An acquired mechanism of antifungal drug resistance simultaneously enables <i>Candida albicans</i> to escape from intrinsic host defenses

<div><p>The opportunistic fungal pathogen <i>Candida albicans</i> frequently produces genetically altered variants to adapt to environmental changes and new host niches in the course of its life-long association with the human host. Gain-of-function mutations in zinc cluster transcription factors, which result in the constitutive upregulation of their target genes, are a common cause of acquired resistance to the widely used antifungal drug fluconazole, especially during long-term therapy of oropharyngeal candidiasis. In this study, we investigated if <i>C</i>. <i>albicans</i> also can develop resistance to the antimicrobial peptide histatin 5, which is secreted in the saliva of humans to protect the oral mucosa from pathogenic microbes. As histatin 5 has been shown to be transported out of <i>C</i>. <i>albicans</i> cells by the Flu1 efflux pump, we screened a library of <i>C</i>. <i>albicans</i> strains that contain artificially activated forms of all zinc cluster transcription factors of this fungus for increased <i>FLU1</i> expression. We found that a hyperactive Mrr1, which confers fluconazole resistance by upregulating the multidrug efflux pump <i>MDR1</i> and other genes, also causes <i>FLU1</i> overexpression. Similarly to the artificially activated Mrr1, naturally occurring gain-of-function mutations in this transcription factor also caused <i>FLU1</i> upregulation and increased histatin 5 resistance. Surprisingly, however, Mrr1-mediated histatin 5 resistance was mainly caused by the upregulation of <i>MDR1</i> instead of <i>FLU1</i>, revealing a previously unrecognized function of the Mdr1 efflux pump. Fluconazole-resistant clinical <i>C</i>. <i>albicans</i> isolates with different Mrr1 gain-of-function mutations were less efficiently killed by histatin 5, and this phenotype was reverted when <i>MRR1</i> was deleted. Therefore, antimycotic therapy can promote the evolution of strains that, as a consequence of drug resistance mutations, simultaneously have acquired increased resistance against an innate host defense mechanism and are thereby better adapted to certain host niches.</p></div

Analysis of <i>MDR1</i> and <i>TPO2</i> expression in strains containing hyperactive zinc cluster transcription factors.

<p>Strains were grown to log phase in YPD medium and gene expression was analyzed by Northern hybridization with specific probes. The <i>ACT1</i> transcript served as control for equal loading on each blot. (A) Strains expressing artificially activated <i>MRR1</i>, <i>MRR2</i>, <i>WAR1</i>, and <i>ZCF35</i>: SC5314 (Parent), SCMRR1GAD1A and -B (<i>MRR1-GAD</i>), SCZCF34GAD1A and -B (<i>MRR2-GAD</i>), SCWAR1GAD1A and -B (<i>WAR1-GAD</i>), SCZCF35GAD1A and -B (<i>ZCF35-GAD</i>). (B) Strains containing GOF mutations in Mrr1: SC5314 (Parent), SCMRR1R24A and -B (Wild type), SCMRR1R34A and -B (P683S), SCMRR1R44A and -B (G997V), SCMRR1R54A and -B (G878E), SCMRR1R64A and -B (Q350L), SCMRR1R74A and -B (N803D), SCMRR1R84A and -B (T360I), SCMRR1R94A and -B (K335N), SCMRR1R982A and -B (K335N/T360I), SCMRR1R104A and -B (T896I). Strains SCMRR1GAD1A and -B (Mrr1-GAD) were included for comparison.</p

Mrr1-mediated Hst 5 resistance does not depend on Flu1.

<p>Wild type and <i>flu1</i>∆ mutants as well as derivatives of these strains containing the artificially activated <i>MRR1</i> were incubated in the absence or presence of 15 μM or 30 μM Hst 5 and the percent killing was determined as described in Materials and Methods. The following strains were used in this experiment: SC5314 (Wild type), SCMRR1GAD1A and -B (Wild type + <i>MRR1-GAD</i>), SCFLU1M4A and -B (<i>flu1</i>∆), SCΔ<i>flu1</i>MRR1GAD1A and -B (<i>flu1</i>∆ + <i>MRR1-GAD</i>). Significant differences (ANOVA) are marked with asterisks (*** <i>P</i> < 0.001); n.s., not significant (<i>P</i> > 0.05).</p

Fluconazole MICs for isogenic strains with different GOF mutations in <i>MRR1</i>.

<p>Fluconazole MICs for isogenic strains with different GOF mutations in <i>MRR1</i>.</p

Clinical <i>C</i>. <i>albicans</i> isolates with Mrr1 GOF mutations exhibit increased Hst 5 resistance.

<p>Strains were incubated in the absence or presence of 30 μM Hst 5 and the percent killing was determined as described in Materials and Methods. (A)-(E) show the results obtained with five different sets of fluconazole-susceptible isolates, matched fluconazole-resistant isolates from the same patients containing the indicated Mrr1 GOF mutations, and <i>mrr1</i>∆ mutants derived from the resistant isolates. The following strains were used in these experiments: G2, G5, G5MRR1M4A and -B (G5<i>mrr1</i>∆), B3, B4, B4MRR1M4A and -B (B4<i>mrr1</i>∆), DSY2285 (2285), DSY2286 (2286), DSY2286MRR1M4A and -B (2286<i>mrr1</i>∆), 5833, 6692, 6692MRR1M4A and -B (6692<i>mrr1</i>∆), 1442, 2271, 2271MRR1M4A and -B (2271<i>mrr1</i>∆). Significant differences (ANOVA) are marked with asterisks (*** <i>P</i> < 0.001); n.s., not significant (<i>P</i> > 0.05).</p

Mrr1-mediated Hst 5 resistance is mainly caused by <i>MDR1</i> overexpression.

<p>Strains were incubated in the presence of the indicated Hst 5 concentrations and the percent killing was determined as described in Materials and Methods. The different mutants were tested on separate occasions and compared with the wild-type controls. (A) Hst 5 sensitivity of strains containing the hyperactive <i>MRR1</i>^G997V allele in wild-type and <i>flu1</i>Δ <i>mdr1</i>Δ <i>tpo2</i>Δ triple mutant backgrounds. (B-D) Hst 5 sensitivity of strains containing the hyperactive <i>MRR1</i>^G997V allele in wild-type and (B) <i>flu1</i>Δ, (C) <i>mdr1</i>Δ, and (D) <i>tpo2</i>Δ single mutant backgrounds. The following strains were used in these experiments: SC5314 (Wild type), SCMRR1R44A and -C (Wild type, <i>MRR1</i>^G997V), SC∆<i>flu1</i>∆<i>mdr1</i>∆<i>tpo2</i>MRR1R44A and -B (<i>flu1</i>Δ <i>mdr1</i>Δ <i>tpo2</i>Δ, <i>MRR1</i>^G997V), SCΔ<i>flu1</i>MRR1R44A and -B (<i>flu1</i>Δ, <i>MRR1</i>^G997V), SCΔ<i>mdr1</i>MRR1R44C and -D (<i>mdr1</i>Δ, <i>MRR1</i>^G997V), SCΔ<i>tpo2</i>MRR1R44A and -B (<i>tpo2</i>Δ, <i>MRR1</i>^G997V). Significant differences (ANOVA) are marked with asterisks (** <i>P</i> < 0.01, *** <i>P</i> < 0.001); n.s., not significant (<i>P</i> > 0.05).</p

MPA resistance conferred by hyperactive Mrr1, Mrr2, War1, and Zcf35 depends on <i>FLU1</i>.

<p>Serial dilutions of the wild type, <i>flu1</i>∆ mutants, and transformants expressing the indicated hyperactive transcription factors were spotted onto SD-CSM agar plates without or with 1 μg/ml MPA or 5 μg/ml fluconazole and grown for 2 days at 30°C. The MICs of fluconazole, as determined in a broth microdilution assay, are given on the right. The following strains were used in these experiments: SC5314 (Wild type), SCFLU1M4A and -B (<i>flu1</i>∆), SCMRR1GAD1A and -B (Wild type + <i>MRR1-GAD</i>), SCΔ<i>flu1</i>MRR1GAD1A and -B (<i>flu1</i>∆ + <i>MRR1-GAD</i>), SCZCF34GAD1A and -B (Wild type + <i>MRR2-GAD</i>), SCΔ<i>flu1</i>MRR2GAD1A and -B (<i>flu1</i>∆ + <i>MRR2-GAD</i>), SCWAR1GAD1A and -B (Wild type + <i>WAR1-GAD</i>), SCΔ<i>flu1</i>WAR1GAD1A and -B (<i>flu1</i>∆ + <i>WAR1-GAD</i>), SCZCF35GAD1A and -B (Wild type + <i>ZCF35-GAD</i>), SCΔ<i>flu1</i>ZCF35GAD1A and -B (<i>flu1</i>∆ + <i>ZCF35-GAD</i>). Strain S2UI1, which carries a mutated <i>IMH3</i> allele that confers MPA resistance <i>(MPA</i>^R), was included as an additional control.</p

Hyperactive forms of the zinc cluster transcription factors Mrr1, Mrr2, War1, and Zcf35 confer increased MPA resistance and mediate <i>FLU1</i> overexpression.

<p>(A) Serial dilutions of the strains were spotted onto SD-CSM agar plates without or with 1 μg/ml MPA. The wild-type parent and derivatives expressing the indicated hyperactive transcription factors (two independent transformants in each case) were used in the experiment: SC5314 (Wild type), SCMRR1GAD1A and -B (<i>MRR1-GAD</i>), SCZCF34GAD1A and -B (<i>MRR2-GAD</i>), SCWAR1GAD1A and -B (<i>WAR1-GAD</i>), SCZCF35GAD1A and -B (<i>ZCF35-GAD</i>). Strain S2UI1, which carries a mutated <i>IMH3</i> allele that confers MPA resistance <i>(MPA</i>^R), was included as an additional control. (B) Strains carrying a P_<i>FLU1</i><i>-GFP</i> reporter fusion and the indicated hyperactive transcription factors were grown to log phase in YPD medium and the mean fluorescence of the cells was determined by flow cytometry. The results obtained with two independently generated reporter strains are shown in each case (means and standard deviations from at least three biological replicates). The background fluorescence of otherwise identical strains without <i>GFP</i> is indicated by the black part of each column. <i>GFP</i> reporter strains: SCFLU1G2A and -B (Wild type), SCFLU1G2-MRR1GAD1A and -B (<i>MRR1-GAD</i>), SCFLU1G2-MRR2GAD1A and -B (<i>MRR2-GAD</i>), SCFLU1G2-WAR1GAD1A and -B (<i>WAR1-GAD</i>), SCFLU1G2-ZCF35GAD1A and -B (<i>ZCF35-GAD</i>). Control strains without <i>GFP</i>: SC5314 (Wild type), SCMRR1GAD1A and -B (<i>MRR1-GAD</i>), SCZCF34GAD1A and -B (<i>MRR2-GAD</i>), SCWAR1GAD1A and -B (<i>WAR1-GAD</i>), SCZCF35GAD1A and -B (<i>ZCF35-GAD</i>). Significant differences from the wild-type control (background-subtracted values) are marked with asterisks (*** <i>P</i> < 0.001; ANOVA).</p

Naturally occuring Mrr1 GOF mutations cause <i>FLU1</i> overexpression and increased Hst 5 resistance.

<p>(A) Activation of the <i>FLU1</i> promoter by different Mrr1 GOF mutations. The two endogenous <i>MRR1</i> alleles of the parental strain SC5314 were replaced by mutated alleles encoding the indicated amino acid exchanges or by a nonmutated control allele (wild type). A P_<i>FLU1</i><i>-GFP</i> reporter fusion was integrated into two independently constructed strains in each case. Strains were grown to log phase in YPD medium and the mean fluorescence of the cells was determined by flow cytometry (means and standard deviations from at least three biological replicates). The background fluorescence of the parental strains without <i>GFP</i> is indicated by the black part of the columns. <i>GFP</i> reporter strains: SCFLU1G2A and -B (Parent), SCMRR1R24FLU1G2A and -B (Wild type), SCMRR1R34FLU1G2A and -B (P683S), SCMRR1R44FLU1G2A and -C (G997V), SCMRR1R54FLU1G2A and -B (G878E), SCMRR1R64FLU1G2A and -B (Q350L), SCMRR1R74FLU1G2A and -B (N803D), SCMRR1R84FLU1G2A and -B (T360I), SCMRR1R94FLU1G2A and -B (K335N), SCMRR1R982FLU1G2A and -B (K335N/T360I), SCMRR1R104FLU1G2A and -B (T896I). Control strains without <i>GFP</i>: SC5314 (Parent), SCMRR1R24A and -B (Wild type), SCMRR1R34A and -B (P683S), SCMRR1R44A and -B (G997V), SCMRR1R54A and -B (G878E), SCMRR1R64A and -B (Q350L), SCMRR1R74A and -B (N803D), SCMRR1R84A and -B (T360I), SCMRR1R94A and -B (K335N), SCMRR1R982A and -B (K335N/T360I), SCMRR1R104A and -B (T896I). Significant differences from the parent control (background-subtracted values, ANOVA) are marked with asterisks (*** <i>P</i> < 0.001); n.s., not significant (<i>P</i> > 0.05). (B) Natural GOF mutations in Mrr1 cause increased Hst 5 resistance. Strains were incubated in the absence or presence of 30 μM Hst 5 and the percent killing was determined as described in Materials and Methods. Two sets of experiments were performed on separate occasions: The top panel shows the comparison of the A-series of mutants with the parental strain SC5314, and the bottom panel shows the comparison of the B-series of mutants with the parental strain SC5314. The following strains were used in these experiments: SC5314 (Parent), SCMRR1R44A and -C (G997V), SCMRR1R54A and -B (G878E), SCMRR1R64A and -B (Q350L), SCMRR1R74A and -B (N803D), SCMRR1R84A and -B (T360I), SCMRR1R104A and -B (T896I). Significant differences from the parent control (ANOVA) are marked with asterisks (*** <i>P</i> < 0.001); n.s., not significant (<i>P</i> > 0.05).</p

A hyperactive Mrr1 causes increased Hst 5 resistance.

<p>The wild-type strain SC5314 (WT, black bars) and derivatives (gray bars) containing artificially activated <i>MRR1</i> (SCMRR1GAD1A and -B), <i>MRR2</i> (SCZCF34GAD1A and -B), <i>WAR1</i> (SCWAR1GAD1A and -B), or <i>ZCF35</i> (SCZCF35GAD1A and -B) were exposed to Hst 5 (3.75 μM to 30 μM) using microdilution plate assays, and the percent killing was calculated as described in Materials and Methods. The four transcription factors were tested on separate occasions, and a different batch of Hst 5 was used for <i>MRR1</i> and <i>ZCF35 vs</i>. <i>MRR2</i> and <i>WAR1</i>. Significant differences from the wild-type control (t-test) are marked with asterisks (* <i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001); n.s., not significant (<i>P</i> > 0.05).</p