An oxindole efflux inhibitor potentiates azoles and impairs virulence in the fungal pathogen Candida auris.
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ABSTRACT:
SUBMITTER: Iyer KR
PROVIDER: S-EPMC7755909 | biostudies-literature | 2020 Dec
REPOSITORIES: biostudies-literature
Publications
An oxindole efflux inhibitor potentiates azoles and impairs virulence in the fungal pathogen Candida auris.
Iyer Kali R KR Camara Kaddy K Daniel-Ivad Martin M Trilles Richard R Pimentel-Elardo Sheila M SM Fossen Jen L JL Marchillo Karen K Liu Zhongle Z Singh Shakti S Muñoz José F JF Kim Sang Hu SH Porco John A JA Cuomo Christina A CA Williams Noelle S NS Ibrahim Ashraf S AS Edwards John E JE Andes David R DR Nodwell Justin R JR Brown Lauren E LE Whitesell Luke L Robbins Nicole N Cowen Leah E LE
Nature communications 20201222 1
Candida auris is an emerging fungal pathogen that exhibits resistance to multiple drugs, including the most commonly prescribed antifungal, fluconazole. Here, we use a combinatorial screening approach to identify a bis-benzodioxolylindolinone (azoffluxin) that synergizes with fluconazole against C. auris. Azoffluxin enhances fluconazole activity through the inhibition of efflux pump Cdr1, thus increasing intracellular fluconazole levels. This activity is conserved across most C. auris clades, wi ...[more]
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Project description:Candida auris is an emerging fungal pathogen capable of causing invasive infections in humans. Since its first appearance around 1996, it has been isolated in countries spanning five continents. C. auris is a yeast that has the potential to cause outbreaks in hospitals, can survive in adverse conditions, including dry surfaces and high temperatures, and has been frequently misidentified by traditional methods. Furthermore, strains have been identified that are resistant to two and even all three of the main classes of antifungals currently in use. Several nuclear genome assemblies of C. auris have been published representing different clades and continents, yet until recently, the mitochondrial genomes (mtDNA chromosomes) of this species and the closely related species of C. haemulonii, C. duobushaemulonii, and C. pseudohaemulonii had not been analyzed in depth. We used reads from PacBio and Illumina sequencing to obtain a de novo reference assembly of the mitochondrial genome of the C. auris clade I isolate B8441 from Pakistan. This assembly has a total size of 28.2 kb and contains 13 core protein-coding genes, 25 tRNAs and the 12S and 16S ribosomal subunits. We then performed a comparative analysis by aligning Illumina reads of 129 other isolates from South Asia, Japan, South Africa, and South America with the B8441 reference. The clades of the phylogenetic tree we obtained from the aligned mtDNA sequences were consistent with those derived from the nuclear genome. The mitochondrial genome revealed a generally low genetic variation within clades, although the South Asian clade displayed two sub-branches including strains from both Pakistan and India. In particular, the 86 isolates from Colombia and Venezuela had mtDNA sequences that were all identical at the base level, i.e., a single conserved haplotype or mitochondrial background that exhibited characteristic differences from the Pakistan reference isolate B8441, such as a unique 25-nt insert that may affect function.
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Project description:Candida auris was first described in 2009, and it has since caused nosocomial outbreaks, invasive infections, and fungaemia across at least 19 countries on five continents. An outbreak of C. auris occurred in a specialized cardiothoracic London hospital between April 2015 and November 2016, which to date has been the largest outbreak in the UK, involving a total of 72 patients. To understand the genetic epidemiology of C. auris infection both within this hospital and within a global context, we sequenced the outbreak isolate genomes using Oxford Nanopore Technologies and Illumina platforms to detect antifungal resistance alleles and reannotate the C. auris genome. Phylogenomic analysis placed the UK outbreak in the India/Pakistan clade, demonstrating an Asian origin; the outbreak showed similar genetic diversity to that of the entire clade, and limited local spatiotemporal clustering was observed. One isolate displayed resistance to both echinocandins and 5-flucytosine; the former was associated with a serine to tyrosine amino acid substitution in the gene FKS1, and the latter was associated with a phenylalanine to isoleucine substitution in the gene FUR1. These mutations add to a growing body of research on multiple antifungal drug targets in this organism. Multiple differential episodic selection of antifungal resistant genotypes has occurred within a genetically heterogenous population across this outbreak, creating a resilient pathogen and making it difficult to define local-scale patterns of transmission and implement outbreak control measures.
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Project description:The emerging "super fungus" Candida auris has become an important threat to human health due to its pandrug resistance and high lethality. Therefore, the development of novel antimicrobial strategy is essential. Antimicrobial photodynamic therapy (aPDT) has excellent performance in clinical applications. However, the relevant study on antifungal activity and the mechanism involved against C. auris remains scarce. Herein, a recyclable and biodegradable polylactic acid-hypocrellin A (PLA-HA) nanofibrous membrane is newly developed. In vitro PLA-HA-aPDT could significantly reduce the survival rate of C. auris plankton and its biofilms, and the fungicidal effect of the membrane is still significant after four repeated uses. Simultaneously, PLA-HA exhibits good biocompatibility and low hemolysis. In vivo experiments show that PLA-HA-aPDT can promote C. auris-infected wound healing, reduce inflammatory response, and without obvious toxic side-effects. Further results reveal that PLA-HA-aPDT could increase endogenous reactive oxygen species (ROS) levels, leading to mitochondrial dysfunction, release of cytochrome C, activation of metacaspase, and nuclear fragmentation, thereby triggering apoptosis of C. auris. Compared with HA, PLA-HA shows stronger controllability and reusability, which can greatly improve the utilization efficiency of HA alone. Taken together, the efficacy, safety and antifungal activity make PLA-HA-aPDT a highly promising antifungal candidate for skin or mucous membrane C. auris infection.
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Project description:Candida auris exhibits resistance to multiple antifungal drug classes and sterilization agents, posing threats to the immunocompromised worldwide. Among the four major geographical clades, the East Asian clade 2 isolates of C. auris are mostly drug susceptible. In this study, we experimentally evolved one such drug-susceptible isolate for multiple generations in the presence of the antifungal compound fluconazole and analyzed changes in the karyotype, DNA sequence, and gene expression profiles in three evolved drug-resistant isolates. Next-generation sequencing and electrophoretic karyotyping confirm the presence of segmental aneuploidy as supernumerary chromosomes originating from centromere-inclusive chromosomal duplication events in two such cases. A 638-kb region and a 675-kb region, both of which originated from chromosome 5 and contained its centromere region, are instances of supernumerary chromosome formation identified in two evolved fluconazole-resistant isolates. Loss of the supernumerary chromosomes from the drug-resistant isolates results in a complete reversal of fluconazole susceptibility. Transcriptome analysis of the third isolate identified overexpression of drug efflux pumps as a possible non-aneuploidy-driven mechanism of drug resistance. Together, this study reveals how both aneuploidy-driven and aneuploidy-independent mechanisms may operate in parallel in an evolving population of C. auris in the presence of an antifungal drug, in spite of starting from the same strain grown under similar conditions, to attain various levels of fluconazole resistance. IMPORTANCE Fungal pathogens develop drug resistance through multiple pathways by acquiring gene mutations, increasing the copy number of genes, or altering gene expression. In this study, we attempt to understand the mechanisms of drug resistance in the recently emerged superbug, C. auris. One approach to studying this aspect is identifying various mechanisms operating in drug-resistant clinical isolates. An alternative approach is to evolve a drug-susceptible isolate in the presence of an antifungal compound and trace the changes that result in drug resistance. Here, we evolve a drug-susceptible isolate of C. auris in the laboratory in the presence of a widely used antifungal compound, fluconazole. In addition to the already known changes like overexpression of drug efflux pumps, this study identifies a novel mechanism of azole resistance by the emergence of additional chromosomes through segmental duplication of chromosomal regions, including centromeres. The centric supernumerary chromosome helps stable amplification of a set of genes with an extra copy to confer fluconazole resistance.
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Project description:Candida auris is amongst the most important emerging fungal pathogens, yet mechanistic insights in its immune recognition and control are lacking. Here, we integrate transcriptional and functional immune cell profiling to uncover innate anti-C. auris defense mechanisms. C. auris induces a specific transcriptome in human mononuclear cells, a stronger cytokine response compared to C. albicans, but a lower macrophage lysis capacity. C. auris-induced innate immune activation is mediated through recognition of C-type lectin receptors, mainly elicited by structurally unique C. auris mannoproteins. In in-vivo experimental models of disseminated candidiasis, C. auris was less virulent than C. albicans. Collectively, these results demonstrate that C. auris is a strong inducer of innate host defense and identify possible targets for adjuvant immunotherapy.
2020-07-23 | GSE154911 | GEO
Project description:The LUFS domain (LUG/LUH, Flo8, single-strand DNA-binding protein [SSBP]) is a well-conserved and apparently ancient region found in diverse proteins and taxa. This domain, which has as its most obvious structural feature a series of three helices, has been identified in transcriptional regulator proteins of animals, plants, and fungi. Recently, in these pages (Wang et al., Protein Sci., 2019, 28:788-793), the first crystal structure of a LUFS domain was reported, for the human SSBP2, a transcriptional repressor. We briefly address how the new insights into LUFS structures might contribute to a better understanding of an important transcriptional activator of yeasts that contains the LUFS domain, Flo8, and consider how a focus on the LUFS domain and its variation could help us to understand etiologies of drug resistance in a recently emerged pathogenic fungus, Candida auris.
| S-EPMC6798126 | biostudies-literature
Project description:Candida auris, the multidrug-resistant human fungal pathogen, emerged as four major distinct geographical clades (clade 1-clade 4) in the past decade. Though isolates of the same species, C. auris clinical strains exhibit clade-specific properties associated with virulence and drug resistance. In this study, we report the identification of unique DNA sequence junctions by mapping clade-specific regions through comparative analysis of whole-genome sequences of strains belonging to different clades. These unique DNA sequence stretches are used to identify C. auris isolates at the clade level in subsequent in silico and experimental analyses. We develop a colony PCR-based clade-identification system (ClaID), which is rapid and specific. In summary, we demonstrate a proof-of-concept for using unique DNA sequence junctions conserved in a clade-specific manner for the rapid identification of each of the four major clades of C. auris. IMPORTANCE C. auris was first isolated in Japan in 2009 as an antifungal drug-susceptible pathogen causing localized infections. Within a decade, it simultaneously evolved in different parts of the world as distinct clades exhibiting resistance to antifungal drugs at varying levels. Recent studies hinted the mixing of isolates belonging to different geographical clades in a single location, suggesting that the area of isolation alone may not indicate the clade status of an isolate. In this study, we compared the genomes of representative strains of the four major clades to identify clade-specific sequences, which were then used to design clade-specific primers. We propose the utilization of whole genome sequence data to extract clade-specific sequences for clade-typing. The colony PCR-based method employed can rapidly distinguish between the four major clades of C. auris, with scope for expanding the panel by adding more primer pairs.
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Project description:Candida auris has simultaneously emerged on five continents as a fungal pathogen causing nosocomial outbreaks. The challenges in the treatment of C. auris infections are the variable antifungal susceptibility profiles among clinical isolates and the development of resistance to single or multiple classes of available antifungal drugs. Here, the in vitro susceptibility to echinocandin antifungal drugs was determined and FKS1 sequencing was performed on 106 C. auris clinical isolates. Four isolates were identified to be resistant to all tested echinocandins (MIC ≥ 4 mg/liter) and harbored an S639F mutation in FKS1 hot spot region 1. All remaining isolates were FKS1 wild type (WT) and echinocandin susceptible, with micafungin being the most potent echinocandin (MIC50 = 0.125 mg/liter). Antifungal susceptibility testing with caspofungin was challenging due to the fact that all FKS1 WT isolates exhibited an Eagle effect (also known as the paradoxical growth effect), which occurred at various intensities. To assess whether the Eagle effect resulted in pharmacodynamic resistance, 8 representative isolates were evaluated for their in vivo drug response in a murine model of invasive candidiasis. All isolates were susceptible to caspofungin at a human therapeutic dose, except for those harboring the S639F mutation. The data suggest that only isolates carrying mutations in FKS1 are echinocandin resistant and that routine in vitro testing of C. auris isolates for susceptibility to caspofungin by the broth microdilution method should be viewed cautiously or avoided.
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Project description:Fungal infections are a major contributor to infectious disease-related deaths worldwide. Recently, global emergence of the fungal pathogen Candida auris has caused considerable concern because most C. auris isolates are resistant to fluconazole, the most commonly administered antifungal, and some isolates are resistant to drugs from all three major antifungal classes. To identify novel agents with bioactivity against C. auris, we screened 2,454 compounds from a diversity-oriented synthesis collection. Of the five hits identified, most shared a common rocaglate core structure and displayed fungicidal activity against C. auris These rocaglate hits inhibited translation in C. auris but not in its pathogenic relative Candida albicans Species specificity was contingent on variation at a single amino acid residue in Tif1, a fungal member of the eukaryotic initiation factor 4A (eIF4A) family of translation initiation factors known to be targeted by rocaglates. Rocaglate-mediated inhibition of translation in C. auris activated a cell death program characterized by loss of mitochondrial membrane potential, increased caspase-like activity, and disrupted vacuolar homeostasis. In a rocaglate-sensitized C. albicans mutant engineered to express translation initiation factor 1 (Tif1) with the variant amino acid that we had identified in C. auris, translation was inhibited but no programmed cell death phenotypes were observed. This surprising finding suggests divergence between these related fungal pathogens in their pathways of cellular responses to translation inhibition. From a therapeutic perspective, the chemical biology that we have uncovered reveals species-specific vulnerability in C. auris and identifies a promising target for development of new, mechanistically distinct antifungals in the battle against this emerging pathogen.IMPORTANCE Emergence of the fungal pathogen Candida auris has ignited intrigue and alarm within the medical community and the public at large. This pathogen is unusually resistant to antifungals, threatening to overwhelm current management options. By screening a library of structurally diverse molecules, we found that C. auris is surprisingly sensitive to translation inhibition by a class of compounds known as rocaglates (also known as flavaglines). Despite the high level of conservation across fungi in their protein synthesis machinery, these compounds inhibited translation initiation and activated a cell death program in C. auris but not in its relative Candida albicans Our findings highlight a surprising divergence across the cell death programs operating in Candida species and underscore the need to understand the specific biology of a pathogen in attempting to develop more-effective treatments against it.
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Project description:Fungi respond to antifungal drugs by increasing their antioxidant stress response. How this impacts antifungal efficacy remains controversial and not well understood. Here we examine the role of catalase activity in the resistance of Saccharomyces cerevisiae to the common antifungals, fluconazole and miconazole, for which we report minimum inhibitory concentrations (MICs) of 104 and 19 μM, respectively. At sub-MIC concentrations, fluconazole and miconazole stimulate catalase activity 2-3-fold but, unexpectedly, deletion of cytosolic catalase (ctt1) makes cells more resistant to these azoles and to clotrimazole, itraconazole and posaconazole. On the other hand, upregulating Ctt1 activity by preconditioning with 0.2 mM H2O2 potentiates miconazole 32-fold and fluconazole 4-fold. Since H2O2 preconditioning does not alter the resistance of ctt1Δ cells, which possess negligible catalase activity, we link azole potentiation with Ctt1 upregulation. In contrast, sod2Δ cells deleted for mitochondrial superoxide dismutase are 4-8-fold more azole sensitive than wild-type cells, revealing that Sod2 activity protects cells against azole toxicity. In fact, the ctt1Δ mutant has double the Sod2 activity of wild-type cells so ctt1 deletion increases azole resistance in part by Sod2 upregulation. Notably, deletion of peroxisomal/mitochondrial cta1 or cytosolic sod1 does not alter fluconazole or miconazole potency.
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