Time course of microbiologic outcome and gene expression in Candida albicans during and following in vitro and in vivo exposure to fluconazole.
Pharmacodynamics (PD) considers the relationship between drug exposure and effect. The two factors that have been used to distinguish the PD behaviors of antimicrobials are the impact of concentration on the extent of organism killing and the duration of persistent microbiologic suppression (postantibiotic effect). The goals of these studies were (i) to examine the relationship between antimicrobial PD and gene expression and (ii) to gain insight into the mechanism of fluconazole effects persisting following exposure. Microarrays were used to estimate the transcriptional response of Candida albicans to a supra-MIC F exposure over time in vitro. Fluconazole at four times the MIC was added to a log-phase C. albicans culture, and cells were collected to determine viable growth and for microarray analyses. We identified differential expression of 18% of all genes for at least one of the time points. More genes were upregulated (n=1,053 [16%]) than downregulated (174 [3%]). Of genes with known function that were upregulated during exposure, most were related to plasma membrane/cell wall synthesis (18%), stress responses (7%), and metabolism (6%). The categories of downregulated genes during exposure included protein synthesis (15%), DNA synthesis/repair (7%), and transport (7%) genes. The majority of genes identified at the postexposure time points were from the protein (17%) and DNA (7%) synthesis categories. In subsequent studies, three genes (CDR1, CDR2, and ERG11) were examined in greater detail (more concentration and time points) following fluconazole exposure in vitro and in vivo. Expression levels from the in vitro and in vivo studies were congruent. CDR1 and CDR2 transcripts were reduced during in vitro fluconazole exposure and during supra-MIC exposure in vivo. However, in the postexposure period, the mRNA abundance of both pumps increased. ERG11 expression increased during exposure and fell in the postexposure period. The expression of the three genes responded in a dose-dependent manner. In sum, the microarray data obtained during and following fluconazole exposure identified genes both known and unknown to be affected by this drug class. The expanded in vitro and in vivo expression data set underscores the importance of considering the time course of exposure in pharmacogenomic investigations.
Department of Medicine, Section of Infectious Diseases and Medical Microbiology and Immunology, University of Wisconsin, 600 Highland Ave., Room H4/572, Madison, WI 53792, USA.
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Lepak
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Nett
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Lincoln
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Marchillo
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D018528:ATP-Binding Cassette Transporters / Q000235:genetics; D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology*; D002176:Candida albicans / Q000187:drug effects* / Q000235:genetics; D015725:Fluconazole / Q000493:pharmacokinetics / Q000494:pharmacology*; D005656:Fungal Proteins / Q000235:genetics; D020869:Gene Expression Profiling; D026901:Membrane Transport Proteins / Q000235:genetics; D051379:Mice; D008826:Microbial Sensitivity Tests; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000032:analysis
D018528:ATP-Binding Cassette Transporters; D000935:Antifungal Agents; C094693:CDR1 protein, Candida albicans; D005656:Fungal Proteins; D026901:Membrane Transport Proteins; D012333:RNA, Messenger; D015725:Fluconazole