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.

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.

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.

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.

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.

Abrogation of Triazole Resistance upon Deletion of CDR1 in a Clinical Isolate of Candida auris.

Candida auris has rapidly emerged as a health care-associated and multidrug-resistant pathogen of global concern. In this work, we examined the relative expression of the four C. auris genes with the highest degree of homology to Candida albicansCDR1 and MDR1 among three triazole-resistant clinical isolates as compared to the triazole-susceptible genome reference clinical isolate. We subsequently utilized a novel Cas9-mediated system for genetic manipulations to delete C. aurisCDR1 and MDR1 in both a triazole-resistant clinical isolate and a susceptible reference strain and observed that MICs for all clinically available triazoles decreased as much as 128-fold in the CDR1 deletion strains. The findings of this work reveal for the first time that C. aurisCDR1 and MDR1 are more highly expressed among triazole-resistant clinical isolates of C. auris and that the overexpression of CDR1 is a significant contributor to clinical triazole resistance.

Humoral immune responses during asthma and influenza co-morbidity in mice.

Humoral immunity serve dual functions of direct pathogen neutralization and enhancement of leukocyte function. Antibody classes are determined by antigen triggers, and the resulting antibodies can contribute to disease pathogenesis and host defense. Although asthma and influenza are immunologically distinct diseases, since we have found that allergic asthma exacerbation promotes antiviral host responses to influenza A virus, we hypothesized that humoral immunity may contribute to allergic host protection during influenza. C57BL/6J mice sensitized and challenged with Aspergillus fumigatus (or not) were infected with pandemic influenza A/CA/04/2009 virus. Negative control groups included naïve mice, and mice with only 'asthma' or influenza. Concentrations of antibodies were quantified by ELISA, and in situ localization of IgA- and IgE-positive cells in the lungs was determined by immunohistochemistry. The number and phenotype of B cells in spleens and mediastinal lymph nodes were determined by flow cytometry at predetermined timepoints after virus infection until viral clearance. Mucosal and systemic antibodies remained elevated in mice with asthma and influenza with prominent production of IgE and IgA compared to influenza-only controls. B cell expansion was prominent in the mediastinal lymph nodes of allergic mice during influenza where most cells produced IgG1 and IgA. Although allergy-skewed B cell responses dominated in mice with allergic airways inflammation during influenza virus infection, virus-specific antibodies were also induced. Future studies are required to identify the mechanisms involved with B cell activation and function in allergic hosts facing respiratory viral infections.