Fig. 6: NP-BTA targets C. albicans glutaminyl-tRNA synthetase.
a Dose−response assay based on twofold serial dilution of NP-BTA for C. albicans (SN95), C. auris (VPCI 673), or C. glabrata (F27). Assays were incubated for 24 h at 30 °C in YPD and growth was normalized relative to the respective no-compound control (see color bar). MIC80 values listed in white. Structure of NP-BTA displayed below heat map. b The double-barcoded C. albicans heterozygous deletion collection was grown in the presence or absence of NP-BTA (0.8 μM). Strains with a solvent/drug log2 ratio greater than 7 median absolute deviations (MADs) above the median were considered significant (see legend). UPTAG reads are shown in light gray, DOWNTAG reads are shown in dark gray. c Dose−response assay based on twofold serial dilution of NP-BTA for C. albicans (CaSS1) or tetO-GLN4/gln4Δ in the absence or presence of 0.05 μg/mL of doxycycline (DOX) as indicated. Assay performed as in (a). d Dose−response assay based on twofold serial dilution of NP-BTA for C. albicans parent (CaLC2749), as well as three independent resistant lineages (R1−R3). Identified Gln4 substitutions are listed. Dose−response assays were performed as in (a). e Homology model of the C-terminal domain of C. albicans Gln4 (beige, cartoon) based on the apo crystal structure of S. cerevisiae Gln4 (PDB: 4H3S; 66% sequence identity). To illustrate the location of the active site, tRNAGln (blue, cartoon) and glutaminyl aminoacyl-adenylate analog 5ʹ-O-[N-(L-glutaminyl)sulphamoyl]adenosine (red spheres; A: adenosine; R: ribose; Gln: glutamine) were placed from the Escherichia coli GlnRS-tRNA-substrate analog complex (PDB: 1QTQ), which aligned to the C. albicans model with an RMSD of 2.2 Å. Amino acids whose substitution confers reduced sensitivity to NP-BTA are shown as red sticks; numbering reflects amino acid position in C. albicans Gln4. Lower inset: Two binding poses of NP-BTA (sticks) were identified in the Gln4 active site after computational docking to the apo structure of S. cerevisiae Gln4 (gray surface). f Protein translation was evaluated as in Fig. 5. Cells were treated for 10 m with 100 μg/mL of anisomycin or 6.25 μM NP-BTA. Histograms depict relative fluorescence intensity (FITC-A) of events, values depict median fluorescence intensity (MFI). Experiment was performed in biological duplicate with similar results. g Relative growth/survival of human kidney-derived cells (HEK293T-luciferase, blue line) and azole-tolerant C. albicans in co-culture (CaCi-2-GFP, CaLC867, red line). Each point depicts the mean of triplicate wells. Error bars, SEM. Four-parameter curve fitting was performed in Prism v8.4. h Depletion of GLN4 by the addition of DOX to the drinking water significantly improved survival relative to other conditions. Log-rank (Mantel-Cox) test, ***P < 0.0001. Log-rank test for trend, P = 0.0003. Source data are provided as a Source Data file.
