cdr1p and ATP catalysis, respectively.

Antifungals: Mechanism of Action and Drug Resistance.

There are currently few antifungals in use which show efficacy against fungal diseases. These antifungals mostly target specific components of fungal plasma membrane or its biosynthetic pathways. However, more recent class of antifungals in use is echinocandins which target the fungal cell wall components. The availability of mostly fungistatic antifungals in clinical use, often led to the development of tolerance to these very drugs by the pathogenic fungal species. Thus, the development of clinical multidrug resistance (MDR) leads to higher tolerance to drugs and its emergence is helped by multiple mechanisms. MDR is indeed a multifactorial phenomenon wherein a resistant organism possesses several mechanisms which contribute to display reduced susceptibility to not only single drug in use but also show collateral resistance to several drugs. Considering the limited availability of antifungals in use and the emergence of MDR in fungal infections, there is a continuous need for the development of novel broad spectrum antifungal drugs with better efficacy. Here, we briefly present an overview of the current understanding of the antifungal drugs in use, their mechanism of action and the emerging possible novel antifungal drugs with great promise.

Candida Efflux ATPases and Antiporters in Clinical Drug Resistance.

An enhanced expression of genes encoding ATP binding cassette (ABC) and major facilitator superfamily (MFS) transport proteins are known to contribute to the development of tolerance to antifungals in pathogenic yeasts. For example, the azole resistant (AR) clinical isolates of the opportunistic human fungal pathogen Candida albicans show an overexpression of CDR1 and/or CaMDR1 belonging to ABC and MFS, superfamilies, respectively. The reduced accumulation (due to rapid efflux) of drugs in AR isolates confirms the role of efflux pump proteins in the development of drug tolerance. Considering the importance of major multidrug transporters, the focus of recent research has been to understand the structure and function of these proteins which could help to design inhibitors/modulators of these pump proteins. This chapter focuses on some aspects of the structure and function of yeast transporter proteins particularly in relation to MDR in Candida.

Mutational Analysis of Intracellular Loops Identify Cross Talk with Nucleotide Binding Domains of Yeast ABC Transporter Cdr1p.

The ABC transporter Cdr1 protein (Cdr1p) of Candida albicans, which plays a major role in antifungal resistance, has two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs) that are interconnected by extracellular (ECLs) and intracellular (ICLs) loops. To examine the communication interface between the NBDs and ICLs of Cdr1p, we subjected all four ICLs to alanine scanning mutagenesis, replacing each of the 85 residues with an alanine. The resulting ICL mutant library was analyzed by biochemical and phenotypic mapping. Only 18% of the mutants from this library displayed enhanced drug susceptibility. Most of the drug-susceptible mutants displayed uncoupling between ATP hydrolysis and drug transport. The two drug-susceptible ICL1 mutants (I574A and S593A) that lay within or close to the predicted coupling helix yielded two chromosomal suppressor mutations that fall near the Q-loop of NBD2 (R935) and in the Walker A motif (G190) of NBD1. Based on a 3D homology model and kinetic analysis of drug transport, our data suggest that large distances between ICL residues and their respective chromosomal suppressor mutations rule out a direct interaction between them. However, they impact the transport cycle by restoring the coupling interface via indirect downstream signaling.

ABC transporter Cdr1p harbors charged residues in the intracellular loop and nucleotide-binding domain critical for protein trafficking and drug resistance.

The ABC transporter Cdr1 protein of Candida albicans, which plays a major role in antifungal resistance, has two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs). The 12 transmembrane helices of TMDs that are interconnected by extracellular and intracellular loops (ICLs) mainly harbor substrate recognition sites where drugs bind while cytoplasmic NBDs hydrolyze ATP which powers drug efflux. The coupling of ATP hydrolysis to drug transport requires proper communication between NBDs and TMDs typically accomplished by ICLs. This study examines the role of cytoplasmic ICLs of Cdr1p by rationally predicting the critical residues on the basis of their interatomic distances. Among nine pairs that fall within a proximity of <4 Å, an ion pair between K577 of ICL1 and E315 of NBD1 was found to be critical. The substitution, swapping and changing of the length or charge of K577 or E315 by directed mutagenesis led to a misfolded, non-rescuable protein entrapped in intracellular structures. Furthermore, the equipositional ionic pair-forming residues from ICL3 and NBD2 (R1260 and E1014) did not impact protein trafficking. These results point to a new role for ICL/NBD interacting residues in PDR ABC transporters in protein folding and trafficking.

Novel role of a family of major facilitator transporters in biofilm development and virulence of Candida albicans.

The QDR (quinidine drug resistance) family of genes encodes transporters belonging to the MFS (major facilitator superfamily) of proteins. We show that QDR transporters, which are localized to the plasma membrane, do not play a role in drug transport. Hence, null mutants of QDR1, QDR2 and QDR3 display no alterations in susceptibility to azoles, polyenes, echinocandins, polyamines or quinolines, or to cell wall inhibitors and many other stresses. However, the deletion of QDR genes, individually or collectively, led to defects in biofilm architecture and thickness. Interestingly, QDR-lacking strains also displayed attenuated virulence, but the strongest effect was observed with qdr2∆, qdr3∆ and in qdr1/2/3∆ strains. Notably, the attenuated virulence and biofilm defects could be reversed upon reintegration of QDR genes. Transcripts profiling confirmed differential expression of many biofilm and virulence-related genes in the deletion strains as compared with wild-type Candida albicans cells. Furthermore, lipidomic analysis of QDR-deletion mutants suggests massive remodelling of lipids, which may affect cell signalling, leading to the defect in biofilm development and attenuation of virulence. In summary, the results of the present study show that QDR paralogues encoding MFS antiporters do not display conserved functional linkage as drug transporters and perform functions that significantly affect the virulence of C. albicans.

In vitro effect of malachite green on Candida albicans involves multiple pathways and transcriptional regulators UPC2 and STP2.

In this study, we show that a chemical dye, malachite green (MG), which is commonly used in the fish industry as an antifungal, antiparasitic, and antibacterial agent, could effectively kill Candida albicans and non-C. albicans species. We have demonstrated that Candida cells are susceptible to MG at a very low concentration (MIC that reduces growth by 50% [MIC(50)], 100 ng ml(-1)) and that the effect of MG is independent of known antifungal targets, such as ergosterol metabolism and major drug efflux pump proteins. Transcriptional profiling in response to MG treatment of C. albicans cells revealed that of a total of 207 responsive genes, 167 genes involved in oxidative stress, virulence, carbohydrate metabolism, heat shock, amino acid metabolism, etc., were upregulated, while 37 genes involved in iron acquisition, filamentous growth, mitochondrial respiration, etc., were downregulated. We confirmed experimentally that Candida cells exposed to MG resort to a fermentative mode of metabolism, perhaps due to defective respiration. In addition, we showed that MG triggers depletion of intracellular iron pools and enhances reactive oxygen species (ROS) levels. These effects could be reversed by the addition of iron or antioxidants, respectively. We provided evidence that the antifungal effect of MG is exerted through the transcription regulators UPC2 (regulating ergosterol biosynthesis and azole resistance) and STP2 (regulating amino acid permease genes). Taken together, our transcriptome, genetic, and biochemical results allowed us to decipher the multiple mechanisms by which MG exerts its anti-Candida effects, leading to a metabolic shift toward fermentation, increased generation of ROS, labile iron deprivation, and cell necrosis.

Alanine scanning of all cysteines and construction of a functional cysteine-less Cdr1p, a multidrug ABC transporter of Candida albicans.

Herein, we discuss the role of the native cysteines present in a major multidrug ABC transporter of Candida albicans, Cdr1p, and describe the construction of this transporter's functional cysteine-less (cysless) protein version for cross-linking studies. In the experiments in which all 23 cysteines were replaced individually, we observed that most of the cysteine replacements were tolerated by the protein, but the replacement of C1056, C1091, C1106, C1294 or C1336 resulted in an enhanced drug susceptibility together with an abrogated drug efflux. Notably, the ATPase activity was uncoupled, which largely remained unaffected in these variants. The substitution of the critical cysteines with serines restored the normal expression and functionality of Cdr1p because serine can effectively mimic the hydrogen bonding properties of cysteine. Finally, we constructed a functional cysless His-tagged Cdr1p in which all the cysteines of the native protein were replaced with alanines and the critical cysteines were replaced with serines. Notably, cysless GFP-tagged variant of Cdr1p was non-functional. The cysless His-tagged variant of Cdr1p is the first example of a cysless ABC transporter in yeast, and it will lead to a greater understanding of the architecture of this important protein and provide insight into the nature of drug binding and interdomain communication.