Hollowayhogan7008

Antifungal resistance mediated by overexpression of ABC transporters is one of the primary roadblocks to effective therapy against Candida infections. Thus, identification and characterization of the ABC transporter repertoire in Candida species are of high relevance. The method described in the chapter is based on our previously developed bioinformatic pipeline for identification of ABC proteins in Candida species. The methodology essentially involves the utilization of a hidden Markov model (HMM) profile of the nucleotide-binding domain (NBD) of ABC proteins to mine these proteins from the proteome of Candida species. Further, a widely used tool to predict membrane protein topology is exploited to identify the true transporter candidates out of the ABC proteins. Even though the chapter specifically focuses on a method to identify ABC transporters in Candida auris , the same can also be applied to any other Candida species.Candida auris is an urgent public health threat characterized by high drug-resistant rates and rapid spread in healthcare settings worldwide. As part of the C. auris response, molecular surveillance has helped public health officials track the global spread and investigate local outbreaks. Here, we describe whole-genome sequencing analysis methods used for routine C. auris molecular surveillance in the United States; methods include reference selection, reference preparation, quality assessment and control of sequencing reads, read alignment, and single-nucleotide polymorphism calling and filtration. We also describe the newly developed pipeline MycoSNP, a portable workflow for performing whole-genome sequencing analysis of fungal organisms including C. auris.Genomic studies of Candida auris are underpinned by the generation of high-quality genome assemblies. These reference genomes have been essential for investigations of the evolution and epidemiology of this emerging fungal pathogen. In addition to genomic epidemiology studies of local outbreaks and analysis of the global emergence of this species, comparisons of genomes of isolates from the five major clades have revealed differences in gene content and genomic structure. Here, we provide a detailed protocol for generating complete genome assemblies for C. auris.Transmission electron microscopy (TEM) is the main technique used to study the ultrastructure of biological samples. Chemical fixation was considered the main method for preserving samples for TEM; however, it is a relatively slow method of fixation and can result in morphological alterations. Cryofixation using high-pressure freezing (HPF) overcomes the limitations of chemical fixation by preserving samples instantly. Here, we describe our HPF methods optimized for visualizing Candida auris at the ultrastructural level.Pathogen-associated molecular patterns (PAMPs) of the fungal cell wall are primary targets for the innate immune system of animals. Therefore, characterizing PAMP exposure of fungal pathogens helps to elucidate how they interact with their hosts at a molecular level. Fluorescent labelling can be used to monitor exposure of multiple fungal cell wall PAMPs in a single experiment. Here, we describe a protocol to simultaneously label chitin, mannan, and β-1,3-glucan in Candida auris to study these PAMPs by fluorescence microscopy and allow high-throughput examination of their exposure by flow cytometry.Extracellular vesicles (EVs) are structures released by a variety of cells from all kingdoms of life. Dovitinib manufacturer EVs are typically involved in communication between tissues and organs, between distinct organisms, or inside microbial communities. The plasticity of these structures is reflected in the range of biological effects they are able to induce or inhibit. The study of fungal EVs is relatively new with the first report in 2007, but investigators have already demonstrated in several model systems that fungal EVs significantly modulate the host immune system and that the immunogenic materials in EV can be harnessed as vaccination platforms. This chapter describes the two main procedures used to isolate EVs from an emerging pathogenic fungus, Candida auris.Unique metabolic features allow fungi to colonize and persist within the human host. Investigations of unique metabolic fingerprints of a pathogenic fungus can provide a more complete understanding of the infection process and an interpretation of associations between genotype and phenotype. Gas chromatography-mass spectrometry (GC-MS) has proved to be one of the most powerful analytical techniques used for qualitative and quantitative detection of cellular metabolites. This technique has been used for comparative metabolomic analyses of both intracellular and secreted metabolites under variable conditions. This book chapter describes the use of GC-MS in the detection of both intracellular and secreted metabolites from Candida auris, a newly emerging fungal pathogen representing a serious global health threat due to its multidrug resistance profile. The identified fungal metabolites are compared using available software in order to assign a correlation between the pattern of accumulation of metabolites and behavior of the organism.The recently emerged human pathogenic yeast Candida auris has become a major global threat. As compared to other Candida species, C. auris often displays a high level of resistance to commonly used antifungals and poses additional therapeutic challenges. There is a great need to understand the molecular basis of its success as a drug-resistant human pathogen. The study of condition-specific gene expression can provide good cues of regulatory circuitry governing high drug resistance. Here, we describe the protocol of quantitative reverse transcription PCR (RT-qPCR) which can be conveniently employed as a highly reproducible method for measuring individual transcripts in C. auris cells.Cell viability assays are useful for assessing the efficacy of antifungal therapeutics and disinfection strategies in vitro. In recent years these assays have been fundamental for the testing of conventional and novel therapies against the nosocomial fungal pathogen Candida auris. Here we provide detailed descriptions of methods for assessing cellular viability of Candida auris in vitro, such as metabolic assays (XTT and resazurin), colony-forming unit counting, live/dead quantitative PCR, and fluorescent staining for microscopic analyses.The recent global emergence of the fungal pathogen Candida auris has caused significant concern given that this pathogen often exhibits resistance to multiple antifungal drug classes. In order to effectively combat C. auris infections, there is a dire need to expand our current antifungal arsenal. Essential proteins often serve as targets for antimicrobial compounds, and thus being able to study essential genes in a pathogen of interest is a critical first step in drug development. To identify and characterize essential genes in microorganisms, researchers must be able to manipulate microbial genomes using a variety of molecular biology techniques. Given the haploid genome of C. auris, genetic alterations have largely been achieved by gene deletion through homologous recombination using a drug resistance marker. However, this approach is not feasible to study essential gene function. Here, we describe a method for the study of essential genes using a tetracycline-repressible promoter replacement system, which can be used to genetically repress essential genes in C. auris and, thus, study their function. This method provides a powerful approach to assess and characterize essential gene function in an emerging fungal pathogen.Reverse genetics is a particularly powerful tool in non-model organisms with known whole-genome sequences enabling the characterization of gene and, thus, protein function via a mutant phenotype. Reverse genetic approaches require genetic manipulation techniques which often need to be specifically developed for non-model organisms; this can be fraught with difficulties. Here, we describe a genetic transformation protocol for the recently emerged human pathogen Candida auris to target the integration of DNA constructs into genomic locations via homology-directed repair using long flanking homologous sequences (>1 kb). We detail the generation of DNA constructs for gene deletion with dominant drug resistance markers via fusion PCR, the transformation of these constructs into chemically competent C. auris cells, and the confirmation of correct integration by PCR. This strategy can be adapted to deliver DNA constructs other than deletion cassettes, including promoter exchanges and protein tags.Candida auris is responsible for recent outbreaks with significant mortality in hospitalized and long-term care patients. As a highly transmissible and multidrug-resistant fungal pathogen, genetic tools are urgently needed for deciphering mechanisms involved in the host-pathogen interactions and potentially identifying new fungal targets for therapeutic development. Here, we provide a reliable transformation protocol based on an efficient electroporation procedure and the use of a mycophenolic acid resistance marker.The paradoxical growth effect (PGE; also known as Eagle effect) is an in vitro phenomenon observed during antifungal susceptibility testing (AFST). In PGE, some fungal isolates grow in medium containing high concentrations of an echinocandin, above the minimal inhibitory concentration (MIC), despite being fully susceptible at lower concentrations. The presence of PGE complicates the assignment of isolates to susceptible or resistant category, especially in the case of newly emerged pathogens like Candida auris, for which susceptibility breakpoints are not established.Here we describe a protocol aiding in the determination of whether a given C. auris isolate is echinocandin-resistant or echinocandin-susceptible but exhibiting paradoxical growth.Susceptibility testing of isolates of Candida auris is helpful as a guide to the selection of the most appropriate antifungal agent for treatment as different clades and strains within clades often demonstrate markedly different susceptibility profiles. Some strains are relatively susceptible to all antifungal drugs, but most demonstrate innate resistance to fluconazole, many are cross-resistant to other azoles and others demonstrate resistance to other classes of antifungal. The finding of multi-drug resistance, where an isolate is resistant to two or more classes of antifungal agent, is not uncommon, and development of resistance during a course of treatment has also been documented. This chapter describes a reference broth microdilution method for susceptibility testing and a commercially available gradient strip method.Candida auris is a multidrug-resistant pathogenic ascomycete yeast of increasing health concern. C. auris colonizes patient's skin and can persist for weeks on surfaces, so it can be transmitted within and between hospitals. The most common diagnostic platforms in microbiology use reference databases that have not yet incorporated C. auris, misidentifying it. This chapter describes how to detect C. auris by qPCR with the GPS™ CanAur MONODOSE dtec-qPCR Test (Alicante, Spain) in less than 45 min, using ready-to-use tubes with all the components dehydrated. This commercial kit was subjected to validation following the guidelines of the UNE-EN ISO/IEC 170252005 and French Standard NF T90-4712010.