Divergent targets of Candida albicans biofilm regulator Bcr1 in vitro and in vivo

Abstract

Candida albicans is a causative agent of oropharyngeal candidiasis (OPC), a biofilm-like infection of the oral mucosa. Biofilm formation depends upon the C. albicans transcription factor Bcr1, and previous studies indicate that Bcr1 is required for OPC in a mouse model of infection. Here we have used a nanoString gene expression measurement platform to elucidate the role of Bcr1 in OPC-related gene expression. We chose for assays a panel of 134 genes that represent a range of morphogenetic and cell cycle functions as well as environmental and stress response pathways. We assayed gene expression in whole infected tongue samples. The results sketch a portrait of C. albicans gene expression in which numerous stress response pathways are activated during OPC. This one set of experiments identifies 64 new genes with significantly altered RNA levels during OPC, thus increasing substantially the number of known genes in this expression class. The bcr1Δ/Δ mutant had a much more limited gene expression defect during OPC infection than previously reported for in vitro growth conditions. Among major functional Bcr1 targets, we observed that ALS3 was Bcr1 dependent in vivo while HWP1 was not. We used null mutants and complemented strains to verify that Bcr1 and Hwp1 are required for OPC infection in this model. The role of Als3 is transient and mild, though significant. Our findings suggest that the versatility of C. albicans as a pathogen may reflect its ability to persist in the face of multiple stresses and underscore that transcriptional circuitry during infection may be distinct from that detailed during in vitro growth.

Fig 1

Comparison of C. albicans RNA levels in mouse tongue samples. RNA was isolated from infected tongues (WT-OPC-1 and WT-OPC-2) or an uninfected tongue and used for RNA level measurements by nanoString analysis. In total, the RNA levels for 134 C. albicans genes were determined. The scatter plot presents each RNA level for one infected tongue (WT-OPC-1) compared to either an independent infected tongue (WT-OPC-2, blue data points) or the uninfected tongue (red data points). These samples all had background subtracted but were not normalized to the level of TDH3 (control) RNA. C. albicans strain DAY185 was used for infections.

Fig 2

Relative C. albicans RNA levels from in vitro growth and OPC infection. NanoString measurements were conducted on three independent cultures of C. albicans grown in vitro (labeled “Spider”) or on three infected mouse tongues (“OPC”). RNA levels for each gene were normalized to internal control TDH3 RNA levels and are presented as ratios to the mean RNA level for each gene in the in vitro samples. In the heat map columns, upregulation is represented by yellow, downregulation is represented by blue, and the magnitude of regulation is reflected by color saturation. Known regulatory pathways that govern each gene are presented in the color bars to the right of each gene name. C. albicans strain DAY185 was used for all assays.

Fig 3

Relative bcr1Δ/Δ mutant RNA levels from in vitro growth and OPC infection. NanoString measurements were conducted on C. albicans strain CJN702 (bcr1Δ/Δ), using three independent cultures grown in vitro (labeled “Spider”) or on three infected mouse tongues (“OPC”). RNA levels for each gene were normalized to internal control TDH3 RNA levels and are presented as ratios to the mean RNA level for each gene in samples from wild-type strain DAY185. Upregulation is represented by yellow; downregulation is represented by blue; the magnitude of regulation is reflected by color saturation.

Fig 4

Contribution of Bcr1, Als3, and Hwp1 to virulence in OPC infection. Immunosuppressed mice were inoculated with C. albicans strains with the genotypes indicated. After 1 day (A) or 5 days (B), mice were sacrificed, and the oral fungal burdens were calculated. Results are medians ± interquartile ranges. Statistical analysis was performed relative to wild-type fungal burdens by the Wilcoxon rank sum test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.0001. The numbers of animals for each determination are given in parentheses across the top of each graph.