**2. Aspartyl proteases**

Aspartyl proteases or acid proteases (optimum activity at acidic pH) are proteins with a signal peptide in the amino-terminal site, at least one aspartic residue in the active site, and 4 cysteins (Hube & Naglik, 2001). The signal peptide is processed in the endoplasmic reticule and the protein is transported to their corresponding cell localization by the secretory pathway. The active site is formed by different amino acids. The consensus pattern described by PROSITE-EXPASY (http://expasy.org/prosite/) is [LIVMFGAC]- [LIVMTADN]-[LIVFSA]-D-[ST]-G-[STAV]-[STAPDENQ]-{GQ}-[LIVMFSTNC]-{EGK}- [LIVMFGTA], and the cysteins help the protein to the three dimensional structure by

intramolecular disulfide bond (Fig. 1). According to the cell localization, aspartyl proteases could be secreted, or destined to vacuole or cell membrane by a GPI-linked site in the carboxyl-terminal residues (Alberch et al. 2006; Jones, 1991; Naglik et al. 2003).

Fig. 1. Typical molecular structure of aspartyl proteases. SP: signal peptide; ASP: aspartic residue in the active site, C: cystein.

## **2.1 Secreted aspartyl proteases (Sap)**

The *C. albicans* secreted aspartyl protease family comprises ten members, eight of which are proper secreted Sap1-Sap8, and two, Sap9 and Sap10, that have been reclassified as GPIanchored aspartyl proteases (Alberch et al. 2006). Nevertheless, Sap9 and Sap10 are clearly more phylogenetically related to Sap than any GPI-anchored aspartyl proteases (Parra et al. 2009). The function of Sap in *C. albicans* has been widely studied, and these proteases are important in proteolysis to get a source of nitrogen, and are differentially regulated depending on the environmental conditions (Schaller et al. 1998; 2003; Taylor et al. 2005; Naglik et al. 2008). *SAP1-*SAP3 are relevant in phenotypic switching during the opaque phase and are not expressed in the WO-1 phase (Morrow et al. 1992; White et al. 1993). Also, they are expressed when yeast colonize and damage reconstructed human epithelium, oral and vaginal, which means that these Sap are important in superficial infections (Schaller et al. 1998; 2003; Copping et al. 2005). *SAP1*-*SAP8* are related to tissular damage (Taylor et al. 2005). *SAP1*, *SAP3* and *SAP8* are expressed in oral and vaginal infections. On the other hand, *SAP4*-*SAP6* are related to systemic infections and only they are expressed in yeast and germ tube at pH 5-7 (Hube et al. 1997; Sanglard et al. 1997; White & Agabian, 1995). Meanwhile *SAP5* is important in epithelial colonization, invasion and infection (Naglik et al. 2008; Lermann & Morschhäuser, 2008).

This kind of proteases are no exclusive of *C. albicans*. Orthologous genes have been described in other closely related species, as *C. dubliniensis* (Sap1-4 and Sap7-10), *C. tropicalis*  (Sapt1-12), *C. guilliermondii* (Sapg1-8), *C. parapsilosis* (Sapp1-14) and *C. lusitaniae* (Sapl1-3)

gene duplications, similitude, synteny, putative transcription factor binding sites and genome traits of the Yps family members are analysed by bioinformatics tools in an

Aspartyl proteases or acid proteases (optimum activity at acidic pH) are proteins with a signal peptide in the amino-terminal site, at least one aspartic residue in the active site, and 4 cysteins (Hube & Naglik, 2001). The signal peptide is processed in the endoplasmic reticule and the protein is transported to their corresponding cell localization by the secretory pathway. The active site is formed by different amino acids. The consensus pattern described by PROSITE-EXPASY (http://expasy.org/prosite/) is [LIVMFGAC]- [LIVMTADN]-[LIVFSA]-D-[ST]-G-[STAV]-[STAPDENQ]-{GQ}-[LIVMFSTNC]-{EGK}- [LIVMFGTA], and the cysteins help the protein to the three dimensional structure by intramolecular disulfide bond (Fig. 1). According to the cell localization, aspartyl proteases could be secreted, or destined to vacuole or cell membrane by a GPI-linked site in the

carboxyl-terminal residues (Alberch et al. 2006; Jones, 1991; Naglik et al. 2003).

Fig. 1. Typical molecular structure of aspartyl proteases. SP: signal peptide; ASP: aspartic

The *C. albicans* secreted aspartyl protease family comprises ten members, eight of which are proper secreted Sap1-Sap8, and two, Sap9 and Sap10, that have been reclassified as GPIanchored aspartyl proteases (Alberch et al. 2006). Nevertheless, Sap9 and Sap10 are clearly more phylogenetically related to Sap than any GPI-anchored aspartyl proteases (Parra et al. 2009). The function of Sap in *C. albicans* has been widely studied, and these proteases are important in proteolysis to get a source of nitrogen, and are differentially regulated depending on the environmental conditions (Schaller et al. 1998; 2003; Taylor et al. 2005; Naglik et al. 2008). *SAP1-*SAP3 are relevant in phenotypic switching during the opaque phase and are not expressed in the WO-1 phase (Morrow et al. 1992; White et al. 1993). Also, they are expressed when yeast colonize and damage reconstructed human epithelium, oral and vaginal, which means that these Sap are important in superficial infections (Schaller et al. 1998; 2003; Copping et al. 2005). *SAP1*-*SAP8* are related to tissular damage (Taylor et al. 2005). *SAP1*, *SAP3* and *SAP8* are expressed in oral and vaginal infections. On the other hand, *SAP4*-*SAP6* are related to systemic infections and only they are expressed in yeast and germ tube at pH 5-7 (Hube et al. 1997; Sanglard et al. 1997; White & Agabian, 1995). Meanwhile *SAP5* is important in epithelial colonization, invasion and infection (Naglik et al.

This kind of proteases are no exclusive of *C. albicans*. Orthologous genes have been described in other closely related species, as *C. dubliniensis* (Sap1-4 and Sap7-10), *C. tropicalis*  (Sapt1-12), *C. guilliermondii* (Sapg1-8), *C. parapsilosis* (Sapp1-14) and *C. lusitaniae* (Sapl1-3)

evolutionary context.

**2. Aspartyl proteases** 

residue in the active site, C: cystein.

**2.1 Secreted aspartyl proteases (Sap)** 

2008; Lermann & Morschhäuser, 2008).

(Parra et al. 2009). Particularly in *C. dubliniensis*, the expression of *SAPD3* and *SAPD4* genes is related to the infection of keratinocyte (HaCAT cells) by yeast. The number and shape of the keratinocyte cells was altered by the infection, but these effects decreased in the presence of pepstatin A, an aspartyl protease inhibitor, suggesting that the Sapd3 and 4 of *C. dubliniensis* could be considered as virulence factors, like their orthologous genes from *C. albicans* (Loaiza-Loeza et al. 2009). The function of these proteases in metabolism and pathogenesis in the rest of pathogenic species is unknown.

According to Dayhoff, protein superfamilies and families are defined as groups of related proteins that exhibit less than 50% and greater than 50% similarity, respectively. Subfamilies were defined as groups of proteins with at least 90% similarity and were often equivalent to clusters of orthologous groups (COGs) (Dayhoff, 1979). Behind this idea, the phylogeny of pathogenic *Candida* spp. Saps allows for the recognition of a superfamily with at least 12 paralogous families and nine orthologous subfamilies. In several Sap families, at least two subfamilies or orthologous groups are proposed (Parra et al. 2009).

## **2.2 Vacuolar aspartyl proteases (PrA)**

The vacuole is a hydrolytic organelle similar to lysosomes in animals and is the site of nonspecific degradation of cytoplasmic proteins (Robinson et al. 1988), proteins delivered via autophagy (Klionsky & Emr, 2000), or plasma membrane proteins turned over via endocytosis (Hicke, 1996). In *S. cerevisiae* the vacuole has been studied and possesses different vacuolar proteases (Table 1).

One of the most important vacuolar proteins is the proteinase A (PrA), encoded by the *PEP4* gene. Mutants in *PEP4* (*pep4*) accumulate multiple zymogens, indicating that PrA initiates processing, maturation and activation of multiple different precursors of PrB, DAP, CPY and PrA, because of their autocatalytic activity and their lack of production of dead cells in nutritional stress. Also, PrA is important in cellular response to starvation, microautophagy, proteolysis involved in cellular and vacuolar protein catabolic process, and sporulation (Palmer, 2007; Jones, 1991; Teichert et al. 1989).

The function of PrA, encoded by the *CaPEP4* gene in the metabolism of *C. albicans*, has also been studied. Null mutants of *CaPEP4* maintain their hydrolytic activity intact, clearly suggesting that *C. albicans* possesses an alternative system that compensates for the lack of this gene (Palmer, 2007). In *C. albicans*, the vacuole is important in cell differentiation, surviving into macrophages, and elimination of drugs as hygromicin B, orthovanadate and rapamicine (Palmer, 2005).

In *C. dubliniensis*, this protein could be important in carbon and nitrogen metabolism and might participate in protein degradation and precursor processing as occurs in *S. cerevisiae*  (Loaiza et al. 2007). The genome-wide environmental stress response expression profile of *C. glabrata* revealed that *CgPEP4* is induced in osmotic stress and glucose starved conditions. Meanwhile, in *S. cerevisiae* no changes in the expression were observed in the same conditions (Gash et al. 2000; Roetzer et al. 2008).

Bioinformatic genomic analysis of *Candida* pathogenic species exhibited that only one version of PrA is harboured by yeast (Table 3), but apparently the *CgPEP4* gene is universally distributed among *C. glabrata* strains, as revealed by PCR multiplex in a collection of 52 *C. glabrata* clinical strains (Table 5; Fig. 3; for PCR conditions see 2.3 section). Phylogenetic analysis was performed by an aligment of PrA homologues identified *in silico*  and those previously characterized. The aligment was conducted using MUSCLE in SeaView 2.4 program (Galtier et al. 1996) with default alignment parameter adjustments.

spp.

PrA (AN)

*C. albicans* 

*C. dubliniensis* 

*C. tropicalis* 

*C. parapsilosis* 

*C. guilliermondii*

*C. glabrata* 

or supercontig.

**Amino acid residues**

CTRG\_01724 422 45.6 4.5

CPAG\_03663 428 46 4.5

PGUG\_04145 409 44 4.3

CLUG\_04124 407 43 4.3

**MM**

Calorf19\_1891 419 45.4 4.5 119-130:

**(kDa) IP MOTIF** 

Cd36\_21670 419 45.4 4.5 14 2

VILDTGSSNLWV 304-315: AAIDTGTSLITL

121-132: VILDTGSSNLWV 306-317: AAIDTGTSLITL

127-138: VILDTGSSNLWV 312–323: AAIDTGTSLITL

109-120: VILDTGSSNLWV 294-305: AAIDTGTSLITL

107-118: VILDTGSSNLWV 292–303: AAIDTGTSLITL

Table 3. Vacuolar aspartyl proteases in pathogenic *Candida* species. (AN): Access number in the respective genome; MM: molecular mass; IP: Isoelectric Point; C: Chromosome or Contig

*C. glabrata* is an opportunistic haploid yeast that suffered evident and extensive reductive evolutionary events. A lot of genes involved in nitrogen metabolism, carbohydrate assimilation (saccharose, galactose, etc.), as well as sulfur, phosphor, thiamine, pyridoxine and nicotinic acid biosynthesis have been lost from the genome (Byrne & Wolfe, 2005;

Evolution of GPI-Aspartyl Proteinases (Yapsines) of *Candida* spp 293

**1.** *S. cerevisiae* **YPL154C** 62 66 66 66 66 65 68 **2.** *C. glabrata* **CAGL0M02211g** 77 55 55 54 54 55 57 **3.** *C. albicans* **orf19\_1891** 77 69 98 90 85 75 78 **4.** *C. dubliniensis* **Cd36\_21670** 77 69 99 91 85 76 79 **5.** *C. tropicalis* **CTRG\_01724** 76 69 97 97 86 75 77 **6. C***. parapsilosis* **CPAG\_03663** 76 67 91 92 93 74 78 **7.** *C. guilliermondii* **PGUG\_04145** 79 69 84 85 85 85 78 **8.** *C. lusitaniae* **CLUG\_04124** 78 69 86 86 87 86 87 Table 2. Similarity and identity (UP/down) between PrA proteins from pathogenic *Candida*

**1 2 3 4 5 6 7 8** 

**Signal peptide (aa)**

20 7

24 2: 1400605-

25 130: 135636-

21 5: 355498-356724 -

19 5: 46984-48204 -

**C** 

1401870-

136919 -

The phylogenetic analyses were performed in the MEGA4 program (Tamura et al. 2007) using Maximun Parsimony evolution. A similitude and identity matrix were computed with the MatGAT4.50.2 software (Campanella et al. 2003). The phylogenetic reconstruction and similarity of PrA reproduce the phylogenetic tree topologies of *Candida* spp. obtained with other genes, suggesting a common ancestral gene (Fig. 2; Table 2). In brief, *C. albicans* was more related to *C. dubliniensis*, followed by *C. tropicalis*, *C. parapsilosis*, *C. guilliermondi* and *C. lusitaniae*. Meanwhile, *C. glabrata* PrA was more related to *S. cerevisiae* PrA than other *Candida* species.


Table 1. Soluble and membrane-bound \* vacuolar proteolytic system of *S. cerevisiae*.

Fig. 2. Maximun Parsimony phylogenetic analysis of vacuolar aspartyl proteases (PrA) superfamily from pathogenic *Candida* spp.

The phylogenetic analyses were performed in the MEGA4 program (Tamura et al. 2007) using Maximun Parsimony evolution. A similitude and identity matrix were computed with the MatGAT4.50.2 software (Campanella et al. 2003). The phylogenetic reconstruction and similarity of PrA reproduce the phylogenetic tree topologies of *Candida* spp. obtained with other genes, suggesting a common ancestral gene (Fig. 2; Table 2). In brief, *C. albicans* was more related to *C. dubliniensis*, followed by *C. tropicalis*, *C. parapsilosis*, *C. guilliermondi* and *C. lusitaniae*. Meanwhile, *C. glabrata* PrA was more related to *S. cerevisiae* PrA than other

**Protein Access number Function Reference** 

Activities of other yeast

Contributes to the proteolytic function of the vacuole

Involved in protein degradation in the vacuole and required for full protein degradation during sporulation

Nitrogen compound metabolic process, proteolysis involved in cellular protein catabolic processes

> *C. albicans (Calorf19 1891) C. dubliniensis (Cd36 21670) C. tropicalis (CTRG 01724) C. parapsilosis (CPAG 03663) C. guilliermondii (PGUG 04145) C. lusitaniae (CLUG 04124) S. cerevisiae (YPL154C)*

 *C. glabrata (CAGL0M02211g)*

CAA33512 Protein processing

/NP\_012819 Catabolic processes

vacuolar hydrolases Parr et al., 2007

Wünschmann et al., 2007

Teichert et al., 1989

Bordallo & Suarez-Rendueles, 1993

*Candida* species.

**Name/systematic name** 

> Proteinase A YPL154C

Carboxypeptidase Y YMR297W

Proteinase B

Carboxypeptidase S

Dipeptidyl aminopeptidase B\* YHR028C

Aminopeptidase

**Gene/** 

PEP4/ PrA

YEL060C PRB1 NM\_001178875

YJL172W CPS X63068/

YKL103C APEI NM\_001179669

superfamily from pathogenic *Candida* spp.

NM\_001183968 / NP\_015171

/NP\_014026

/NP\_010854

CAA44790

Table 1. Soluble and membrane-bound \* vacuolar proteolytic system of *S. cerevisiae*.

Fig. 2. Maximun Parsimony phylogenetic analysis of vacuolar aspartyl proteases (PrA)

DAP-B X15484/

CPY NM\_001182806


Table 2. Similarity and identity (UP/down) between PrA proteins from pathogenic *Candida* spp.


Table 3. Vacuolar aspartyl proteases in pathogenic *Candida* species. (AN): Access number in the respective genome; MM: molecular mass; IP: Isoelectric Point; C: Chromosome or Contig or supercontig.

*C. glabrata* is an opportunistic haploid yeast that suffered evident and extensive reductive evolutionary events. A lot of genes involved in nitrogen metabolism, carbohydrate assimilation (saccharose, galactose, etc.), as well as sulfur, phosphor, thiamine, pyridoxine and nicotinic acid biosynthesis have been lost from the genome (Byrne & Wolfe, 2005;

Evolution of GPI-Aspartyl Proteinases (Yapsines) of *Candida* spp 295

(Kalkanci et al. 2005). Given the number of *CgYPS* in *C. glabrata* and their potential role in pathogenesis, it is important to establish the universality of *CgYPS* in *C. glabrata*

**Factor** *C. glabrata C. albicans* **Reference** Infection sites Oral, vaginal, bloodstream Fidel et al. 1999

Krcmery, 1999

& Haynes, 2000

Biasoli et al. 2002

2002

Ghannoum, 2000

Schaller et al. 2002

al. 2007a

2005

et al. 2001

Albrecht et al. 2006; Kaur et al. 2007 This work

Mortality in systemic infection urinary tract Abi-Said et al. 1997;

Virulence in animal models High Arendrup et al. 2002

Adherence to denture material Lower Higher Luo & Samaranayake,

Phenotypic switching Low High Brockert et al. 2003

Human-defensin resistance Strong Weak Joly et al. 2004; Feng et al.

Histatin resistance Partially resistant Susceptible Helmerhorst et al. 2005 Azole resistance High Low Sanglard et al. 1999

*SAP* genes 0 10 Parra et al. 2009

Table 4. Comparison of virulence factors of *C. glabrata* and *C. albicans* (modified from Li,

Our group explored the *CgYPS* gene distribution among clinical isolates (n=52) and type strains CBS138 and BG6 (N=2) by an original multiplex PCR procedure (Table 5). The yeasts were routinely grown on YPD broth and DNA was extracted using a previously reported protocol (Hoffman & Winston 1987). PCR was performed in a DNA thermal cycler 9600

adherence 20 *EPA* genes *ALS* proteins Castaño et al. 2005; Hoyer

Filamentation Present Lachke et al. 2002; Csank

Biofilm formation High Castaño et al. 2006

keratinocytes Lower Higher Nikawa et al. 1995;

activity Absent Present Chakrabarti et al. 1991

keratinocytes Weak Strong Schaller et al. 2002; Li et

dependent High Samaranayake et al. 1994;

True hyphae and pseudohyphae

populations.

Adherence to oral

Extracellular proteinase

IL-8 induction in oral

GM-CSF induction in oral

Molecules involved in

2007b).

Phospholipase activity Isolation site

keratinocytes Pseudohyphae

*YPS* genes 12 0

Wolfe, 2006). This species produces between 15-20% of reported systemic yeast infections (Almirante et al. 2005; Manzano-Gayosso et al. 2000; Trick et al. 2002; Méan et al. 2008). *C. glabrata* is the most common yeast species isolated from patients with cancer, organ transplantation and fluconazole therapy (Safdar et al. 2001; Bodey et al. 2002). The mortality associated with *C. glabrata* in systemic infections of cancer patients is 50% and almost 100% in transplant patients (Anaissie et al. 1992; Goodman et al. 1992; Krcmery et al. 1998). This scenario is related to indiscriminate antifungal use, and to the innate resistance of *C. glabrata* (Sobel, 2006).

According to Table 4, virulence factors of *C. albicans* and *C. glabrata* are quite different. However, an evident feature is the difference in number and kind of aspartyl proteases. A total of 12 *YPS* genes, but no *SAP* genes have been detected in *C. glabrata.* Contrarily, a total of 10 *SAP* genes, but no *YPS* genes have been recognized in *C. albicans.* Clearly, the phylogenetic trees constructed with ribosomal or other gene groups include the majority of the clinical relevant *Candida* species, with exception of *C. glabrata*, which is grouped in another cluster with non-pathogenic yeasts, as *S. cerevisiae* and *Kluyveromyces* spp. This evidence suggests that the aspartyl proteases in *Candida* spp. have evolved independently as virulence factors at least two times, and possibly the amplification by duplication of *SAP*  and *YPS* gene superfamilies in clinically relevant species is an example of convergent evolution.

A physiological approach could possibly contribute to the understanding of which *C. glabrata YPS* (*CgYPS*) genes are covering the functions of each secreted aspartyl protease of *C. albicans* under different conditions. Evidently, the comparison of virulence strategies, expression profiles, complementation of mutants, among other experiments, could suggest common and particular features and roles for all *SAP* and *YPS* genes. For now, the questions remain open. Have the function of *C. glabrata CgYPS* and *SAP C. albicans* genes functionally converged?

The transcription profile of 11 *CgYPS* was studied when yeast were ingested by macrophages. Apparently, *CgYPS* are important in survival and virulence of the the yeast in macrophages, damage to mousses, Epa1 protein processing, and cell wall integrity, as occur in *S. cerevisiae*, which possesses *5 ScYPS* (*ScYPS1-ScYPS3, ScYPS6* and *ScYPS7*) (Kaur et al*.*  2007; Krysan et al. 2005). They are important to cell wall synthesis and glucan homeostasis, mainly *ScYPS1* and *ScYPS7*. It seems that *ScYPS3* does not have functions associated with the cell wall (Krysan et al. 2005).

*C. albicans SAP9* and *C. glabrata CgYPS1* genes complement the defects in the cell wall provoked by *yps1* of *S. cerevisiae*. One important difference is that *SAP9* complement *yps1* only when *SAP9* is under a heterologous and constitutive promoter from *S. cerevisiae*, while *CgYPS1* complements the mutation, using its promoter (Krysan et al. 2005), evidence that supports the othologous status proposed above for these gene pairs. As happened with *ScYPS1, SAP9* gene expression increases during the stationary phase and damage of the cell wall (Monod et al. 1998; Copping et al. 2005), and protects the yeast from caspofungin (an inhibitor of 1,3-glucan synthesis) (Lesage et al. 2004). Also, inhibitors of ScYps1p disable the specificity of both proteins, ScYps1p and Sap9 (Cawley et al. 2003).

Distribution of the *SAP* gene superfamily among *C. albicans* strains is universal (Gilfillan et al. 1998; Bautista et al. 2003; Parra et al. 2009), although one study concludes that the distribution of *SAP* genes in clinical strains depends on infection associated with isolation

Wolfe, 2006). This species produces between 15-20% of reported systemic yeast infections (Almirante et al. 2005; Manzano-Gayosso et al. 2000; Trick et al. 2002; Méan et al. 2008). *C. glabrata* is the most common yeast species isolated from patients with cancer, organ transplantation and fluconazole therapy (Safdar et al. 2001; Bodey et al. 2002). The mortality associated with *C. glabrata* in systemic infections of cancer patients is 50% and almost 100% in transplant patients (Anaissie et al. 1992; Goodman et al. 1992; Krcmery et al. 1998). This scenario is related to indiscriminate antifungal use, and to the innate resistance of *C. glabrata*

According to Table 4, virulence factors of *C. albicans* and *C. glabrata* are quite different. However, an evident feature is the difference in number and kind of aspartyl proteases. A total of 12 *YPS* genes, but no *SAP* genes have been detected in *C. glabrata.* Contrarily, a total of 10 *SAP* genes, but no *YPS* genes have been recognized in *C. albicans.* Clearly, the phylogenetic trees constructed with ribosomal or other gene groups include the majority of the clinical relevant *Candida* species, with exception of *C. glabrata*, which is grouped in another cluster with non-pathogenic yeasts, as *S. cerevisiae* and *Kluyveromyces* spp. This evidence suggests that the aspartyl proteases in *Candida* spp. have evolved independently as virulence factors at least two times, and possibly the amplification by duplication of *SAP*  and *YPS* gene superfamilies in clinically relevant species is an example of convergent

A physiological approach could possibly contribute to the understanding of which *C. glabrata YPS* (*CgYPS*) genes are covering the functions of each secreted aspartyl protease of *C. albicans* under different conditions. Evidently, the comparison of virulence strategies, expression profiles, complementation of mutants, among other experiments, could suggest common and particular features and roles for all *SAP* and *YPS* genes. For now, the questions remain open. Have the function of *C. glabrata CgYPS* and *SAP C. albicans* genes functionally

The transcription profile of 11 *CgYPS* was studied when yeast were ingested by macrophages. Apparently, *CgYPS* are important in survival and virulence of the the yeast in macrophages, damage to mousses, Epa1 protein processing, and cell wall integrity, as occur in *S. cerevisiae*, which possesses *5 ScYPS* (*ScYPS1-ScYPS3, ScYPS6* and *ScYPS7*) (Kaur et al*.*  2007; Krysan et al. 2005). They are important to cell wall synthesis and glucan homeostasis, mainly *ScYPS1* and *ScYPS7*. It seems that *ScYPS3* does not have functions associated with

*C. albicans SAP9* and *C. glabrata CgYPS1* genes complement the defects in the cell wall provoked by *yps1* of *S. cerevisiae*. One important difference is that *SAP9* complement *yps1* only when *SAP9* is under a heterologous and constitutive promoter from *S. cerevisiae*, while *CgYPS1* complements the mutation, using its promoter (Krysan et al. 2005), evidence that supports the othologous status proposed above for these gene pairs. As happened with *ScYPS1, SAP9* gene expression increases during the stationary phase and damage of the cell wall (Monod et al. 1998; Copping et al. 2005), and protects the yeast from caspofungin (an inhibitor of 1,3-glucan synthesis) (Lesage et al. 2004). Also, inhibitors of ScYps1p disable

Distribution of the *SAP* gene superfamily among *C. albicans* strains is universal (Gilfillan et al. 1998; Bautista et al. 2003; Parra et al. 2009), although one study concludes that the distribution of *SAP* genes in clinical strains depends on infection associated with isolation

the specificity of both proteins, ScYps1p and Sap9 (Cawley et al. 2003).

(Sobel, 2006).

evolution.

converged?

the cell wall (Krysan et al. 2005).

(Kalkanci et al. 2005). Given the number of *CgYPS* in *C. glabrata* and their potential role in pathogenesis, it is important to establish the universality of *CgYPS* in *C. glabrata* populations.


Table 4. Comparison of virulence factors of *C. glabrata* and *C. albicans* (modified from Li, 2007b).

Our group explored the *CgYPS* gene distribution among clinical isolates (n=52) and type strains CBS138 and BG6 (N=2) by an original multiplex PCR procedure (Table 5). The yeasts were routinely grown on YPD broth and DNA was extracted using a previously reported protocol (Hoffman & Winston 1987). PCR was performed in a DNA thermal cycler 9600

Evolution of GPI-Aspartyl Proteinases (Yapsines) of *Candida* spp 297

The universality of the 12 *CgYPS* genes among all *C. glabrata* clinical isolates and type strains was confirmed (Fig. 3), which suggests that all *CgYPS* are important to yeast life cycle as pathogen or commensal, and probably are differentially regulated according to each

**100** 

**F)**

**100 pb**

**CGL1**

**CGL2**

**CGL3**

**CGL4**

**CGL5**

**CGL6**

**CGL7**

**C)**

**477** *CgYPS1* **596** *CgYPS7*

> **464** *CgYPS9* **526** *CgYPS8* **566** *CgYPS12*

**pb**

**CGL1**

**CGL3**

**CGL2**

**CGL4**

**CGL5**

**CGL6**

**CGL7**

**653** *CgYPS3* **502** *CgYPS4*

> **526** *CgYPS10* **631** *CgYPS6*

environmental condition, as occurs with *C. albicans SAP.*

**421** *CgYPS11* **536** *CgYPS2*

> **545** *CgPEP4* **619** *CgYPS5*

*CgYPS8, CgYPS12* and *CgYPS9*; F, *CgYPS6* and *CgPEP10*.

**2.3** *YPS genes* **in clinically relevant** *Candida* **species** 

content were calculated using Antheprot 2000 version 5.2 (Table 6).

**100 pb**

**CGL2**

**100 pb**

**E)**

**CGL1**

**CGL2**

**CGL3**

**CGL4**

**CGL5**

Fig. 3. Amplification of *CgYPS C. glabrata* gene fragments by multiplex PCR. A, *CgYPS2* and *CgYPS11*; B, *CgYPS7* and *CgYPS1*; C, *CgYPS3* and *CgYPS4*; D, *CgYPS3* and *CgPEP4*; E,

The genome sequence projects of *Candida* species allows for the exploration of whether *YPS* genes are harboured in these opportunistic pathogen yeasts. *C. dubliniensis* sequences were obtained from the Sanger Institute Microorganisms Sequencing Group (http://www.sanger.ac.uk/sequencing/Candida/dubliniensis/). Sequences from *C. guilliermondii*, *C. lusitaniae*, *C. tropicalis* and *C. parapsilosis* were obtained from (http://www.broad.mit.edu/annotation/genome/candida\_group/MultiHome.html). The GenBank database (http://www.ncbi.nlm.nih.gov) was also used. The detection was made by using the previous *YPS* and *SAP* genes detected in *S. cerevisiae* (http://www.yeastgenome.org), *C. glabrata* (http://cbi.labri.fr/Genolevures/elt/CAGL) and *C. albicans* (http://www.candidagenome.org) genomes, and the proteins detected by BLAST analysis in NCBI. Also, the different patterns of motif that could be obtained were used as a new query. In *C. lusitaniae* and *C. guilliermondii* only one *YPS* was detected. Meanwhile in *C. dubliniensis* and *C. albicans* four *YPS* genes were detected, in *C. tropicalis* two, and in *C. parapsilosis* six. Theoretical isoelectric point, molecular weight and amino acid

Prediction of motif sequences was performed with PROSITE (http://www.expasy.org) (Falquet et al. 2002). Some of the proteins possess a typical molecular structure of aspartyl proteases, but others have some differences in composition (Fig. 4; Table 6). Some of them possess high Ser/Thr content in the amino terminal, suggesting that this zone is exposed at the surface of the protein. The presence of Ser/Thr in the carboxyl terminal in almost all *YPS*

**CGL6**

**CGL7**

**CGL3**

**CGL4**

**CGL5**

**CGL6**

**CGL7**

**B) CGL1**

**100 pb**

**A)**

**CGL1**

**100 pb**

**CGL2**

**CGL3**

**CGL4**

**CGL5**

**CGL6**

**CGL7**

**D) CGL1**

**CGL2**

**CGL3**

**CGL4**

**CGL5**

**CGL6**

**CGL7**

(Applied Biosystems, Foster City, CA). Amplification reactions (25 μL) were performed using a buffer containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 2 mM MgCl2, 0.2 mM of each deoxynucleoside triphosphate, 0.6 μM each primers, 4 ng/μL of genomic DNA, and 1.5 U/μL of *Taq* polymerase (Invitrogen). The PCR conditions included a denaturation step for 3 min at 94°C, followed by 38 amplification cycles consisting of 1 min at 94°C, 1 min annealing temperature and 1 min at 72°C. A final extension step was performed for 7 min at 72°C. Fig. 3 shows the amplification products of *CgYPS* gene fragments of some representative *C. glabrata* clinical strains electrophoresed in 1% agarose gels. Similar PCR conditions were used to study the universal distribution of PrA.


Table 5. Primer pairs used for conventional multiplex PCR of *C. glabrata YPS* genes. Bp, Base pair; Tm, Melting temperature.

(Applied Biosystems, Foster City, CA). Amplification reactions (25 μL) were performed using a buffer containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 2 mM MgCl2, 0.2 mM of each deoxynucleoside triphosphate, 0.6 μM each primers, 4 ng/μL of genomic DNA, and 1.5 U/μL of *Taq* polymerase (Invitrogen). The PCR conditions included a denaturation step for 3 min at 94°C, followed by 38 amplification cycles consisting of 1 min at 94°C, 1 min annealing temperature and 1 min at 72°C. A final extension step was performed for 7 min at 72°C. Fig. 3 shows the amplification products of *CgYPS* gene fragments of some representative *C. glabrata* clinical strains electrophoresed in 1% agarose gels. Similar PCR

> **Expected amplified fragment (bp)**

+1779 a +1803 477 55

+1743 a +1770 536 61

+1580 a +1603 652 58

+1411 a +1435 502 55

+1528 a +1553 619 <sup>58</sup>

+1542 a +1518 631 <sup>58</sup>

+1404 a +1427 596 <sup>55</sup>

+1457 a +1482 626 <sup>58</sup>

+1510 a +1535 464 <sup>58</sup>

+1479 a +1504 526 <sup>58</sup>

+1495 a +1521 421 <sup>61</sup>

+1542 a +1567 566 <sup>58</sup>

+1208 - +1236 545 <sup>58</sup>

+1326 a +1348

+1234 a +1262

+951 a +973

+933 a +956

+934 a +959

+887 a +910

+831 a +855

+856 a +878

+1071 a +1096

+978 a +1003

+1100 a +1125

+1001 a +1025

+691 - +716

**Tm (ºC)** 

conditions were used to study the universal distribution of PrA.

*CgYPS1*  CAGL0M04191g

*CgYPS2* CAGL0E01419g

*CgYPS3* CAGL0E01727g

*CgYPS4*  CAGL0E01749g

*CgYPS5*  CAGL0E01771g

*CgYPS6*  CAGL0E01793g

*CgYPS7*  CAGL0A02431g

*CgYPS8*  CAGL0E01815g

*CgYPS9*  CAGL0E01837g

*CgYPS10*  CAGL0E01859g

*CgYPS11*  CAGL0E01881g

*CgYPS12*  CAGL0J02288g

*CgPEP4*  CAGL0M02211g

pair; Tm, Melting temperature.

**Gene Primer Location** 

F:5´-TTCTGGTGACAGTTGTATCTTGG-3´ R:5´-GATAAATGAAACCAAAAGACCAGCG-3´

F:5´-ACTCAACTTGTTTTTAACTTCGGTGGTGC-3´ R:5´-TAGCATGGAGAGTAGGATGTTAAACACC-3´

F:5´-AAAGCAAGTCGTCGATGTCATCG-3´ R:5´-TTGCAACTAACACTAAAGTGGTGC-3´

F:5´-TTCTGTGTTACCAGCAAAGGTTGC-3´ R:5´-TTAATGTAGTTCTCTTACGGAGAGC-3´

F:5´-TATACATATATGCCAAGCAGCGTTGC-3´ R:5´-AACAAGGCAGTAACTGCTGATAAAGC-3´

F:5´-ACCAGAAGGTAGCTGCATTAATCG-3´ R:5´-AATGGTAGCTAATATGGCAGCAACG-3´

F: 5´-TATGGGACCAATCTATATAACGTCC-3´ R: 5´-TAAGTAGCATACGGTATGTAGCCC-3´

F: 5´-TTGGGATTACAGGGTAATGATGC-3´ R: 5´-AACTCTTTTTTGAAGGTCAAAACGCG-3´

F: 5´-TTCCGTAAATGTGACTGATTTCATGG-3´ R: 5´-ATCATAATGAGTATGGCAGAGTTGGC-3´

F: 5´-TAATAAGACGGAAGCCATCAGACTGC-3´ R: 5´-TTGTAATTGCTGCTAGTACTAGGACG-3´

F: 5´-TTGGTGTCCCATACAAGGAAATGGTC-3´ R: 5´-AATCCACAAG ACCAGCAACA GGATAGC-3´

F: 5´-AATTGCACATGAAGATTCCGTTGCG-3´ R: 5´-TATCAGTTATTGTAGCAGTTACTGGC-3´

F 5´ -TATCTGAAGAGTGTCAATGACCCAGC-3´ R 5´-TACAGCCTCAGCTAAACTGACAACATTGG-3´

Table 5. Primer pairs used for conventional multiplex PCR of *C. glabrata YPS* genes. Bp, Base

The universality of the 12 *CgYPS* genes among all *C. glabrata* clinical isolates and type strains was confirmed (Fig. 3), which suggests that all *CgYPS* are important to yeast life cycle as pathogen or commensal, and probably are differentially regulated according to each environmental condition, as occurs with *C. albicans SAP.*

Fig. 3. Amplification of *CgYPS C. glabrata* gene fragments by multiplex PCR. A, *CgYPS2* and *CgYPS11*; B, *CgYPS7* and *CgYPS1*; C, *CgYPS3* and *CgYPS4*; D, *CgYPS3* and *CgPEP4*; E, *CgYPS8, CgYPS12* and *CgYPS9*; F, *CgYPS6* and *CgPEP10*.
