mrr1 complementation plasmids  (New England Biolabs)

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

Journal: mBio

Article Title: Balancing Positive and Negative Selection: In Vivo Evolution of Candida lusitaniae MRR1

doi: 10.1128/mBio.03328-20

Figure Lengend Snippet: Constitutively active and low-activity Mrr1 variants naturally evolved in a single C. lusitaniae population. (A) Maximum likelihood-based phylogeny constructed from SNPs identified in the whole-genome sequences of 20 C. lusitaniae isolates; modified from Demers et al. . Select branchpoints are marked with the Mrr1 variants (text colored to match branches) present in subsequent isolates. Mrr1 variants are identified by amino acid changes that resulted from SNPs or indels; * indicates a stop codon. The one-nucleotide indel in codons P1174 (insertion) and K912 (deletion) cause frameshift mutations that resulted in early termination, denoted with “(t),” at N1176 and L927, respectively. Gray star at the root of the tree denotes the “ancestral” MRR1 sequence, which lacks any of the mutations listed. U04 and U05, which are used in panels B and C, are highlighted. FLZ MICs (μg/ml) as determined in reference are listed. (B) FLZ MICs for unaltered, mrr1 Δ and MRR1 complemented strains in the FLZ-resistant U04 (native allele MRR1 Y813C ) strain background. (C) Same as in panel B, but in the FLZ-sensitive U05 strain background (native allele MRR1 L1191H+Q1197 * ). Strains containing the same MRR1 allele in panels B and C are represented by circles of the same color. Data shown represent at least four independent assays on different days. Each sample was statistically compared to every other sample; the same lowercase letters indicate samples that are not significantly different, and different lowercase letters indicate significant differences ( P < 0.0001 [B] or P < 0.001 [C]) as determined by one-way ANOVA with Tukey’s multiple-comparison test of log 2 -transformed values. (D) Differentially expressed genes between strains harboring the constitutively active Mrr1-Y813C variant (U04 and U04 mrr1 Δ +MRR1 Y813C ) and those lacking MRR1 (U04 mrr1 Δ) or harboring low-activity variants (U04 mrr1 Δ +MRR1 ancestral and U04 mrr1 Δ +MRR1 L1191H+Q1197 * ) when grown in liquid YPD medium; statistical cutoffs used were FDR of <0.05 and fold change of ≥2 (see Table S1 in the supplemental material). Normalized counts per million (CPM) from RNA-Seq are scaled by row (gene) with hierarchical clustering by Euclidean distance. Complemented strains are denoted by their respective MRR1 allele. Predicted C. albicans homologs are listed next to C. lusitaniae gene names ( Table S1 ).

Article Snippet: Plasmids from E. coli were isolated using a Zyppy plasmid miniprep kit (Zymo Research) and subsequently verified by Sanger sequencing at the Dartmouth College Genomics and Molecular Biology Shared Resources Core. pMQ30 MRR1 complementation plasmids were linearized with NotI-HF (New England BioLabs), cleaned up with 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 or U05 mrr1 Δ, as described below.

Techniques: Activity Assay, Construct, Modification, Sequencing, Transformation Assay, Variant Assay, RNA Sequencing Assay

Journal: mBio

Article Title: Balancing Positive and Negative Selection: In Vivo Evolution of Candida lusitaniae MRR1

doi: 10.1128/mBio.03328-20

Figure Lengend Snippet: Premature stop codons in Mrr1 differentially impact MDR1 induction by benomyl. (A) Mean FLZ MICs for each of the 20 clinical C. lusitaniae isolates in Fig. 1A separated by the number of nonsynonymous mutations within MRR1 (as defined in reference ); mean of each group is shown. Two-tailed unpaired t test of log 2 -transformed MIC values; ***, P < 0.001. (B) Schematic of C. lusitaniae MRR1 annotated with putative regulatory domains determined by sequence analysis and homology to C. albicans and locations of truncating (top) and putative activating (bottom) mutations. Putative domains include a DNA binding domain with a zinc cluster motif (Zn 2 Cys 6 ; amino acids 33 to 61), a transcriptional regulatory middle homology region (MHR; amino acids ∼607 to 1023), an inhibitory domain (ID; amino acids 1123 to 1217), and an activating domain (AD; amino acids 1218 to 1265). L927 and N1176 are the locations of stop codons caused by indels in codons K912 and P1174, respectively. (C and D) MDR1 expression normalized to ACT1 in the absence (solid bars) or presence (striped bars) of 50 μg/ml benomyl. Means ± standard deviations (SDs) of representative data in biological triplicates are shown; similar trends observed on at least three different days. Two-way ANOVA with Sidak’s multiple-comparison test; **, P < 0.01; ****, P < 0.0001; ns, not significant. In panel D, strain names are highlighted corresponding to the number of mutations in MRR1 , yellow for one and green for two, as in Fig. 2B . The colors of the circles (A), lines (B), and bars (D) correspond to MRR1 alleles shown Fig. 1A .

Article Snippet: Plasmids from E. coli were isolated using a Zyppy plasmid miniprep kit (Zymo Research) and subsequently verified by Sanger sequencing at the Dartmouth College Genomics and Molecular Biology Shared Resources Core. pMQ30 MRR1 complementation plasmids were linearized with NotI-HF (New England BioLabs), cleaned up with 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 or U05 mrr1 Δ, as described below.

Techniques: Two Tailed Test, Transformation Assay, Sequencing, Binding Assay, Expressing

Journal: mBio

Article Title: Balancing Positive and Negative Selection: In Vivo Evolution of Candida lusitaniae MRR1

doi: 10.1128/mBio.03328-20

Figure Lengend Snippet: Premature stop codons repeatedly arose in constitutively active Mrr1 variants resulting in reduced Mrr1 activity but, in some cases, restored Mrr1 inducibility. Schematic of inferred evolution of MRR1 alleles in the L1191H+Q1197* (A) and Y1126N (B) lineages. (C) FLZ MICs for U04, U04 mrr1 Δ and MRR1 complemented strains in the U04 mrr1 Δ background. Means ± SDs from three independent assays on different days shown. Each sample was statistically compared to every other sample; the same lowercase letters indicate samples that are not significantly different, and different lowercase letters indicate significant differences ( P < 0.01) as determined by one-way ANOVA with Tukey’s multiple-comparison test of log 2 -transformed values. (D) MDR1 expression normalized to ACT1 from culture grown in YPD (bars, left y axis). Means ± SDs from three independent assays on different days; data from each day were normalized to the expression of U04 mrr1 Δ+ MRR1 ancestral . Each sample was statistically compared to every other sample; the same lowercase letters indicate samples that are not significantly different, and different lowercase letters indicate significant differences (b to d, P < 0.05; all other pairwise comparisons, P < 0.01) as determined by one-way ANOVA with Tukey’s multiple-comparison testing of log 2 -transformed data. Overlaid with log 2 -transformed mean ± SD fold change (FC) in normalized MDR1 expression following exposure to 50 μg/ml benomyl (squares, right y axis); full data presented with statistics in Fig. S3D . (C and D) FLZ MICs and MDR1/ACT1 expression data are colored to match; the sample names are shown on the x axis of panel D. Comparison of mean basal MDR1 (E), MGD1 (F), or FLU1 (G) expression from panel D and Fig. S3B or C , respectively, excluding strains lacking functional MRR1 , and mean ± SD log 2 -transformed FC of the induction following benomyl exposure from Fig. S3D to ; circles colored to match those in panel C. Goodness of fit r 2 value for nonlinear regression shown.

Article Snippet: Plasmids from E. coli were isolated using a Zyppy plasmid miniprep kit (Zymo Research) and subsequently verified by Sanger sequencing at the Dartmouth College Genomics and Molecular Biology Shared Resources Core. pMQ30 MRR1 complementation plasmids were linearized with NotI-HF (New England BioLabs), cleaned up with 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 or U05 mrr1 Δ, as described below.

Techniques: Activity Assay, Transformation Assay, Expressing, Functional Assay

Journal: mBio

Article Title: Balancing Positive and Negative Selection: In Vivo Evolution of Candida lusitaniae MRR1

doi: 10.1128/mBio.03328-20

Figure Lengend Snippet: Constitutive Mrr1 activity decreases resistance to H 2 O 2 in multiple Candida species. (A) Growth curve of U04 mrr1 Δ+ MRR1 Y813C (teal) and U04 mrr1 Δ+ MRR1 Y813C mdr1 Δ (black) grown at 37°C in YNB medium supplemented with the indicated carbon source: glucose (circles), amino acids (triangles), or glycerol (squares). Means from representative data acquired in triplicates shown. (B) Quantification of cytokines IL-8 and IL-1β in BAL fluid from the CF patient with (red) or seven patients without (black) C. lusitaniae in their lungs. Two-way ANOVA with Sidak’s multiple-comparison testing found no significant differences. (C) Serial dilution assays of C. lusitaniae , C. albicans , and C. dubliniensis strains on YPD or YPD supplemented with the indicated concentration of diamide or H 2 O 2 . Strain names in bold font were shown to contain GOF mutations in Mrr1 resulting in increased FLZ resistance ( Fig. 3C and references , , and ). Plates were imaged after 24 or 48 h of growth at 37°C, as indicated. (D) Percent growth in well-aerated 5 ml YPD plus 1 mM H 2 O 2 was calculated relative to that of the vehicle only control after 22 to 24 h growth at 37°C. These data represent six independent assays performed on different days. Significance determined by paired t test; **, P < 0.01.

Article Snippet: Plasmids from E. coli were isolated using a Zyppy plasmid miniprep kit (Zymo Research) and subsequently verified by Sanger sequencing at the Dartmouth College Genomics and Molecular Biology Shared Resources Core. pMQ30 MRR1 complementation plasmids were linearized with NotI-HF (New England BioLabs), cleaned up with 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 or U05 mrr1 Δ, as described below.

Techniques: Activity Assay, Serial Dilution, Concentration Assay

Journal: mBio

Article Title: Balancing Positive and Negative Selection: In Vivo Evolution of Candida lusitaniae MRR1

doi: 10.1128/mBio.03328-20

Figure Lengend Snippet: Trade-off between FLZ and H 2 O 2 resistance persists in evolving C. lusitaniae populations during a chronic lung infection. (A) Schematic of sampling timeline (top) and histogram of the number of isolates that (i) were mostly uninhibited on FLZ, but were inhibited by H 2 O 2 (red), (ii) were mostly uninhibited on H 2 O 2 but were inhibited by FLZ (blue), or (iii) were uninhibited under both conditions (black). For the schematic, the gray bar represents the 6 to 10 months before the BAL during which this patient was identified as being colonized by non- albicans Candida (NAC) species. C. lusitaniae was determined to be the dominate microbe in the upper and lower lobe (UL and LL, respectively) BAL samples (red arrow), which marks the start of the green bar. Sp1 was obtained 1 month before the BAL and was retrospectively also found to contain abundant C. lusitaniae . Sp1.5, Sp2, Sp4, Sp5, and Sp6 were obtained 3, 9, 32, 35, and 38 months, respectively, after the BAL and all contained C. lusitaniae . A 4-month course of FLZ therapy was given after the BAL. Scale bar indicates 6 months. Multiple isolates were collected from each sample/timepoint ( n = 38 to 80) and assayed for growth on YPD supplemented with 8 μg/ml FLZ or 4 mM H 2 O 2 . Growth was scored as completely inhibited, partially inhibited or uninhibited compared to that of a YPD-only control. (B) Model for the evolution of C. lusitaniae MRR1 in this population. Whole-genome sequencing and mutation analyses suggest that following the initial infection with C. lusitaniae harboring the Mrr1-ancestral variant, a combination of exposure to different stimuli that changed overtime or by locations within the CF lung environment led to the selection for a heterogeneous population. Multiple constitutively active Mrr1 variants arose, and while some persisted over time, others were subsequently mutated again. The secondary mutations causing premature stop codons (represented by shortened bars) resulted in reversion to low Mrr1 activity that was inducible or complete loss of Mrr1 activity. The balance between selective pressures resulted in a heterogeneous population of isolates with varied resistance ( R ) to biologically and clinically important compounds.

Article Snippet: Plasmids from E. coli were isolated using a Zyppy plasmid miniprep kit (Zymo Research) and subsequently verified by Sanger sequencing at the Dartmouth College Genomics and Molecular Biology Shared Resources Core. pMQ30 MRR1 complementation plasmids were linearized with NotI-HF (New England BioLabs), cleaned up with 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 or U05 mrr1 Δ, as described below.

Techniques: Infection, Sampling, Sequencing, Mutagenesis, Variant Assay, Selection, Activity Assay