Identification of Genes Upregulated by the Transcription Factor Bcr1 That Are Involved in Impermeability, Impenetrability, and Drug Resistance of Candida albicans a/ α Biofilms

Abstract

Candida albicans forms two types of biofilm, depending upon the configuration of the mating type locus. Although architecturally similar, a/␣ biofilms are impermeable, impenetrable, and drug resistant, whereas a/a and ␣/␣ biofilms lack these traits. The difference appears to be the result of an alternative matrix. Overexpression in a/a cells of BCR1, a master regulator of the a/␣ matrix, conferred impermeability, impenetrability, and drug resistance to a/a biofilms. Deletion of BCR1 in a/␣ cells resulted in the loss of these a/␣-specific biofilm traits. Using BCR1 overexpression in a/a cells, we screened 107 genes of interest and identified 8 that were upregulated by Bcr1. When each was overexpressed in a/a biofilms, the three a/␣ traits were partially conferred, and when each was deleted in a/␣ cells, the traits were partially lost. Five of the eight genes have been implicated in iron homeostasis, and six encode proteins that are either in the wall or plasma membrane or secreted. All six possess sites for O-linked and N-linked glycosylation that, like glycosylphosphatidylinositol (GPI) anchors, can cross-link to the wall and matrix, suggesting that they may exert a structural role in conferring impermeability, impenetrability, and drug resistance, in addition to their physiological functions. The fact that in a screen of 107 genes, all 8 of the Bcr1-upregulated genes identified play a role in impermeability, impenetrability, and drug resistance suggests that the formation of the a/␣ matrix is highly complex and involves a larger number of genes than the initial ones identified here.

Complex Haploinsufficiency-Based Genetic Analysis of the NDR/Lats Kinase Cbk1 Provides Insight into Its Multiple Functions in Candida albicans

“…To test this hypothesis, we determined the effect of a bcr1D/D mutation of CHK1 expression during oxidative stress; as shown in Figure 4C, CHK1 expression is fivefold lower in the bcr1D/D mutant relative to WT. This is consistent with previous data indicating that overexpression of BCR1 increases CHK1 expression (Srikantha et al 2013). However, the cbk1D/D mutant has a larger effect (20-fold) on CHK1 expression than the bcr1D/D mutant, suggesting that other transcriptional regulators may also contribute to Cbk1-regulated expression of CHK1.…”

“…As discussed above, overexpression of the Cbk1 target BCR1 increases expression of CHK1 (Srikantha et al 2013). In addition, Cbk1 has been shown to phosphorylate Bcr1 in the setting of biofilm formation (Gutierrez-Escribino et al 2012).…”

“…We have previously shown that the RAM and PKA pathways interact (Bharucha et al 2011). In addition, CHK1 is induced by overexpression of the transcription factor BCR1 in a/a biofilms (Srikantha et al 2013); Bcr1 is a known target of Cbk1 in biofilms. Based on these previous observations, the genetic interaction between CHK1 and CBK1 seemed quite reasonable.…”

Plasticity of Candida albicans Biofilms

“…However, MTL-homozygous biofilms formed by white cells are, on average, 25% thinner than MTL-heterozygous biofilms (183,251). Most of the difference is erased if minority (10%) MTL-homozygous opaque cells of the opposite mating type are added to the white cell population (38).…”

“…Profiling can be performed by assessing genome-wide transcription by using microarrays (195,200,(244)(245)(246)(247)(248), NanoString nCounter technology (249,250), and RNA sequencing (RNA-Seq) (197,200) or by using quantitative reverse transcription-PCR (RT-PCR) or Northern blot analysis of a smaller, select group of genes (251). Such comparisons are often made between biofilm preparations and planktonic cultures.…”