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CSU52, a novel regulator functions as a repressor of L-sorbose utilization in Candida albicans

Background and Objectives: Monosomy of chromosome 5 associated with utilization of non-canonical sugar L-sorbose is one of the well-studied aneuploidies in Candida albicans. Stress-induced ploidy changes are crucial determinants for pathogenicity and genetic diversity in C. albicans. The five scattered regulatory regions (A, B, C, 135, and 139) comprising of two functionally redundant pathways (SUR1 and SUR2) were found to be responsible for the growth on L-sorbose. So far, three genes such as CSU51, CSU53 and CSU57 have been identified in region A, region 135 and region C, respectively. In this study we have verified the role of region B in this regulatory pathway. Materials and Methods: We employed a combinatorial gene deletion approach to verify the role of region B followed by co-over expression studies and qRT-PCR to identify the regulatory role of this region. Results: We confirmed the role of region B in the regulation of SOU1 gene expression. The qRT-PCR results showed that regulation occurs at transcriptional level along with other two regions in SUR1 pathway. A previously uncharacterized open reading frame in region B has been implicated in this regulation and designated as CSU52. Integrating multiple copies of CSU52 in the genome at tandem, suppresses the growth of recipient strain on L-sorbose, establishing it as a repressor of SOU1 gene. Conclusion: This finding completes the identification of regulators in SUR1 pathway. This result paves the way to study the underlying molecular mechanisms of SOU1 gene regulation that in-turn helps to understand stress induced aneuploidy.


INTRODUCTION
Candida albicans is a common human fungal commensal that resides in mucosal membranes of chromosome loss depending upon the environmental cues (5). Factors contributing to this process include exposure to antifungals, growth on alternative carbon sources, high temperature, and host-pathogen interactions result in aneuploidy (5)(6)(7).
C. albicans, naturally disomic, produces spontaneous mutants, thus acquiring the ability to assimilate alternate carbon sources like L-sorbose and D-arabinose. These acquired assimilatory phenotypes have been linked to specific chromosomal alterations. Monosomy of chromosomes 5 (Chr5) and 6 (Chr6) are the chromosomal changes found to be associated with growth on L-sorbose and D-arabinose, respectively (5,8). The SOU1 gene present on chromosome 4 (Chr4) encodes a NADPH-dependent sorbose reductase. This enzyme catalyzes the first step in the L-sorbose metabolic pathway by converting L-sorbose to D-sorbitol and subsequently routed to the glycolytic pathway (9). The copy number of chromosome 5 regulates the expression of the SOU1 gene and the utilization of L-sorbose. Candida strain monosomic to Chr5 can grow on L-sorbose (Sou + ) otherwise it remains Sou - (8). Also, overexpression of SOU1 in a Chr5 disomic strain using a low copy number plasmid makes the strain Sou + (8,9). These observations suggest that the Chr5 and SOU1 gene ratio is the key to growth on L-sorbose (8,9). Therefore, the loss and gain of Chr5 homolog make the strain Sou + or Sou -, suggesting that Chr5 harbors repressors of L-sorbose utilization. Systematic truncations established that these negative regulators are located in a region of approximately 341 kb on the right arm of Chr5 (8). Further analysis identified five dispersed regions, such as A, B, C, 135, and 139 which might harbor repressors for L-Sorbose utilization. These five regions were classified into two independent functionally redundant pathways-one involving regions A, B, and 135 and the other involving regions A, C, and 139 where region A is part of both the pathways. The genes CSU51 and CSU53 have already been identified from regions A and 135, respectively (8,10). However, region B has not been investigated and the possible repressor present in this region was not identified. In this study, we have adopted a combinatorial gene deletion approach to implicate region B in the regulation of L-sorbose utilization and to identify the gene present in this region. Using this strategy, we have identified a previously uncharacterized ORF (open reading frame) orf19.1105.3 in region B for L-sorbose utilization and designated it as CSU52. The coding region of CSU52 is 258 bp long (855689-855432, coordinates on Chr5) (www.candidagenome.org). This gene does not have any homologs in any other sequenced Candida species, suggesting its unique function in L-sorbose regulation. This finding completes the identification and implication of all the three genes present in three different regions of the SUR1 pathway (Sorbose Utilization Regulatory) of L-sorbose utilization in C. albicans.
YPD (1% yeast extract, 2% peptone, and 2% dextrose) and SD (synthetic dextrose) media were used to grow the Candida strains and were prepared as described (16). SD media with D-sorbitol was used for Candida transformations. SD media with 2% L-Sorbose (Calbiochem, USA) was used for phenotypic analyses. Uridine (50 µg/mL) was added whenever necessary, and mycophenolic acid (MPA) (8 µg/mL) was added for the selection of transformants carrying IMH3 marker. Mycophenolic acid (Sigma, USA) stock solution (10 mg/ml) was prepared in dimethyl sulfoxide (DMSO). YT media (0.5% NaCl, 0.5% yeast extract and 1% tryptone) was used for growing E. coli strains. Ampicillin (100 µg/mL) was added to YT media for growing E. coli strains containing plasmids. 2% Agar was added while preparing solid media. A stock solution of zymolyase (Sigma, USA) of concentration 1 mg/ml was made in 20% glycerol and stored at -20°C. All the C. albicans and bacterial strains were stored in 15% glycerol and maintained at -80°C. C. albicans and E. coli strains were grown at 30°C and 37°C, respectively. Molecular biology methods. All basic molecular biology techniques such as PCR amplification, cloning, restriction digestion, and gel elutions were done as described (13). Plasmid isolation was carried out using the alkaline lysis method (13). The calcium chloride method was used for E. coli transformation (13). C. albicans transformation was done by the spheroplast method as described (17).

Plasmid cassettes for internal deletions and overexpression.
Plasmid vectors pUC19 and pSFU1 were used for making deletion cassettes (14,15). Briefly, 500-1000bp upstream and downstream flanking sequences of the region to be deleted were amplified by PCR from CAF4-2 genomic DNA. The amplified sequences were cloned into pTZ57R/T vector using InsTA PCR cloning kit following the manufacturer's instructions (Thermo Fisher Scientific, Vilnius, Lithuania). Subsequently, PCR products were cloned into the pUC19-based vector at BamHI-SacI and Ps-tI-HindIII sites, whereas KpnI-XhoI and NotI-Sa-cI sites were used for pSFU1. Candida strains were transformed with the resulting deletion constructs after digesting with appropriate restriction enzymes.
For co-overexpression studies, CSU52 and SOU1 genes were cloned together in the replicative plasmid pRC2312. CSU52 was amplified as a 649bp PCR product using oligonucleotides KC448/KC449 and cloned into TA cloning pTZ57R/T vector. The CSU52 gene was released from the TA vector as a PstI fragment and cloned into the plasmid pKA444 (SOU1 gene in pRC2312) (12) generating a co-overexpression vector pKA648. As a positive control for suppression assay, pKA534 (pRC2312 with CSU51 and SOU1) was used (12). The plasmid pKA712 (12) was used for multiple copy integration of CSU52 gene. The CSU52 gene was cloned at the PstI site of pKA712 generating plasmid pKA833, for tandem integration of multiple copies of CSU52. Plasmids used in this study are listed in (Table 1).

PCR-based verification of gene deletions.
Specific PCR primers were used to confirm accurate gene deletions. Primers were designed from the marker gene and outside the flanking sequences on both 5ˈ and 3ˈ ends of the chromosomal DNA. PCR amplifications were performed for both the 5ˈ and 3ˈ ends to confirm the marker-genomic DNA junctions. The location and orientation of primers used for verification of gene deletions are illustrated ( Fig. 1A and 1B). PCR products were run on 0.8% agarose gel along with molecular size markers, stained with ethidium bromide, and visualized using gel documentation system (Bio-Rad Laboratories, USA). Primers used in this study are listed in (Tables 2-4).
L-sorbose phenotypic assay. Spot dilution assay was performed on L-sorbose plates to determine the phenotypes of mutants generated by gene deletions as described (8). Strains were streaked from glycerol stocks onto YPD plates with uridine and were allowed to grow 16-24 h at 30ºC. Fresh cells were collected from the plate and washed thrice with distilled water to remove traces of glucose. Cells were then counted using a hemocytometer, and approximately 10 6 cells were taken, serially diluted, and spotted on L-sorbose plate where glucose plate served as a control. Images of glucose plates were taken at 24-36 h of spotting, whereas, L-sorbose plates were monitored and imaged for four days to observe the confluent growth of Sou + colonies. Uridine was added to both glucose and L-sorbose plates except for maintaining replicative plasmids.
Gene expression studies. Real-time quantitative PCR was used to analyze the changes in SOU1 gene expression. Total RNA was isolated from the mutant and wild type strains using the acid phenol method (18), and RNA purity was assessed by NanoDrop spectrophotometer (Eppendorf, Germany). RNA was treated with DNaseI (Thermo Fischer Scientific, Vilnius, Lithuania) to remove genomic DNA contamination, followed by PCR amplification to verify complete DNA removal. cDNA was synthesized from total RNA using Revert Aid first-strand cDNA synthesis kit (Thermo Fisher Scientific). cDNA synthesized was either stored at -80ºC or proceeded for qRT-PCR.
Real-time quantitative PCR was performed using SYBR Green/Rox qPCR Master-mix (Thermo Fischer Scientific, Vilnius, Lithuania) in a Bio-Rad CFX 96 system. Briefly, PCR reaction mixture consists of 10 µl SYBR green master mix, 2 µl each of forward  The cassette consists of FRT, FLP under SAP2 promoter (SAP2P), ACT1 terminator (ACT1T), and URA3 as the selection marker. Primers P1 and P2 are designed for 5ˈend verification, where P1 is present on Chr5, upstream of 5ˈFS (flanking sequence), and P2 is the marker primer located within the SAP2 promoter. Primers P3 and P4 are for 3ˈend verification, where P3 is the marker primer located in URA3 and P4 is present downstream of 3ˈFS on the chromosome. (B) Vector pUC19-based deletion cassette: Primers P5 and P6 are for 5ˈ end verification. Here, P5 is the on chromosome upstream of 5ˈFS, and P6 is the marker primer. P7 and P8 are for 3ˈ end verification. P8 is present downstream of 3ˈFS and P7 is the marker primer. The primers designed on the chromosome vary with the region to be deleted. and reverse primers (0.3mM final concentration) and 2 µl of cDNA (25 ng/µl), finally made up to 20 µl total volume with nuclease-free DEPC water. ACT1 and RDN18 (18S rRNA) genes were used as internal controls. An in-built 2 step amplification protocol (CFX_ 2 step amplification protocol) was used with additional melt curve analysis to verify the presence of a single amplified product. Briefly, initial denaturation at 95ºC for 3 min, followed by 95ºC for 10 sec and 55ºC for 30 sec for 40 cycles. Following the amplification, melt curves were generated at 65ºC to 95ºC for 5 sec with an increment of 0.5ºC. Gene expression changes were measured as fold change using the ΔΔCt method (19).

Minimum inhibitory concentration (MIC) assay.
Minimum inhibitory concentration (MIC) for the mutants generated was performed following the Clinical and Laboratory Standards Institute (CLSI) protocols (20). Briefly, fresh cells were collected from YPD plates with uridine and approximately 5 × 10 6      bai, India), was placed in the middle of the plate and incubated at 30°C. The minimum inhibitory concentration was recorded after 24 hrs of incubation; also the plate photographs were taken using Quantity One Software (Bio-Rad Laboratories, USA). Minimum inhibitory concentrations were determined in µg/ml against the caspofungin.

RESULTS
Deletion and phenotypic analyses of genes/regions in the SUR1 pathway. The SUR1 pathway contains three regions A, B, and 135, responsible for negatively regulating the expression of SOU1, and subsequently, the utilization of L-sorbose as a sole carbon source. The genes present in regions A and 135 have been identified as CSU51 and CSU53, respectively. However, the gene present in region B remains uncharacterized. Schematic representation of regions in the SUR1 pathway has been shown in (Fig.  2). To identify the gene/ORF in region B responsible for the regulation of L-sorbose utilization, we performed a series of systematic deletions of the genes/ regions.

Single-copy deletion of the genes/regions and phenotypic analyses.
To confirm the role of region B in the regulation of L-sorbose utilization, we have first deleted a single copy of the genes/regions individually and assessed the Sou phenotype. The CSU51 gene was deleted as described (12). The region 135 was deleted by transforming Candida strain with the plasmid pKA140. The 5ˈ and 3ˈ junctions of deletion of region 135 were confirmed by PCR using oligonucleotides KC16/KC114 and KC17/KC116, respectively ( Fig. 3A; lanes 8 and 10). Similarly, region B was deleted using the plasmid pKA822, and the transformants were verified using oligonucleotides KC102/ KC14 and KC17/KC119, respectively ( Fig. 3A; lanes  3 and 5). Therefore, single copies of all the three regions/genes were deleted one at a time in Candida strain, keeping the second copy intact. The strains harboring these individual deletions were spotted on L-sorbose plates, and the growth was monitored. It has been observed that the Candida strains carrying these individual deletions produced Sou-phenotype (Fig.  3B). This finding strongly suggests the possibility of combinatorial effects of the genes located in these regions for the regulation of L-sorbose utilization.
Combinational deletions of the regions and phenotypic verification. As the strains carrying dele- tions of single copies failed to produce Sou + phenotype, it becomes evident that more than one region/ gene is to be deleted for utilization of L-sorbose as a carbon source. Therefore, we have designed the experiments to delete two regions/genes simultaneously in the same strain to assess L-sorbose utilization. As there are three regions/genes, three possible combinations of double deletions such as CSU51 plus region B, CSU51 plus region 135, and region B plus region 135 have been carried out. For the deletion of CSU51 and region B simultaneously, Candida strain was transformed with plasmid pKA139 after digesting with appropriate enzymes. The ura + transformants were screened by PCR. The oligonucleotides KC16/KC14 and KC17/KC146 were used for verification of 5ˈ and 3ˈjunctions, respectively (see Fig. 4A; lanes 1 and 5). For generating double deletions of (CSU51 +region135) and regions (B +135), we initially deleted region 135 using a deletion cassette with IMH3 as the selection marker. The transformants were selected on SD plates containing mycophenolic acid (MPA) and they were re-streaked on MPA plates for confirmation. The MPA R (MPA-resistant) transformants were screened by PCR by verifying both 5ˈ and 3ˈjunctions ( Fig. 4B; lanes 1 and 5). A single-copy deletant of region 135 has been designated as C85. Subsequently, both CSU51 and region B were independently deleted in the Candida strain C85 using deletion cassettes pKA34 and pKA822, respectively, generating the stains carrying deletions (CSU51+region 135) and regions (B+135).
Finally, we generated a strain where all the three regions/genes, CSU51, B, and 135, were deleted together in the same strain. For this purpose, both CSU51 and region B were knocked out simultaneously in Candida strain C85 using deletion cassette pKA139 as described above. The transformants, which were both ura + and MPA R , were verified using PCR. The oligonucleotides KC16/KC14 and KC17/KC146 were used for verification of 5ˈ and 3ˈjunctions, respectively ( Fig. 4A; lanes 1 and 5).
A minimum of three PCR verified positive colo-nies were taken and checked for Sou phenotype. One representative colony for each deletion was taken and spotted on the L-sorbose plate along with appropriate positive and negative controls. It has been found that even double knockouts did not produce Sou + phenotype (Fig. 5A). However, upon deleting all the three regions together, the strain acquired Sou + phenotype (Fig. 5B). Therefore, it clearly established that all three regions/genes act in a concerted manner in the SUR1 pathway. This result also proves that region B plays a significant role in L-sorbose regulation.
SOU1 gene expression analysis. It was previously reported that Candida strain could be Sou + either by loss of one homolog of chromosome 5 (monosomy) or by incorporation of extra copies of the SOU1 gene through replicative plasmid (8). Herein we have analyzed the expression of structural gene SOU1 to comprehend whether the regulation works at the transcriptional level or not. Quantitative real-time PCR was done for the strains such as CAF4-2, double deletion of regions/genes (CSU51 plus 135), triple  deletion of regions/genes (CSU51, B and 135), and strain C571 (positive control) to measure the expression of SOU1 gene. This analysis revealed basal SOU1 expression in CAF4-2 and double deletions of regions/genes (CSU51 plus 135) where C571 (positive control) showed 2.7 times more expression of SOU1 compared to CAF4-2. However, the strain with all the three regions deleted showed 1.5 times more expression than the negative control (Fig. 6). This result suggests the involvement of region B (CSU52) in regulating SOU1 gene expression at the transcriptional level.
Co-overexpression analysis of CSU52 with SOU1. It has been shown earlier that overexpressing the SOU1 gene in wild-type Candida strain using a replicative plasmid pRC2312 produced confluent growth on the L-sorbose plate. However, the strain containing CSU51 and SOU1 together remained Soudue to the repressing effect of CSU51 (8). Herein, we have applied the same approach to verify the repressing ability of CSU52. For this purpose, Candida strain CAF4-2 was transformed with the plasmids, pKA648 (CSU52+SOU1 in pRC2312), pKA534 (CSU51+SOU1 in pRC2312), pKA444 (SOU1 in pRC2312) and pRC2312 (empty vector). The ura + transformants were spotted on L-sorbose taking glucose plates as control. CAF4-2 transformed with empty vector pRC2312 (negative control) failed to grow, while the strain with additional copies of SOU1 exhibited confluent growth. The positive control (CSU51+SOU1) for repression did not grow on L-sorbose plate as reported earlier (8). However, transformants containing the plasmid pKA648 (CSU52+SOU1) could not suppress the growth on the L-sorbose plate. This indicates that CSU52 may encode a weak repressor compared to CSU51 (Fig.  7A). This observation is expected, as CSU51 is essential for both the regulatory pathways required for negative regulation of L-sorbose utilization and acts as the master molecule. As the copy number of IRAN repressors and SOU1 is the key in this regulatory system, we suspected that the expression of CSU52 is insufficient to repress SOU1. To test this, we integrated multiple copies of CSU52 by exploiting the property of the plasmid pRC2312, which can generate 7-13 copies of tandem integration into Candida genome and produce much larger colonies compared to strain with autonomously replicating plasmids (12). For tandem integration of multiple copies of CSU52, Candida strain C89 was transformed with pKA833 (CSU52 in pKA712), and ura + integrated colonies were selected, and newly generated strain has been designated as C587. The strain C587 was subsequently transformed with pKA444 (SOU1 in pRC2312) and pRC2312 (vector control). Simultaneously, the strain C89 was transformed with pKA444 and pRC2312. The ura + transformants were spotted on L-sorbose plate, taking glucose plate as a control. As expected, the negative controls (vector only) were Sou-while C89 transformed with pKA444 (SOU1 in pRC2312) grew well on the L-sorbose plate. However, the strain C587 transformed with pKA444 (SOU1 in pRC2312) showed very little growth on L-sorbose plate. This elegant experiment clearly showed that transformed with vector pRC2312, [CSU52] transformed CSU52 can suppress the Sou + phenotype (Fig. 7B). This result strongly suggests that the CSU52 gene encodes a potential repressor for the regulation of L-sorbose utilization. Further, it also establishes that the copy number of repressors is indeed the key to the regulation of the SOU1 gene.
MIC values for the mutant strains. It was reported, that L sorbose resembles with echinocandin class of antifungals in repressing cell wall β 1-3 glucan synthesis (21). Here, we have verified the MIC values for caspofungin on the mutants generated. The minimum inhibitory concentrations for the mutant strains varied from 0.094 µg/ml -0.25 µg/ml (Fig. 8). The observed MIC values suggested that the mutants were not resistant against caspofungin compared to wild type strain.

DISCUSSION
L-Sorbose metabolism in C. albicans was previously shown to be an ideal process to study chromosomal changes induced by stress conditions and the effects of gene copy number alterations. The growth of Candida strains is correlated with the loss of one with pKA444 (SOU1 gene in pRC2312), C89 transformed with vector pRC2312, C89 transformed with pKA444 (SOU1 gene in pRC2312).
homolog of chromosome 5, suggesting the presence of repressors regulating SOU1 gene expression required for utilization of L-sorbose as a carbon source (8,9). A systematic chromosomal truncations approach combined with large internal deletions identified five scattered regions containing putative repressors on the right-hand side of the centromere on Chr5 for the regulation of L-sorbose utilization. These five regions have been categorized into two independent functionally redundant pathways -SUR1 and SUR2 (Sorbose Utilization Regulatory pathways 1 and 2). Each pathway involves three regions, with region A being common to both the pathways. The SUR1 pathway consists of three regions (A, B, and 135), whereas the SUR2 pathway contains regions A, C and 139. The regions A and 135 harbor CSU51 and CSU53, respectively which are shown to repress the SOU1 gene expression and utilization of L-sorbose (8,10). In this study, we focused on identifying the remaining repressor in region B to understand the different players in the SUR1 pathway.
Neither single-copy independent deletions of all three regions (A, B, and 135) nor combinatorial deletions of two regions together (A+B, A+135, B+135) resulted in Sou + phenotype. Rather deletion of all the three regions/genes is required to produce Sou + phenotype. These deletion studies revealed beyond doubt that region B has an essential role in L-sorbose utilization by C. albicans. The uncharacterized ORF, orf19.1105.3 located in region B, has been designated as CSU52. The CSU52 gene is unable to suppress the Sou + phenotype upon co-overexpression with SOU1 in a replicative plasmid, unlike CSU51 (8). However, tandem integration of multiple copies of the CSU52 gene is able to suppress the growth on L-sorbose plate. We obtained similar results for CSU57, which is functioning in the SUR2 pathway (12). The qRT-PCR result showed that CSU52 functions at the transcriptional level of SOU1 gene expression similar to CSU57 (12). Except for CSU51 that appears to be the master molecule, each of the repressors is necessary but insufficient to suppress SOU1 individually. Moreover, the copy number of these repressors plays a crucial role in regulating the SOU1 gene expression. Differential suppression of Sou phenotype by these repressors strongly suggests their function-al hierarchy where CSU51 is at the centre, and other repressors such as CSU52 and CSU57 might act as additional factors. However, to relieve the repression, at least three repressors must be reduced to half of the copy number of wild-type Candida strain. In summary, this study proves that L-sorbose metabolism is regulated by multiple pathways involving chromosomal changes and gene copy numbers. All the regulatory proteins are critical in performing the function and they act in a concerted manner with scope for hierarchy where CSU51 acts as a master molecule. While CSU51 was found to be involved in echinocandin drug tolerance (21), so far, none of the other repressors has been reported to perform any function apart from regulating L-sorbose utilization.
The MIC values obtained for the generated strains against caspofungin are similar to that of wild type strain and did not show any resistant phenotype. Although, CSU52 (Orf19.1105.3) expression has been found to be enhanced upon heat shock in HSP90 depleted cells (22). The interaction between HSP90 and CSU52 is still unclear, as HSP90 involved in plethora of cellular functions.
The underlying molecular mechanisms of how CSU51 orchestrates SOU1 suppression employing two independent pathways are potential areas for future studies. As L-sorbose does not occur naturally in the niches inhabited by C. albicans, these genes may represent a more general stress response mechanism, the elucidation of which requires further investigation.