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

Divergent Targets of Candida albicans Biofilm Regulator Bcr1 In Vitro and In Vivo Saranna Fanning,a Wenjie Xu,a Norma Solis,b Carol A. Woolford,a Scott G. Filler,b and Aaron P. Mitchella Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA,a and Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, California, USAb 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 bcr1⌬/⌬ 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. C andida albicans is a major invasive fungal pathogen of humans and can cause both mucosal and disseminated infections. Infections of the oral mucosa in particular, called oropharyngeal candidiasis (OPC), affect HIV patients (41), diabetes patients (51), and head and neck cancer patients receiving radiation therapy (1, 39, 48). Our goal is to define the attributes of C. albicans that make it a successful oral pathogen. Mucosal infections may be considered biofilms, in that the pathogen adheres to a surface and produces an extracellular matrix (15, 39). This analogy has prompted investigations that test the hypothesis that genes required for biofilm formation in vitro may be required for mucosal infection as well. Findings from these studies have underscored the utility of this perspective, in that there are several common genetic requirements for the formation of abiotic surface biofilms and mucosal infections (10, 11, 15, 19). One of the central regulators of biofilm formation is the zinc finger transcription factor Bcr1. It was identified in screens for mutants defective in biofilm formation on abiotic surfaces (33– 35) and in adherence to a silicone substrate (14). Bcr1-dependent genes have been defined under in vitro growth conditions (14, 33–35). Many are cell surface protein genes, including ALS1, ALS3, and HWP1. These three genes are major functional Bcr1 targets, in that they are required for abiotic surface biofilm formation, and their overexpression restores biofilm formation in bcr1⌬/⌬ mutant backgrounds (33, 34). Transcription factors like Bcr1 have long been used to define the functional basis of pathogenicity traits (8, 27, 42). One strength of this approach comes from the fact that virulence potential may arise from expression of gene families or other gene sets with overlapping functions. Because transcription factors often control functionally related target genes, a single transcription factor defect can abolish a function that is carried out by redundant genes. One weakness of this approach comes from the fact 896 ec.asm.org Eukaryotic Cell that the spectrum of transcription factor target genes may be contingent upon environmental conditions. Thus, the gene expression impact of a transcription factor in an infection setting may be different from its impact in vitro. In fact, there are now several examples in which a target gene is expressed in colonization or infection samples independently of a transcriptional regulator that was defined by in vitro assays (24). Moreover, this limitation is not restricted to transcription factors, since almost any genetic perturbation has gene expression consequences (22) that may contribute to a mutant phenotype. Clearly it is critical to assess pathogen gene expression in vivo during infection, and several prior studies have done so (2, 18, 21, 29, 36, 38, 47, 49, 52, 54). Relevant to oral C. albicans infection, there has been a quantitative reverse transcription (QRT)-PCR analysis of C. albicans gene expression in a gnotobiotic pig OPC model (50), which revealed that ECE1 RNA accumulated at very high levels during infection. There have also been microarray analyses of both reconstituted human epithelial (RHE) infection and OPC patient samples (31, 54). The most highly upregulated genes in these contexts, compared to in vitro yeast extract-peptone dextrose (YPD)-grown cells, included the cell surface protein genes ALS3 and HWP1. All three of these genes—ECE1, ALS3, and HWP—require Bcr1for expression under in vitro growth conditions (35). These findings suggested that Bcr1 may be required for Received 28 March 2012 Accepted 23 April 2012 Published ahead of print 27 April 2012 Address correspondence to Aaron P. Mitchell, . Supplemental material for this article may be found at http://ec.asm.org/. Copyright © 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/EC.00103-12 p. 896 –904 July 2012 Volume 11 Number 7 Divergent Bcr1 Targets In Vitro and In Vivo TABLE 1 Candida albicans strains used in this study Strain name Genotype Reference BWP17 ura3⌬::␭imm434 arg4::hisG his1::hisG ura3⌬::␭imm434 arg4::hisG his1::hisG ura3⌬::␭imm434 HIS1::his1::hisG ARG4::URA3::arg4::hisG ura3⌬::␭imm434 his1::hisG arg4::hisG ura3⌬::␭imm434 ARG4::pARG4-URA3 ura3⌬::␭imm434 ARG4 ura3⌬::␭imm434 arg4::hisG his1::hisG::pHIS1-BCR1 bcr1:: ARG4 ura3⌬::␭imm434 arg4::hisG his1::hisG bcr1::URA3 ura3⌬::␭imm434 arg4::hisG his1::hisG::pHIS1 bcr1::ARG4 ura3⌬::␭imm434 arg4::hisG his1::hisG bcr1::URA3 ura3⌬::␭imm434 hwp1::hisG eno1::URA3 ura3⌬::␭imm434 hwp1::hisG ENO1 ura3⌬::␭imm434 hwp1::hisG eno1::URA3 ura3⌬::␭imm434 HWP1 ENO1 ura3⌬::␭imm434::URA3-IRO1 als3::ARG4 arg4::hisG his1::hisG ura3⌬::␭imm434 als3::HIS1 arg4::hisG his1::hisG ura3⌬::␭imm434::URA3-IRO1 als3::ARG4::ALS3 arg4::hisG his1::hisG ura3⌬::␭imm434 als3::HIS1 arg4::hisG his1::hisG 53 DAY185 CAI4-URA3 CJN698 CJN702 CAH7-1A1E2 CAHR3 CAYF178U CAQTP178U OPC. Indeed (...truncated)