Time course of microbiologic outcome and gene expression in Candida albicans during and following in vitro and in vivo exposure to fluconazole

Abstract

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.

FIG. 1.

Impact of fluconazole exposure on growth of C. albicans K1 in vitro. Each symbol represents the mean and standard deviation from three experiments. Solid symbols represent control growth in the absence of fluconazole. Open symbols represent data for drug-exposed cells. The solid horizontal bar represents the duration of fluconazole exposure.

FIG. 2.

Categories and percentages of C. albicans genes upregulated and downregulated during fluconazole exposure in vitro compared to gene expression in unexposed cells at the same time and phase of growth.

FIG. 3.

Categories and percentages of C. albicans genes upregulated and downregulated in the post-fluconazole exposure period in vitro compared to gene expression in unexposed cells at the same time and phase of growth.

FIG. 4.

Impact of five fluconazole concentrations on growth of C. albicans K1 in vitro. Each shape represents a fluconazole concentration relative to the MIC. Each symbol represents the mean and standard deviation from three experiments. Solid symbols represent control growth in the absence of fluconazole. Open symbols represent data for drug-exposed cells. The solid horizontal bar represents the duration of fluconazole exposure.

FIG. 5.

mRNA abundances of CDR2 (top left panel) and ERG11 (top right panel) in response to five escalating fluconazole concentrations in vitro by RT-PCR and Northern blotting, respectively. Data expressed as changes in mRNA abundance are ratios of mRNAs estimated in exposed relative to unexposed C. albicans cells in the same phase of growth. The solid bars represent the duration of fluconazole exposure. The first symbol represents expression during fluconazole exposure, and the second symbol represents expression following fluconazole removal. The left panel represents data obtained by quantitative real-time RT-PCR, and the right panel represents data obtained from Northern blots. The bottom panels represent mRNA abundances of CDR1 in response to five escalating fluconazole concentrations in vitro. Data expressed as changes in mRNA abundance are ratios of mRNAs estimated in exposed relative to unexposed C. albicans cells in the same phase of growth. The solid bars represent the duration of fluconazole exposure. The first symbol represents expression during fluconazole exposure, and the second symbol represents expression following fluconazole removal. The left bottom panel represents data obtained by RT-PCR, and the right bottom panel represents data obtained from Northern blots.

FIG. 6.

Impact of three fluconazole dose levels on growth of C. albicans K1 in vivo. Each symbol represents the mean and standard deviation from three experiments. Circles represent control growth in the absence of fluconazole. The other symbols represent data for drug-exposed cells. The solid horizontal bar represents the duration that levels remained above the MIC in serum.

FIG. 7.

mRNA abundances of CDR1 (left panel) and CDR2 (right panel) in response to three escalating fluconazole dose levels in vivo by quantitative real-time RT-PCR. Data expressed changes in mRNA abundance are ratios of mRNAs estimated in exposed relative to unexposed C. albicans cells in the same phase of growth. The solid bars represent the duration that fluconazole levels in serum remained above the MIC.