In vivo NMR as a tool for probing molecular structure and dynamics in intact Chlamydomonas reinhardtii cells

GO biological process enrichment for the 882 deletion strains below the threshold of detection by microarray in the BCprot relative to the gene universe of strains present in at least one deletion collectio

Genetic and Genomic Architecture of the Evolution of Resistance to Antifungal Drug Combinations

<div><p>The evolution of drug resistance in fungal pathogens compromises the efficacy of the limited number of antifungal drugs. Drug combinations have emerged as a powerful strategy to enhance antifungal efficacy and abrogate drug resistance, but the impact on the evolution of drug resistance remains largely unexplored. Targeting the molecular chaperone Hsp90 or its downstream effector, the protein phosphatase calcineurin, abrogates resistance to the most widely deployed antifungals, the azoles, which inhibit ergosterol biosynthesis. Here, we evolved experimental populations of the model yeast <i>Saccharomyces cerevisiae</i> and the leading human fungal pathogen <i>Candida albicans</i> with azole and an inhibitor of Hsp90, geldanamycin, or calcineurin, FK506. To recapitulate a clinical context where Hsp90 or calcineurin inhibitors could be utilized in combination with azoles to render resistant pathogens responsive to treatment, the evolution experiment was initiated with strains that are resistant to azoles in a manner that depends on Hsp90 and calcineurin. Of the 290 lineages initiated, most went extinct, yet 14 evolved resistance to the drug combination. Drug target mutations that conferred resistance to geldanamycin or FK506 were identified and validated in five evolved lineages. Whole-genome sequencing identified mutations in a gene encoding a transcriptional activator of drug efflux pumps, <i>PDR1</i>, and a gene encoding a transcriptional repressor of ergosterol biosynthesis genes, <i>MOT3</i>, that transformed azole resistance of two lineages from dependent on calcineurin to independent of this regulator. Resistance also arose by mutation that truncated the catalytic subunit of calcineurin, and by mutation in <i>LCB1</i>, encoding a sphingolipid biosynthetic enzyme. Genome analysis revealed extensive aneuploidy in four of the <i>C. albicans</i> lineages. Thus, we identify molecular determinants of the transition of azole resistance from calcineurin dependence to independence and establish multiple mechanisms by which resistance to drug combinations evolves, providing a foundation for predicting and preventing the evolution of drug resistance.</p></div

Mutations in <i>HSP90</i> confer resistance to azole and geldanamycin in two <i>S. cerevisiae</i> lineages and in one <i>C. albicans</i> lineage.

<p> (A) Sc-G-12 (right panel) and Sc-G-14 (left panel) are both resistant to azole and geldanamycin and slightly cross-resistant to azole and radicicol, relative to their parental strains (above). (B) Resistance to azole and geldanamycin in Sc-G-14 is attributable to <i>HSC82^I117N</i>. Replacing the ancestral allele with the <i>HSC82^I117N</i> allele expressed on a plasmid increases resistance of the ancestral strain to the level observed in Sc-G-14, while replacing the <i>HSC82^I117N</i> allele with the ancestral allele on a plasmid abrogates resistance of Sc-G-14. (C) Deletion of <i>HSC82</i> in Sc-G-12 or its parental strain phenocopies resistance of Sc-G-12, suggesting that <i>HSC82^K385*</i> confers resistance by loss of function of <i>HSC82</i>. (D) Ca-G-10 has increased resistance to azole and geldanamycin but no cross-resistance to azole and FK506 or azole and radicicol. (E) Resistance to azole and geldanamycin in Ca-G-10 is attributable to <i>HSP90^D91Y</i>. Replacing the native <i>HSP90</i> allele in parental strain with <i>HSP90^D91Y</i> phenocopied resistance of Ca-G-10. Conversely, resistance of Ca-G-10 was abrogated when <i>HSP90^D91Y</i> was replaced with the ancestral <i>HSP90</i> allele. Resistance assays were performed and analyzed is in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003390#pgen-1003390-g002" target="_blank">Figure 2</a>, after incubation at 30°C for 2 days (D) or 3 days (A–C, E). Assays were performed in YPD (A, C–E) or SD with amino acid supplements (B). GdA = geldanamycin; RAD = radicicol; and FL = fluconazole.</p

Non-synonymous <i>S. cerevisiae</i> single nucleotide variants.

<p>Non-synonymous <i>S. cerevisiae</i> single nucleotide variants.</p

Cross-resistance profiles provide a strategy to predict resistance mechanisms.

<p>(A) Strains evolved in azole and FK506 were tested for cross-resistance to azole and the calcineurin inhibitor cyclosporin A as well as azole and the Hsp90 inhibitor geldanamycin. (B) Candidate resistance mechanisms based on specific cross-resistance profiles of strains evolved with azole and FK506. (C) Strains evolved in azole and geldanamycin were tested for cross-resistance to azole and the Hsp90 inhibitor radicicol as well as azole and the calcineurin inhibitor FK506. (D) Candidate resistance mechanisms based on specific cross-resistance profiles of strains evolved with azole and geldanamycin. GdA = geldanamycin; RAD = radicicol; and CsA = cyclosporin A.</p

Non-synonymous <i>C. albicans</i> single nucleotide variants.

<p>Non-synonymous <i>C. albicans</i> single nucleotide variants.</p

Design and outcome of the experimental evolution of resistance to drug combinations.

<p>A) Experimental populations were initiated with <i>S. cerevisiae</i> and <i>C. albicans</i> strains resistant to azoles due to <i>erg3</i> loss of function. This resistance mechanism is contingent on Hsp90 and calcineurin, such that inhibition of either of these cellular stress response regulators results in cell death (<b>t₀</b>). Propagation of these strains in the presence of azole and the Hsp90 inhibitor geldanamycin or azole and the calcineurin inhibitor FK506 at concentrations that exert selection pressure for resistance to the drug combination results in the evolution of resistance to geldanamycin or FK506 (<b>t_1a</b>) or the evolution of an azole resistance mechanism that is independent of Hsp90 or calcineurin (<b>t_1b</b>) among extant lineages. B) Single colony founders were used to initiate evolution experiments in 24- or 96-well plates containing control and treatment wells. Controls consisted of: no drug, azole alone, geldanamycin alone, or FK506 alone, where drug concentrations were not inhibitory. Treatment wells consisted of combinations of azole and geldanamycin or FK506, selected based on dose response matrices (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003390#pgen.1003390.s002" target="_blank">Figure S2</a>). <b>C</b>) Experimental evolution of resistance to azole and geldanamycin or azole and FK506 yielded 14 resistant lineages out of 290 initiated. <i>Ca</i> = <i>Candida albicans; Sc</i> = <i>Saccharomyces cerevisiae</i>.</p

Evolution experiment treatments and conditions.

<p>Evolution experiment treatments and conditions.</p

Comparing predicted ancestral and predicted extant nucleosome occupancy.

<p>(A) The relationship between nucleosome occupancy scores as predicted for nucleotides positioned at nucleosome dyads in humans and the corresponding nucleotides in the ancestor of humans and chimps (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003373#s4" target="_blank">Materials and Methods</a> for details on the prediction algorithm). (B) The distance between each human dyad and the nucleotide with the highest occupancy score within a ±100 nt window around that dyad as calculated for human sequence (D_h) and ancestral sequence (D_a) assuming human dyad positions. Only dyads where a single substitution had occurred within the ±100 nt window along the human lineage were considered. (C) Distribution of differences (ΔD) between D_a and D_h as defined in the text.</p