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Gain of function mutations in CgPDR1 of Candida glabrata not only mediate antifungal resistance but also enhance virulence.

Ferrari S, Ischer F, Calabrese D, Posteraro B, Sanguinetti M, Fadda G, Rohde B, Bauser C, Bader O, Sanglard D - PLoS Pathog. (2009)

Bottom Line: Moreover, C. glabrata isolates complemented with CgPDR1 hyperactive alleles were not only more virulent in mice than those with wild type alleles, but they also gained fitness in the same animal model.The presence of CgPDR1 hyperactive alleles also contributed to fluconazole treatment failure in the mouse model.In conclusion, this study shows for the first time that CgPDR1 mutations are not only responsible for in vitro/in vivo azole resistance but that they can also confer a selective advantage under host conditions.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology, University of Lausanne and University Hospital Center, Lausanne, Switzerland.

ABSTRACT
CgPdr1p is a Candida glabrata Zn(2)-Cys(6) transcription factor involved in the regulation of the ABC-transporter genes CgCDR1, CgCDR2, and CgSNQ2, which are mediators of azole resistance. Single-point mutations in CgPDR1 are known to increase the expression of at least CgCDR1 and CgCDR2 and thus to contribute to azole resistance of clinical isolates. In this study, we investigated the incidence of CgPDR1 mutations in a large collection of clinical isolates and tested their relevance, not only to azole resistance in vitro and in vivo, but also to virulence. The comparison of CgPDR1 alleles from azole-susceptible and azole-resistant matched isolates enabled the identification of 57 amino acid substitutions, each positioned in distinct CgPDR1 alleles. These substitutions, which could be grouped into three different "hot spots," were gain of function (GOF) mutations since they conferred hyperactivity to CgPdr1p revealed by constitutive high expression of ABC-transporter genes. Interestingly, the major transporters involved in azole resistance (CgCDR1, CgCDR2, and CgSNQ2) were not always coordinately expressed in presence of specific CgPDR1 GOF mutations, thus suggesting that these are rather trans-acting elements (GOF in CgPDR1) than cis-acting elements (promoters) that lead to azole resistance by upregulating specific combinations of ABC-transporter genes. Moreover, C. glabrata isolates complemented with CgPDR1 hyperactive alleles were not only more virulent in mice than those with wild type alleles, but they also gained fitness in the same animal model. The presence of CgPDR1 hyperactive alleles also contributed to fluconazole treatment failure in the mouse model. In conclusion, this study shows for the first time that CgPDR1 mutations are not only responsible for in vitro/in vivo azole resistance but that they can also confer a selective advantage under host conditions.

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Reconstitution of CgPDR1 GOF alleles in C. glabrata.(A) Fluconazole susceptibility testing of DSY562 pdr1Δ mutant strain (SFY93) expressing different CgPDR1 alleles, which were named according to their strain number origin and by indicating the amino acid substitution (in superscript) associated with a specific strain number. The following strains correspond to the indicated genotypes: DSY562 pdr1Δ+486:SFY98; 489L328F: SFY99; 738: SFY100; 739R376W: SFY101; 2253: SFY102; 2254D1082G:SFY103; 2235: SFY104; 2234T588A: SFY105; 701: SFY106; 704T607S: SFY107; 529:SFY108; 530E1083Q: SFY109; 753: SFY110; 754Y584C: SFY111; 726: SFY112; 727D876Y: SFY113; BPY55P882L: SFY116. (B) Expression of CgCDR1, CgCDR2, and CgSNQ2 in the DSY562 pdr1Δ mutant strain (SFY93) expressing different CgPDR1 alleles, named according to their strain number origin. Quantification was performed by real-time RT-PCR. The values, which are averages of four separated experiments, represent the increase in gene expression relative to DSY562 (set at 1.00). Error bars show standard deviations.
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ppat-1000268-g006: Reconstitution of CgPDR1 GOF alleles in C. glabrata.(A) Fluconazole susceptibility testing of DSY562 pdr1Δ mutant strain (SFY93) expressing different CgPDR1 alleles, which were named according to their strain number origin and by indicating the amino acid substitution (in superscript) associated with a specific strain number. The following strains correspond to the indicated genotypes: DSY562 pdr1Δ+486:SFY98; 489L328F: SFY99; 738: SFY100; 739R376W: SFY101; 2253: SFY102; 2254D1082G:SFY103; 2235: SFY104; 2234T588A: SFY105; 701: SFY106; 704T607S: SFY107; 529:SFY108; 530E1083Q: SFY109; 753: SFY110; 754Y584C: SFY111; 726: SFY112; 727D876Y: SFY113; BPY55P882L: SFY116. (B) Expression of CgCDR1, CgCDR2, and CgSNQ2 in the DSY562 pdr1Δ mutant strain (SFY93) expressing different CgPDR1 alleles, named according to their strain number origin. Quantification was performed by real-time RT-PCR. The values, which are averages of four separated experiments, represent the increase in gene expression relative to DSY562 (set at 1.00). Error bars show standard deviations.

Mentions: Selected CgPDR1 alleles from eight other pairs of isolates (Table 1) were reintroduced at the CgPDR1 genomic locus in an azole-susceptible background lacking CgPDR1. CgPDR1 alleles from each pair of isolates only differ by a point mutation leading to a single amino acid substitution in either the inhibitory domain, the MHR or the activation domain of CgPdr1p. These mutations, which were specific for azole-resistant isolates, restored azole resistance in a pdr1Δ mutant (fluconazole MICs from 64–128 µg/ml, Figure 6A). Since only alleles containing these mutations conferred CgCDR1 constitutive high expression (from 4- to 150-fold expression increase, Figure 6B), these mutations could be assigned as GOF mutations. Moreover, single amino acid substitutions in either the inhibitory domain, the MHR or the activation domain could confer drug resistance. Once again, altered CgPDR1 expression could not account for azole resistance, since the CgPDR1 mRNA levels were similar between the clinical strains and the revertant strains expressing their corresponding CgPDR1 alleles (Figure S3).


Gain of function mutations in CgPDR1 of Candida glabrata not only mediate antifungal resistance but also enhance virulence.

Ferrari S, Ischer F, Calabrese D, Posteraro B, Sanguinetti M, Fadda G, Rohde B, Bauser C, Bader O, Sanglard D - PLoS Pathog. (2009)

Reconstitution of CgPDR1 GOF alleles in C. glabrata.(A) Fluconazole susceptibility testing of DSY562 pdr1Δ mutant strain (SFY93) expressing different CgPDR1 alleles, which were named according to their strain number origin and by indicating the amino acid substitution (in superscript) associated with a specific strain number. The following strains correspond to the indicated genotypes: DSY562 pdr1Δ+486:SFY98; 489L328F: SFY99; 738: SFY100; 739R376W: SFY101; 2253: SFY102; 2254D1082G:SFY103; 2235: SFY104; 2234T588A: SFY105; 701: SFY106; 704T607S: SFY107; 529:SFY108; 530E1083Q: SFY109; 753: SFY110; 754Y584C: SFY111; 726: SFY112; 727D876Y: SFY113; BPY55P882L: SFY116. (B) Expression of CgCDR1, CgCDR2, and CgSNQ2 in the DSY562 pdr1Δ mutant strain (SFY93) expressing different CgPDR1 alleles, named according to their strain number origin. Quantification was performed by real-time RT-PCR. The values, which are averages of four separated experiments, represent the increase in gene expression relative to DSY562 (set at 1.00). Error bars show standard deviations.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2607542&req=5

ppat-1000268-g006: Reconstitution of CgPDR1 GOF alleles in C. glabrata.(A) Fluconazole susceptibility testing of DSY562 pdr1Δ mutant strain (SFY93) expressing different CgPDR1 alleles, which were named according to their strain number origin and by indicating the amino acid substitution (in superscript) associated with a specific strain number. The following strains correspond to the indicated genotypes: DSY562 pdr1Δ+486:SFY98; 489L328F: SFY99; 738: SFY100; 739R376W: SFY101; 2253: SFY102; 2254D1082G:SFY103; 2235: SFY104; 2234T588A: SFY105; 701: SFY106; 704T607S: SFY107; 529:SFY108; 530E1083Q: SFY109; 753: SFY110; 754Y584C: SFY111; 726: SFY112; 727D876Y: SFY113; BPY55P882L: SFY116. (B) Expression of CgCDR1, CgCDR2, and CgSNQ2 in the DSY562 pdr1Δ mutant strain (SFY93) expressing different CgPDR1 alleles, named according to their strain number origin. Quantification was performed by real-time RT-PCR. The values, which are averages of four separated experiments, represent the increase in gene expression relative to DSY562 (set at 1.00). Error bars show standard deviations.
Mentions: Selected CgPDR1 alleles from eight other pairs of isolates (Table 1) were reintroduced at the CgPDR1 genomic locus in an azole-susceptible background lacking CgPDR1. CgPDR1 alleles from each pair of isolates only differ by a point mutation leading to a single amino acid substitution in either the inhibitory domain, the MHR or the activation domain of CgPdr1p. These mutations, which were specific for azole-resistant isolates, restored azole resistance in a pdr1Δ mutant (fluconazole MICs from 64–128 µg/ml, Figure 6A). Since only alleles containing these mutations conferred CgCDR1 constitutive high expression (from 4- to 150-fold expression increase, Figure 6B), these mutations could be assigned as GOF mutations. Moreover, single amino acid substitutions in either the inhibitory domain, the MHR or the activation domain could confer drug resistance. Once again, altered CgPDR1 expression could not account for azole resistance, since the CgPDR1 mRNA levels were similar between the clinical strains and the revertant strains expressing their corresponding CgPDR1 alleles (Figure S3).

Bottom Line: Moreover, C. glabrata isolates complemented with CgPDR1 hyperactive alleles were not only more virulent in mice than those with wild type alleles, but they also gained fitness in the same animal model.The presence of CgPDR1 hyperactive alleles also contributed to fluconazole treatment failure in the mouse model.In conclusion, this study shows for the first time that CgPDR1 mutations are not only responsible for in vitro/in vivo azole resistance but that they can also confer a selective advantage under host conditions.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology, University of Lausanne and University Hospital Center, Lausanne, Switzerland.

ABSTRACT
CgPdr1p is a Candida glabrata Zn(2)-Cys(6) transcription factor involved in the regulation of the ABC-transporter genes CgCDR1, CgCDR2, and CgSNQ2, which are mediators of azole resistance. Single-point mutations in CgPDR1 are known to increase the expression of at least CgCDR1 and CgCDR2 and thus to contribute to azole resistance of clinical isolates. In this study, we investigated the incidence of CgPDR1 mutations in a large collection of clinical isolates and tested their relevance, not only to azole resistance in vitro and in vivo, but also to virulence. The comparison of CgPDR1 alleles from azole-susceptible and azole-resistant matched isolates enabled the identification of 57 amino acid substitutions, each positioned in distinct CgPDR1 alleles. These substitutions, which could be grouped into three different "hot spots," were gain of function (GOF) mutations since they conferred hyperactivity to CgPdr1p revealed by constitutive high expression of ABC-transporter genes. Interestingly, the major transporters involved in azole resistance (CgCDR1, CgCDR2, and CgSNQ2) were not always coordinately expressed in presence of specific CgPDR1 GOF mutations, thus suggesting that these are rather trans-acting elements (GOF in CgPDR1) than cis-acting elements (promoters) that lead to azole resistance by upregulating specific combinations of ABC-transporter genes. Moreover, C. glabrata isolates complemented with CgPDR1 hyperactive alleles were not only more virulent in mice than those with wild type alleles, but they also gained fitness in the same animal model. The presence of CgPDR1 hyperactive alleles also contributed to fluconazole treatment failure in the mouse model. In conclusion, this study shows for the first time that CgPDR1 mutations are not only responsible for in vitro/in vivo azole resistance but that they can also confer a selective advantage under host conditions.

Show MeSH
Related in: MedlinePlus