Analysis of clinical Candida parapsilosis isolates reveals copy number variation in key fluconazole resistance genes.

We used whole-genome sequencing to analyze a collection of 35 fluconazole-resistant and 7 susceptible Candida parapsilosis isolates together with coverage analysis and GWAS techniques to identify new mechanisms of fluconazole resistance. Phylogenetic analysis shows that although the collection is diverse, two persistent clinical lineages were identified. We identified copy number variation (CNV) of two genes, ERG11 and CDR1B, in resistant isolates. Two strains have a CNV at the ERG11 locus; the entire ORF is amplified in one, and only the promoter region is amplified in the other. We show that the annotated telomeric gene CDR1B is actually an artifactual in silico fusion of two highly similar neighboring CDR genes due to an assembly error in the C. parapsilosis CDC317 reference genome. We report highly variable copy numbers of the CDR1B region across the collection. Several strains have increased the expansion of the two genes into a tandem array of new chimeric genes. Other strains have experienced a deletion between the two genes creating a single gene with a reciprocal chimerism. We find translocations, duplications, and gene conversion across the CDR gene family in the C. parapsilosis species complex, showing that it is a highly dynamic family.

A secondary mechanism of action for triazole antifungals in Aspergillus fumigatus mediated by hmg1.

Triazole antifungals function as ergosterol biosynthesis inhibitors and are frontline therapy for invasive fungal infections, such as invasive aspergillosis. The primary mechanism of action of triazoles is through the specific inhibition of a cytochrome P450 14-α-sterol demethylase enzyme, Cyp51A/B, resulting in depletion of cellular ergosterol. Here, we uncover a clinically relevant secondary mechanism of action for triazoles within the ergosterol biosynthesis pathway. We provide evidence that triazole-mediated inhibition of Cyp51A/B activity generates sterol intermediate perturbations that are likely decoded by the sterol sensing functions of HMG-CoA reductase and Insulin-Induced Gene orthologs as increased pathway activity. This, in turn, results in negative feedback regulation of HMG-CoA reductase, the rate-limiting step of sterol biosynthesis. We also provide evidence that HMG-CoA reductase sterol sensing domain mutations previously identified as generating resistance in clinical isolates of Aspergillus fumigatus partially disrupt this triazole-induced feedback. Therefore, our data point to a secondary mechanism of action for the triazoles: induction of HMG-CoA reductase negative feedback for downregulation of ergosterol biosynthesis pathway activity. Abrogation of this feedback through acquired mutations in the HMG-CoA reductase sterol sensing domain diminishes triazole antifungal activity against fungal pathogens and underpins HMG-CoA reductase-mediated resistance.

Mutations in TAC1 and ERG11 are major drivers of triazole antifungal resistance in clinical isolates of Candida parapsilosis.

OBJECTIVES: The aim of this study was to determine how mutations in CpERG11 and CpTAC1 contribute to fluconazole resistance in a collection of clinical isolates. METHODS: Sequences of CpERG11 and CpTAC1 were determined for 35 resistant Candida parapsilosis clinical isolates. A plasmid-based CRISPR-Cas9 system was used to introduce mutations leading to amino acid substitution in CpTac1 and CpErg11. Triazole susceptibility was determined by broth microdilution and E-test. Differential expression of genes mediated by CpTAC1 mutation was determined by RNA sequencing, and relative expression of individual transporter genes was assessed with RT-qPCR. RESULTS: Six isolates carried a mutation in CpTAC1 in combination with the CpERG11 mutation, leading to the CpErg11Y132F substitution. When introduced into susceptible isolates, this CpERG11 mutation led to a 4- to 8-fold increase in fluconazole minimum inhibitory concentrations (MIC; 0.125 µg/mL vs. 0.5 µg/mL, 0.125 µg/mL vs. 0.5 µg/mL, and 0.5 µg/mL vs. 4 µg/mL). When introduced into a susceptible isolate, the CpTAC1 mutation leading to the G650E substitution resulted in an 8-fold increase in fluconazole MIC (0.25 µg/mL vs. 2 µg/mL), whereas correction of this mutation in resistant isolates led to a 16-fold reduction in MIC (32 µg/mL vs. 2 µg/mL). CpCDR1, CpCDR1B, and CpCDR1C were overexpressed in the presence CpTac1G650E. Disruption of these genes in combination resulted in a 4-fold reduction in fluconazole MIC (32 µg/mL vs. 8 µg/mL). DISCUSSION: These results define the specific contribution made by the Y132F substitution in CpERG11 and demonstrate a role for activating mutations in CpTAC1 in triazole resistance in C. parapsilosis.

The sterol C-24 methyltransferase encoding gene, erg6, is essential for viability of Aspergillus species.

Ergosterol is a critical component of fungal plasma membranes. Although many currently available antifungal compounds target the ergosterol biosynthesis pathway for antifungal effect, current knowledge regarding ergosterol synthesis remains incomplete for filamentous fungal pathogens like Aspergillus fumigatus. Here, we show for the first time that the lipid droplet-associated sterol C-24 methyltransferase, Erg6, is essential for A. fumigatus viability. We further show that this essentiality extends to additional Aspergillus species, including A. lentulus, A. terreus, and A. nidulans. Neither the overexpression of a putative erg6 paralog, smt1, nor the exogenous addition of ergosterol could rescue erg6 deficiency. Importantly, Erg6 downregulation results in a dramatic decrease in ergosterol and accumulation in lanosterol and is further characterized by diminished sterol-rich plasma membrane domains (SRDs) at hyphal tips. Unexpectedly, erg6 repressed strains demonstrate wild-type susceptibility against the ergosterol-active triazole and polyene antifungals. Finally, repressing erg6 expression reduced fungal burden accumulation in a murine model of invasive aspergillosis. Taken together, our studies suggest that Erg6, which shows little homology to mammalian proteins, is potentially an attractive antifungal drug target for therapy of Aspergillus infections.

hapE and hmg1 Mutations Are Drivers of cyp51A-Independent Pan-Triazole Resistance in an Aspergillus fumigatus Clinical Isolate.

Aspergillus fumigatus is a ubiquitous environmental mold that can cause severe disease in immunocompromised patients and chronic disease in individuals with underlying lung conditions. Triazoles are the most widely used class of antifungal drugs to treat A. fumigatus infections, but their use in the clinic is threatened by the emergence of triazole-resistant isolates worldwide, reinforcing the need for a better understanding of resistance mechanisms. The predominant mechanisms of A. fumigatus triazole resistance involve mutations affecting the promoter region or coding sequence of the target enzyme of the triazoles, Cyp51A. However, triazole-resistant isolates without cyp51A-associated mutations are frequently identified. In this study, we investigate a pan-triazole-resistant clinical isolate, DI15-105, that simultaneously carries the mutations hapEP88L and hmg1F262del, with no mutations in cyp51A. Using a Cas9-mediated gene-editing system, hapEP88L and hmg1F262del mutations were reverted in DI15-105. Here, we show that the combination of these mutations accounts for pan-triazole resistance in DI15-105. To our knowledge, DI15-105 is the first clinical isolate reported to simultaneously carry mutations in hapE and hmg1 and only the second with the hapEP88L mutation. IMPORTANCE Triazole resistance is an important cause of treatment failure and high mortality rates for A. fumigatus human infections. Although Cyp51A-associated mutations are frequently identified as the cause of A. fumigatus triazole resistance, they do not explain the resistance phenotypes for several isolates. In this study, we demonstrate that hapE and hmg1 mutations additively contribute to pan-triazole resistance in an A. fumigatus clinical isolate lacking cyp51-associated mutations. Our results exemplify the importance of and the need for a better understanding of cyp51A-independent triazole resistance mechanisms.

Analysis of clinical Candida parapsilosis isolates reveals copy number variation in key fluconazole resistance genes.

We used whole-genome sequencing to analyse a collection of 35 fluconazole resistant and 7 susceptible Candida parapsilosis isolates together with coverage analysis and GWAS techniques to identify new mechanisms of fluconazole resistance. Phylogenetic analysis shows that although the collection is diverse, two probable outbreak groups were identified. We identified copy number variation of two genes, ERG11 and CDR1B, in resistant isolates. Two strains have a CNV at the ERG11 locus; the entire ORF is amplified in one, and only the promoter region is amplified in the other. We show the annotated telomeric gene CDR1B is actually an artefactual in silico fusion of two highly similar neighbouring CDR genes due to an assembly error in the C. parapsilosis CDC317 reference genome. We report highly variable copy numbers of the CDR1B region across the collection. Several strains have increased expansion of the two genes into a tandem array of new chimeric genes. Other strains have experienced a deletion between the two genes creating a single gene with a reciprocal chimerism. We find translocations, duplications, and gene conversion across the CDR gene family in the C. parapsilosis species complex, showing that it is a highly dynamic family.

The molecular and genetic basis of antifungal resistance in the emerging fungal pathogen Candida auris.

Fungal infections are responsible for significant morbidity and mortality. Resistance to the limited number of agents in the antifungal armamentarium among pathogenic fungi represents a growing public health threat. Particularly concerning is the emerging fungal pathogen Candida auris that frequently exhibits resistance to the triazole class of antifungals and amphotericin B, and for which isolates resistant to all of the major antifungal classes have been reported. In this brief review, we provide an overview of what is currently known about the molecular and genetic basis for antifungal resistance in this fungal pathogen.

Candida parapsilosis Mdr1B and Cdr1B Are Drivers of Mrr1-Mediated Clinical Fluconazole Resistance.

Candida parapsilosis is a common cause of invasive candidiasis worldwide and is the most commonly is7olated Candida species among pediatric and neonatal populations. Previous work has demonstrated that nonsynonymous mutations in the gene encoding the putative transcription factor CpMrr1 can influence fluconazole susceptibility. However, the direct contribution of these mutations and how they influence fluconazole resistance in clinical isolates are poorly understood. We identified 7 nonsynonymous CpMRR1 mutations in 12 isolates from within a collection of 35 fluconazole-resistant clinical isolates. The mutations leading to the A854V, R479K, and I283R substitutions were further examined and found to be activating mutations leading to increased fluconazole resistance. In addition to CpMDR1, we identified two other genes, one encoding a major facilitator superfamily (MFS) transporter (CpMDR1B, CPAR2_603010) and one encoding an ATP-binding cassette (ABC) transporter (CpCDR1B, CPAR2_304370), as being upregulated in isolates carrying CpMRR1-activating mutations. Overexpression of CpMDR1 in a susceptible strain and disruption in resistant clinical isolates that overexpress CpMDR1 had little to no effect on fluconazole susceptibility. Conversely, overexpression of either CpMDR1B or CpCDR1B increased resistance, and disruption in clinical isolates overexpressing these genes decreased fluconazole resistance. Our findings suggest that activating mutations in CpMRR1 represent important genetic determinants of fluconazole resistance in clinical isolates of C. parapsilosis, and unlike what is observed in Candida albicans, this is primarily driven by upregulation of both MFS (CpMdr1B) and ABC (CpCdr1B) transporters.

In vivo emergence of high-level resistance during treatment reveals the first identified mechanism of amphotericin B resistance in Candida auris.

OBJECTIVE: Candida auris has emerged as a health-care-associated and multidrug-resistant fungal pathogen of great clinical concern. As many as 50% of C. auris clinical isolates are reported to be resistant to amphotericin B, but no mechanisms contributing to this resistance have been identified. Here we describe a clinical case in which high-level amphotericin B resistance was acquired in vivo during therapy and undertake molecular and genetic studies to identify and characterize the genetic determinant of resistance. METHODS: Whole-genome sequencing was performed on four C. auris isolates obtained from a single patient case. Cas9-mediated genetic manipulations were then used to generate mutant strains harbouring mutations of interest, and these strains were subsequently subjected to amphotericin B susceptibility testing and comprehensive sterol profiling. RESULTS: A novel mutation in the C. auris sterol-methyltransferase gene ERG6 was found to be associated with amphotericin B resistance, and this mutation alone conferred a >32-fold increase in amphotericin B resistance. Comprehensive sterol profiling revealed an abrogation of ergosterol biosynthesis and a corresponding accumulation of cholesta-type sterols in isolates and strains harbouring the clinically derived ERG6 mutation. CONCLUSIONS: Together these findings definitively demonstrate mutations in C. auris ERG6 as the first identified mechanism of clinical amphotericin B resistance in C. auris and represent a significant step forward in the understanding of antifungal resistance in this emerging public health threat.

Delineation of the Direct Contribution of Candida auris ERG11 Mutations to Clinical Triazole Resistance.

Resistance to fluconazole is one of clinical characteristics most frequently challenging the treatment of invasive Candida auris infections, and is observed among >90% of all characterized clinical isolates. In this work, the native C. auris ERG11 allele in a previously characterized fluconazole-susceptible clinical isolate was replaced with the ERG11 alleles from three highly fluconazole-resistant clinical isolates (MIC ≥256 mg/L), encoding the amino acid substitutions VF125AL, Y132F, and K143R, using Cas9-ribonucleoprotein (RNP) mediated transformation system. Reciprocally, the ERG11WT allele from the same fluconazole-susceptible clinical isolate, lacking any resistance-associated mutation, was introduced into a previously characterized fluconazole-resistant clinical isolate, replacing the native ERG11K143R allele, using the same methods. The resulting collection of strains was subjected to comprehensive triazole susceptibility testing, and the direct impact each of these clinically-derived ERG11 mutations on triazole MIC was determined. Introduction of each of the three mutant ERG11 alleles was observed to increase fluconazole and voriconazole MIC by 8- to 16-fold. The MIC for the other clinically available triazoles were not significantly impacted by any ERG11 mutation. In the fluconazole-resistant clinical isolate background, correction of the K143R encoding mutation led to a similar 16-fold decrease in fluconazole MIC, and 8-fold decrease in voriconazole MIC, while the MIC of other triazoles were minimally changed. Taken together, these findings demonstrate that mutations in C. auris ERG11 significantly contribute to fluconazole and voriconazole resistance, but alone cannot explain the substantially elevated MIC observed among clinical isolates of C. auris. IMPORTANCE Candida auris is an emerging multidrug-resistant and health care-associated pathogen of urgent clinical concern. The triazoles are the most widely prescribed antifungal agents worldwide and are commonly utilized for the treatment of invasive Candida infections. Greater than 90% of all C. auris clinical isolates are observed to be resistant to fluconazole, and nearly all fluconazole-resistant isolates of C. auris are found to have one of three mutations (encoding VF125AL, Y132F, or K143R) in the gene encoding the target of the triazoles, ERG11. However, the direct contribution of these mutations in ERG11 to fluconazole resistance and the impact these mutations may have the susceptibility of the other triazoles remains unknown. The present study seeks to address this knowledge gap and potentially inform the future application the triazole antifungals for the treatment of infections caused by C. auris.

Loss-of-Function ROX1 Mutations Suppress the Fluconazole Susceptibility of upc2AΔ Mutation in Candida glabrata, Implicating Additional Positive Regulators of Ergosterol Biosynthesis.

Two of the major classes of antifungal drugs in clinical use target ergosterol biosynthesis. Despite its importance, our understanding of the transcriptional regulation of ergosterol biosynthesis genes in pathogenic fungi is essentially limited to the role of hypoxia and sterol-stress-induced transcription factors such as Upc2 and Upc2A as well as homologs of sterol response element binding (SREB) factors. To identify additional regulators of ergosterol biosynthesis in Candida glabrata, an important human fungal pathogen with reduced susceptibility to ergosterol biosynthesis inhibitors relative to other Candida spp., we used a serial passaging strategy to isolate suppressors of the fluconazole hypersusceptibility of a upc2AΔ deletion mutant. This led to the identification of loss-of-function mutations in two genes: ROX1, the homolog of a hypoxia gene transcriptional suppressor in Saccharomyces cerevisiae, and CST6, a transcription factor that is involved in the regulation of carbon dioxide response in C. glabrata. Here, we describe a detailed analysis of the genetic interaction of ROX1 and UPC2A. In the presence of fluconazole, loss of Rox1 function restores ERG11 expression to the upc2AΔ mutant and inhibits the expression of ERG3 and ERG6, leading to increased levels of ergosterol and decreased levels of the toxic sterol 14α methyl-ergosta-8,24(28)-dien-3ß, 6α-diol, relative to the upc2AΔ mutant. Our observations establish that Rox1 is a negative regulator of ERG gene biosynthesis and indicate that a least one additional positive transcriptional regulator of ERG gene biosynthesis must be present in C. glabrata. IMPORTANCE Candida glabrata is one of the most important human fungal pathogens and has reduced susceptibility to azole-class inhibitors of ergosterol biosynthesis. Although ergosterol is the target of two of the three classes of antifungal drugs, relatively little is known about the regulation of this critical cellular pathway. Sterols are both essential components of the eukaryotic plasma membrane and potential toxins; therefore, sterol homeostasis is critical for cell function. Here, we identified two new negative regulators in C. glabrata of ergosterol (ERG) biosynthesis gene expression. Our results also indicate that in addition to Upc2A, the only known activator of ERG genes, additional positive regulators of this pathway must exist.

The Candida glabrata Upc2A transcription factor is a global regulator of antifungal drug resistance pathways.

The most commonly used antifungal drugs are the azole compounds, which interfere with biosynthesis of the fungal-specific sterol: ergosterol. The pathogenic yeast Candida glabrata commonly acquires resistance to azole drugs like fluconazole via mutations in a gene encoding a transcription factor called PDR1. These PDR1 mutations lead to overproduction of drug transporter proteins like the ATP-binding cassette transporter Cdr1. In other Candida species, mutant forms of a transcription factor called Upc2 are associated with azole resistance, owing to the important role of this protein in control of expression of genes encoding enzymes involved in the ergosterol biosynthetic pathway. Recently, the C. glabrata Upc2A factor was demonstrated to be required for normal azole resistance, even in the presence of a hyperactive mutant form of PDR1. Using genome-scale approaches, we define the network of genes bound and regulated by Upc2A. By analogy to a previously described hyperactive UPC2 mutation found in Saccharomyces cerevisiae, we generated a similar form of Upc2A in C. glabrata called G898D Upc2A. Analysis of Upc2A genomic binding sites demonstrated that wild-type Upc2A binding to target genes was strongly induced by fluconazole while G898D Upc2A bound similarly, irrespective of drug treatment. Transcriptomic analyses revealed that, in addition to the well-described ERG genes, a large group of genes encoding components of the translational apparatus along with membrane proteins were responsive to Upc2A. These Upc2A-regulated membrane protein-encoding genes are often targets of the Pdr1 transcription factor, demonstrating the high degree of overlap between these two regulatory networks. Finally, we provide evidence that Upc2A impacts the Pdr1-Cdr1 system and also modulates resistance to caspofungin. These studies provide a new perspective of Upc2A as a master regulator of lipid and membrane protein biosynthesis.

Mutations in TAC1B: a Novel Genetic Determinant of Clinical Fluconazole Resistance in Candida auris.

Candida auris has emerged as a multidrug-resistant pathogen of great clinical concern. Approximately 90% of clinical C. auris isolates are resistant to fluconazole, the most commonly prescribed antifungal agent, and yet it remains unknown what mechanisms underpin this fluconazole resistance. To identify novel mechanisms contributing to fluconazole resistance in C. auris, fluconazole-susceptible C. auris clinical isolate AR0387 was passaged in media supplemented with fluconazole to generate derivative strains which had acquired increased fluconazole resistance in vitro Comparative analyses of comprehensive sterol profiles, [3H]fluconazole uptake, sequencing of C. auris genes homologous to genes known to contribute to fluconazole resistance in other species of Candida, and relative expression levels of C. aurisERG11, CDR1, and MDR1 were performed. All fluconazole-evolved derivative strains were found to have acquired mutations in the zinc-cluster transcription factor-encoding gene TAC1B and to show a corresponding increase in CDR1 expression relative to the parental clinical isolate, AR0387. Mutations in TAC1B were also identified in a set of 304 globally distributed C. auris clinical isolates representing each of the four major clades. Introduction of the most common mutation found among fluconazole-resistant clinical isolates of C. auris into fluconazole-susceptible isolate AR0387 was confirmed to increase fluconazole resistance by 8-fold, and the correction of the same mutation in a fluconazole-resistant isolate, AR0390, decreased fluconazole MIC by 16-fold. Taken together, these data demonstrate that C. auris can rapidly acquire resistance to fluconazole in vitro and that mutations in TAC1B significantly contribute to clinical fluconazole resistance.IMPORTANCECandida auris is an emerging multidrug-resistant pathogen of global concern, known to be responsible for outbreaks on six continents and to be commonly resistant to antifungals. While the vast majority of clinical C. auris isolates are highly resistant to fluconazole, an essential part of the available antifungal arsenal, very little is known about the mechanisms contributing to resistance. In this work, we show that mutations in the transcription factor TAC1B significantly contribute to clinical fluconazole resistance. These studies demonstrated that mutations in TAC1B can arise rapidly in vitro upon exposure to fluconazole and that a multitude of resistance-associated TAC1B mutations are present among the majority of fluconazole-resistant C. auris isolates from a global collection and appear specific to a subset of lineages or clades. Thus, identification of this novel genetic determinant of resistance significantly adds to the understanding of clinical antifungal resistance in C. auris.

Characterization of the Efflux Capability and Substrate Specificity of Aspergillus fumigatus PDR5-like ABC Transporters Expressed in Saccharomyces cerevisiae.

This research analyzed six Aspergillus fumigatus genes encoding putative efflux proteins for their roles as transporters. TheA. fumigatus genes abcA, abcC, abcF, abcG, abcH, and abcI were cloned into plasmids and overexpressed in a Saccharomyces cerevisiae strain in which the highly active endogenous ABC transporter gene PDR5 was deleted. The activity of each transporter was measured by efflux of rhodamine 6G and accumulation of alanine ß-naphthylamide. The transporters AbcA, AbcC, and AbcF had the strongest efflux activities of these compounds. All of the strains with plasmid-expressed transporters had more efflux activity than did the PDR5-deleted background strain. We performed broth microdilution drug susceptibility testing and agar spot assays using an array of compounds and antifungal drugs to determine the transporter specificity and drug susceptibility of the strains. The transporters AbcC and AbcF showed the broadest range of substrate specificity, while AbcG and AbcH had the narrowest range of substrates. Strains expressing the AbcA, AbcC, AbcF, or AbcI transporter were more resistant to fluconazole than was the PDR5-deleted background strain. Strains expressing AbcC and AbcF were additionally more resistant to clotrimazole, itraconazole, ketoconazole, and posaconazole than was the background strain. Finally, we analyzed the expression levels of the genes by reverse transcription-quantitative PCR (RT-qPCR) in triazole-susceptible and -resistant A. fumigatus clinical isolates. All of these transporters are expressed at a measurable level, and transporter expression varied significantly between strains, demonstrating the high degree of phenotypic variation, plasticity, and divergence of which this species is capable.IMPORTANCE One mechanism behind drug resistance is altered export out of the cell. This work is a multifaceted analysis of membrane efflux transporters in the human fungal pathogen A. fumigatus Bioinformatics evidence infers that there is a relatively large number of genes in A. fumigatus that encode ABC efflux transporters. However, very few of these transporters have been directly characterized and analyzed for their potential role in drug resistance.Our objective was to determine if these undercharacterized proteins function as efflux transporters and then to better define whether their efflux substrates include antifungal drugs used to treat fungal infections. We chose six A. fumigatus potential plasma membrane ABC transporter genes for analysis and found that all six genes produced functional transporter proteins. We used two fungal systems to look for correlations between transporter function and drug resistance. These transporters have the potential to produce drug-resistant phenotypes in A. fumigatus Continued characterization of these and other transporters may assist in the development of efflux inhibitor drugs.

Lyophilized Iron Oxide Nanoparticles Encapsulated in Amphotericin B: A Novel Targeted Nano Drug Delivery System for the Treatment of Systemic Fungal Infections.

We formulated and tested a targeted nanodrug delivery system to help treat life-threatening invasive fungal infections, such as cryptococcal meningitis. Various designs of iron oxide nanoparticles (IONP) (34-40 nm) coated with bovine serum albumin and coated and targeted with amphotericin B (AMB-IONP), were formulated by applying a layer-by-layer approach. The nanoparticles were monodispersed and spherical in shape, and the lead formulation was found to be in an optimum range for nanomedicine with size (≤36 nm), zeta potential (-20 mV), and poly dispersity index (≤0.2), and the drug loading was 13.6 ± 6.9 µg of AMB/mg of IONP. The drug release profile indicated a burst release of up to 3 h, followed by a sustained drug release of up to 72 h. The lead showed a time-dependent cellular uptake in C. albicans and C. glabrata clinical isolates, and exhibited an improved efficacy (16-25-fold) over a marketed conventional AMB-deoxycholate product in susceptibility testing. Intracellular trafficking of AMB-IONP by TEM and confocal laser scanning microscopy confirmed the successful delivery of the AMB payload at and/or inside the fungal cells leading to potential therapeutic advantages over the AMB-deoxycholate product. A short-term stability study at 5 °C and 25 °C for up to two months showed that the lyophilized form was stable.

Molecular and genetic basis of azole antifungal resistance in the opportunistic pathogenic fungus Candida albicans.

Candida albicans is an opportunistic yeast and the major human fungal pathogen in the USA, as well as in many other regions of the world. Infections with C. albicans can range from superficial mucosal and dermatological infections to life-threatening infections of the bloodstream and vital organs. The azole antifungals remain an important mainstay treatment of candidiasis and therefore the investigation and understanding of the evolution, frequency and mechanisms of azole resistance are vital to improving treatment strategies against this organism. Here the organism C. albicans and the genetic changes and molecular bases underlying the currently known resistance mechanisms to the azole antifungal class are reviewed, including up-regulated expression of efflux pumps, changes in the expression and amino acid composition of the azole target Erg11 and alterations to the organism's typical sterol biosynthesis pathways. Additionally, we update what is known about activating mutations in the zinc cluster transcription factor (ZCF) genes regulating many of these resistance mechanisms and review azole import as a potential contributor to azole resistance. Lastly, investigations of azole tolerance in C. albicans and its implicated clinical significance are reviewed.

Impact of the Major Candida glabrata Triazole Resistance Determinants on the Activity of the Novel Investigational Tetrazoles VT-1598 and VT-1161.

VT-1161 and VT-1598 are promising investigational tetrazole antifungals that have shown in vitro and in vivo activity against Candida and other fungi. Candida glabrata is a problematic opportunistic pathogen that is associated with high mortality in invasive infection, as well as both intrinsic and rapidly acquired antifungal resistance. The MICs of VT-1161 and VT-1598 were determined by CLSI methodology to evaluate their in vitro activities against clinical C. glabrata isolates and strains containing individual deletions of the zinc cluster transcription factor genes PDR1 and UPC2A as well as the efflux transporter genes CDR1, PDH1, and SNQ2 Overall, both tetrazoles demonstrated relative activities comparable to those of the tested triazole antifungals against clinical C. glabrata isolates (MIC range, 0.25 to 2 mg/liter and 0.5 to 2 µg/ml for VT-1161 and VT-1598, respectively). Deletion of the PDR1 gene in fluconazole-resistant matched clinical isolate SM3 abolished the decreased susceptibility phenotype completely for both VT-1161 and VT-1598, similarly to the triazoles. UPC2A deletion also increased susceptibility to both triazoles and tetrazoles but to a lesser extent than PDR1 deletion. Of the three major transporter genes regulated by Pdr1, CDR1 deletion resulted in the largest MIC reductions for all agents tested, while PDH1 and SNQ2 deletion individually impacted MICs very little. Overall, both VT-1161 and VT-1598 have comparable activities to those of the available triazoles, and decreased susceptibility to these tetrazoles in C. glabrata is driven by many of the same known resistance mechanisms.

Mutations in hmg1, Challenging the Paradigm of Clinical Triazole Resistance in Aspergillus fumigatus.

Aspergillus fumigatus is the predominant pathogen of invasive aspergillosis, a disease state credited with over 200,000 life-threatening infections each year. The triazole class of antifungals are clinically essential to the treatment of invasive aspergillosis, both as frontline and as salvage therapy. Unfortunately, resistance to the triazoles among A. fumigatus isolates is now increasingly reported worldwide, and a large proportion of this resistance remains unexplained. In this work, we characterize the contributions of previously identified mechanisms of triazole resistance, including mutations in the sterol-demethylase-encoding gene cyp51A, overexpression of sterol-demethylase genes, and overexpression of the efflux pump-encoding gene abcC, among a large collection of highly triazole-resistant clinical A. fumigatus isolates. Upon revealing that these mechanisms alone cannot substantiate the majority of triazole resistance exhibited by this collection, we subsequently describe the identification and characterization of a novel genetic determinant of triazole resistance. Mutations in the 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase-encoding gene, hmg1, were identified in a majority of triazole-resistant clinical isolates in our collection. Introduction of three different hmg1 mutations, predicted to encode residue alterations in the conserved sterol sensing domain of Hmg1, resulted in significantly increased resistance to the triazole class of agents. Additionally, correction of a hmg1 mutation in a pan-triazole-resistant clinical isolate of A. fumigatus with a novel Cas9-ribonucleoprotein-mediated system was shown to restore clinical susceptibility to all triazole agents. Mutations in hmg1 were also shown to lead to the accumulation of ergosterol precursors, such as eburicol, by sterol profiling, while not altering the expression of sterol-demethylase genes.IMPORTANCEAspergillus fumigatus is the predominant pathogen of invasive aspergillosis, a disease state credited with over 200,000 life-threatening infections annually. The triazole class of antifungals are clinically essential to the treatment of invasive aspergillosis. Unfortunately, resistance to the triazoles among A. fumigatus isolates is now increasingly reported worldwide. In this work, we challenge the current paradigm of clinical triazole resistance in A. fumigatus, by first demonstrating that previously characterized mechanisms of resistance have nominal impact on triazole susceptibility and subsequently identifying a novel mechanism of resistance with a profound impact on clinical triazole susceptibility. We demonstrate that mutations in the HMG-CoA reductase gene, hmg1, are common among resistant clinical isolates and that hmg1 mutations confer resistance to all clinically available triazole antifungals.