shuttle plasmid mediated complementation  (ATCC)

Journal: mBio

Article Title: Coordinated regulation of Mdr1- and Cdr1-mediated protection from antifungals by the Mrr1 transcription factor in emerging Candida spp.

doi: 10.1128/mbio.01323-25

Figure Lengend Snippet: Biochemical and phenotypic analysis of HF-tagged Mrr1 and binding profiles of constitutively active Mrr1. ( A ) Log 2 counts per million (CPM) values of MDR1 ( CLUG_01938_39 ), CDR1 ( CLUG_03113 ), and FLU1 ( CLUG_05825 ) from RNA-seq analysis of Demers et al . comparing U04 clinical isolate (native allele MRR1 Y813C ) and U04 mrr1 ∆ complemented with either MRR1 Y813C or MRR1 ancestral . Ordinary one-way analysis of variance (ANOVA) and Dunnett’s multiple comparisons testing with a single pooled variance were used to evaluate the statistical significance for each gene. ****, P < 0.0001. ( B ) Western blot of whole cell protein lysates of U04 strains expressing N-terminal 6×His-3×FLAG-tagged Mrr1 (HF-Mrr1) variants. HF-Mrr1 was probed using an α-FLAG antibody. Mean ± SD of HF-Mrr1 band intensities normalized to total protein ( n = 4 biological replicates). ( C ) FLZ minimum inhibitory concentration (MIC) of U04 clinical isolate (native allele MRR1 Y813C ) and U04 mrr1 ∆ complemented with untagged or HF-MRR1 was determined by broth microdilution assays. The data represent the mean ± SD from three independent experiments. No more than a twofold difference in MICs was observed between data from strains with untagged Mrr1 variants and data from strains with their respective HF-tagged counterparts. ( A–C ) Strains with constitutive Mrr1 activity are in bold. ( D–F ) HF-Mrr1 Y813C cleavage under targets and release using nuclease (CUT&RUN) read coverage plots normalized per 20 bp bin size. Chromosomal positions of regions containing MDR1, CDR1, and FLU1 and adjacent genes are represented to scale with boxes and arrows. Peaks from HF-Mrr1 Y813C -bound DNA recovered by an α-FLAG antibody and for the non-specific binding control recovered via IgG are shown. The signal indicates the average read density in α-FLAG relative to IgG within the peak region. Two independent experiments were performed, and the results of both are shown.

Article Snippet: MRR1 complementation plasmids were linearized with the NotI-HF restriction enzyme (New England BioLabs), cleaned up using the Zymo DNA Clean & Concentrator kit (Zymo Research), and eluted in molecular biology grade water (Corning) before transformation of 2 μg into C. lusitaniae strain U04 mrr1 Δ as described below.

Techniques: Binding Assay, RNA Sequencing, Western Blot, Expressing, Concentration Assay, Activity Assay, Control

Journal: mBio

Article Title: Coordinated regulation of Mdr1- and Cdr1-mediated protection from antifungals by the Mrr1 transcription factor in emerging Candida spp.

doi: 10.1128/mbio.01323-25

Figure Lengend Snippet: Effects of Mrr1 activity and MDR1, CDR1, and FLU1 on susceptibility to clinically relevant azoles. Structures and MICs (µg/mL) of FLZ ( A ), voriconazole (VOR) ( B ), ketoconazole (KTZ) ( C ), itraconazole (ITZ) ( D ), and isavuconazole (ISA) ( E ) are shown. MICs were determined using broth microdilution assays for strain U04 (native allele MRR1 Y813C ) and U04 mutants: mrr1 ∆+ MRR1 ancestral , mrr1 ∆+ MRR1 Y813C , and mdr1 , cdr1, and flu1 deletion mutants in the mrr1 ∆+ MRR1 Y813C background. Heatmaps represent the optical density (600 nm) of the azole-treated wells normalized to the respective no-drug controls. Drug concentrations in µg/mL are shown on the x -axis. The average from three independent experiments performed on different days is shown. Strains with constitutive Mrr1 activity are in bold.

Article Snippet: MRR1 complementation plasmids were linearized with the NotI-HF restriction enzyme (New England BioLabs), cleaned up using the Zymo DNA Clean & Concentrator kit (Zymo Research), and eluted in molecular biology grade water (Corning) before transformation of 2 μg into C. lusitaniae strain U04 mrr1 Δ as described below.

Techniques: Activity Assay

Journal: mBio

Article Title: Coordinated regulation of Mdr1- and Cdr1-mediated protection from antifungals by the Mrr1 transcription factor in emerging Candida spp.

doi: 10.1128/mbio.01323-25

Figure Lengend Snippet: Effects of Mrr1 activity and MDR1, CDR1, and FLU1 on susceptibility to broad-spectrum antifungals. ( A ) Log 2 transformed fold difference of MIC for diverse antifungals for strains U04 (native allele MRR1 Y813C ) and its mrr1 ∆+ MRR1 ancestral and mrr1 ∆+ MRR1 Y813C derivatives determined using broth microdilution assays. Data were normalized to that for mrr1 ∆+ MRR1 ancestral strain. ( B ) Log 2 transformed fold difference in MIC values of mdr1∆ , cdr1∆, and flu1∆ mutants normalized to their parent U04 mrr1 ∆+ MRR1 Y813C . The data represent the mean ± SD from at least three independent experiments performed on different days. Strains with constitutive Mrr1 activity are in bold. Ordinary one-way ANOVA and Dunnett’s multiple comparisons testing with a single pooled variance were used to evaluate the statistical significance of log2-transformed MIC values of the different strains to either mrr1 ∆+ MRR1 ancestral ( A ) or mrr1 ∆+ MRR1 Y813C ( B ). All significant comparisons are shown; *, P < 0.05, **, P < 0.01, ***, P < 0.001, and ****, P < 0.0001.

Article Snippet: MRR1 complementation plasmids were linearized with the NotI-HF restriction enzyme (New England BioLabs), cleaned up using the Zymo DNA Clean & Concentrator kit (Zymo Research), and eluted in molecular biology grade water (Corning) before transformation of 2 μg into C. lusitaniae strain U04 mrr1 Δ as described below.

Techniques: Activity Assay, Transformation Assay

Journal: mBio

Article Title: Coordinated regulation of Mdr1- and Cdr1-mediated protection from antifungals by the Mrr1 transcription factor in emerging Candida spp.

doi: 10.1128/mbio.01323-25

Figure Lengend Snippet: The Mrr1 regulon of C. lusitaniae . ( A ) Venn diagram shows the overlap between differentially expressed (DE) genes from RNA-seq in Demers et al. and ORFs with HF-Mrr1 Y813C peaks in their intergenic regions from CUT&RUN. The 25 differentially regulated genes that have HF-Mrr1 Y813C peaks in either the 5′ or 3′ regions are listed in . ( B, C ) HF-Mrr1 Y813C CUT&RUN read coverage plots normalized per 20 bp bin size. Chromosomal positions of regions containing MGD1 and MRR1 and adjacent genes are represented to scale with boxes and arrows. Peaks from HF-Mrr1 Y813C -bound DNA recovered by an α-FLAG antibody and for the non-specific binding control recovered via IgG are shown. The signal indicates the average read density in α-FLAG relative to IgG within the peak region. Two independent experiments were performed, and the results of both are shown.

Article Snippet: MRR1 complementation plasmids were linearized with the NotI-HF restriction enzyme (New England BioLabs), cleaned up using the Zymo DNA Clean & Concentrator kit (Zymo Research), and eluted in molecular biology grade water (Corning) before transformation of 2 μg into C. lusitaniae strain U04 mrr1 Δ as described below.

Techniques: RNA Sequencing, Binding Assay, Control

Journal: mBio

Article Title: Coordinated regulation of Mdr1- and Cdr1-mediated protection from antifungals by the Mrr1 transcription factor in emerging Candida spp.

doi: 10.1128/mbio.01323-25

Figure Lengend Snippet: The consensus Mrr1-binding DNA motif of C. lusitaniae . ( A ) Sequence logo of the consensus motif detected within 100 bp of CUT&RUN peak summits by STREME. E-value is an estimate of motif significance. The 9-nt c onsensus M rr1- b inding m otif (cMBM) is boxed in yellow. ( B ) cMBM location (blue hatches) in the ~890 bp upstream intergenic regions of MDR1 in different C. lusitaniae strains. The boxed region was used as the basis for cMBM-containing DNA probes. ( C ) Sequence of the double-stranded DNA probes used for analytical size exclusion chromatography (SEC) ( D ) and electrophoretic mobility shift assays (EMSA) ( E ). The location in the upstream intergenic region of MDR1 is shown. ( D ) Analytical SEC chromatogram of recombinant Mrr1 1-196 protein, 50 bp probe, or the Mrr1 1-196 -50-bp probe (4:1) samples. The molecular weight standards used for SEC calibration are marked at their respective elution volumes. ( E ) EMSAs of recombinant Mrr1 1-196 with either Cy5.5-labeled cMBM probe or Cy5.5-labeled mut-cMBM probe. A representative image from three independent experiments is shown. ( F ) cMBM location (blue hatches) in the 1 kb upstream intergenic regions of the MDR1 homologs in C. parapsilosis CDC317, C. auris B11205 , C. albicans SC5314, and C. lusitaniae ATCC 42720. The C. lusitaniae ATCC 42720 upstream intergenic region is 893 bp. The phylogenetic tree was constructed using the MDR1 nucleotide sequences.

Article Snippet: MRR1 complementation plasmids were linearized with the NotI-HF restriction enzyme (New England BioLabs), cleaned up using the Zymo DNA Clean & Concentrator kit (Zymo Research), and eluted in molecular biology grade water (Corning) before transformation of 2 μg into C. lusitaniae strain U04 mrr1 Δ as described below.

Techniques: Binding Assay, Sequencing, Size-exclusion Chromatography, Electrophoretic Mobility Shift Assay, Recombinant, Molecular Weight, Labeling, Construct

Journal: mBio

Article Title: Coordinated regulation of Mdr1- and Cdr1-mediated protection from antifungals by the Mrr1 transcription factor in emerging Candida spp.

doi: 10.1128/mbio.01323-25

Figure Lengend Snippet: Local and global binding profiles of constitutively active and low-activity Mrr1. ( A–C ) HF-Mrr1 ancestral CUT&RUN read coverage plots normalized per 20 bp bin size. Chromosomal positions of regions containing MDR1, CDR1, and FLU1 and adjacent genes are represented to scale with boxes and arrows. Peaks from HF-Mrr1 ancestral -bound DNA recovered by an α-FLAG antibody and for the non-specific binding control recovered via IgG are shown. Signal indicates the average read density in α-FLAG relative to IgG within the peak region. ( D ) Circos plot showing global CUT&RUN-determined Mrr1-binding peaks of HF-Mrr1 Y813C (in blue) and HF-Mrr1 ancestral (in gray) in the C. lusitaniae L17 genome. Mrr1-binding peaks with a signal ≥2-fold compared to their respective IgG backgrounds and up to 1 kb away from the nearest ORF from experiment-1 were used (see ). The genomic positions of the 25 differentially expressed genes that constitute the Mrr1-regulon are marked with the L17 gene IDs.

Article Snippet: MRR1 complementation plasmids were linearized with the NotI-HF restriction enzyme (New England BioLabs), cleaned up using the Zymo DNA Clean & Concentrator kit (Zymo Research), and eluted in molecular biology grade water (Corning) before transformation of 2 μg into C. lusitaniae strain U04 mrr1 Δ as described below.

Techniques: Binding Assay, Activity Assay, Control

Journal: mBio

Article Title: Coordinated regulation of Mdr1- and Cdr1-mediated protection from antifungals by the Mrr1 transcription factor in emerging Candida spp.

doi: 10.1128/mbio.01323-25

Figure Lengend Snippet: Evolution of naturally acquired MRR1 mutations and binding profiles of low-activity Mrr1. ( A ) Schematic of the clinically evolved MRR1 alleles reported in Demers et al . . The asterisk indicates a nonsense mutation. Alleles in blue and orange encode for constitutively active and low-activity Mrr1 variants, respectively. ( B ) FLZ MIC of U04 clinical isolate (native allele MRR1 Y813C ) and U04 mrr1 ∆ complemented with MRR1 L1191H or MRR1 L1Q1* (L1191H + Q1197*) was determined by broth microdilution assays. The data shown represent the mean ± SD from three independent experiments. Strains with constitutive Mrr1 activity are in bold. ( C–E ) HF-Mrr1 L1Q1* CUT&RUN read coverage plots normalized per 20 bp bin size. Chromosomal positions of regions containing MDR1, CDR1, and FLU1 and adjacent genes are represented to scale with boxes and arrows. Peaks from HF-Mrr1 L1Q1* -bound DNA recovered by an α-FLAG antibody and for the non-specific binding control recovered via IgG are shown. The signal indicates the average read density in α-FLAG relative to IgG within the peak region.

Article Snippet: MRR1 complementation plasmids were linearized with the NotI-HF restriction enzyme (New England BioLabs), cleaned up using the Zymo DNA Clean & Concentrator kit (Zymo Research), and eluted in molecular biology grade water (Corning) before transformation of 2 μg into C. lusitaniae strain U04 mrr1 Δ as described below.

Techniques: Binding Assay, Activity Assay, Mutagenesis, Control

Journal: mBio

Article Title: Coordinated regulation of Mdr1- and Cdr1-mediated protection from antifungals by the Mrr1 transcription factor in emerging Candida spp.

doi: 10.1128/mbio.01323-25

Figure Lengend Snippet: Models for Mrr1 regulation in Candida spp. ( A ) Evolution of transporter regulation in Candida spp. Genes having characterized GOF mutations are in blue. The shapes indicate the type of experimental data used to support the model, including protein-DNA studies (this study; , , ), expression and phenotypic studies ( , , , , , , ). ( B ) Possible mechanism(s) for gene induction by Mrr1 based on published studies ( , , , , ). The mechanisms that impact Mrr1-mediated gene expression may vary between promoters and conditions within a strain, and there may be differences across strains and species.

Article Snippet: MRR1 complementation plasmids were linearized with the NotI-HF restriction enzyme (New England BioLabs), cleaned up using the Zymo DNA Clean & Concentrator kit (Zymo Research), and eluted in molecular biology grade water (Corning) before transformation of 2 μg into C. lusitaniae strain U04 mrr1 Δ as described below.

Techniques: Expressing, Gene Expression