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Brazilian Journal of Oral Sciences, Vol. 5, No. 16, Jan - March, 2006, pp. 944-952

Candida albicans proteinases

Rita de Cássia Mardegan¹Mary Ann Foglio²Reginaldo Bruno Gonçalves³José Francisco Höfling⁴

¹DDS, MSc, Graduate Student at the University of Campinas – Piracicaba Dental School -Department of Oral Diagnosis, São Paulo, Brazil ²DDS, PhD, Professor - Research Center for Chemistry, Biology and Agriculture (CPQBA) -Department of Phytochemical, University of Campinas, São Paulo, Brazil ³DDS, PhD, Professor at the University of Campinas - Piracicaba Dental School-Department of Oral Diagnosis, São Paulo, Brazil,. ⁴BS, MS, PhD, Professor at the University of Campinas - Piracicaba Dental School -Department of Oral Diagnosis, São Paulo, Brazil.
Correspondence to: José Francisco Höfling Division of Microbiology and Immunology, Department of Oral Diagnostic Piracicaba Dental School –UNICAMP Av. Limeira, 901 13414-903 - Piracicaba - SP - Brazil Phone: 55 19 3412 5321/ 5322/5323 Fax: 55 19 3412 521 E-mail: hofling@fop.unicamp.br

Received for publication: November 10, 2005
Accepted: February 14, 2006

Code Number: os06003

Abstract

Candida species are ubiquitous commensal yeast that usually reside as part of an individual´s normal mucosal microflora and can be detected in approximately 50% of the population in this form. However, if the balance of the normal flora is disrupted or the immune defences are compromised, Candida species can invade mucosal surfaces and cause disease manifestations. Determining exactly how this transformation from commensal to pathogen takes place and how it can be prevented is a continuing challenger for the medical mycology field. Attributes that contribute to Candida albicans virulence include adhesion, hyphal formation, phenotypic switching and extra cellular hydrolytic enzyme production. The extra cellular hydrolytic enzyme, especially the secreted aspartyl proteinases (Saps), are one a few gene products that have been shown to directly contribute to C. albicans pathogenicity. Given the limited number of suitable and effective antifungal drugs, the continuing increase in the incidence of Candida infections, together with increasing drug resistance, highlights the need to discover new and better agents that target fundamental biological processes and or pathogenic determinants of C. albicans.

Key Words: proteinases, virulence, candida

Introduction

Candida infections are common, debilitating and often recurring fungal diseases and a problem of significant clinical importance. The frequency of Candida infections has increased in recent years and it has been accompanied by a significant rise in morbidity and mortality¹. The secretion of aspartic proteases by Candida spp was demonstrated to be one of the virulence determinants². Candida albicans is classified as the most virulent human pathogen in the genus Candida and may cause severe mucosal and life-threatening systemic infections in immunocompromissed hosts. The physiological status of the host is the primary factor governing the etiology of candidiasis¹.

All pathogenic microorganisms have developed mechanisms that allow successful colonization or infection of the host². As a result, most pathogens, including Candida species, have developed an effective battery of putative virulence factors and specific strategies to assist in their ability to colonize host tissues, cause disease, and overcome host defences. The virulence factors expressed or required by Candida species, and in particular Candida albicans, to cause infections may well vary depending on the type of infection, and the nature of the host response³. Many factors have been suggested to be virulence attributes for C. albicans, hyphal formation, surface recognition molecules, phenotypic switching, and extracellular hydrolytic enzyme production have been the most widely studies in recent years⁴. One factor that contributes to the process of virulence is hydrolytic enzyme production, which is known to play a central role in the pathogenicyty of bacteria², protozoa⁵, and pathogenic yeasts⁶. Although many microorganisms possess a variety of hydrolytic enzymes, but proteinases are by far the most commonly associated with virulence⁴.

Extracellular proteases of eukaryotic microbial pathogens have attracted the attention of many laboratories because of their potential role in pathogenesis.

Extracellular proteinases of saprophytic microorganisms are primarily secreted to breakdown or decompose complex material into nutrients readily available to the cells or to complete other environmental bacteria, parasites or fungi. However, pathogenic microorganisms (bacterias, parasites and fungi) appear to have adapted this biochemical property to fulfill a number of specialized functions during the infective process in human, animals and plants⁷.

All proteinases catalyse the hydrolysis of peptide bonds (CO-NH) in proteins but can differ markedly in specificity and mechanism of action⁸. The Enzyme Nomenclature distinguished four classes of proteinases: serine, cysteine, aspartyl proteinases, and metalloproteinases. Aspartyl proteinases are ubiquitous in nature and are involved in a myriad of biochemical processes³. These more direct virulence functions may include digesting or distorting host cell membranes to facilitate adhesion and tissue invasion, which has been demonstrated in plants⁹ and insects¹⁰, or damaging cells and molecules of the host immune system to avoid or resist antimicrobial attack by the host¹¹.

Most studies investigating the role of extracellular hydrolytic enzymes in fungal pathogenicity have focused on human pathogenic fungi, including Aspergillus fumigatus¹², Cryptococcus neoformans¹³, and C. albicans^14-17.

The Candida albicans secreted aspartyl proteinases (SAP) the most significant extracellular hydrolytic enzymes produced by C. albicans are the secreted aspartyl proteinases (Sap), phospholipase B enzyme, and lipases. C. albicans is not the only Candida species known to produce extracellular proteinases. Many of the pathogenic Candida species have been shown SAP genes, including C. dubliniensis¹⁸C. tropicalis and C. parapsilosis¹⁹. Less pathogenic or nonpathogenic Candida species do not appear to produce significant amounts of proteinase, even though they may possess aspartyl proteinase genes¹⁹. Finally, one should note that all Candida secreted proteinases belong to the same class of enzyme: the aspartyl proteinases. Neither extracellular serine nor cysteine proteinases nor metalloproteinases have been identified in pathogenic Candida species. The complexity of Sap involvement in C. albicans virulence have been highlighted by the fact that Sap production is associated with a number of other putative virulence attributes of C. albicans including, hyphal formation, adhesion, phenotypic switching, dimorphism, and the secretion of hydrolytic enzymes such as aspartyl proteinases and phospholipases³.

Saps are encoded by a multigene family encompassing at least ten different highly regulated genes (SAP 1 –SAP 10). The existence of 10 SAP genes and their controlled expression and regulation raises a number of questions concerning the role and functions of these proteinases during the infective process. The presence of 10 SAP genes, their temporal activation during distinct stages of infection, and their coordinate constitute a robust body of evidence showing that individual members of this gene family have a major role in the adaptive response of C. albicans to its environment, including its host. For instance, a gene SAP1 encoding an extracellular proteinase was cloned in 1991 and was thought to be responsible for the observed secretory aspartic proteinase (Sap) activity of C. albicans²⁰. Shortly after SAP1 was discovered, more genes encoding extracellular aspartic proteinases were identified, by PCR-based cloning strategies²¹, by using SAP1 as a probe^22-23, by sequencing the promoter region of SAP1²⁴ or by BLAST searches and sequence alignments in Candida genome databases. The deduced proteins are all aspartic proteinases and share a number of Sap-specific characteristics. All ten SAP genes encode preproenzymes approximately 60-200 amino acids longer than the mature enzyme. The N-terminal secretion signal is cleaved by a signal peptidase in the endoplasmic reticulum (ER). The peptide is removed to activate the proteinases by the subtilisin-like Kex2 proteinase in the Golgi before being transported via vesicles to the cell surface for secretion or glycosylphosphatidylinositol (GPI)-anchoring. Although Kex2 may be a key regulatory proteinase of Saps²⁵, other alternative processing pathways are thought to exist²⁶, and autoactivation was shown to occur extracellularly for Sap1-3 and Sap6 at certain pH values²⁷. The mature enzyme contains sequence motifs typical for all aspartic proteinases, including the two conserved aspartate residues of the active site. Conserved cysteine residues are propably implicated in maintaining the three-dimensional structure of the enzyme²⁸. Unlike Sap1-8, Sap9 and Sap10 both have C-terminal consensus sequences typical for GPI proteins.

While in the early nineties efforts concentrated on the collection of sequence data, the increasing numbers of cloned SAP genes soon shifted the interest towards the role and function of the SAP gene family during the infection process. The fact that the presence of SAP gene family was unique to only the most pathogenic Candida species, such as C. albicans²⁹, C. dubliniensis¹⁸, C. tropicalis³⁰ and C. parapsilosis¹⁹, but was absent in non-pathogenic yeast S. cerevisiae, supported the view that these proteinases may be involved in virulence. Less common clinical isolates such as C. kefyr, C. glabrata and C. guilliermondii appear to be nonproteolytic when tested in culture medium with bovine serum albumin (BSA) as the sole nitrogen source^31-33. However, in the light of the discovery that only a single isoenzime, Sap2, was necessary and sufficient to allow rapid growth of C. albicans in media containing protein as the sole source of nitrogen^34-35 it remained a mystery as to why this fungus possessed a whole family of SAP genes. As one possible explanation may be that different proteinases are required to act upon different host proteins and tissues in vivo, a number of studies dealt with the possible targets of Saps.

However, the Saps may have also adapted and involved to have more direct virulence functions. For example, saps could contribute to host tissue adhesion and invasion by degrading or distorting host cell surface structures and intercellular substances, or by destroying cells and molecules of the host immune system to avoid or resist antimicrobial attack¹⁶. The degradation of human proteins and structural analysis in determining Sap substrate specificity was demonstrated first by Staib³⁶ when yeast cells were grown in media containing BSA as the sole source of protein. Three years later, Remold et al.³⁷, attributed this activity to the production of an extracellular proteinase. Since then and up to the early 1990s, a plethora of studies reported on the purification and biochemical properties of an extracellular factors such as pH and temperature on proteolytic activity. The culture conditions used to induce proteinase activity in these early reports have subsequently been shown to favor SAP2 expression^34-35. Therefore, any attempts to determine the substrate specificities and potential targets of the Sap family in vivo were based on the activity of Sap2 in vitro. At present in vivo is similar to that shown in vitro or whether the substrates for Sap 2 are similar or different from those of the other proteinases in the Sap. family. The full range of substrate specificities for all the secreted proteinases has not been adequately studied, but the in vitro proteolytic properties of Sap 2 have been described in some detail.

One the most noticeable properties of Sap 2 is the variety of proteins it can cleave. The contribution of this broad activity to Candida pathogenesis, along with other virulence attributes of C. albicans, that commonly colonizes the epithelial surface (stage1) and causes superficial infections (stage2), but under conditions when the host is compromised, the fungus establishes deep-seated infections (stage3) by penetrating further into the epithelial tissue. Occasionally, C. albicans causes disseminated infections (stage4), which allow the fungus to colonize and infect other host tissues and can be fatal. This infective process involves numerous virulence attributes including adhesions, hydrolytic enzyme production (Sap proteins, phospholipases ad lipases), hypha formation and phenotypic switching. Sap2 (and possibly other Saps proteins) is know to degrade many human proteins, including mucin, extracellular matrix proteins, numerous immune system molecules, endothelial cell proteins, and coagulation and clotting factors. Therefore, the action of Sap proteins could be involved in all four stages of infection and probably greatly enhances the pathogenic ability of Candida albicans³⁸. This infective process involves numerous virulence attributes including adhesions, hydrolytic enzyme production, hypha formation and phenotypic switching. Sap2, and possibly other Saps proteins, is known to degrade many human proteins, including mucin, extracellular matrix proteins, numerous immune system molecules, endothelial cells proteins, and coagulation and clotting factors including molecules that protect mucosal surfaces such as mucin^39-40 and secretory immunoglobulin A (IgA)⁴¹. Not only could this provide essential nitrogen for growth, but also it could enhance attachment, colonization and penetration of host tissue by the removal of host barriers. Digestion of secretory IgA is particularly noteworthy because it is considerably more resistant to proteolysis than are monomeric or serum immunoglobulins, is able to neutralize many toxins and enzymes⁴², and can inhibit C. albicans attachment to buccal epithelial cells⁴³. Wu and Samaranayake⁴⁴ noted that reduction of total salivary protein concentration correlated with the degree of Sap expression, suggesting that Candida Sap protein degrade salivary proteins in the oral cavity. Sap2 can also degrade molecules of the extracellular matrix such as keratin, collagen and vimetin^6,28,31. C albicans proteinases may also evade host defenses by directly degrading molecules such as salivary lactoferrina, lactoperoxidase, cathepsin D (an intracellular lysosomal enzyme of leukocytes) and complement^28,45-46. Moreover, the coordinated regulation of the SAP genes with other virulence factors described, provide C. albicans a biological advantage to specifically enhace the pathogenic ability of the fungus. These studies suggested that Sap 2, in contrast to the highly substrate-specific enzyme produced by certain bacteria, has very broad substrate specificity and may multiple targets in vivo. Many of the early proteinase studies focused on the influence of culture conditions on Sap expression and proteolytic activity in vitro²⁸. However, after the discovery of a SAP gene family, it became apparent that this enzyme family had a more significant and complex contribution to C. albicans pathogenicity. C. albicans is a polymorphic pathogen, which can exist in a yeast or a hiphal state and can undergo phenotypic switching. However, the question of why, if a single proteinase has such a range of activities and functions, does C. albicans need a family of ten SAP genes?

Magee et al.²⁹ postulated that the Sap isoenzymes might have a variety of functions in vivo, which may be called upon at different sites, and during different stages and types of C. albicans infection.

Therefore, it seemed logical to assume that due to the large number of proteinases present in C. albicans, the SAP gene family may be differentially expressed in the different morphological forms. As a result, the relationship between proteinase production and hyphal production, phenotypic switching, and other putative virulence attributes of C. albicans was investigated.

Under most proteinase-inducing conditions in the laboratory, the major proteinase gene expressed in C. albicans yeast forms is SAP2, which was found to be regulated by a positive feedback mechanism: peptides resulting from proteolysis of high-molecular-mass proteins were proposed to lead to the induction of SAP2 gene expression³⁴. In contrast, SAP1 and SAP3 were discovered to be differentially expressed during phenotypic switching in certain strains^23,47. However, studies indicated the regulation of SAP3 during switching was not absolute and SAP4-6 genes were almost exclusively expressed during hyphal formation at neutral pH, even in defined protein-free media^34-35. The expression of SAP8 is temperature-regulated⁴⁸.

In vitro experimental models of oral⁴⁹ and cutaneous⁵⁰ C. albicans infections suggested that SAP1-3 were the main proteinases expressed during superficial infections. In contrast to mucosal infection models, experimental models of systemic C. albicans infections correlated SAP4-6 expression with systemic disease⁵¹.

As a result, nearly all the studies have implicated the proteinases in C. albicans virulence in one of the following seven ways, with perhaps the most definitive data obtained from the behavior of the various SAP-disrupted strains: (1) correlation between Sap production in vitro and Candida virulence, (2) degradation of human proteins and structural analysis in determining Sap substrate specificity, (3) association of Sap production with other virulence processes of C. albicans, (4) Sap protein production and sap immune responses in animal and human infections, (5) SAP gene expression during Candida infections, (6) modulation of C. albicans virulence by aspartyl proteinase inhibitors, and (7) the use of SAP- disrupted mutants to analyze C. albicans virulence³.

Correlation between Aspartyl proteinases and virulence

Most of the studies have concentrated on C. abicans strains isolated from the oral cavity or the vaginal lumen and on the effect of HIV infection on proteinase production and C. albicans strain selection. Candida albicans strains from different patient group with various clinical infections have been isolated, and the level of Sap activity produced in vitro has been correlated with virulence. In oral candidiasis, the increased Sap activity occurred in C. albicans strains isolated from HIV-positive patients compared with HIV-negative C. albicans carriers^52-53. Candida albicans from HIV-positive patients with oropharyngeal candidiasis produced more proteinase activity than did C. albicans from HIV-negative asymptomatic oral carriers or HIV-negative subjects with oral candidiasis⁵⁴.

All reports showed that C. albicans isolates from symptomatic patients with vaginal candidiasis were significantly more proteolytic than isolates from asymptomatic vaginal carriers^55-56. The study by the same group found that C. albicans strains isolated from HIVpositive women with vaginitis produced significantly higher levels of Sap (fourfold) that did C. albicans strains isolated from HIV-positive asymptomatic carries or HIV-negative subjects with candidal vaginitis⁵⁷. These studies using oral and vaginal clinical isolates showed a positive correlation between the level of Sap production in vitro and the virulence of C. albicans.

Proteinase and adherence to host cell

Adherence of C. albicans to host cell is a complex, multifactorial process involving several types of candidal adhesions on a morphogenically changing cell surface, and one mechanism through which Candida adherence might be promoted is via the production of proteinases. A more recent report correlated proteinase production with increase adherence to buccal epithelial cells and death of mice⁵⁸. One study to link proteinase production to adherence in Candida albicans showed that strongly proteolytic strains of C. albicans adhered significantly more strongly to human buccal epithelial cells in vitro than did strains producing less proteinase. A more recent report correlated proteinase production with increased adherence to buccal epithelial cells and death of mice: the higher-Sap-producing strains shower greater levels of tissue colonization in the liver, kidneys, and spleen⁵⁸. Although the precise mechanisms by which Sap proteins contribute to the adherence process are not clear, two hypotheses are currently favored. In the first, Candida albicans proteinase could act as ligands to surface moieties on host cells, which does not necessarily require activity of the enzymes. In the second, C. albicans utilizes Sap proteins as active enzymes to modify target proteins or ligands on the fungal surface or on host cell which may alter surface hidrophobicity or lead to conformational changes, thus allowing better adhesion of the fungus¹⁴. In summary, laboratory studies have indicated that the C. albicans SAP gene family is differentially expressed in the yeast, hyphal, and phenotypically switched states and may contribute to C. albicans adherence. Therefore, it is quite possible that the SAP genes expressed by C. albicans cells in the laboratory may not equate to the SAP genes expressed in vivo. Determination of exactly which SAP genes are expressed by the two morphological forms and during phenotypic switching at the single-cell level in vivo may provide a significant step forward in elucidating the complex interaction between the host environment and SAP gene regulation³.

In Sap protein production and Sap immune responses in animal and human infections several studies have provided strong evidence demonstrating the models of disseminated candidiasis revealed the presence of Sap proteins on the surface of C. albicans cells in murine kidneys⁵⁸. The presence of Sap protein has also been demonstrated during phagocytosis of C. albicans by leukocytes. Macdonald and Odds⁵⁹ were the first to show that the resistance of C. albicans to phagocytosis was associated with Sap expression. Some years later, it was observed that C. albicans and C. tropicalis yeast cells that resisted phagocytic killing germinated intracellularly and expressed Sap on their surface⁶⁰.

The discovery that C. albicans possessed a multitude of proteinase genes that were differentially expresses under a variety of environmental conditions in vitro^43-44 led to the attractive proposition that different members of the Sap family might also be differentially expressed in vivo and might contribute to different C. albicans infections. This concept, together with the knowledge that C. albicans inhabits a diverse number of host niches, was the driving force behind subsequent studies that investigated SAP gene expression in several models to ascertain which proteinases were expressed in which infections. The precise roles and functions of the C. albicans proteinases during human infections are currently not clear. Naglik et al.⁶¹ published the only detailed study of humans, in which SAP1 to SAP7 expression in the oral cavities of both patients with oral candidiasis (n=10) and asymptomatic candida carriers (n=8) was analyzed. SAP2 and SAP4 to SAP6 (SAP4 and SAP6 were detected together as a subfamily and not individually) were the predominant proteinase genes expressed in the oral cavity of infected patients and Candida carries, while SAP1 and SAP3 transcripts were observed only in patients. SAP7 mRNA expression, which had never previously been demonstrated in vitro or in vivo, was readily detected in both carriers and patients. The results indicate that not only are certain hydrolytic enzymes preferentially expressed in the oral cavity and vaginal lumen but also individual SAP genes are more frequently expressed during active C. albicans infection than during carriage⁶². Schaller et al.⁶³ also analyzed SAP1 to SAP4and SAP8 expression in two patients with oral candidiasis: an HIV-negative female and HIVpositive male, both suffering from pseudomembranous candidiasis. This analysis was undertaken to confirm the SAP expression data obtained using an in vitro model of oral candidiasis based on reconstituted human epithelium (RHE) models could be used as surrogates for human infections. However, since only two patients samples were analyzed, no conclusions could be drawn regarding which SAP genes were associated with human oral candidiasis. Future experiments using patients’samples should bring us one step closer to identifying the gene and proteins (SAP or otherwise) that are directly involved in Candida pathogenesis in human. Although this may simply allude to the intricate and complex relationship between Sap production, Candida pathogenesis, and the host, a number of other explanations are possible. In addition to these disparities, the environmental conditions alone at the different infection sites, including pH, substrate availability, and ionic content, may differ markedly and could partly account for the observed differences in SAP expression between the studies. With these considerations in mind, it is entirely probable that C. albicans has adapted to these niches sites in different hosts by expressing a different set of SAP genes.

Candida albicans virulence and proteinase inhibitors

Drugs-resistant Candida infections are likely to pose a serious therapeutic challenge over the next few decades. Consequently, discoveries of novel drugs classes are crucial aims of antifungal research. Modulation of C. albicans virulence by proteinase inhibitors is one of the opportunities for development of new compounds active against Candida species includes the development of drugs directed against the Candida proteinases. This is particularly pertinent now that the three-dimensional structure of one member of the Sap family (SAP2) has been determined^64-65. In last years, the role of Candida proteinases in pathogenesis and their potential as antifungal targets have driven the use of aspartyl proteinase inhibitors (PIs) in Candida research. These studies using the classical aspartyl PI pepstatin, HIV PIs, and computer-assisted structure-based designed inhibitors have provide direct evidence demonstrating the contribution of Sap proteins to C. albicans virulence and have indicated that the proteinase family may be a possible target for antifungal research.

The contribution of proteinases to C. albicans adherence, invasion, and infection has long been advocated, but it was not until the use of the classical aspartyl PI pepstatin that many of these associations were confirmed. With use de pepstatin in vitro assays showed that the digestion of mucin could be inhibited by pepstatin, indicating that Candida Sap proteins may degrade mucosal barrier proteins⁴⁶. This may allow C. albicans to gain access to the oral and gastrointestinal mucosa and may consequently indicate a role for Candida proteinases in dissemination from these colonized sites. Likewise, the addition of pepstatin inhibited the in vitro digestion of soluble and immobilized extracellular matrix proteins produced by a human endothelial cell line⁶⁶. Again, this suggests that Candida Sap proteins contribute to cell damage and invasion of the subendothelial extracellular matrix, which in turn could facilitate dissemination via the circulatory system. These studies indicate that proteinase inhibition by pepstatin can reduce the ability of Candida albicans to colonize and invade host tissues and illustrate the potential benefits of pepstatin in vivo, chiefly in mucosa models.

Since the Candida proteinases and the HIV proteinase are members of the same aspartyl proteinase family, these finding led to the hypothesis that HIV PIs may also act against Candida aspartyl proteinases in vivo and consequently prevent or reduce candidal infections directly^67-68. A total of four HIV Pis, namely Ritonavir, Saquinavir, Indinavir and Nelfinavir, have been investigated for their ability to inhibit Candida proteinase activity^69-72. Ritonavir was consistency found to be the most potent inhibitor of C albicans Sap 2 activity^68-69,72, while Saquinavir, Indinavir and Nelfinavir inhibited Sap2 activity to differing extents.

The HIV PIs have been tested in artificial, animal and humans models to determine their effectiveness at attenuating C. albicans infection. Using an RHE model of oral candidiasis, Saquinavir, but not Indinavir strongly attenuated the ability of C. albicans to cause tissue damage⁷³. Since there is good evidence that tissue damage in this model may result from the activity of Sap1 to Sap3 subfamily⁷⁴, the data support the evidence that Sap 1 to Sap 3 contribute to the development of mucosal tissue damage. Notwithstanding the basic scientific question of whether the HIV PIs can inhibit proteinase activity per se or C. albicans infection in experimental models, the underlying principle in performing the above studies is to determine whether the HIV PIs could be used therapeutically to treat Candida infections in humans. Only one report has addressed this issue to date, and this study claimed that the anti-Sap effect of the HIV PIs appeared to be associated with the clinical resolution of oral candidiasis⁷⁵.

The identification of novel Candida Sap inhibitors, either through design by modification of existing inhibitors or by isolation of the inhibitors in their natural state from microbes, has been reported. Pichova et al.⁷² designed a series of peptidomimetic inhibitors derived from the structure of Pepstatin A, and showed that most of the inhibitors were essentially equally active against four purified secreted proteinases isolated from C. albicans (Sap2), C. tropicalis (Sapt1), C. parapsilosis (Sapp1), and C. lusitaniae (Sap1). Unfortunately, these peptidomimetic inhibitors were not tested in animal models, but one might hypothesize that since they are based on pepstatin, similar ineffective protective properties may be observed during systemic Candida infections to found previously^76-79. Other groups have identified natural molecules isolated from microorganisms with inhibitory activity against the C. albicans proteinase. These include compounds from Streptomyces species^80-81, numerous xanthones from Tovomita krokovvi⁸², the serratene triterpenes from Lycopodium cernuum⁸³, and phenolic compounds from Miconia myriantha⁸³. The in vivo efficacy of all these compounds was not investigated; however, the IC₅₀s alone indicated that these compounds were not serious candidates as C. albicans Sap inhibitors. Studies using PIs have been instrumental in demonstrating the direct contribution of the Sap proteins to C. albicans virulence and have confirmed their status as true virulence factors of C. albicans. More conclusive studies are required in which all group of inhibitors are tested for their ability to inhibit the activity of all members of the C. albicans proteinase family and to attenuate mucosal and systemic C. albicans infections in numerous experimental models. Only then will sufficient information be available to determine the efficacy of these agents at preventing C. albicans infection. Future studies will judge whether the C. albicans proteinase are genuine targets for treatment therapies of mucosal or systemic infections and will underline the therapeutic potential of drugs that are targeted against virulence traits rather than essential primary metabolic or biosynthetic processes³.

The presence of SAP gene family in C. albicans clearly provides the fungus with an efficient and flexible proteolytic system that may prove vital to its success as an opportunistic pathogen. Sap production is probably a well-regulated process that is activated at specific time during colonization and infection to obtain maximum benefits for C. albicans. This review which demonstrate the multiple functions of the SAP genes and have established the proteinases as a very versatile and multifunctional virulence gene family of C. albicans and the importance of futures studies us to identify new and no doubt surprising correlations between Sap gene expression and Candida biology and virulence.

In time, this may lead to the development of new prophylactic and therapeutic strategies specific and investigating the mechanism underlying the Sap response to antifungal exposure is important for candidiasis treatment.

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