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. Author manuscript; available in PMC: 2013 Aug 1.
Published in final edited form as: Mol Microbiol. 2012 Jul 5;85(3):557–573. doi: 10.1111/j.1365-2958.2012.08127.x

BRG1 and NRG1 Form a Novel Feedback Circuit Regulating C. albicans Hypha Formation and Virulence

Ian A Cleary 1, Anna L Lazzell 1, Carlos Monteagudo 2, Derek P Thomas 3, Stephen P Saville 1,*
PMCID: PMC3402693  NIHMSID: NIHMS384952  PMID: 22757963

Summary

In the opportunistic fungal pathogen Candida albicans both cellular morphology and the capacity to cause disease are regulated by the transcriptional repressor Nrg1p. One of the genes repressed by Nrg1p is BRG1, which encodes a putative GATA family transcription factor. Deletion of both copies of this gene prevents hypha formation. We discovered that BRG1 over-expression is sufficient to overcome Nrg1p-mediated repression and drive the morphogenetic shift from yeast to hyphae even in the absence of environmental stimuli. We further observed that expression of BRG1 influences the stability of the NRG1 transcript, thus controlling filamentation through a feedback loop. Analysis of this phenomenon revealed that BRG1 expression is required for the induction of an antisense NRG1 transcript. This is the first demonstration of a role for mRNA stability in regulating the key C. albicans virulence trait: the ability to form hyphae.

Keywords: C. albicans, filamentation, antisense, NRG1, hyphae, virulence

Introduction

The opportunistic fungal pathogen Candida albicans inhabits many niches in the human host environment, both as a commensal and as a pathogen capable of causing serious disease. Its ability to grow in different cellular morphologies, including yeast cells, pseudohyphae and hyphae, is a pivotal aspect of its capacity to move from the commensal to the disease–causing state. C. albicans is the number one cause of hospital-acquired fungal infections, with mortality rates from systemic infections ranging from 30 to 50% (Viudes et al., 2002, Wey et al., 1988). The incidence of C. albicans infections has risen with the increased use of implanted medical devices and the greater prevalence of immunocompromised patients such as transplant recipients, chemotherapy patients and those with HIV/AIDS. Since many infections stem from biofilms formed on surgically implanted devices (Boland et al., 2010), and hypha formation is absolutely required for the formation of biofilms, understanding the genetic mechanisms governing changes in cellular morphology is an important avenue of investigation for improving mortality rates and treatment outcomes. Moreover, C. albicans strains that grow only in the yeast (Lo et al., 1997, Stoldt et al., 1997, Saville et al., 2003) or filamentous forms (Braun et al., 2000, Braun & Johnson, 1997) are avirulent in the murine model of systemic candidiasis.

The various environmental stimuli which induce C. albicans hypha formation are transmitted through several signalling pathways (Sudbery, 2011), including the cyclic AMP/protein kinase A and mitogen-activated protein kinase (MAPK) pathways (Biswas et al., 2007). At the base of these pathways are genes such as EFG1, CPH1, RIM101 and CZF1 that encode transcriptional regulators required for filamentation. Acting opposite these genes are repressors, including RFG1, NRG1 and TUP1, which prevent filamentous growth in the absence of appropriate signals. Many components of the signalling pathways have been identified but the initial signal transduction elements, potential cross-talk between the pathways, and the specific methods of effector regulation remain less well understood.

Nrg1p is a key repressor of C. albicans filamentation and hypha-specific gene expression. Mutant strains lacking NRG1 are constitutively filamentous (Braun et al., 2001, Murad et al., 2001) whilst NRG1 over-expression is sufficient to block hypha formation altogether (Cleary & Saville, 2010, Saville et al., 2003). Both NRG1 mRNA (Braun et al., 2001, Murad et al., 2001) and protein (Lu et al., 2011) levels fall during hyphal induction and this process appears to depend on the cyclic AMP/protein kinase A pathway (Lu et al., 2011).

Small RNA molecules have emerged as important regulators of eukaryotic gene expression since the RNA interference (RNAi) system was first characterized in Caenorhabditis elegans (Fire et al., 1998). Numerous small, non-coding, regulatory RNAs have now been identified and fall into three main classes: small interfering RNAs (siRNA) found throughout eukaryotes, microRNAs (miRNA) found in higher eukaryotes and piwi-interacting RNAs (piRNA) found only in animals (Ketting, 2011, Farazi et al., 2008). The siRNAs arise from dsRNA molecules that can be produced from single RNA molecules that form stem-loop structures, from homologous pairing of complementary antisense transcripts (especially repetitive transcripts), or via the action of RNA-dependent RNA polymerases (Ketting, 2011). The enzyme dicer (a protein with RNAse III activity) cleaves the dsRNA molecules, which are then loaded onto an argonaute protein to form the RNA-induced silencing complex (RISC) (Cenik & Zamore, 2011). Complementary basepairing between the RISC RNA and the target RNA leads to specific gene silencing (Tomari & Zamore, 2005, Farazi et al., 2008). The target RNA can be cleaved via the RNaseH activity of argonaute or of other RISC components (Liu et al., 2004, Cenik & Zamore, 2011).

RNAi may have arisen in an ancestral eukaryote as a defence mechanism to control the expression and proliferation of transposons and viruses (Ding & Voinnet, 2007). This ancient machinery has been adapted to regulate development in various multicellular organisms (Ketting, 2011). RNAi is conserved in most eukaryotic lineages, but is conspicuously absent from the budding yeast Saccharomyces cerevisiae, which lacks homologues of both dicer and argonaute. It has recently been proposed that S. cerevisiae and other fungal species that have lost RNAi may have done so in order to harbour the double-stranded RNA virus system killer, which is sensitive to dsRNA processing. Acquisition of killer provides resistance to killer-produced protein toxin and maintenance of this viral system confers a competitive survival advantage versus toxin-sensitive cells (Drinnenberg et al., 2011). Nevertheless, RNAi is still present in many fungal species and is used for a range of purposes (reviewed in (Dang et al., 2011). Two key proteins necessary for RNAi, argonaute and a dicer-like protein with RNAse III activity, have recently been identified in C. albicans (Drinnenberg et al., 2009). Experiments attempting to harness this machinery for gene knockdown have so far proved unsuccessful (Staab et al., 2011) and to date the only confirmed role for RNAi components is that dicer activity is required for ribosome biogenesis (Bernstein et al., 2012). In the filamentous fungus Neurospora crassa, RNAi machinery is responsible for quelling, the suppression of repeated DNA sequence expression (Catalanotto et al., 2000, Catalanotto et al., 2004, Romano & Macino, 1992). N. crassa also appears to have evolved several novel small RNA molecules, including microRNA-like RNAs (milRNAs) that resemble the miRNAs found in plants and dicer-independent siRNAs whose method of biogenesis is as yet unknown (Lee et al., 2010).

In this study, we demonstrate that upregulation of a specific antisense transcript in C. albicans is controlled by expression of the GATA-family transcription factor BRG1, and further that expression of this gene is required to modulate expression of the key morphogenesis and virulence regulator NRG1. Our results describe a novel feedback component of the genetic machinery regulating C. albicans filamentation and virulence.

Results

Strain Construction

Large-scale genetic screens (Homann et al., 2009, Noble et al., 2010) revealed that a predicted DNA binding protein, encoded by orf19.4056, was required for hypha formation. We deleted both copies of BRG1 in more than one genetic background and, in agreement with the phenotypes previously described (Homann et al., 2009, Noble et al., 2010), strains lacking BRG1 are unable to form hyphae under numerous inducing conditions (Fig. 1B, S2, S3). Recent articles describing studies conducted concurrently with our own have confirmed its DNA binding activity and described a role for this protein in biofilm formation (Du et al., 2012, Nobile et al., 2012). As part of our ongoing analysis of the genetic mechanisms governing hypha formation in C. albicans, we discovered that BRG1 is a target of Nrg1p-mediated repression: its expression is moderately derepressed in the nrg1Δ mutant compared to a wild-type strain and its induction is blocked when NRG1 is over-expressed in our tet-NRG1 strain SSY50-B (Fig. 1A).

Figure 1. BRG1 is repressed by Nrg1p and its over-expression leads to increased invasion of solid medium.

Figure 1

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A. Quantitative real-time PCR analysis identified BRG1 as a target of Nrg1p-mediated repression. Expression of BRG1 is derepressed in the nrg1Δ strain (BCa23-3) compared to a wild-type strain (SC5314) in yeast conditions (YPD 28°C 6 h, left panel). Conversely, BRG1 expression is repressed when NRG1 is over-expressed in our tet-NRG1 strain (SSY50-B, No DOX) in hypha-inducing conditions (RPMI-1640 37°C 3 h, middle panel). Levels were normalized by ACT1 and expressed relative to the wild-type strain (left panel) or to the doxycycline-containing culture (right panel) therefore those results are 1.0. Error bars represent the standard error of the mean.

B. Cells from an overnight culture were washed in sterile PBS, diluted 1:20 into fresh, pre-warmed YPD+10% FBS and incubated with shaking at 37°C for 3 h. The brg1Δ strain (ICY326) fails to form hyphae.

C. A copy of BRG1 under the control of the tetO promoter was integrated at the RPS1 locus in a wild-type background (THE1-CIp10) and in our tet-NRG1 strain (SSY50-B) to construct strains ICY171 and ICY175, respectively. Cells were streaked on YPD plates with or without doxycycline (DOX) and the plates incubated at 30°C for 48 hours. The plates were photographed prior to (upper panels) and after (lower panels) surface cells were removed by gentle washing under running water. Cells which grew solely on the surface of the medium washed away while cells that had invaded the agar remained.

D. Cells from an overnight culture were washed in sterile PBS, counted and spotted onto solid Lee medium (buffered to pH 4 or 7) in the presence or absence of doxycycline and incubated at 30°C or 37°C for 2 days. A wrinkled colony morphology indicates the presence of filamentous cells.

To investigate the role of BRG1 in C. albicans filamentous growth, we constructed a novel tet-regulatable strain where an additional copy of BRG1, under the control of the bacterially-derived tetO promoter, was integrated at the RPS1 locus of the transactivator-containing strain THE1. In order to more closely examine the interplay between Brg1p and NRG1, we constructed additional strains in which we either added a tet-regulated allele of BRG1 to our tet-NRG1 strain or deleted both copies of BRG1 from the wild-type (SC5314) and tet-NRG1 strains.

Over-expression of BRG1 stimulates hyphal growth

We examined the influence of BRG1 over-expression on C. albicans morphology under a variety of environmental conditions. When incubated in yeast growth conditions (YPD, 30°C), strains over-expressing BRG1 (ICY171 and ICY175) were highly wrinkled on solid media, unlike the smooth phenotype of the parental strains (Fig. 1C). The parental strains were easily removed from the surface of the agar by gentle washing, whereas strains over-expressing BRG1 were resistant to washing, indicating invasion of the medium. This change in morphology was abolished by the inclusion of doxycycline (DOX) in the medium to switch off expression from the tet-regulated allele (Fig. 1C). C. albicans does not normally filament on acidic Lee pH 4 medium. BRG1 over-expression again resulted in a wrinkled colony appearance (Fig. 1D) whilst the parental strain remained smooth. Over-expression of BRG1 was also able to promote hyphal growth in non-inducing liquid media such as YPD at 28°C where cells with long, parallel-sided walls divided by septa, hallmarks of C. albicans hyphae, were readily apparent in non-inducing conditions (Fig. 2A). Consistent with this hyphal morphology, expression of the hypha-specific genes, ALS3, HWP1 and ECE1 was elevated during BRG1 over-expression (No DOX) compared to the control (Plus DOX) culture, (Fig. S1). Over-expression of BRG1 is therefore able to induce hypha formation in the absence of environmental stimuli.

Figure 2. Over-expressing BRG1 promotes C. albicans hyphal growth and biofilm formation.

Figure 2

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A. The THE1-CIp10 control and tet-BRG1 (ICY171) strains were grown under yeast conditions (YPD at 28°C) with or without doxycycline. Over-expression of BRG1 stimulates the production of hyphae. Cell walls were visualized with calcofluor white and nuclei with DAPI.

B. The tet-NRG1 (SSY50-B) and tet-NRG1+tet-BRG1 (ICY175) strains were grown in hypha inducing conditions (YPD at 37°C). BRG1 over-expression restores the ability of the tet-NRG1 strain to filament in the absence of DOX. Cell walls were visualized with calcofluor white and nuclei with DAPI.

C. To examine biofilm formation, cells from the indicated strains were incubated in SD medium at 30°C for 24h in 96-well microtitre plates. The bottom of the wells was photographed after non-adherent cells were washed away. A dense mass of cells covering the bottom of the well is visible when biofilms formed.

BRG1 over-expression restores the ability of the tet-NRG1 strain to filament

We previously demonstrated that over-expressing NRG1 is sufficient to block hyphal induction in numerous in vitro conditions and in vivo (Cleary & Saville, 2010, Saville et al., 2003). We therefore tested the capacity of BRG1 over-expression to restore the ability of the tet-NRG1 strain to form hyphae. Whereas the parental strain grew only as yeast cells in hypha stimulating conditions in the absence of DOX, simultaneous BRG1 over-expression (in strain ICY175) restored the ability to form hyphae in liquid medium under inducing conditions (Fig. 2B). Over-expression of BRG1 also restored hyphal growth on solid Lee medium pH 7 at 37°C (Fig. 1D) as well as solid Spider medium, liquid RPMI-1640, YPD+10% FBS and under embedded conditions (data not shown). As in the THE1-CIp10 background, over-expression of BRG1 in the tet-NRG1 strain also stimulated hyphal growth in yeast conditions (Fig. 1C). Thus over-expression of BRG1, is sufficient to overcome the effect of NRG1 over-expression and allow hypha formation.

Biofilm growth is affected by BRG1 over-expression

An important clinical aspect of Candida growth is the ability to form biofilms (Ramage et al., 2005). Biofilm formation is impaired or absent when the cells are maintained in the yeast form, either through growth in non-inducing conditions or by over-expression of NRG1 (Fig. 2C). Since hyphal growth is required for biofilm formation, we predicted that, in addition to affecting the morphology of individual cells, BRG1 over-expression would likely influence biofilm formation. Using the 96-well microtitre plate-based method (Pierce et al., 2008), we tested the ability of several strains to form biofilms under yeast growth conditions. The control strain THE1-CIp10 showed the presence of only sporadic yeast cells after washing had removed non-adherent cells. The nrg1Δ strain, which is constitutively filamentous, forms pseudohyphae in yeast growth conditions, and it too displayed few adhered cells with large areas of the well remaining bare. Conversely, over-expression of BRG1 resulted in robust biofilm formation with a dense mass of hyphal cells (Fig. 2C). Over-expression of NRG1 normally blocks hyphal growth and therefore the ability to form biofilms in inducing conditions. BRG1 over-expression was able to overcome this repression and promote biofilm formation under yeast (Fig. 2C) and inducing (not shown) conditions.

Brg1p had previously been identified as necessary for hyphal growth (Homann et al., 2009, Noble et al., 2010). Here we have demonstrated that its over-expression is sufficient to drive hyphal growth in the absence of inducing signals and, most importantly, to overcome Nrg1p-mediated inhibition of hypa formation in every in vitro condition tested.

BRG1 over-expression influences virulence in a disseminated model

After establishing that BRG1 expression can have a profound effect on C. albicans morphology in vitro, we tested its ability to affect virulence in vivo in the widely-used murine model of haematogenously disseminated candidiasis. Surprisingly, over-expression of BRG1 attenuated virulence (Fig. 3A), with significant changes in the survival curves at both high and low infecting doses. Histological examination of kidneys from infected animals indicated the presence of abundant filamentous fungal cells both with (No DOX) and without (Plus DOX) BRG1 over-expression. Neutrophil infiltration was also evident in both samples. Interestingly, tissues retrieved from an animal that survived the fungal challenge during BRG1 over-expression nevertheless showed evidence of damage and blood vessel occlusion, as well as a subcapsular scar, consistent with the earlier presence of an invasive C. albicans infection (Fig. 3A, far right panel).

Figure 3. BRG1 over-expression influences C. albicans virulence.

Figure 3

Figure 3

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A. BRG1 Over-expression attenuates virulence in a wild-type strain. Approximately 4.6×105 (dark lines) or 2.4×105 (light lines) yeast cells of C. albicans strain tet-BRG1 (ICY171) were injected into mice. Survival differences between Plus DOX and No DOX groups at both high and low doses were significantly different (p=0.0016 and p=0.0018, respectively). Examination of kidneys retrieved from mice that succumbed to the infection revealed extensive C. albicans growth (GMS staining, upper panels), and infiltration of numerous neutrophils (H&E staining, lower panels). Interestingly, examination of the kidney retrieved from a mouse that survived the infection (No DOX) revealed a subcapsular scar (H&E staining, far right panel) indicative of earlier tissue damage.

B. For timed sacrifice experiments, approximately 1.7×106 (6 h sacrifice) or 3.9×105 (3 day sacrifice) yeast cells of the tet-BRG1 strain (ICY171) were injected into mice. The indicated tissues were homogenized and plated onto Sabouraud agar to determine the fungal burdens (group median indicated by a bar). There was a significant reduction (p=0.0212) in kidney fungal burdens upon BRG1 over-expression (6 h sacrifice). Examination of kidneys retrieved after 3 days from the BRG1 over-expression group (No DOX) revealed kidney necrosis and significant vascular thrombosis (H&E staining, upper panels) associated with invasive C. albicans growth (GMS staining, lower panel).

C. BRG1 Over-expression results in attenuated virulence in the tet-NRG1 background. Approximately 6.0×105 (dark lines) or 2.4×105 (light lines) yeast cells of the tet-NRG1+tet-BRG1 strain (ICY175) were injected into mice. The dashed line represents the survival data produced by the parental tet-NRG1 strain (SSY50-B) when NRG1 levels are elevated (Saville et al., 2003). Survival differences between the Plus DOX and No DOX groups were not statistically significant at the high dose (p=0.1573) but were at the low dose (p=0.0031). Examination of kidneys retrieved from infected mice revealed extensive lesions containing hyphal cells irrespective of the presence or absence of doxycycline (H&E staining, upper panels; GMS staining, lower panels). This contrasts starkly with the tet-NRG1 strain that grows only as yeast cells in the kidney in the absence of DOX (Saville et al., 2003).

To determine whether BRG1 over-expression had a particular influence on dissemination and early-stage proliferation, we performed timed sacrifice experiments and measured the fungal burden residing in the brain, spleen and kidney at 6 h and 3 days post-infection. There were no significant differences between the groups in the burdens found in the brain or spleen after 6 h. There was, however, a statistically significant lower burden in the kidneys of animals not treated with doxycycline (BRG1 over-expression). After 3 days there were no statistically significant differences between the median fungal burdens of any tissue retrieved from the treated and untreated animals. There was, however, a marked increase in the variability of the number of fungal cells recovered from the No DOX tissues compared to Plus DOX control tissues which exhibited very little variation between animals. Furthermore, histological examination of kidneys retrieved from No DOX animals three days post infection revealed marked necrosis and vascular thrombosis with C. albicans associated with thrombi in some vessels (Fig. 3B).

Taken together, these data indicate that over-expression of BRG1 still allows C. albicans to disseminate to different tissues, cause damage to kidney cells and stimulate an immune response as evidenced by neutrophil influx to sites of infection. The variation in fungal burden observed in the No DOX group after 3 days parallels the variable results from our virulence study: some animals from the BRG1 over-expression group died at nearly the same rate as the control group, whilst some survived, albeit with scarring as a result of the fungal infection. Results from the earliest time point examined (6 h) suggest that BRG1 over-expression does have an effect on the initial dissemination to, or replication within, the kidney. In particular, BRG1 over-expression appeared to be associated with prominent parenchymal necrosis mostly related to vascular thrombosis within the kidney.

BRG1 over-expression in the tet-NRG1 background results in attenuated virulence

We have demonstrated previously that NRG1 over-expression is able to block both filamentation and virulence of C. albicans (Saville et al., 2003) and have shown above that BRG1 over-expression is able to overcome this inhibition in vitro. In the haematogenously disseminated infection model, however, over-expression of BRG1 in the tet-NRG1 background still results in attenuated virulence. At the lower dose, the survival curve was shifted significantly and some animals survived the challenge (Fig. 3C). To confirm that the strain was capable of forming hyphae in vivo, as it had in vitro, we examined tissue sections from infected kidneys. Extensive hyphal growth was observed in both the doxycycline treated and untreated samples, in contrast to the presence of only yeast cells previously observed when NRG1 alone was over-expressed (Saville et al., 2003). The ability of Brg1p to drive hyphal induction is therefore as effective in the tissues as it was in vitro. These results reinforce the key role played by Brg1p in C. albicans virulence, but BRG1 over-expression does not restore wild-type virulence to the tet-NRG1 strain.

BRG1 over-expression affects NRG1 transcript abundance

To better understand the mechanism by which Brg1p stimulates hyphal growth, we first examined its pattern of expression during normal hyphal induction in response to serum using a wild-type strain (SC5314). We observed that BRG1 expression was virtually absent in yeast cells, but its transcription was rapidly and strongly upregulated in response to environmental stimulus early during hyphal induction (Fig. 4A). Its transcript levels then declined after this initial rise. This pattern is opposite to that of NRG1, which falls during the initial stages of hyphal induction. In an attempt to understand the filamentous phenotype resulting from BRG1 over-expression, we examined NRG1 transcript levels when BRG1 was over-expressed. We observed that BRG1 over-expression in yeast growth conditions resulted in decreased levels of NRG1 transcript (Fig. 4C). Surprisingly, this same inverse relationship was observed in strain ICY175, which harbours tet-regulated alleles of both NRG1 and BRG1 (Fig. 4D). Because the wild-type and tet-regulated NRG1 transcripts have different 5’ untranslated regions, due to the insertion of the tetO promoter sequence, their individual patterns of expression can be distinguished by northern blot analysis (Saville et al., 2003). BRG1 over-expression resulted in a reduction of both wild-type NRG1 and tet-NRG1 transcript levels (Fig. 4D). In fact, this effect was so great that the abundance was reduced to such an extent that there was overall less NRG1 transcript present during BRG1 over-expression than when transcription from the tet-NRG1 allele had been abolished by the addition of doxycycline. The observation that the abundance of both the wild-type and tet-NRG1 transcripts were affected by BRG1 over-expression meant it was unlikely that NRG1 promoter activity was being impaired. In particular, the bacterially-derived tetO promoter should not be affected by eukaryotic transcription factors (Gossen & Bujard, 1992). Moreover, if Brg1p directly influenced transcription from the tetO promoter then it would quickly shut off its own expression, whereas our northern blot analyses show abundant transcript produced by the tet-BRG1 allele (Fig. 4C, D).

Figure 4. Analysis of BRG1 and NRG1 expression during hyphal induction.

Figure 4

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BRG1 and NRG1 transcription was examined during hyphal induction (YPD+10% FBS, 37°C) by northern blot analyses. RNA was isolated, transferred to a Nytran membrane and probed with DNA fragments specific to the BRG1 or NRG1 coding sequence. Ethidium bromide-stained rRNA bands are shown as a loading control. Numbers represent time post-induction.

A. In a wild-type strain (SC5314), BRG1 transcript is undetectable in yeast cells (Time 0), is abundant 30 minutes after induction and subsequently declines. Expression of NRG1 is high in yeast cells (Time 0), falls during the first hour of hyphal induction and gradually returns to pre-induction levels.

B. In the brg1Δ strain (ICY326), no BRG1 transcript is detectable and during hyphal induction, NRG1 levels remain high.

C. In the tet-BRG1 strain (ICY171), when BRG1 expression is induced from a tet-regulated allele, BRG1 levels rise steadily over time, whilst NRG1 levels fall. When BRG1 is not over-expressed, through the addition of DOX to the medium, NRG1 levels remain stable (YPD 28°C).

D. When BRG1 is over-expressed in yeast conditions (YPD 28°C) in the tet-BRG1 strains ICY171 and ICY175, NRG1 levels fall. Note that in ICY175, transcription from both the endogenous and tet-regulated (arrow) NRG1 alleles is affected. (-) No DOX, (+) Plus DOX.

E. Quantitative real-time PCR analysis confirmed that NRG1 mRNA destabilization depends on BRG1. The tet-NRG1 (SSY50-B) and brg1Δ (ICY277) strains were grown overnight (YPD 28°C No DOX) then diluted intro fresh medium and grown under hypha inducing conditions (YPD+10% FBS 37°C Plus DOX). In the tet-NRG1 strain, NRG1 levels fall substantially by 30 minutes after induction whilst in the brg1Δ strain NRG1 levels remain stable. Levels were normalized by ACT1 and expressed relative to the respective time zero samples (therefore those results are 1.0). Error bars represent the standard error of the mean.

F. During hyphal induction (YPD+10% FBS 37°C 2 h) NRG1 transcript is most abundant in the brg1Δ (ICY326) strain. Levels are returned to those observed in the BRG1/brg1Δ heterozygote (ICY318) by reintegrating a copy of BRG1 at its native locus (ICY330).

Expression of BRG1 influences NRG1 transcript stability

Since the abundance of both NRG1 transcripts was altered, this suggested that Brg1p was affecting the stability of the NRG1 mRNA transcript rather than reducing its production. We predicted that, if this were the case, NRG1 transcript abundance should remain high in a strain lacking BRG1. We therefore deleted both copies of BRG1 in wild-type (SC5314) and tet-NRG1 (SSY50-B) strains to form strains ICY326 and ICY277, respectively. The tet-NRG1 (SSY50-B) and brg1Δ (ICY277) strains were grown overnight in the absence of doxycycline, then diluted 1/20 into fresh medium (YPD+10%FBS) containing doxycycline in order to stop new NRG1 production from the tetO allele (expression from the wild-type allele present in both strains should be regulated normally). In the tet-NRG1 strain (SSY50-B), NRG1 levels rapidly declined by 30 minutes post induction (Fig 4E) whereas in brg1Δ strain (ICY277), which differs solely in the deletion of both copies of BRG1, NRG1 levels remained steady. This demonstrated that BRG1 expression mediates NRG1 transcript destabilisation. During longer hyphal induction time-course experiments, NRG1 levels remained high in our brg1Δ strains (Fig. 4B, S4A). Returning one copy of BRG1 to its chromosomal location restored normal NRG1 regulation (Fig. 4F, S4B).

Since the wild-type NRG1 and tet-NRG1 transcripts have different 5’ UTRs, we felt comfortable ruling out a role for the 5’ UTR in mediating BRG1-dependent transcript instability. We therefore used two different approaches to analyze the role of BRG1 in affecting NRG1 transcript stability. In the first approach, we replaced the NRG1 coding sequence with a lacZ reporter gene, which has been used previously in C. albicans (Uhl & Johnson, 2001). The reporter was placed under the transcriptional control of the tetO promoter. If BRG1 expression affects the stability of any transcript produced from the NRG1 locus, then tet-lacZ levels should be reduced, as was the tet-NRG1 transcript. If, on the other hand, the transcriptional regulation is dependent on the NRG1 coding sequence, then BRG1 over-expression should not affect the reporter. Transcription of lacZ and production of β-galactosidase, as indicated by the conversion of colourless X-gal in the medium into the blue precipitate, was unaffected by BRG1 over-expression (Fig. 5A). Moreover, expression of lacZ, as measured by quantitative real-time PCR, was elevated irrespective of BRG1 over-expression (Fig. 5B). These results revealed that Brg1p does not alter transcription at the NRG1 locus via upstream or downstream sequence elements.

Figure 5. Examination of NRG1 transcript stability.

Figure 5

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A. One copy of NRG1 was replaced with lacZ under the control of the tetO promoter (ICY281). In this strain reporter activity, as indicated by the appearance of the blue precipitate, is modulated through the addition of doxycycline to the medium. In the tet-lacZ tet-BRG1 strain (ICY307) simultaneous over-expression of an ectopic copy of BRG1 under the control of the tetO promoter results in a wrinkled morphology as in the tet-BRG1 strain (ICY171), but does not affect expression of the lacZ reporter inserted at NRG1.

B. lacZ expression was measured using quantitative real-time PCR. In agreement with the phenotypic results, over-expression of BRG1 (ICY307) reduced NRG1 expression but not lacZ expression. Levels were normalized by ACT1 and expressed relative to the tet-lacZ strain (ICY281) time zero samples (therefore those results are 1.0). Error bars represent the standard error of the mean.

C. Constitutive expression of NRG1 from an ectopic ACT1 promoter-regulated allele (strain ICY293) is sufficient to block hyphal induction in YPD at 37°C. Addition of a tet-regulated BRG1 allele, to form strain ICY295, overcame this Nrg1p-mediated repression.

D. Northern blot analysis. The abundance of both the endogenous and larger ectopic NRG1 (arrow) transcripts depends on BRG1 over-expression. Strains were grown in YPD at 37°C for 3 h. Ethidium bromide-stained rRNA bands are shown as a loading control. (-) No DOX. (+) Plus DOX

Transcript stability can also be modulated by 3’ UTR interactions (Wilusz & Wilusz, 2004). We therefore integrated an additional copy of the NRG1 coding sequence under the control of the constitutive ACT1 promoter at the RPS1 locus to produce strain ICY293. This new strain was unable to form hyphae under inducing conditions (Fig. 5C), indicating that the ectopic ACT1p-NRG1 allele was transcribed and functional. Northern blot analysis revealed that the transcript produced from this allele was slightly longer than that produced from the wild-type gene (Fig. 5D, arrow), presumably due to the addition of the RPS1 downstream sequence rather than the usual NRG1 3’ UTR. An ectopic tet-BRG1 allele was then introduced into strain ICY293 to form strain ICY295. This tet-BRG1 allele restored hyphal growth, overcoming the inhibitory effect of the constitutive NRG1 allele (Fig. 5C). Despite the altered 3’UTR sequence of the ACT1p-NRG1 allele, BRG1 over-expression affected the stability of this transcript as well as that produced from the wild-type alleles (Fig. 5D), demonstrating that the instability of the NRG1 transcript does not depend on 3’UTR sequences.

NRG1 transcript stability depends on the coding sequence

If neither the promoter of the NRG1 locus nor the 5’ or 3’ UTRs are involved in NRG1 mRNA stability, then the effect must be specific to the NRG1 coding sequence itself. This suggested that an antisense NRG1 transcript was being produced, possibly leading to dsRNA formation and subsequent sense mRNA destruction using the RNAi machinery recently shown to exist in C. albicans (Drinnenberg et al., 2009). To investigate whether this was the case, we performed a series of strand-specific RT-PCR reactions. First strand synthesis was performed using RNA isolated from the wild-type (SC5314) and brg1Δ (ICY277) strains grown in YPD+10% FBS for 30 minutes and single primers that would bind either to the sense NRG1 transcript or to an antisense transcript (if present). In addition, total cDNA was synthesized from the poly-adenylated RNA population, which should include both sense and antisense transcripts (Cai et al., 2004, Ho et al., 2010), using oligodT priming. For each cDNA sample prepared, a control PCR reaction was performed using primers specific to the housekeeping gene EFB1. This gene contains an intron, enabling discrimination between genomic DNA derived (462 bp) and cDNA derived (97 bp) products. All of our cDNA samples synthesized with strand-specific primers lacked either of these bands, demonstrating that the templates were free of gDNA or non-specific cDNA contamination (Fig. 6A). Because the position or length of any antisense transcript cannot be simply predicted, we used several different primers for cDNA synthesis (Fig. 6A, Table S2). Primers NRG1_FOR, NRG1_S2 and NRG1_S4 are capable of binding to antisense NRG1 sequences (if present), whereas primers NRG_A2 and NRG1_REV bind to sense strand templates. PCR reactions were performed on these templates using the indicated primer pairs. For each set of PCR reactions, gDNA and oligodT primed cDNA were used as positive control templates. Reverse-transcription using primer NRG1_S2 produced a template, as indicated by the amplification product resulting from the subsequent PCR reactions using primer NRG1-S with NRG1-A, NRG1_A4 (Fig 6A) or NRG1_A3 (Fig. S5A). We were thus able to detect the presence of an antisense transcript produced roughly from the middle portion of NRG1 encompassing positions 363-669 of the coding sequence (Fig. 6A). Conversely, these same primer combinations failed to produce a product when the cDNA synthesis was primed with NRG1_FOR or NRG1_S4. Thus it appears that the antisense transcript does not extend to either end of the coding sequence. As expected, both the sense and antisense transcripts were absent from the nrg1Δ strain BCa23-3 (data not shown). We were also able to detect the antisense transcript in our brg1Δ strain (Fig 6A), suggesting that it is produced constitutively at a low level, but upregulated by BRG1 expression during hyphal induction. To investigate this possibility, we employed the more sensitive technique of quantitative, real-time PCR to examine antisense NRG1 production during the early stages of hyphal induction. We induced hyphae in both the wild-type and brg1Δ strains using YPD+10% FBS at 37°C and isolated RNA from cells at time 0 and 20 and 30 minutes after induction. Confirming our earlier northern blot analysis, BRG1 expression was virtually absent from yeast cells (time 0). However, BRG1 expression rises quickly and dramatically upon exposure to inducing signals (Fig. 6B) while, during the same time, NRG1 sense transcript levels fall. Conversely, there is an initial increase in the level of antisense NRG1 transcript present followed by a decline (Fig. 6B). These differing patterns of NRG1 sense and antisense expression preclude the possibility that our antisense primers were not strand specific. In the non-filamenting brg1Δ strain, NRG1 sense transcript levels remain constant in the same inducing growth conditions. More importantly, antisense NRG1 transcript levels also remain constant, demonstrating that its induction depends on BRG1 (Fig. 6B).

Figure 6. RT-PCR and quantitative real-time PCR analysis of antisense NRG1 production.

Figure 6

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A. RNA was isolated from the wild-type (SC5314) and the brg1Δ (ICY326) strains grown under hypha-inducing conditions (YPD+10% FBS, 37°C) for 30 minutes. RNA was treated with DNaseI and used as a template for reverse transcription with either strand-specific primers or oligodT, as indicated above each lane. Different primer combinations (indicated to the right of each panel) were used in PCR reactions with these single stranded cDNA templates. Aliquots of each PCR reaction were electrophoresed on an agarose gel and visualized with ethidium bromide. The housekeeping gene EFB1 serves as a control for the presence of genomic DNA or total cDNA. A no reverse transcriptase sample serves as a control for the RT-PCR. The diagram at the bottom indicates the relative positions of the primers used in this experiment (not to scale). F=NRG1_FOR, S2=NRG1_S2, S=NRG1-S S4=NRG_S4, A=NRG1-A, A2=NRG1_A2, A3=NRG1_A3, A4=NRG1_A4, R=NRG1_REV, dT=oligodT, G=gDNA, N=no reverse transcriptase control.

B. The wild-type (dark bars) and the brg1Δ (light bars) strains were grown under hypha-inducing conditions (YPD+10% FBS, 37°C). Samples were removed prior to, as well as 20 and 30 minutes after induction and cDNA synthesized with oligodT for ACT1 and BRG1 real-time PCR reactions, and NRG1_S2 and NRG1_REV for the antisense and sense reactions, respectively. Levels were normalized by ACT1 and expressed relative to the yeast control culture (wild-type time 0 sample, therefore its results are 1.0). Error bars represent the standard error of the mean. BRG1 expression is rapidly upregulated in the early stages of hyphal induction (upper panel). The NRG1 sense transcript falls in the wild-type, but remains constant in the brg1Δ strain. Conversely, the antisense NRG1 transcript rises concurrently with BRG1 in the wild-type (SC5314) but remains similar to time 0 levels in the brg1Δ strain (ICY326).

C. BRG1 expression does more than stimulate antisense NRG1 production. In yeast conditions, the nrg1Δ strain (BCa23-3) is constitutively pseudohyphal. Strains over-expressing BRG1 (ICY171 and ICY175), however, form hyphae under the same conditions. Cell walls were visualized with calcofluor white and nuclei with DAPI.

We hypothesized that our observed NRG1 antisense induction and concomitant NRG1 sense strand destruction might be mediated via an RNAi mechanism. To test this possibility, we measured NRG1 sense transcript levels during hyphal induction in the recently constructed ago1Δ strain (Bernstein et al., 2012). Surprisingly, NRG1 sense transcript levels rapidly declined after 20 minutes (Fig. S5C), similar to results obtained using a wild-type strain. It seems, therefore, that the phenomenon we have observed operates independently of argonaute activity. Attempts to mimic our BRG1 over-expression results by expressing an antisense transcript which encompassed the whole NRG1 coding sequence failed to alter the morphology of the cells or NRG1 sense transcript levels (not shown). However, this is consistent with the negative results reported previously reported in C. albicans (Staab et al., 2011) and also in S. cerevisiae (which lacks RNAi) where it was found that only expression of an antisense of a precise length was able to regulate gene expression (Gelfand et al., 2011).

One possible mechanism via which antisense transcription can affect sense transcript levels is through promoter exclusion/transcriptional interference. Since we were unable to detect an antisense transcript extending to the translational start site of NRG1, we thought promoter exclusion was unlikely. However, in an attempt to rule out this possibility, we analyzed NRG1 antisense production using our tet-BRG1 induction time course samples (Fig. 4C). We predicted that if NRG1 sense transcription were blocked because of the production of an antisense transcript then, as NRG1 sense levels declined, we would see a corresponding accumulation of antisense transcript. Instead we observed that in later time points of BRG1 induction, the antisense levels were lower than those in the control sample (Fig S5B). This is similar to the 30 minute time point during serum induction, where antisense levels have begun to decline from their peak. These data suggest that promoter exclusion is not the mechanism underlying our observed expression patterns, but instead suggests a mechanism involving destruction of both the sense and antisense transcripts.

Discussion

In this study we describe the dramatic impact caused by altered expression of a single gene on C. albicans morphology and a novel regulatory mechanism governing this process. As part of large scale phenotypic screens, orf19.4056 (BRG1) was shown to be required for hyphal growth and biofilm formation (Homann et al., 2009, Noble et al., 2010, Du et al., 2012, Nobile et al., 2012). We identified BRG1 as a hypha-specific gene and a target of Nrg1p-mediated repression. Over-expression of BRG1 in vitro was sufficient to overcome the effects of NRG1 over-expression and restore the ability to form hyphae and biofilms in numerous conditions.

A growing body of evidence suggests that yeast form cells are key for dissemination: they adhere better than filaments to endothelial cells in vitro under conditions of flow (Grubb et al., 2009) and are able to escape the bloodstream (Saville et al., 2003, Bendel et al., 2003, Chen et al., 2006). Since we injected fungal cells in the yeast form, we expected efficient escape from the bloodstream. The results from timed sacrifice experiments examining the early stages of infection suggest that driving hyphal growth by over-expressing BRG1 might affect C. albicans dissemination, as there was a reduced burden in the kidney after 6 h post-injection. Animals from which the infection had been cleared nonetheless showed tissue scarring, indicating that the fungus had successfully penetrated the organs and damaged host cells. Kidneys retrieved from mice infected with C. albicans cells either with or without BRG1 over-expression showed neutrophil infiltration. Perhaps this apparent deficiency in early dissemination to the kidney provides a greater opportunity for a successful host response, enabling animals to clear the infection and survive. Although the hyphae resulting from BRG1 over-expression appear phenotypically normal in vitro, this morphological change resulted in attenuated virulence in both wild-type and tet-NRG1 genetic backgrounds. Our transcriptional analyses reveal that BRG1 expression normally rises quickly in response to serum, but falls after its initial induction. Its sustained expression is apparently detrimental to virulence so perhaps additional virulence factors are only activated once BRG1 expression begins to fall.

Although several regulators of hyphal growth have been identified in C. albicans, our understanding of the mechanism controlling their expression remains incomplete. We are particularly ignorant of how the expression of the repressors of filamentation is regulated. The experiments described here have established that BRG1 over-expression affects NRG1 transcript abundance and this process occurs irrespective of upstream or downstream sequences or the genomic location of the NRG1 gene: these observations indicate a mechanism that depends specifically on the NRG1 coding sequence. We also established that BRG1 expression induces the production of an antisense NRG1 transcript. The antisense product does not span the full length of the sense NRG1 ORF and its induction correlates with NRG1 sense transcript instability.

A recent analysis of gene expression in biofilms has determined that the consensus BRG1 binding site upstream of biofilm-associated genes is C/GA/CGGTAC/A (Nobile et al., 2012). This sequence occurs over 6,000 times throughout the C. albicans genome (Skrzypek et al., 2010), and no exact matches fall within NRG1. Seven predicted sites consisting of the consensus core sequence (GGTA) with variant flanking nucleotides are present within NRG1: three are found on the antisense strand upstream of the region where we have detected the antisense transcript. These may be potential Brg1p binding sites, but, until further studies have been performed, we do not yet know whether Brg1p induces production of the antisense transcript directly or indirectly.

The identification of eukaryotic antisense sequences is becoming increasingly common. New coding and non-coding transcripts, including sense-antisense gene pairs exhibiting anti-correlated expression, have been identified in C. albicans using high-resolution tiling arrays (Sellam et al., 2010) and second generation sequencing methodologies such as RNA-Seq (Tuch et al., 2010, Bruno et al., 2010). All of these prior studies examined later time points in the hyphal induction process (at least one hour) and would thus not detect the NRG1 antisense transcript that we observed due to its rapid induction and transient nature.

A recent development in C. albicans has been the identification of the machinery required for RNAi (Drinnenberg et al., 2009). However AGO1, which encodes the RISC complex protein argonaute, is not required for hyphal growth (Bernstein et al., 2012) nor for NRG1 sense transcript instability (Fig. S5C). This points to an argonaute-independent mechanism of regulation, but our observed pattern of sense and antisense transcript destruction strongly suggests the involvement of dsRNA formation (Fig. 7A). Although we have not yet determined the exact process via which the NRG1 transcript is destroyed, it is worth nothing that regulatory non-coding RNAs are abundant in S. cerevisiae, which lacks RNAi altogether (Chen & Neiman, 2011, Tisseur et al., 2011).

Figure 7. A model for Brg1p function in C. albicans hyphal induction.

Figure 7

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BRG1 is an important component of hyphal induction in C. albicans. A. Under yeast conditions, BRG1 is repressed in an Nrg1p-mediated fashion. During hyphal induction, BRG1 expression is rapidly and substantially increased. This leads to Brg1p-dependent induction of an antisense NRG1 transcript (wavy line) and subsequent destruction of the NRG1 sense transcript (thin arrow) by an, as yet unknown, argonaute-independent mechanism (dashed lines). B. Brg1p acts in a feedback mechanism with NRG1 to regulate hypha-specific gene (HSGs) expression. BRG1 expression is upregulated by appropriate environmental stimuli, resulting in decreased NRG1 transcript levels and stimulation of hypha-specific gene expression. As BRG1 levels fall after the initial phase of induction, NRG1 levels are able to rise and the system is reset.

We know that Brg1p must have other functions beyond its role in NRG1 mRNA depletion since its over-expression results in hypha formation in yeast growth conditions rather than the pseudohyphae formed by an nrg1Δ strain (Fig. 6C). Our observation that over-expressing either NRG1 or BRG1 attenuates virulence demonstrates the importance of the balance between these two key regulators of hyphal growth. BRG1 is both necessary and sufficient for hypha formation in this important opportunistic pathogen, while previous work had demonstrated that modulation of NRG1 expression is also required for filamentation and virulence. Based on our results that show NRG1 sense mRNA stability and the production of antisense NRG1 transcript both depend on BRG1 expression, we propose a novel addition to the model of C. albicans hyphal induction (Fig. 7B): antisense regulation of NRG1 transcript stability.

Experimental Procedures

Strains and media

The yeast strains and plasmids used in this study are listed in Tables 1 and S1 respectively. Strains were routinely maintained as -80°C frozen stocks and grown on yeast extract-peptone-dextrose (YPD). Please refer to the Supporting Information for strain construction descriptions. Expression from the tetO promoter was abolished by the addition of 20 μg ml–1 doxycycline to the growth medium. Visual screens for β-galactosidase reporter activity in C. albicans were carried out as described previously by streaking colonies onto X-Gal Modified Medium (XMM) plates (Uhl & Johnson, 2001). All plasmid manipulations were performed with Escherichia coli strain DH5α with selection on Luria-Bertani plates containing 100mg ml-1 ampicillin when necessary.

Table 1.

Strains used in this study.

Strain Parent Genotvne1 Reference
SC5314 wild-type (Gillum et al., 1984)
BCa23-3 CAI4 nrg1::hisG- URA3-hisG/nrg1::hisG (Braun et al., 2001)
THE1 ade2::hisG/ade2::hisG ura3::λ imm434/ura3:: λ imm434 ENO1/eno1::ENO1-tetR-ScHAP4AD-3xHA-ADE2 (Nakayama et al., 2000)
SSY50-B THE1 NRG1/nrg1:: URA3-tetO-NRG1 (Saville et al., 2003)
THE1-CIp10 THE1 RPS1/RPS1:: URA3 (Bernardo et al.,2008)
ICY171 THE1-CIp10 RPS1:: URA3/RPS1: :SAT1-tetO-BRG1 This study.
ICY175 SSY50-B RPS1/RPS1: :SAT1-tetO-BRG1 This study.
ICY194 SC5314 BRG1/brg1Δ: :SAT1-FLIP2 This study.
ICY318 ICY194 BRG1/brg1 Δ : :FRT This study.
ICY322 ICY318 brg1Δ::FRT/brg1Δ: :SAT1-FLIP This study.
ICY326 ICY322 brg1 Δ ::FRT/brg1 Δ ::FRT This study.
ICY330 ICY326 brg1 Δ ::FRT BRG1::SAT1-FLIP This study.
ICY197 SSY50-B BRG1/brg1 Δ ::SAT1-FLIP This study.
ICY200 ICY197 BRG1/brg1 Δ ::FRT This study.
ICY276 ICY200 brg1 Δ ::FRT/brg1 Δ ::SAT1-FLIP This study.
ICY277 ICY276 brg1Δ: :FRT/brg1Δ::FRT This study.
ICY314 ICY277 brg1 Δ ::FRT/BRG1::SAT1-FLIP This study.
ICY293 THE1 RPS1/RPS1: :SAT1-ACT1p-NRG1 This study.
ICY295 ICY293 RPS1:: URA3-tetO-BRG1 This study.
ICY281 THE1 NRG1/nrg1Δ:: URA3-tetO-lacZ This study.
ICY307 ICY281 RPS1/RPS1: :SAT1-tetO-BRG1 This study.
VY561 ago1::FRT/ago1::FRT (Bernsteinet al., 2012)
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1

The full genotype is that of the parental strain with additional modifications as indicated.

2

SAT1-FLIP denotes the SAT1 flipper cassette.

Filamentation Assays

For filamentation assays in liquid media, strains were grown overnight at 28°C, washed in sterile PBS and diluted 1:20 into fresh media and incubated with shaking. For environmental induction of hyphae, cells were grown either in RPMI-1640 supplemented with L-glutamine and buffered with MOPS (Angus Buffers and Chemicals), YPD or YPD+10% fetal bovine serum FBS (Lonza) media and incubated with shaking at 37°C.

Filamentation assays on solid media were performed as described (Cleary & Saville, 2010). Strains were either streaked directly onto plates or strains were grown overnight at 28°C, washed in sterile PBS, cells counted using a haemocytometer and appropriate dilutions spread or spotted onto plates (YPD, Spider medium (Liu et al., 1994), Lee medium (Lee et al., 1975) pH 4 or pH 7, N-acetylglucosamine (GlcNAc) medium (Hubbard et al., 1985) or embedded in molten YPD.

Biofilm Formation

Biofilms were formed as previously described (Pierce et al., 2008) with the following modifications. Briefly, cells from an overnight culture were washed in sterile PBS, counted using a haemocytometer and resuspended in growth medium (YNB, Spider or RPMI-1640) to the desired density. Aliquots of 100μl (1×105 cells) were placed in wells of a 96-well microtitre plates. The plates were incubated at 30°C for 2 h and then the wells were washed with sterile PBS to remove non-adherent cells. Incubation was continued with fresh medium for 24 hours at 30°C, after which the wells were washed and photographed. All morphological tests were performed in at least biological triplicate.

Fluorescence microscopy

C. albicans cells were stained using calcofluor white (Sigma), and nuclei were revealed through the use of Vectashield mounting medium containing DAPI (4’,6-diamidino-2-phenylindole; Vector Labs). A DMR epi-fluorescence microscope (Leica) equipped with a digital camera was used to visualize fluorescence and capture cell images.

Murine virulence assays

For injection, cultures of the BRG1 over-expression strains were grown overnight at 28°C in YPD. Cells were harvested by centrifugation and washed three times in sterile pyrogen-free saline. Cells were counted using a haemocytometer, and appropriate dilutions made so that the required dosage of cells could be injected in a final volume of 200μl into the lateral tail veins of 6- to 8-week-old female BALB/c mice. Confirmation of the number and viability of cells present in the infecting inocula was performed by plate count. The following doses were used for the indicated experiments: for over-expression virulence tests (Fig. 3A), approximately 4.6×105 or 2.4×105 CFU of strain ICY171; for timed sacrifice experiments (Fig. 3B) approximately 1.7×106 (6 h sacrifice) or 3.9×105 (3 day sacrifice) CFU of strain ICY171; for over-expression virulence experiments in the tet-NRG1 background (Fig. 3C) approximately 6.0×105 or 2.4×105 CFU of strain ICY175.

Groups of five mice were used for each condition. Days on which the animals died were recorded; severely moribund animals were humanely sacrificed to minimize suffering and recorded as having died the following day. In all experiments, one kidney was processed for histopathology, whereas the other kidney, the brain, and the spleen were homogenized and fungal loads determined by plating dilutions onto Sabouraud agar plates. All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of The University of Texas at San Antonio (an AAALAC accredited institution) and performed in accordance with institutional regulations. Mice were allowed a one week acclimatization period before experiments were started.

Statistical analyses

For statistical analyses of survival curves, the Log-rank (Mantel-Cox) test was used to detect statistically significant differences between groups. For statistical analyses of fungal burdens (cfu), the Mann–Whitney test was used to detect statistically significant differences between doxycycline-treated and untreated animals. Analyses were performed using Prism 5 (GraphPad Software Inc).

Histopathology

Kidneys excised from deceased or sacrificed mice were fixed in 10% buffered formalin and stored at 4°C until required. Kidneys were embedded in paraffin, tissue slices were cut and stained with Grocott-Gomori methenamine-silver (GMS) (Grocott, 1955) or hematoxylin and eosin stain (H&E) prior to microscopic evaluation .

Strand-Specific Reverse Transcription

Strand-specific reverse transcription was based on a described method (Ho et al., 2010). Briefly, RNA was isolated as described (Cleary et al., 2010) using the MasterPure™ Yeast RNA Extraction Kit (Epicentre Biotechnologies) and treated with amplification grade DNaseI (Invitrogen) to remove any genomic DNA. First strand synthesis was initiated with strand-specific primers or oligodT (Invitrogen) using the MasterScript cDNA synthesis kit (5prime). Actinomycin D (Sigma) was added to a final concentration of 6 ng ul-1 to reduce non-specific synthesis (Sellam et al., 2010).

RT-PCR

Various primer pairs (Table S2) were used in conjunction with GoTaq® Green Master Mix (Promega) to amplify products from strand-specific and oligodT –primed RT products. All RT-PCR experiments were performed in at least biological triplicate.

Quantitative PCR

The cDNA templates used for BRG1 expression profiling were synthesized with random hexamers (Applied Biosystems). For quantification of the NRG1 sense and antisense transcripts cDNA was synthesized with strand-specific primers or oligodT (Invitrogen) using the MasterScript cDNA synthesis kit (5prime). Primer pairs (Table S2) were used in conjunction with GoTaq® qPCR Master Mix (Promega) and twin.tec real-time 96 well PCR plates (Eppendorf) in an ABI 7300 Real Time PCR System (Applied Biosystems). Dissociation curves were analyzed for all reactions to verify the presence of single peaks/products. Expression levels were analyzed using ABI 7300 System SDS Software (Applied Biosystems). All quantitative real-time PCR experiments were performed in biological triplicate.

Supplementary Material

Supp Material S1

Acknowledgements

We would like to thank Joachim Morschhaüser for providing plasmid pSFS2, Mark Paget for providing plasmid pMT3000, Alistair Brown for providing plasmid CIp10, Hironobu Nakayama for providing strain THE1 and plasmid p97CAU1, Alexander Johnson for providing strain BCa23-3 and plasmid pAU22, Sam Lee for providing strain THE1-CIp10 and Gerald Fink for providing the ago1Δ strain VY561. We would also like to thank Sara M. Reinhard, Kristi Barker, Fatemeh Sanjar and Sarah Bubeck for technical assistance. The work presented here was funded in part by NIH Grant RO1 AI063256-01 to SPS from the National Institute of Allergy and Infectious Diseases (NIAID).

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