**1. Introduction**

288 Gene Duplication

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Identification and genetic mapping of variant forms of puroindoline b expressed in

The *Candida* genus is a polyphyletic genus with at least 150 species. Nine are recognized opportunistic pathogens of humans and animals. *C. albicans* is the species most frequently isolated from human infections, followed by Candida non-Candida species (CNCA), as *C. glabrata*, *C. tropicalis*, C. *dubliniensis*, *C. parapsilosis*, *C. guilliermondii*, *C. lusitaniae, C. kefyr* and *C. krusei* (Méan et al. 2008; Pfaller & Diekema, 2007; Almirante et al. 2005; Manzano-Gayosso et al. 2000).

Some works describe the phylogenetic relationships of *Candida* genus and illustrate the limited relationship between the pathogenic *Candida* spp. The genus has been divided into: the CTG clade, which includes yeast that encodes CTG as serine instead of leucine (*C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis* and *C. lusitaniae*); and the WGD clade, which includes yeast that has undergone a genome duplication event (*Saccharomyces* spp*., Kluyveromyces* spp. and *C. glabrata*). Evidently, *C. glabrata* is more related to non-pathogenic yeasts, as *Saccharomyces cerevisiae*, than to the other pathogenic species (Scannell et al. 2007).

*C. albicans* is a normal microorganism in humans, and colonise up to 70% of skin, mucoses, and faeces of individuals with no apparent detriment to health. However, in some circumstances, either through environmental factors or a weakening of the host immune system, a proliferation and infection by *C. albicans* arise inducing candidosis (Wei et al. 2011).

Biofilm formation, adhesion, cavitation, phenotypic switching, dimorphism, interaction with the host immune system, invasion and tissue damage are virulence virulence factors for *C. albicans*. All these factors are related to the secreted aspartyl proteases (Sap) family, which is considered an important virulence factor and is studied as a possible target for therapeutic drug design (Naglik et al. 2004; Chaffin et al. 1998; Hube, 1998; Naglik et al. 2003, 2004, 2008).

The topic of this chapter is to understand the molecular characteristics, evolution and putative functions of glycosylphosphatidylinositol (GPI)-linked aspartyl proteases (Yps), a protein superfamily distributed among all pathogenic *Candida* species. Cell location motifs,

Evolution of GPI-Aspartyl Proteinases (Yapsines) of *Candida* spp 291

(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

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

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

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

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

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

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.

pathogenesis in the rest of pathogenic species is unknown.

**2.2 Vacuolar aspartyl proteases (PrA)** 

different vacuolar proteases (Table 1).

rapamicine (Palmer, 2005).

(Palmer, 2007; Jones, 1991; Teichert et al. 1989).

conditions (Gash et al. 2000; Roetzer et al. 2008).

subfamilies or orthologous groups are proposed (Parra et al. 2009).

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