Mapping of functional domains and characterization of the transcription factor Cph1 that mediate morphogenesis in Candida albicans

Fungal Genetics and Biology 83 (2015) 45–57

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Mapping of functional domains and characterization of the transcription factor Cph1 that mediate morphogenesis in Candida albicans Protiti Maiti a,b, Priyanka Ghorai b, Sumit Ghosh b,1, Mohan Kamthan b,2, Rakesh Kumar Tyagi a,⇑, Asis Datta a,b,⇑ a b

Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi 110067, India National Institute of Plant Genome Research, New Delhi 110067, India

a r t i c l e

i n f o

Article history: Received 26 February 2015 Revised 9 August 2015 Accepted 15 August 2015 Available online 17 August 2015 Keywords: MAP kinase pathway Transcription factor Morphogenesis Candida albicans

a b s t r a c t Cph1, a transcription factor of the Mitogen Activated Protein (MAP) kinase pathway, regulates morphogenesis in human fungal pathogen Candida albicans. Here, by following a systemic deletion approach, we have identified functional domains and motifs of Cph1 that are involved in transcription factor activity and cellular morphogenesis. We found that the N-terminal homeodomain is essential for the DNA binding activity; however, C-terminal domain and polyglutamine motif (PQ) are indispensable for the transcriptional activation function. Complementation analysis of the cph1D null mutant using various deletion derivatives revealed functional significance of the N- and C-terminal domains and PQ motif in filamentation process, chlamydospore formation and sensitivity to the cell wall interfering compounds. Genome-wide identification of the Cph1 binding site and quantitative RT-PCR transcript analysis in cph1D null mutant revealed that a number of genes which are associated with the filamentous growth, maintaining cell wall organization and mitochondrial function, and the genes of the pH response pathway are the transcriptional targets of Cph1. The data also suggest that Cph1 may function as a positive or negative regulator depending on the morphological state and physiological conditions. Moreover, differential expression of the upstream MAP kinase pathway genes in wild type and cph1D null mutant indicated the existence of a feedback regulation. Ó 2015 Elsevier Inc. All rights reserved.

1. Introduction Candida albicans is an opportunistic fungal pathogen that can cause lethal infection in humans (Berman and Sudbery, 2002; Au et al., 2007). C. albicans has notable capability of adapting itself to the varying physiological conditions of the host microniches that render it a versatile pathogen (Biswas et al., 2007). C. albicans, being a close relative, is similar in many ways to the budding yeast Saccharomyces cerevisiae. However, there are many unique aspects that make it an important model organism for the study of pathogenesis and signal transduction pathways in fungal pathogens (Sudbery, 2011; Gow et al., 2011). Different aspects of C. albicans ⇑ Corresponding authors at: Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi 110067, India (R.K. Tyagi) and National Institute of Plant Genome Research, New Delhi 110067, India (A. Datta). E-mail addresses: [email protected] (R.K. Tyagi), [email protected] (A. Datta). 1 Present address: CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow 226015, India. 2 Present address: CSIR-Indian Institute of Toxicology Research, Lucknow 226001, India. http://dx.doi.org/10.1016/j.fgb.2015.08.004 1087-1845/Ó 2015 Elsevier Inc. All rights reserved.

pathogenicity including structural organization and dynamics of the genome, virulence factors, drug resistance and biofilm formation have been studied extensively in the past few years (Biswas et al., 2007; Sudbery, 2011). A number of genes that are involved in fungal virulence and antifungal resistance were identified and characterized during the last decade (Watamoto et al., 2011; Ramesh et al., 2011). However, even after the whole genome sequencing and annotation, a lot remains unknown about the cellular and molecular aspect of the pathogenicity in this systemic pathogen. One of the unique features of C. albicans is its ability to grow in different morphogenetic forms: unicellular yeast to filamentous pseudohyphal or true hyphal form (Biswas et al., 2007; Brown et al., 2007). Other than this it can form chlamydospores during unfavourable conditions or undergo white to opaque switching to initiate mating process (Nobile et al., 2003; Johnson, 2003). Different morphogenetic forms differ in their cell and colony morphologies, prevalent signal transduction pathways, metabolic rate and often also preferred physiological niches (Sudbery, 2011; Shapiro et al., 2011; Brown et al., 2007). The yeast-tohyphal transition has been extensively studied in C. albicans

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because of its correlation with the pathogenicity (Lo et al., 1997; Sudbery, 2011). During infection, yeast cells adhere to the epithelial cells and then rapidly change into hyphal forms. Hypha-specific cell wall proteins like Hwp1, Als1, Als3, Als5, Int1 and Eap1 are responsible for the adhesion of the hyphal cells with the host epithelia (Sudbery, 2011; Calderone and Fonzi, 2001). Either endocytosis or active penetration of the plasma membrane by the hyphal cells is required for inclusion and penetration into the epithelia, endothelia and host tissue (Gow et al., 2002). Signal transduction pathways that positively and negatively regulate the yeast-to-hyphal switch in C. albicans are known (Shapiro et al., 2011). Positive regulation of yeast-to-hyphal morphogenetic switch is controlled by an array of transcription factors like Cph1 (also known as Acpr), Efg1, Rim101, Tec1, Cph2 and Czf1, while negative regulation is brought about by the proteins like Tup1, Nrg1 and Rfg1 (Sudbery, 2011). One of the signaling cascades that regulate morphogenesis in C. albicans is the Cek1-mediated MAP kinase pathway. Cph1, the terminal transcription factor of this MAP kinase pathway, was previously identified in our laboratory as a transcriptional regulator that can complement the mating defect in S. cerevisiae (Malathi et al., 1994). Deletion of this gene in C. albicans resulted in delayed filamentation (Liu et al., 1994; Huang et al., 2008). Other major signaling pathways controlling morphogenesis in C. albicans are the cAMP–PKA pathway, the pH response pathway, including the transcription factor Rim101, the Hog1-mediated MAPK pathway and the PKC pathway for maintenance of the cell integrity (Biswas et al., 2007). Cph1 can act in concert with another regulator of morphogenesis, Efg1, the transcription factor of the cAMP–PKA pathway. Both these pathways are regulated by the Ras1 protein (Biswas et al., 2007; Shapiro et al., 2011). The cph1efg1D double mutant exhibited loss of function of almost the entire transcription machinery regulating filamentation and the mutant was avirulent in the mouse model of candidiasis (Lo et al., 1997). Recently, Cph1 has been implicated in maintenance of cell wall organization, pseudohypha formation in response to oxidative stress, biofilm formation and in regulation of pheromone response in both white and opaque phase cells (Eisman et al., 2006; Srinivasa et al., 2012; Lin et al., 2013). Although Cph1 protein has been an area of interest over the past decade, our knowledge of the precise function under various physiological conditions is far from complete. Even though a number of studies have been directed to determine the biological function of Cph1, our understanding about the structure–function relationships of this transcription factor is fragmentary. The functional domains/motifs of the protein are yet to be experimentally

validated. Here, we studied structure–function relationships by systematically deleting seven domains/motifs of Cph1 and by carrying out comparative functional analyses of the deletion mutants. This work led to the identification of different domains/motifs of Cph1 that contribute to the transcription factor activity and emphasized on the functional role of Cph1 in controlling various cellular processes. 2. Materials and methods 2.1. Strains and growth conditions C. albicans strains used are listed in Table 1. Strains were regularly maintained on YPD medium or on SD minimal medium at 30 °C. Transformation of C. albicans strains was carried out using electroporation method (De Backer et al., 1999). For studies on hyphal induction in solid media, strains were grown for 6 to 7 days at 37 °C either on Spider (Liu et al., 1994), SLAD (Gimeno and Finkm, 1992), or Lee’s (Lee et al., 1975) media. For hyphal induction in liquid media, strains were grown in either Spider or synthetic succinate medium at 37 °C (Lane et al., 2001). To induce chlamydospore formation, cells were streaked out lightly on chlamydospore induction medium (cornmeal agar, 0.5% Tween 80), covered with a coverslip, and incubated at 25 °C for 7 days. Sensitivity of the strains to the cell wall interfering compound like Congo Red was tested in supplemented YPD plates. Serial dilutions (1/10) of exponentially growing cells were spotted to examine the growth pattern of the respective strains after 24 h of incubation at 37 °C (Eisman et al., 2006). For the Nikkomycin Z sensitivity disk assay, exponentially growing cells were plated onto SD plates and 5 ll of Nikkomycin Z solution (50 lg/ml and 100 lg/ml) was added to 5 mm diameter Whatman paper disks that were placed on the surface of agar plates. After 12 h of incubation at 37 °C cell growth inhibition was evaluated (McCarthy et al., 1985). For the purpose of study of hyphal induction in the presence of H2O2, the cells were grown on solid YPD and synthetic succinate media supplemented with 1–10 mM H2O2. The liquid media were supplemented with 1 mM H2O2 or 25 mM ascorbic acid. 2.2. Construction of strains expressing CPH1 deletions Combination of PCR based mutagenesis and conventional restriction digestion–ligation methods were followed for the creation of deletion cassettes. The primers used for PCR amplification were designed to have the desired deletions and the final URA3

Table 1 List of the strains used in this study. Organism

Strain

Genotype

Reference

C. albicans

SC5314 A11-1-1 A11-1-1-1 REV-1 CLJ19 CP-1

Wild type Like CAI4, but acpr::hisG/acpr::hisG-URA3-hisG Like CAI4, but acpr::hisG/acpr::hisG Like A11-1-1-1, but acpr::hisG/acpr::[ACPR-URA3] ura3/ura3 cph1D::hisG/cph1D::hisG with pYPB1-ADH1pt-CPH1 ura3/::imm434::URA3/ura3::imm434 iro1::IRO1/iro1::imm434 acpr::HisG/his1::hisG acpr::Leu2/leu2 arg4/arg4 like REV-1, but encoding (2–27)D of Cph1 like REV-1, but encoding (41–215)D of Cph1 like REV-1, but encoding (220–222)D of Cph1 like REV-1, but encoding (293–298)D of Cph1 like REV-1, but encoding (555–563)D and (574–586)D of Cph1 like REV-1, but encoding (564–573)D Cph1 like REV-1, but encoding (588–655)D Cph1

Gillum et al. (1984) This study This study This study Huang et al. (2008) Noble et al. (2010)

MATa, trp1-901, leu2-3, 112, ura3-52, his3-200, gal4D, gal80D, LYS2::GAL1UASGAL1TATA-HIS3, GAL2UAS-GAL2TATA-ADE2, URA3::MEL1UAS-MEL1TATA-lacZ MATa, ura3-52, his3-200, ade2-101, trp1-901, leu2-3, 112, gal4D, gal80D, met-, URA3::GAL1UAS-GAL1TATA-LacZ MEL1

Clontech

NT1D NT2D DTGD IDD PQD IHD CTD S. cerevisiae

AH109 Y187

This This This This This This This

study study study study study study study

Clontech

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tagged deletion cassettes were further cloned into pGEMT-easy vector (Table S1). The deletion vectors were then digested with NotI and the linearized deletion cassette was integrated into the C. albicans A11-1-1-1 (URA3 cured cph1D null mutant) genome by homologous recombination. The resultant deletion mutants were having a single copy of the intact allele or the allele with respective deletions. Proper genomic integration was verified by Southern blotting using XbaI and ClaI restriction enzymes and PCR amplification followed by sequencing. 2.3. Yeast one hybrid assay The vectors pHIS2.1 and pGADT7 (Clontech) were used for this purpose. The promoter of the C. albicans hypha specific gene, HWP1 was cloned into pHIS2.1 vector and the resulting plasmid (pHIS2.1HWP-Pr) was transformed into S. cerevisiae strain Y187 that is auxotrophic for tryptophan, leucine and histidine. The CPH1 ORF and its deletion variants were cloned into pGADT7 vector (with ADH1 promoter) and the plasmids were transformed individually into the Y187 strain harboring pHIS2.1-HWP-Pr plasmid. The respective positive transformants were then streaked in SD plates lacking trp, leu and his; however, supplemented with 0.25 mM 3-AT and growth was observed after 3–4 days of incubation at 30 °C. 2.4. Transcriptional activation assay in yeast The CPH1 complete ORF and the deletion variants were cloned into pGBKT7 vector (Clontech) and the plasmids were transformed individually into S. cerevisiae strain AH109, carrying HIS3, ADE2 and LacZ reporter genes under the control of GAL4 promoter. The Cph1 protein and the deletion variants were expressed in fusion with the GAL4 DNA binding domain under the control of the ADH1 promoter. Transcriptional activation assay was performed by streaking the respective colonies in SD plates lacking trp, ade and his and kept at 30 °C for 3–4 days. The b-galactosidase assay was performed on the basis of blue coloration in a colony filter lift assay using 5-bromo-4-chloro-3-in-dolyl-b-D-galactoside (X-gal) as a substrate. However, for b-galactosidase assay in liquid culture o-nitrophenyl b-D-galactopyranoside (ONPG) was used. 2.5. RNA isolation and quantitative RT-PCR analysis RNA was isolated according to the Tripure method (Invitrogen). RNA quality was checked by running 1.2% agarose gel and by determining the A260/280 and A260/230 ratios. Two microgram of total RNAs, quantified using a nanodrop (ND 1000, Thermo Scientific) were reverse transcribed using superscript II (Invitrogen) at 42 °C for 50 min in 20 ll reaction volume following the manufacturer’s instructions. cDNAs were diluted three times before using in quantitative RT-PCR analysis. Quantitative RT-PCR reactions (10 ll) consisted of 5 ll 2X Power SYBR Green PCR Master Mix (Applied Biosystems), 0.5 ll of diluted cDNA and 500 nM of each forward and reverse gene specific primers. Quantitative RT-PCR was performed using 7900 HT Fast Real Time PCR (Applied Biosystems). Gene specific primers were designed using Primer Express (Applied Biosystems). List of the genes and oligonucleotide primers used are shown in Table S1. Melting curves were analyzed at the dissociation step to examine the specificity of amplification. Relative gene expression was analyzed following the 2
DDCT method, using ACT1 transcript as endogenous control. 2.6. Chitin staining and quantitative chitin assay The cells were grown in YPD broth until OD600 reached 0.8, washed with sterile MilliQ water and resuspended in YPD or SS medium with or without Nikkomycin Z and further grown for

47

6 h. Then, 1 ll of the 10 mg/ml stock of freshly prepared Calcofluor White was added to 1 ml of culture and incubated for 10 min in dark. The cells were then washed twice with sterile MilliQ water and microscopic observations were made using a Nikon 80i Fluorescent microscope. The chitin content was measured as described previously (Soulie et al., 2003) with few modifications. Dried biomass (10 mg) was mixed in 1 ml 2 N H2SO4 and heated for 20 h at 105 °C. Further, 0.2 ml of aliquot was adjusted to pH 3 using 2 N KOH. After adjusting the sample volume to 2 ml with MilliQ water, 1 ml Na2CO3 solution (1.5 N in 4% acetyl acetone) was added, and heated to 100 °C for 20 min. After cooling to room temperature for 1 h, 7 ml ethanol and 1 ml Ehrlich’s reagent (10 mM dimethylaminobenzaldehyde in 50% ethanol and 6 N HCl) were added to the sample. Hexosamine content was determined (OD at 520 nm), using GlcNAc as standard. 2.7. Reactive Oxygen Species (ROS) staining The cells were grown in YPD broth until OD600 reached 0.8, washed with sterile MilliQ water and resuspended to SS medium and grown for another 6 h. ROS production was determined by incubating the cells with 10 lM dihydroethidium (Sigma) for 15 min at 37 °C under dark and further washing of the cells twice with potassium phosphate buffer. microscopic observations were made using a Nikon 80i Fluorescent microscope. 2.8. Identification of Cph1 binding sites at the promoter region of C. albicans genes The upstream (1 kb from ATG) non-overlapping sequences of C. albicans ORFs (total 6218) were retrieved from the Candida Genome Database (www.candidagenome.org). Training data set was generated using promoter regions (
1 to
1000 bases upstream of ATG) of the genes regulated by CPH1 (24–26). These included HWP1, RBT4, FUS1, MSB2, OPI1, CTK1, GPA2, FAB3, REG1, BUD20, PIR1, HIT1, GZF3, RHO1, CEK1, PGA18, BET5, ECE1, AAT1, FAV1, ORC4, SST2, CPH1, MSN4, FRK26, RSN1, CPP1, PRP45, ADH6, AMD3, SAP4, SAP5, SAP6, ADE2, DBP10, ZCF33, EFG1, GTS1, AST2. Weight matrix was constructed using CONSENSUS program (RSAT; http://rsat.ulb.ac.be). All the parameter was set as default, except the Cph1 binding motif TGAAACA (Lane et al., 2001) was used as seed sequence and reverse strand was included as separate sequence. The Logo was prepared using Weblogo program (http://weblogo.berkeley.edu/logo.cgi). Further, by applying a threshold score of 7.00 (7.9 showed the actual match to TGAAACA), non-overlapping upstream sequences of entire C. albicans ORFs were analyzed through Matrix-scan program (RSAT; http://rsat. ulb.ac.be). A list of C. albicans ORFs having TGAAACA motif within their promoter is provided in Table S2. 3. Results 3.1. Identification of the domains/motifs of Cph1 Cph1 encodes a 656 amino acid homeodomain transcription factor that is an orthologue of S. cerevisiae Ste12. To gain insight into the functional domains/motifs present in Cph1, in silico analyses were carried out using the following programmes; Motif Search (www.genome.jp/tools/motif/) and Motif scan (myhits.isb-sib.ch/ cgi-bin/motif_scan). The analyses revealed that the N-terminal region, comprising 44–154 amino acids, is similar to the DNA binding domain of STE-like transcription factor. This was in accordance with a previous report exhibiting 71% identity of the N-terminal region of Cph1 with the N-terminal 40–204 amino acids of Ste12 (Malathi et al., 1994). The N-terminal region has been shown to

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be necessary for the DNA binding function of Ste12 (Yuan and Fields, 1991). In silico analyses also indicated the presence of two polyglutamine (PolyQ) stretches (555–563 and 574–586 amino acids) near the C-terminal region of the Cph1 (Table S3). These polyQ tracts were separated by a ten amino acid region that we named as inhibitory (IH) motif. PolyQ tracts have been known to act as transcriptional activators in a wide range of transcription factors from yeast to mammals and are found to be present in well known transcription factors like Ap2, Oct2 and Efg1 (Clerc et al., 1988; Williams and Tjian, 1991; Noffz et al., 2008; Atanesyan et al., 2012). Another striking feature of the Cph1 is the presence of a consensus sequence [Asp-Thr-Gly (DTG), 220–222 amino acids] that is characteristic of the active site of the acid proteases. Aspartic proteases, including the retroviral proteases, generally have these conserved residues for the active site geometry and catalytic function (Sayer et al., 2008). However, N-terminal region of the Cph1 does not correspond to the N-terminal region of the known secretory acid proteases and the signal peptide typical of a secretory protein was absent. Induction motif (ID) comprises of the amino acids 293–298 that are conserved among Cph1 and

A

Ste12, and Stek1 of Kluyveromyces lactis (Malathi et al., 1994). These residues are required for the pheromone-inducible transcriptional activation function of Ste12 (Song et al., 1991). Apart from these, in silico analyses revealed the presence of a few phosphorylation as well as glycosylation and myristoylation sites. Depending on in silico analysis and based on the domains/motifs of the known homeodomain transcription factors, seven putative motifs/domains of Cph1 were selected of the functional domain mapping. These domains were named as NT1D, NT2D, DTGD, IDD, PQD, IHD and CTD. (Fig. 1A and Table S3). 3.2. Identification and expression analysis of candidate Cph1-regulated genes of different cellular processes and signaling pathways To have an overview of the genes of different signaling and metabolic pathways that are transcriptionally regulated by Cph1, a genome-wide in silico analysis of the Cph1 binding site (TGAAACA) at the promoter region of the C. albicans genes was carried out (Fig. S1A–D and Supplemental Table S2). A total of 1869 ORFs of C. albicans were identified with the occurrence of

B Growth on SD-Trp-Leu-His+0.25mM 3AT CT∆

Growth on SD-Trp-Ade-His NT2∆

NT2∆

N-Terminal region 1, 2-27 aa (NT1∆) N-Terminal region 2, 41-215 aa (NT2∆) DTG motif, 220-222 aa (DTG∆) ID motif, 293-298 aa (ID∆) PolyQ motifs, 555-563 & 574-586 aa (PQ∆) IH motif 564-573 aa (IH∆) C-Terminal region, 588- 655 aa (CT∆)

C

Vector

COOH

ID∆

Acpr/Cph1 (656 aa)

NH2

D

DTG∆ β-Galactosidase filter assay NT2∆

E

Relative β-galactosidase activity (%)

PQ∆ 120

PQ∆

100 80 60 40 20 0

Fig. 1. Cph1 domains and motifs that are required for the DNA binding and transcriptional activation function. (A) The putative domains/motifs of Cph1 are diagrammatically represented. (B) Identification of the domain responsible for the site-specific DNA binding activity. HWP1 promoter was inserted upstream of HIS3 reporter in pHIS2.1 vector and co-transformed into S. cerevisiae strain Y187 along with pGADT7 vector that carried complete Cph1 ORF (AT) or different deletion variants (NT1D, NT2D, DTGD, IDD, CTD, IHD and PQD) upstream of GAL4 transcription activation domain. Transformants were grown on SD medium lacking Trp, Leu and His, however, containing 0.25 mM 3AT to determine HIS3 reporter gene activation. Empty pGADT7 vector co-transformed with pHIS2.1-HWP1 served as the vector control. Complete loss of DNA binding activity was observed in NT2D variant while partial loss was observed in NT1D variant. (C-E) For transcriptional activation assay complete ORF or different deletion variants were expressed as N-terminal GAL4 DNA binding domain-fused recombinant proteins in S. cerevisiae strain AH109 which carried reporter genes (HIS3, ADE2 and LacZ) under the control of GAL4 promoter. Transformants were grown on SD medium lacking Trp, His and Ade (C). Activation of the b-galactosidase reporter gene was determined by filter assay using 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (D). Relative b-galactosidase activity was determined using o-nitrophenyl b-D-galactopyranoside substrate (E). Complete loss of transcriptional activation was observed after deletion of the N-terminal (NT1, NT2) and C-terminal regions (CT), and PQ motif. The IHD variant was partially compromised in transcriptional activation function.

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1.4

Relative gene expression

A

WT

Null mutant

1.2 1 0.8

* **

0.6

**

**

**

**

0.4 0.2 0

WT

C

Null mutant

1 0.8 0.6 0.4 0.2 0

* **

** **

**

Relative gene expression

1.2

Relative gene expression

B

1.4

WT

Null mutant

1.2 1 0.8 0.6

* **

** **

0.4 0.2 0

Fig. 2. Expression analysis of the genes those are hypha-specific, cell wall-related, and involved in maintaining mitochondrial integrity and function. Wild type and cph1D null mutant (A11-1-1) were initially grown in YPD medium until mid log phase, resuspended in SS medium and incubated at 37 °C for 6 h. Quantitative RTPCR expression analysis of the hypha-specific genes (A), cell wall-related genes (B), mitochondrial integrity and function-related genes (C). Data are mean ± SE (n = 3– 6). Asterisks indicate statistically significant difference at ⁄⁄P < 0.01 and ⁄P < 0.05.

4.5 4

Relative gene expression

TGAAACA motif within 1 kb promoter regions. Filamentation process in C. albicans involves co-ordinated action of a large number of genes associated with various signaling and metabolic pathways (Biswas et al., 2007). The transcriptional regulation of these large set of genes by Cph1 is conceivable because of its vital role in mediating filamentous growth of C. albicans. In order to get an idea of the putative function of the candidate Cph1-regulated genes, they were categorized based on GO biological processes and cellular component (Fig. S1C and D). A total of 198 candidate Cph1-regulated genes were found to be associated with the filamentous growth of C. albicans. Some of them include ACE2, STE13, ADR1, AGE3, PHO4, SNF4, NDH51, FRG15, RIM20, GIR2 and MED20. Many of the candidate Cph1-regulated genes (total 39) were cell wall-related; suggesting a key role of Cph1 in regulating cell wall organization in C. albicans. Some of these genes include ABG1, ACE2, BPH1, CSH2, CMP1, CNB1, CRH12, DIT1, DIT2, ECM25, HST7 and HWP1. Quantitative RT-PCR analysis further validated Cph1-regulated expression of the hypha-specific and cell wall-related genes (Fig. 2A and B). Interestingly, some of the components of the CEK1-mediated MAP kinase pathway were found to have Cph1 binding sites at their promoter (Supplemental Table S2). These included STE2, STE3, STE18, STE50 and HST7. Moreover, using chromatin immunoprecipitation assay, STE50 and CEK1 were found to be the putative transcriptional targets of Cph1 (Borneman et al., 2007). To check the possibility of Cph1-mediated regulation of the upstream MAP kinase pathway components, their expression levels in wild type, cph1D null mutant and over-expressor strains were compared (Fig. 3). Quantitative RT-PCR analysis revealed more than 1.5-fold higher transcript expressions for the MAP kinase pathway genes STE2, STE3, STE11, STE18, STE50, HST7 and CEK1 in cph1D null mutant as compared to the wild type. Moreover, transcript levels of STE50, CST20 and CEK1 were more than 1.5-fold higher in

WT Null Mutant Overexpressor

3.5 3 2.5 2 1.5 1 0.5 0

CEK1 CST20 HST7

STE2

STE3

STE11 STE18 STE50

Fig. 3. Expression analysis of the upstream MAP kinase pathway genes in wild type and cph1D null mutant (A11-1-1). Strains were initially grown in YPD medium until mid log phase, resuspended in SS medium and incubated at 37 °C for 6 h. Data are mean ± SE (n = 4).

over-expressor strain compared to the wild type. In C. albicans, Cph1 activation requires a cascade of MAP kinase signaling involving kinases STE11, HST7 and CEK1 (Fig. S2; Monge et al., 2006). Therefore, these results suggested the existence of a Cph1mediated feedback regulation of the MAP kinase pathway. 3.3. Motifs/domains involved in DNA binding and transcriptional activation function Yeast one hybrid (Y1H) assay was performed to identify Cph1 domains/motifs responsible for the DNA binding activity. HWP1 promoter (1 kb upstream of ATG) was chosen as DNA bait for the assay because promoter of this hypha-specific gene was found to have three Cph1 binding sites (TGAAACA) and also transcript level of this gene was down-regulated in cph1D null mutant (Fig. 2A and Table S2). To check specificity of the Y1H assay, the ability of the full-length Cph1 to activate HWP1 promoter-driven expression of the HIS3 reporter in S. cerevisiae strain (Y187) was determined. Y1H assay was carried out in the presence of 3-amino-1,2,4triazole (3AT) to prevent leaky expression of the HIS3 reporter (Fig. 1B). Efficient growth of the transformed yeasts in the absence of histidine, indicated activation of the HIS3 reporter gene expression by Cph1 which otherwise was not observed in case of the vector control. This demonstrated specificity of the Y1H assay and excluded the possibility of endogenous Cph1 homologue (Ste12)mediated activation of the HIS3 reporter gene in S. cerevisiae. However, HIS3 reporter gene activation was not observed when NT2-deleted Cph1 was expressed and yeast cells were completely unable to grow in drop-out plate lacking histidine (Fig. 1B). While NT1-deleted Cph1 could incompletely activate the reporter gene and suppressed growth of the yeast cells was observed. In contrast, deletion of the C-terminal (CT) region, DTG, ID, PQ and IH motifs had no effect on the HIS3 reporter gene activation, thus, DNA binding activity of the Cph1 (Fig. 1B). These results indicated that the N-terminal region of the Cph1 is responsible for the DNA binding activity. In silico analysis predicted the presence of a homeodomain at the N-terminal region (41–215 amino acids) that displayed high level similarity to the homeodomain of Ste12. Therefore, it can be concluded that the domain essential for the site-specific DNA binding included the NT2 (41–215 amino acids) as well as the extreme N-terminal region (NT1, 2–27 amino acids) of the Cph1. For the transcriptional activation assay, full-length ORF as well as different domains/motifs-deleted Cph1 were expressed as Nterminal GAL4 DNA binding domain-fused recombinant proteins

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SPIDER

Lee’s

SLAD

SS medium

B

NT1∆ PQ∆ IH∆ CT∆

A11 -1-1 CP-1 REV-1

Null mutants

Deletion mutants

NT2∆

Overexpressor

A11 -1-1 CP-1 ID∆ CT∆

IH∆

PQ∆

Deletion mutants

DTG∆

NT2∆

NT1∆

REV-1

Null mutants

Overexpressor

WT

WT

A

Fig. 4. Comparative morphological analysis of the wild type (SC5314), CPH1 over-expressor (CLJ19), null mutants (A11-1-1 and CP-1), revertant (REV-1) and deletion mutants (NT1D, NT2D, DTGD, IDD, CTD, IHD and PQD) in various hypha inducing media. After initial culture in YPD until mid log phase, cells were either plated onto Spider, SLAD and Lee’s agar media, and incubated at 37 °C for 7 days (A) or resuspended in liquid synthetic succinate (SS) medium and incubated at 37 °C for 5 h (B). Experiments were repeated three times with similar results. Scale bars, 2 mm (A) or 10 lm (B).

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in yeast strain AH109 that carried three reporter genes (HIS3, ADE2 and LacZ) under the control of GAL4 promoter. On triple dropout plates (SD medium lacking trp, his and ade), yeast cells expressing intact ORF as well as DTG and ID-deleted Cph1 variants were able to grow efficiently, indicating transcriptional activation of the reporter genes (Fig. 1C). In contrast, deletion of NT1, NT2 and CT regions and PQ motif resulted in complete loss of the transcriptional activation function of the Cph1. Although, yeast cellsexpressing IH-deleted Cph1 were able to grow on triple dropout plate, their colonies were pink in color (Fig. 1C). This indicated that the ADE2 reporter was not properly activated in yeast cellsexpressing IH-deleted Cph1 and therefore, complete transcriptional activation was absent. To further confirm these results, lacZ reporter activation was checked through b-galactosidase assays using X-Gal and ONPG substrates (Fig. 1D and E). The desired blue coloration and lacZ activation were observed in yeast colonies expressing the intact as well as DTG and ID motif-deleted Cph1. However, a slight blue coloration and partial activation of the lacZ was displayed by the pink colonies of yeast cells-expressing IHdeleted Cph1 (Fig. 1D and E). Altogether, these results suggested that NT1, NT2, PQ, and CT domains/motifs are indispensable for the transcriptional activation function of the Cph1. However, IH motif was not indispensable, but required for the full transcriptional activation function of the Cph1. PolyQ stretches were previously shown to be part of the transcriptional activation domain in some transcription factors (Atanesyan et al., 2012). Therefore, C-terminal region of Cph1, including the PQ and IH motifs, could be regarded as part of the transcriptional activation domain. The N-terminal region-deleted Cph1 proteins (NT1D, NT2D) were also compromised in transcriptional activation function. This may be due to the structural alteration at the C-terminal transcriptional activation domain because of the deletion of the N-terminal region. Further protein structure studies will be helpful to understand the effect(s) of these N-terminal deletions to the C-terminal domain.

3.4. Effects of Cph1 deletions on morphogenesis C. albicans transformants with different deletion variants for the Cph1, under the cph1D null mutant background (A11-1-1-1), were analyzed for their ability to undergo different morphological changes like yeast-to-hyphal transition and chlamydospore formation. The revertant strain (REV-1), containing a wild-type CPH1 allele and the A11-1-1 null mutant were used as the experimental controls. The URA3 marker was used for the gene disruption as well as creating revertant strains. It had been reported earlier that ectopic expression of the URA3 marker can confer filamentation as well as virulence defects in C. albicans (Cheng et al., 2003; Brand et al., 2004; Noble and Johnson, 2005). Therefore, another cph1D null mutant strain, CP-1 (Noble et al., 2010) developed with SN-152 (his-leu-arg-) genotype background was used as the control to determine whether integration of the URA3 marker at the CPH1 locus was the reason for the observed morphological changes. However, resemblance of both the cph1D null mutants indicated that ectopic expression of the URA3 marker from the CPH1 locus did not contribute to the observed morphological defects in the null mutant (Figs. 4A and B and 5). From comparative morphological analysis of the wild type, CPH1 over-expressor, null mutant and deletion mutants in solid (Spider, SLAD and Lee’s) and liquid synthetic succinate (SS) media, we found that all the mutants that were compromised in either/or both DNA binding and transcriptional activation (i.e. NT1D, NT2D, PQD, IHD and CTD) also displayed delayed or reduced hypha formation (Fig. 4A and B). These deletion mutants exhibited colony morphologies that were similar to the null mutant. DTGD and IDD variants which did not show defect in Cph1 function, resembled revertant in filamentation (Figs. 1B–E and 4A). Cph1 over-expressor strain exhibited enhanced hyphae formation in all the growth media tested. The only liquid medium that was able to confer filamentation defect in cph1D null mutant and the deletion mutants was SS

Null mutants WT

Overexpressor

A11-1-1

CP-1

Revertant

Deletion mutants NT1∆

NT2∆

PQ∆

IH∆

CT∆

Fig. 5. Comparative analysis of chlamydospore formation in wild type, CPH1 overexpressor, null mutants and deletion mutants. For inducing chlamydospore formation, strains were grown under anaerobic conditions in cornmeal agar plates for 7 days at 25 °C as described in Section 2.1. cph1D null mutants and deletion mutants exhibited defects in chlamydospore formation.

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YPD

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YPD + Congo red (100μg/ml)

10-1 10-2 10-3 10-4 10-5

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Overexpressor

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CP-1

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WT

Fig. 6. Sensitivity of cph1D null mutants and deletion mutants to the cell wall-interfering compounds. (A) Wild type, CPH1 over-expressor, null mutants, revertant and deletion mutants were grown in YPD broth until mid log phase, serially diluted and spotted onto YPD media with or without Congo Red. (B) For Nikkomycin Z assay, cells from mid log phase growth were plated onto SD media and disk diffusion assay was performed. Scale bars, 5 mm. Experiments were repeated three times with similar results.

medium (Fig. 4B), unlike Spider, SLAD and Lee’s (Fig. S3 and data not shown). However, the wild type strain displayed delayed hyphae formation in SS medium compared to the other hyphaeinducing media. Similar observations on liquid Spider, SLAD and Lee’s were also reported earlier for the null mutant (Lane et al., 2001; Huang et al., 2008). The high level expression of CPH1 in SS medium further supported its involvement in morphogenetic transition (Fig. S4A–C). However, we do not exclude the role of Cph1 in metabolism of non-fermentable carbon source like succinate. In SS medium, only CPH1 over-expressor strain displayed true hyphae formation, while wild type and revertant strains mostly exhibited pseudohypha formation. cph1D null mutants and the deletion mutants, NT1D, NT2D, PQD, IHD and CTD were locked in the budding yeast state (Fig. 4B). These results were in agreement with the morphogenesis on the solid media that these domains/motifs are essential for proper functioning of the Cph1 (Fig. 4A). In other liquid media, cph1D null mutants as well as deletion mutants did not show significant defects in filamentation, but CPH1 over-expressor strain displayed enhanced hypha formation (Fig. S3 and data not shown). Colony morphology or hyphal induction of revertant strain (carrying a single CPH1 allele) did not completely resemble to the wild type that carries two CPH1 alleles (Fig. 4A and B), therefore, CPH1 transcript level was analyzed in wild type, cph1D null mutant, revertant and also in overexpressor strain (Fig. S5). CPH1 transcript level in revertant was about 50% of that detected in wild

type. However, about 18-fold higher transcript level was detected in overexpressor strain compared to the wild type. These results substantiated the incomplete rescue of the morphological phenotypes in the revertant strain (Fig. 4A and B). The morphology of the mutant strains differed considerably from the wild type in corn meal agar medium under chlamydospore inducing conditions (Fig. 5). cph1D null mutants and deletion mutants displayed reduced number of chlamydoconidia as compared to the wild type. In cph1D null mutant, blastoconidia were arranged in a grape like fashion associated with long unbranched hyphae and chlamydospores were present infrequently on the tip of short lateral suspensor cells (Fig. 5). These results suggest that Cph1 is required for the normal chlamydospore formation in C. albicans. There are several genes like SUV3, MDS3, ISW2, RIM13, RIM101, AAH1 (Nobile et al., 2003) HOG1, (Eisman et al., 2006), EFG1 (Palige et al., 2013), NRG1 (Staib and Morschhäuser, 2005) that genetically control the process of chlamydospore formation in C. albicans. The defect in chlamydospore formation observed in cph1D null mutant closely resembled the pH response pathway mutants, rim13D, rim101D and mds3D (Nobile et al., 2003). Moreover, the expression of RIM13 was down-regulated in cph1D null mutant, suggesting that RIM13 may be under the transcriptional control of Cph1 (Fig. 2A). Also, genome-wide analysis revealed Cph1 binding site (TGAAACA) within the promoter of RIM13 and other pH response pathway genes including RIM 8, RIM 9 and RIM 20 (Table S2). Besides, other

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nikkomycin Z (Kingsbury et al., 2012). Surprisingly, all the mutants showed sensitivity to nikkomycin Z, in contrast to the wild type, revertant and CPH1 over-expressor strains (Fig. 6B). To further confirm the effect of nikkomycin Z on chitin synthesis in the mutants, we examined the level of chitin with calcofluor white staining of C. albicans after treatment with nikkomycin Z (Fig. 7A) and also by quantitative determination of the cell wall chitin level (Fig. 7B). In wild type and CPH1 over-expressor, chitin was evenly distributed in the cell wall as well as in septum, while reduced chitin staining was observed in cell wall of the cph1D null mutant in the presence of nikkomycin Z (Fig. 7A). Similarly, reduced chitin level was quantified in cph1D null mutant compared to wild type in the presence of nikkomycin Z (Fig. 7B). These results confirmed that the reduced cell wall chitin level leads to the nikkomycin Z sensitivity (Rao et al., 2013). Further, to substantiate these results, the expression levels of the genes involved in chitin synthesis (CHS1, CHS8 and UAP1) and hypha-specific genes (HWP1 and ECE1) were determined in wild type and cph1D null mutant through quantitative RT-PCR analysis (Fig. 7C–E). When nikkomycin Z was added to the medium, induced expression of these genes was observed in wild type strain (Fig. 7D). This may be a counteraction mechanism of the cells to overcome the chitin synthase activity inhibition brought about by nikkomycin Z. However, these genes showed reduced expression in cph1D null mutant compared to the wild type in the presence of nikkomycin Z (Fig. 7E). These

chlamydospore-regulating genes such as SUV3, AAH1, NRG1, PGA55, DIT2 and MET15 were also found to have Cph1 binding sites at their promoter regions (Table S2). Therefore, Cph1 could affect chlamydospore formation by regulating the expression of these genes directly and/or indirectly through some other Cph1-regulated pathway/process. 3.5. Sensitivity of the deletion mutants to the cell wall interfering compounds It has been shown that C. albicans mutants for Cek1 MAP kinase and other MAPK pathway components are sensitive toward the cell wall assembly inhibitors such as congo red and calcofluor white (Eisman et al., 2006). A comparative analysis of the growth pattern of the wild type, CPH1 over-expressor, null mutant and deletion mutants revealed that all the strains except wild type exhibited growth impairment in media supplemented with congo red. CPH1 deletion mutants and over-expressor behaved similar to the null mutant; while, in revertant strain growth impairment was complemented with a wild type allele of the CPH1 (Fig. 6A). We next tested nikkomycin Z sensitivity of the mutants. Nikkomycin Z inhibits chitin synthase activity thus prevents the formation of septum and cell wall chitin (Kim et al., 2002). Generally the mutants that are sensitive to the cell wall assembly inhibitors such as congo red and calcofluor white do not show sensitivity toward

WT

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GlcNAc (μg/mg dry weight)

B

1.4 1.2

WT+ NZ Null mutant + NZ

1 0.8 0.6 0.4 0.2 0

Fig. 7. Cell wall chitin analysis in wild type, CPH1 overexpressor and null mutant. (A) Cells were initially grown in YPD medium until mid log phase, resuspended into fresh YPD medium supplemented with 50 lM Nikkomycin Z (NZ) and further grown for 6 h at 37 °C. Microscopic observations were made after staining the cells with Calcofluor White to visualize the difference in cell wall chitin content. Scale bars, 5 lm. Experiments were repeated three times with similar results. (B) Chitin content was measured as the amount of glucosamine liberated from cells after acid hydrolysis. ‘
’ and ‘+’ signs denote with and without NZ. Data are mean ± SE (n = 3). (C–E) Expression analysis of the cell wall-related genes in wild type and cph1D null mutant (A11-1-1) after Nikkomycin Z treatment. Strains were grown in SD medium without (C) and with NZ (D, E). Expression levels of the genes were determined by quantitative RT PCR analysis. Data are mean ± SE (n = 4).

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results provided a mechanistic view of the nikkomycin Z sensitivity of the cph1D null mutant and deletion mutants and suggested the involvement of Cph1 in nikkomycin Z-induced up-regulation of the hyphae-specific and chitin synthesis-related genes in C. albicans. Surprisingly, transcript levels of the CHS1, CHS8, UAP1, HWP1 and ECE1 were found to be higher in the cph1D null mutant as compared to the wild type under normal growth condition (Fig. 7C), suggesting their negative regulation by the Cph1. The induced expression of CHS1, CHS8 and UAP1 was possibly responsible for the increased chitin content in the cell wall of cph1D null mutant (Fig. 7A and B) and, thus, sensitivity to congo red (Fig. 6A). 3.6. Effects of oxidative stress on Cph1-mediated filamentation process SS medium was the only liquid medium, where cph1D null mutant and deletion mutants showed filamentation defect (Fig. 4B). We next examined whether Cph1-mediated filamentation induction in SS medium was related to the oxidative stress in cells. We hypothesized this because succinate might contribute to the mitochondrion hyperactivity which may result in the formation of ROS in cells. Moreover, the cellular ROS burst can be the outcome of mitochondrial dysfunction (Leadsham et al., 2013). In the genome wide study of the Cph1 binding sites, we found that 14.7% of the putative Cph1-regulated genes having TGAAACA motifs in their promoters were related to the cellular component mitochondrion (Table S2). Interestingly, wild type strain showed

YPD

YPD + H2O2

YPD + AA

SS

SS + H2O2

SS + AA

Null mutant

WT

A

increased sensitivity to hydrogen peroxide (H2O2) when cultured on SS medium, compared to YPD (Fig. S6). This suggested that cells which were grown in SS medium were already under oxidative stress possibly because of the hyperactivity of the mitochondria in the presence of succinate substrate that resulted in enhanced ROS production. This was also evident from the comparative analysis of the dihydroethidium (an indicator of superoxide)-stained cells grown in YPD, SS and SS medium supplemented with ascorbic acid (Fig. 8B). Both the wild type and cph1D null mutant acquired intense dihydroethidium staining when grown in SS medium compared to YPD and addition of ROS scavenger ascorbic acid resulted in reduced ROS accumulation. However in SS medium, cph1D null mutant showed intense dihydroethidium staining in comparison to the wild type; suggesting hyper-accumulation of ROS. To better understand the role of ROS in Cph1-mediated filamentation process, we tested filamentation of the wild type and null mutant in the presence of ROS (in this case H2O2) and ascorbic acid, a ROS scavenger. In YPD medium, wild type strain was able to form pseudohyphae in response to H2O2, while null mutant remained in yeast state (Fig. 8A). Ascorbic acid had no effect on morphology of either strain on YPD medium. A recent report also suggested the role of Cph1 in pseudohyphae formation in response to subtoxic levels of H2O2 (Srinivasa et al., 2012). Interestingly, addition of H2O2 to the SS medium resulted in impaired filamentation in wild type (Fig. 8A). Moreover, when SS medium was supplemented with ascorbic acid, germ tube formation was initiated in null mutant

YPD

SS

SS + AA

Null mutant

WT

B

Fig. 8. Comparative analysis of the morphogenesis of wild type and cph1D null mutant (A11-1-1) in YPD broth and synthetic succinate (SS) medium in the presence of H2O2 and antioxidant ascorbic acid (AA). (A) Cells were initially grown in YPD until mid log phase and resuspended into the respective media supplemented with 1 mM H2O2 and 25 mM AA. Microscopic observations were made at 4 h after the treatment. (B) Dihydroethidium staining of the cells to determine H2O2 level. Experiments were repeated three times with similar results. Scale bars, 10 lm.

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after 4 h of incubation, however in case of wild type delayed filamentation was observed (Fig. 8A). A total of 274 putative Cph1-regulated genes were categorized into Gene Ontology (GO) cellular component mitochondrion (Table S2) and quantitative RT-PCR analysis revealed down-regulation of the genes involved in mitochondrial function in cph1D null mutant, thus validated their regulation by Cph1 (Fig. 2C). All these results suggested a possible role of Cph1 in mediating ROS homeostasis in cells and maintenance of the mitochondrial function under stressful condition.

4. Discussion Cph1. is regarded as the master regulator of mating process and morphogenesis in response to various environmental signals (Malathi et al., 1994; Liu et al., 1994; Biswas et al., 2007; Shapiro et al., 2011; Lin et al., 2013). We have taken a systemic deletion approach to identify different functional domains/motifs of Cph1 and determined the role of Cph1 in controlling cellular process and morphogenesis under various stressful conditions. Using HWP1 promoter as bait, the N-terminal region of Cph1 was found to be indispensable for the DNA binding activity (Fig. 1B). N-terminal region resembled helix-turn-helix homeodomain (Table S3); further substantiating its involvement in sequencespecific DNA binding. However, both the N-terminal and C-terminal regions were found to be essential for the transcriptional activation function (Fig. 1C–E). The polyglutamine stretches (PDQ) at the C-terminal region were found to be vital for the transcriptional activation and deletion of amino acids 563–574 (IHD) lying between two polyQ tracts resulted in reduced transcriptional activation potential (Fig. 1C–E). All these observations suggested that C-terminal region of the Cph1 forms the transcriptional activation domain. Polyglutamine or polyproline residues were identified as important motifs of transcriptional activation domain of other eukaryotic transcription factors as well (Atanesyan et al., 2012). However, the unlikely observation was the absence of transcriptional activation function in the N-terminal deletions (NT1D and NT2D) required for the DNA binding (Fig. 1C–E). This may be due to the reason that deletion of the N-terminal domain led to inappropriate folding and thus, function of the C-terminal transcriptional activation domain. Further protein structural studies will be useful to support this view. Transformation of the cph1D null mutant with different domains/motifs-deleted CPH1, revealed that domains/motifs which were essential for the transcription factor activity, were also important for the morphogenesis, cell wall organization and chlamydospore formation (Figs. 1A–E, 4A and B, 5 and 6A and B). DTG and ID motifs were not involved in DNA binding and/or transcriptional activation activity and their deletion did not have any phenotypic defect in C. albicans (Figs. 1A–E and 4A). Genomewide identification of the Cph1 binding site and quantitative RTPCR expression analysis in cph1D null mutant showed that a large number of genes involved in filamentation process, cell wall organization and chlamydospore formation are the potential transcriptional targets of the Cph1 (Figs. 2A–C and S1A–D and Table S2). In C. albicans, the pH response pathway is implicated in yeastto-hyphal switch in response to neutral or alkaline pH, repression of acidic pH regulated genes and chlamydospore formation (Davis et al., 2000). The genes of the pH response pathway including RIM8, RIM9, RIM20 and RIM13 were found to be the putative transcriptional target of the Cph1 (Table S2). Rim101 is the terminal transcription factor of this signaling pathway that needs proteolytic cleavage by proteins like Rim13 and Rim8 to acquire its functionally active form (Nobile et al. (2008). Interestingly, we found that the expression of the RIM13 was down-regulated in cph1D null

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mutant and chlamydospore formation defect observed in cph1D null mutant was similar to the rim13D, rim101D and mds3D mutant (Fig. 2A; Nobile et al., 2003). Therefore, it is quite possible that Cph1 affects chlamydospore formation by regulating the genes of the pH response pathway, and/or other genes that affect chlamydospore formation such as SUV3, NRG1, PGA55, AAH1, DIT2, MET15 (Nobile et al., 2003; Staib and Morschhäuser, 2005; Melo et al., 2008; Palige et al., 2013; Eisman et al., 2006) and have Cph1 binding sites in their promoter (Table S2). cph1D null mutant and deletion mutants were sensitive to the cell wall perturbing compounds like congo red and nikkomycin Z (Fig. 6A and B). The combinatorial effect of reduced expression of UAP1 that is required for UDP-GlcNAc synthesis, chitin synthases (CHS1 and CHS2) and the cell wall-specific protein (HYR1 and HWP1) was possibly responsible for the cell wall defect observed in cph1D mutants (Fig. 2A and B; Bailey et al., 1996; Mio et al., 1996, 1998). Congo red sensitivity of the CPH1 over-expressor strain could be due to the increased cell wall chitin because of the upregulation of chitin synthases (Fig. 6A). We observed that Cph1 regulates expression of the genes responsible for chitin synthesis (CHS1, CHS8 and UAP1) as well as outer cell wall GPI anchored hypha-specific protein (HWP1) and another hyphaspecific protein (ECE1) in a differential manner (Fig. 7C–E). Under normal growth condition, Cph1 negatively regulated the expression of the chitin synthase genes and, thus, deletion of the CPH1 gene resulted in up-regulation of these genes (Fig. 7C), and higher cell wall chitin level (Fig. 7A and B). This explained the basis of congo red sensitivity of the null mutant and the deletion mutants (Fig. 6A). However, in the presence of nikkomycin Z, transcript levels of these genes were up-regulated in wild type and transcript levels were found to be higher than that observed in null mutant (Fig. 7D and E). Moreover, calcofluor white staining and chitin quantification revealed decreased level of the cell wall chitin in null mutant as compared to the wild type in the presence of nikkomycin Z (Fig. 7A and B). These results further confirmed the sensitivity of the null mutant and deletion mutants toward nikkomycin Z (Fig. 6B) which is a competitive inhibitor of chitin synthases (Kim et al., 2002). In wild type strain which is resistant to nikkomycin Z, up-regulation of the genes involved in chitin synthesis may be a part of drug response machinery to counteract the cell wall synthesis inhibition exerted by nikkomycin Z (Fig. 7D). The role of Cph1 in mediating antifungal drug response is expected because 5.5% of putative Cph1-regulated genes were categorized in GO term ‘response to drug’ (Fig. S1C). These results suggested that Cph1 may affect the expression of the same set of genes positively or negatively depending on the physiological condition and morphological state of the cells. Whether Cph1 is to act as a repressor or activator is possibly modulated by another factor(s) that need(s) to be determined. The data also suggest the involvement of Cph1 in feed-back regulation of the upstream MAP kinase pathway components (Fig. 3). Cph1 was essential for morphogenesis in all solid media tested (Fig. 4A). However, cph1D null mutant and deletion mutants displayed morphogenetic defects only in SS liquid medium (Figs. 4B and S3). The pseudohyphae formation by the wild type strain in SS medium could be due to the intracellular ROS generated because of the hyperactivity of the mitochondria in the presence of succinate. The involvement of Cph1 in regulating morphogenesis in response to oxidative stress is expected since wild type strain showed pseudohyphae formation in the presence of H2O2 which was not observed in case of cph1D null mutant (Fig. 8A; Srinivasa et al., 2012). Interestingly, ascorbic acid complemented pseudohyphae formation defect of the cph1D null mutant in SS medium (Fig. 8A). This provided evidence for the involvement of ROS in conferring morphogenetic defect to the null mutant in SS medium. However, in presence of ascorbic acid, wild type strain

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showed delayed filamentation (Fig. 8A); suggesting that filament induction in SS medium was possibly because of ROS generated due to the mitochondrial hyperactivity. In contrast, filamentation defect of the null mutant in SS medium might be due to the generation toxic levels of ROS because of the mitochondrial dysfunction (Fig. 8A and B). This might be also the reason for the suppression of filamentation of wild type in SS medium after H2O2 treatment (Fig. 8A). Identification of a large number of putative Cph1regulated genes which belong to the GO category ‘mitochondrion’ and down-regulation of the genes involved in mitochondrial function in cph1D null mutant further supported this hypothesis (Figs. 2C and S1D). Previous reports revealed the role of mitochondria in producing intracellular ROS in response to antifungal agents and proper functioning of the mitochondria is absolute necessity for hypha production (Helmerhorst et al., 2001; Nobile et al., 2003). There are few instances where Cph1 like proteins were related to ROS homeostasis in fungal pathogens. In the citrus pathogen Elsinoë fawcettii, a Cph1 orthologue (EfSte12) was reported to be involved in ROS driven production of the phytotoxin elsinochrome that is required for the host-pathogen interaction (Daub et al., 2013). Recently, in Sordaria macrospora, a Cph1 orthologue has been shown to be a part of the genetic pathway involving the NADPH oxidase Nox2 and its regulator Nor1 in controlling sexual development and ascospore formation in response to ROS (Dirschnabel et al., 2014). Taken together, these results suggest the roles of Cph1 in mediating cellular ROS homeostasis and controlling morphogenesis under stressful conditions. Acknowledgments This work was financially supported by the Department of Biotechnology, Ministry of Science and Technology, Government of India. PM and PG acknowledge Council of Scientific and Industrial Research for the research fellowship. Authors thank Prof. Malcolm Whiteway, Biotechnology Research Institute, Montreal, and Fungal Genomics Stock Center for the C. albicans strains CLJ19 and CP-1, respectively. Authors are thankful to Anil Kumar for help in chitin analysis. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.fgb.2015.08.004. References Atanesyan, L., Gunther, V., Dichtl, B., Georgiev, O., Schaffner, W., 2012. Polyglutamine tracts as modulators of transcriptional activation from yeast to mammals. Biol. Chem. 393, 63–70. Au, L., Guduru, K., Lipscomb, G., Kelly, S.P., 2007. Candida endophthalmitis: a critical diagnosis in the critically ill. Clin. Ophthalmol. 1, 551–554. Bailey, D.A., Feldmann, P.J., Bovey, M., Gow, N.A., Brown, A.J., 1996. The Candida albicans HYR1 gene, which is activated in response to hyphal development, belongs to a gene family encoding yeast cell wall proteins. J. Bacteriol. 178, 5353–5360. Berman, J., Sudbery, P.E., 2002. Candida albicans: a molecular revolution built on lessons from budding yeast. Nat. Rev. Genet. 3, 918–930. Biswas, S., Van Dijk, P., Datta, A., 2007. Environmental sensing and signal transduction pathways regulating morphopathogenic determinants of Candida albicans. Microbiol. Mol. Biol. Rev. 71, 348–376. Borneman, A.R., Gianoulis, T.A., Zhang, Z.D., Yu, H., Rozowsky, J., Seringhaus, M.R., Wang, L.Y., Gerstein, M., Snyder, M., 2007. Divergence of transcription factor binding sites across related yeast species. Science 317, 815–819. Brand, A., MacCallum, D.M., Brown, A.J.P., Gow, N.A.R., Odds, F.C., 2004. Ectopic expression of URA3 can influence the virulence phenotypes and proteome of Candida albicans but can be overcome by targeted reintegration of URA3 at RP10 locus. Eukaryot. Cell 3, 900–909. Brown, A.J.P., Argimon, S., Gow, N.A.R., 2007. Signal Transduction and Morphogenesis in Candida albicans, . second ed.. In: Howard, R.J., Gow, N.A.R. (Eds.), Biology of the Fungal Cell, The Mycota VIII second ed., vol. 8 Springer, Berlin Heidelberg, pp. 167–194.

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