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Candida albicans Secreted Aspartyl Proteinases in Virulence and Pathogenesis

Department of Oral Medicine, Pathology & Immunology, GKT Dental Institute, Kings College London, London, United Kingdom,¹ Robert Koch Institut, D-13353 Berlin, Germany²

SUMMARY

INTRODUCTION

PATHOGENESIS AND VIRULENCE OF CANDIDA INFECTIONS

HYDROLYTIC ENZYMES

Extracellular Proteinases of Pathogenic Fungi

SECRETED ASPARTYL PROTEINASES OF CANDIDA

Molecular and Biochemical Properties of the Candida SAP Family

Basic structure of the C. albicans SAP family.

Processing, activation, and regulation of the C. albicans proteinases.

Biochemical properties of the C. albicans proteinases.

SECRETED ASPARTYL PROTEINASES AND C. ALBICANS PATHOGENESIS

Correlation between Sap Production In Vitro and Candida Virulence

Main focus points.

Sap production by clinical C. albicans isolates from humans.

C. albicans strain selection in HIV infection.

Degradation of Human Proteins and Structural Analysis in Determining Sap Substrate Specificity

Main focus points.

Broad substrate specificity of Sap2.

Deducing proteinase specificity via three-dimensional structure and molecular modeling.

Discussion.

Association of Sap Production with Other Virulence Processes of C. albicans

Main focus points.

Sap production and C. albicans adherence.

(i) How do Sap proteins contribute to adherence?

Sap production and yeast-to-hypha transition.

(i) Coordinate regulation of hypha-formation and SAP4 to SAP6 expression.

Sap production and phenotypic switching.

(i) Which SAP genes are regulated by phenotypic switching?

Discussion.

Sap Protein Production and Sap Immune Responses in Animal and Human Infections

Main focus points.

Sap protein production during C. albicans infections.

Sap localization to the cell wall.

Antibodies against Sap induced by C. albicans infections.

(i) Antibodies produced during systemic infection.

(ii) Antibodies produced during mucosal infection.

Functional anti-Sap antibodies.

(i) Systemic infections.

(ii) Mucosal infections.

(iii) Inhibitory antibodies.

(iv) Sap B-cell epitopes.

SAP Gene Expression during Candida Infections

Main focus points.

Human infections.

In vitro artificial experimental infections based on RHE.

Animal experimental models.

(i) Mucosal models.

(ii) Systemic models.

Universal expression of the SAP4 to SAP6 subfamily at mucosal surfaces.

Discussion and future directions for SAP expression studies.

Modulation of C. albicans Virulence by Aspartyl Proteinase Inhibitors

Main focus points.

Pepstatin.

(i) Use of pepstatin in vitro.

(ii) Use of pepstatin in vivo.

HIV PIs.

(i) Inhibition of Candida proteinase activity by HIV PIs in vitro.

(ii) Effectiveness of HIV PIs against Candida infection in RHE and animal models.

(iii) Effectiveness of HIV PIs against Candida infection in humans.

Computer-assisted structure-based designed inhibitors.

Other inhibitors of Candida Sap proteins.

Discussion and future directions for PI studies.

Use of SAP-Disrupted Mutants To Analyze C. albicans Virulence

Main focus points.

Contribution of Sap proteins to mucosal infections using SAP-deficient strains.

Contribution of Sap proteins to systemic infections using SAP-deficient strains.

Contribution of Sap proteins to evasion of host immune responses using SAP-deficient strains.

Conclusions from SAP-deficient mutant studies.

FUNCTIONAL GENOMICS AND CANDIDA

Candida DNA Microarrays

FUTURE DIRECTIONS IN SAP RESEARCH

ACKNOWLEDGMENTS

REFERENCES

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INTRODUCTION

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Medical mycology is a relatively new field within the area of medical microbiology. Fungal diseases became recognized as being of clinical importance in the second half of the last century, mainly due to advances in medical technologies. However, within the last 20 years, the advent of the AIDS epidemic has opened up the clinical mycology field. The discovery that reduction of the CD4⁺ lymphocyte population of the cell-mediated immune system could predispose patients to a multitude of opportunistic fungal infections uncovered a whole new area of host susceptibility and disease. As a result, a notable increase in basic research on pathogenic fungi, predominantly Candida species, Cryptococcus neoformans, and Aspergillus fumigatus, has taken place (162). The outcome of this research has led to the unraveling of many fundamental biological processes that take place in the main fungal pathogens, particularly Candida albicans.

Candida infections are a problem of growing clinical importance. The incidence of infections has increased dramatically over the past two to three decades, and this trend will inevitably continue into the 21st century. C. albicans is the most common fungal pathogen of humans and has become the fourth leading cause of nosocomial infections (59, 167). At the most serious level, mortality rates from systemic candidiasis are high. However, the majority of patients, notably immunosuppressed individuals with human immunodeficiency virus (HIV) infection, experience some form of superficial mucosal candidiasis, most commonly thrush, and many suffer from recurrent infections. In addition, nearly three-quarters of all healthy women experience at least one vaginal yeast infection and about 5% endure recurrent bouts of disease (211, 212).

Candida species usually reside as commensal organisms as part of an individual's normal 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 defenses are compromised, Candida species often become pathogenic. Determining exactly how this transformation from commensal to pathogen takes place and how it can be prevented is a continuing challenge for the medical mycology field. 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.

PATHOGENESIS AND VIRULENCE OF CANDIDA INFECTIONS

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The physiological status of the host is the primary factor governing the etiology of candidiasis. However, the observation that only slight alterations in the host can turn normally harmless commensal yeasts into agents able to inflict severely debilitating illness points to the pathogenic potential of Candida species. Indeed, it appears that the transition from harmless commensal to unrelenting pathogen is a fine line and one that is attributable to an extensive repertoire of virulence determinants selectively expressed under suitable predisposing conditions (232).

All pathogenic microorganisms have developed mechanisms that allow successful colonization or infection of the host (69). 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 defenses. The virulence factors expressed or required by Candida species, and in particular C. albicans, to cause infections may well vary depending on the type of infection (i.e., mucosal or systemic), the site and stage of infection, and the nature of the host response. It seems apparent that a panel of virulence attributes are involved in the infective process, but no single factor accounts for Candida virulence and not all expressed virulence attributes may be necessary for a particular stage of infection (40, 161). Although 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 studied in recent years (24). The reader is guided to several excellent reviews on the topics of hyphal formation, surface recognition molecules, and phenotypic switching listed in Table 1.

HYDROLYTIC ENZYMES

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One factor that contributes to the process of virulence is hydrolytic enzyme production, which is known to play a central role in the pathogenicity of bacteria (69), protozoa (141), and pathogenic yeasts (163). Although many microorganisms possess a variety of hydrolytic enzymes, proteinases are by far the most commonly associated with virulence.

All proteinases catalyze the hydrolysis of peptide bonds (CO—NH) in proteins but can differ markedly in specificity and mechanism of action (7). Proteinases are classified on the basis of their catalytic mechanism and not according to their anatomical origin, substrate specificity, or physiological function. In 1978, Enzyme Nomenclature distinguished four classes of proteinases: serine, cysteine, and aspartyl proteinases and metalloproteinases. Examples of serine proteinases are the divergent trypsin, chymotrypsin, and subtilisin subfamilies; cysteine proteinases include streptococcal proteinase and papain; and metalloproteinases include collagenases and microvillus proteinases (6). Aspartyl proteinases are ubiquitous in nature and are involved in a myriad of biochemical processes (41). Well-known aspartyl proteinases include the HIV aspartyl proteinase, and pepsin and renin in humans.

Most studies investigating the role of extracellular hydrolytic enzymes in fungal pathogenicity have focused on human-pathogenic fungi, including the filamentous fungus Aspergillus fumigatus (104, 176, 208), the dermatophytes Trichophyton rubrum (5) and Trichophyton mentagrophytes (236), and the dimorphic yeasts Cryptococcus neoformans (21), Coccidioides immitis (253), and C. albicans (50, 75, 94, 97, 145). While little is known about the extracellular proteinases of most dimorphic human pathogenic fungi, the proteolytic system of C. albicans is well described.

SECRETED ASPARTYL PROTEINASES OF CANDIDA

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The three most significant extracellular hydrolytic enzymes produced by C. albicans are the secreted aspartyl proteinases (Sap), phospholipase B enzymes, and lipases. Of these, the Sap proteins, encoded by a family of 10 SAP genes (66, 146, 147), have been the most comprehensively studied as key virulence determinants of C. albicans and are the subject of this review. For more information on phospholipases and lipases, the reader is guided to references 75 and 98.

C. albicans is not the only Candida species known to produce extracellular proteinases. Many of the pathogenic Candida species have been shown to posses SAP genes, including C. dubliniensis (76), C. tropicalis (147, 234, 254), and C. parapsilosis (53, 147), all of which produce active extracellular proteinases in vitro (76, 185). C. tropicalis is thought to possess four SAP genes (254), whereas C. parapsilosis possesses at least two SAP genes (53). Little published information is available with regard to the importance of Sap proteins in the virulence of C. dubliniensis. However, since C. dubliniensis probably possesses at least nine SAP genes (76) (J. R. Naglik, unpublished data), it is highly likely that proteinase production contributes to the virulence of this fungus. Less pathogenic or nonpathogenic Candida species do not appear to produce significant amounts of proteinase, even though they may possess aspartyl proteinase genes (see "Correlation between Sap production in vitro and Candida virulence" below). Finally, one should note that all secreted 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.

View larger version (17K): [in this window] [in a new window]   FIG. 1. Dendrogram displaying the relationship by sequence homology of the C. albicans Sap isoenzyme family. Three distinct groups are clustered within the family. Sap1 to Sap3 are up to 67% identical, and Sap4 to Sap6 are up to 89% identical, while Sap7 is only 20 to 27% identical to other Sap proteins. Sap9 and Sap10 both have C-terminal consensus sequences typical for GPI proteins and constitute the third distinct group. Similar Sap families exist in C. dubliniensis and C. tropicalis. Reprinted from reference 225a with permission.

Processing, activation, and regulation of the C. albicans proteinases. The pathway of proteinase synthesis starts in the nucleus, from where the newly synthesized mRNA is transferred to the cytoplasm and translated into the preproenzyme on the rough endoplasmic reticulum. The N-terminal signal peptide is removed in the rough endoplasmic reticulum by a signal peptidase (242), and the proenzyme transferred to the Golgi apparatus, where it is further processed after Lys-Arg sequences by a Kex2 proteinase (159, 235). Alternative but less efficient processing pathways for Saps are thought to exist (9, 115). Once activated, the enzyme is packaged into secretory vesicles and transported to the plasma membrane and either remains attached to the cell membrane, is incorporated into the cell wall via a GPI anchor (Sap9 and Sap10), or is released into the extracellular space.

Since the SAP gene family encode preproenzymes, the regulation of proteinase expression can be controlled either at the mRNA or protein level. Comparisons of Sap protein and mRNA levels at identical time points (246) and kinetic studies of proteinase secretion by protein labeling and immunoprecipitation (pulse chase experiments) (91) suggested that proteinase synthesis and secretion were tightly coupled, strongly implying that regulation of Sap activity occurred predominantly at the mRNA level. Studies using the proteinase inhibitor pepstatin A demonstrated that SAP2 expression in C. albicans was also regulated via a positive-feedback mechanism, since the proteolytic products of Sap2 and peptides of 8 amino acid residues or more induced this gene (96, 125).

Biochemical properties of the C. albicans proteinases. The Sap proteinases have been purified and characterized either by direct purification from Candida culture supernatants (reviewed in reference 94) or by expression in recombinant Pichia pastoris (16, 254) or Escherichia coli (115). This has provided a more detailed insight into the characteristics of the Sap isoenzymes, whose general biochemical properties are described in Table 3.

Based on present data (see the following sections), it is highly probable that the main roles of the C. albicans proteinases are to provide nutrition for the cells, to aid penetration and invasion, and to evade immune responses. However, the biochemical and proteolytic properties of the Sap7 to Sap10 enzymes are not presently known, and thus the full functional repertoire of the Sap family has yet to be elucidated.

For more details, the reader is guided to review articles on discovery and characterization of the SAP gene family; structure, processing, activation and regulation; purification, activity and enzymatic properties; and in vitro SAP gene expression in culture medium (50, 88, 94, 95, 97, 98, 145, 163, 185, 227). The present review is restricted to studies addressing the relationship between proteinase production and C. albicans pathogenesis.

SECRETED ASPARTYL PROTEINASES AND C. ALBICANS PATHOGENESIS

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Over the past two decades, a plethora of studies have contributed to our understanding and knowledge of the SAP gene family. The existence of 10 SAP genes in C. albicans and their controlled expression and regulation raises a number of questions concerning the roles and functions of these proteinases during the infective process. The complexity of Sap involvement in C. albicans virulence has 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, and phenotypic switching. Although the consequences of proteinase secretion during human infections is not precisely known, the roles and functions of the Sap family can perhaps be deduced from in vitro and in vivo animal model data. 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: (i) correlation between Sap production in vitro and Candida virulence, (ii) degradation of human proteins and structural analysis in determining Sap substrate specificity, (iii) association of Sap production with other virulence processes of C. albicans, (iv) Sap protein production and Sap immune responses in animal and human infections, (v) SAP gene expression during Candida infections, (vi) Modulation of C. albicans virulence by aspartyl proteinase inhibitors, and (vii) The use of SAP-disrupted mutants to analyze C. albicans virulence.

This review critically discusses the data relevant to each of these seven criteria, with specific emphasis on how this proteinase family could contribute to Candida virulence and pathogenesis in human infections. Each of the seven criteria is discussed in its own section.

(i) The virulence of C. albicans species appears to correlate with the level of Sap activity in vitro and may correlate with the number of SAP genes.

(ii) Infected patients (oral or vaginal) harbor C. albicans strains that are significantly more proteolytic than are isolates from asymptomatic carriers.

(iii) HIV infection appears to lead to the selection of C. albicans strains with heightened virulence attributes such as proteinase production.

Numerous studies have correlated extracellular proteolytic activity in vitro with the virulence of Candida species and have shown that only the most virulent species such as C. albicans, C. tropicalis and C. parapsilosis produce more proteinases in vitro than do less virulent species (185). 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 (131, 174, 189). The apparent clear-cut correlation between proteinase production and virulence may be due in part to the sensitivity of the assays used to determine proteolytic activity. For example, the BSA-agar method, which has been routinely used over the years (163), is a relatively insensitive hydrolysis assay that is unlikely to detect low levels of proteinase activity. This notion was supported when a sensitive, rapid fluorescence-based assay was developed that was able to detect aspartyl proteinase activity in all Candida species tested, in the order C. albicans > C. tropicalis > C. kefyr > C. lusitaniae > C. krusei (26). Since the proteolytic activity could be inhibited with pepstatin, this study demonstrated that some non-C. albicans species that were previously thought not to possess Sap activity are in fact proteolytic. It should be noted that although more virulent Candida species may in fact produce more detectable proteolytic activity in vitro, this does not necessarily imply that the production of Sap enzymes is the sole reason for virulence.

Many Candida species possess aspartyl proteinase genes. Monod et al. (147) used Southern analysis with SAP1 as a probe to demonstrate the presence of four bands with sequence similarity in EcoRI-digested genomic DNA of C. guilliermondii, although this yeast does not produce proteinase in vitro in BSA-containing medium. However, even though C. guilliermondii and possibly other Candida species may possess SAP-like genes, it is not known whether they are functional in vivo. Although these less pathogenic Candida species may be capable of producing secreted proteinases, in general the amount of Sap activity produced in vitro and possibly the number of SAP genes appear to be directly correlated to the virulence of the Candida species.

Sap production by clinical C. albicans isolates from humans. C. albicans strains from different patient groups with various clinical infections have been isolated, and the level of Sap activity produced in vitro has been correlated with virulence (Table 4). Most of these studies have concentrated on C. albicans strains isolated from the vaginal lumen or the oral cavity and on the effect of HIV infection on proteinase production and C. albicans strain selection.

A similar approach has been applied to oral isolates of C. albicans, mainly from HIV-positive individuals. C. albicans isolates from 100 HIV-positive patients with oropharyngeal candidiasis produced significantly more proteinase activity than did isolates from 122 patients without HIV infection (50 with oral candidiasis and 72 asymptomatic Candida carriers) (166). The higher level of proteinase activity correlated with the increased level of cell surface-associated and secreted Sap, as revealed by cytofluorometry and Western blotting, respectively. A similar study, but using fewer patients, reported a comparable increase in Sap activity in C. albicans strains isolated from the oral cavities of 44 HIV-positive patients (advanced disease) with oral candidiasis compared with that in 30 HIV-negative C. albicans carriers (46). However, since a control group of HIV-negative subjects with oral candidiasis was not included, it is unclear whether the observed increase in Sap production resulted from the advanced HIV status of the individuals or from the Candida infection. Likewise, Wu et al. (252) found that oral C. albicans isolates from HIV-positive subjects (n = 18) produced significantly more proteinase than did isolates from HIV-negative individuals (n = 18) when they were investigated in a BSA agar plate assay. Finally, high Sap-producing oral (48) and vaginal (49) C. albicans strains isolated from HIV-positive patients were more pathogenic in the mouse and rat vaginitis models, respectively, than were lesser Sap-producing C. albicans strains from HIV-negative patients (Table 4).

In summary, 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. Whether these observations reflect an elevated "fitness" or a specific adapted response of C. albicans strains during infection is not clear, but the data tentatively support a role for the proteinases during the infective process in vivo.

C. albicans strain selection in HIV infection. There is mounting evidence that Candida species colonizing the oral cavities of HIV-infected individuals are subject to selective pressures that may lead to the emergence of strains with altered genotypic and phenotypic characteristics and enhanced expression of known and putative virulence determinants. Studies have shown that the genotype of the infecting Candida cells in HIV infection is stable, and, as a result, HIV-infected patients tend to be colonized by a single endogenous strain of Candida that persists throughout recurrent bouts of oral candidiasis, even after antifungal therapy (34, 130, 143, 169, 175, 204, 245, 248). However, these and other studies also suggest that in the majority of AIDS patients the original commensal strains are replaced and that this replacement of genotypes occurs only once, early in the course of HIV infection, producing a genetically conserved population (140, 204).

Both Ollert et al. (166) and De Bernardis et al. (46) showed that the increase in Sap activity was observed only in patients with advanced HIV infection and not in those with earlier stages of HIV infection or HIV-negative subjects. This indicated that more virulent biotypes of C. albicans with heightened proteinase production might be selected in HIV-infected patients. However, it should be pointed out that this selection appeared to occur before patients developed AIDS and was independent of CD4⁺ counts.

One intriguing possibility for the observed differences in Sap production between HIV-positive and HIV-negative patients may be due to the direct binding of HIV proteins to Candida cells. Treatment of C. albicans with gp160, but not with gp120, led to an elevation of free and cell-bound aspartyl proteinase (82). In addition, culture supernatants obtained from C. albicans treated with gp160 or gp41, but not with gp120, showed a strong increase in proteinase activity. Why or how HIV gp160 or gp41, but not gp120, influences proteinase production and whether they modulate Sap secretion directly or indirectly through another mechanism remain to be elucidated. HIV infection might also promote C. albicans virulence in another way, since the HIV transactivating protein Tat binds RGD sequences present on the surface of C. albicans to induce hyphal production (81), a process known to be linked with virulence and the expression of the SAP4 to SAP6 subfamily (96, 246).

The pathobiological effects of HIV infection, including possible epithelial cell surface changes (170), reduced salivary flow rate (203), and alterations in the oral microflora (171), might also influence the candidal microenvironment. Together with impaired humoral or cell-mediated mucosal immunity and/or impaired nonspecific host defenses, these selective pressures in HIV infection are likely to contribute to the selection of Candida strains, some of which may possess altered or heightened virulence attributes such as proteinase production (232). However, the mechanism by which these selective pressures contribute to strain selection in HIV infection remains to be elucidated and may prove particularly challenging to resolve.

(i) Sap2 has very broad substrate specificity and can degrade many human proteins.

(ii) The crystal structure of Sap2 indicates that the C. albicans proteinase family is unique among the aspartyl proteinases.

(iii) Computer modeling suggests that the electrostatic charge of the different Sap proteins may contribute to different substrate specificities and tissue targeting.

The first observation of proteolytic activity in C. albicans was demonstrated by Staib (222) when yeast cells were grown in media containing BSA as the sole source of protein. Three years later, Remold et al. (177) 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 proteinase from C. albicans and the effect of environmental factors such as pH and temperature on proteolytic activity (Table 3). The culture conditions used to induce proteinase activity in these early reports have subsequently been shown to favor SAP2 expression (96, 246). 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, it is not clear whether the digestion of substrates by Sap2 in vivo is similar to that shown in vitro or whether the substrates for Sap2 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 Sap2 have been described in some detail.

Broad substrate specificity of Sap2. One of the most noticeable properties of Sap2 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, is illustrated in Fig. 2. Sap2 is known to degrade many human proteins including molecules that protect mucosal surfaces such as mucin (36, 52) and secretory immunoglobulin A (IgA) (78, 184). 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 enzymes (109), and can inhibit C. albicans attachment to buccal epithelial cells (243). Supporting these data, Wu and Samaranayake (251) noted that reduction of total salivary protein concentration correlated with the degree of Sap expression, suggesting that Candida Sap proteins degrade salivary proteins in the oral cavity. Sap2 can also degrade molecules of the extracellular matrix such as keratin, collagen and vimentin (94, 163, 174). Morschhauser et al. (152) showed that induction of C. albicans proteinase caused digestion of soluble and immobilized extracellular matrix proteins produced by a human endothelial cell line, suggesting that Sap proteins may facilitate the dissemination of C. albicans via the circulatory system.

View larger version (48K): [in this window] [in a new window]   FIG. 2. Schematic diagram illustrating the contribution of the various virulence attributes to C. albicans pathogenicity. C. albicans commonly colonizes the epithelial surface (stage 1) and causes superficial infections (stage 2), but under conditions when the host is compromised, the fungus establishes deep-seated infections (stage 3) by penetrating further into the epithelial tissue. Occasionally, C. albicans causes disseminated infections (stage 4), which allow the fungus to colonize and infect other host tissues and can be fatal. This infective process involves numerous virulence attributes including adhesins, hydrolytic enzyme production (Sap proteins, phospholipases, and lipases), hypha formation, and phenotypic switching. Sap2 (and possibly other Sap proteins) is known 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 C. albicans. Modified from reference 160 with permission.

Deducing proteinase specificity via three-dimensional structure and molecular modeling. The substrate specificity of Sap2 is noticeably very broad, and some researchers thought that this broad specificity could be deduced from its three-dimensional structure. Accordingly, two reports on the crystal structure of Sap2 complexed with a potent inhibitor (A-70450 [see "Modulation of C. albicans virulence by aspartyl proteinase inhibitors" below]) were published (1, 39), which indicated that the Sap2 structure conforms to the classical aspartyl proteinase fold typified by pepsin. However, comparisons of Sap2 with pepsin have revealed a number of major differences that may contribute to the broad substrate specificity of Sap2 and which make the C. albicans proteinase family unique among the aspartyl proteinases (1, 39). Specifically, Sap2 has an enlarged and well-defined cavity for binding the third residue N-terminal to the cleaved bond in the substrate and two "flaps" overlying this cavity, the latter observation being a hallmark of the Sap family.

At present, the structure of Sap2 alone has been determined, and although other members of the Sap family are known to contribute to C. albicans virulence, very few data are available regarding their structures or substrate specificities. To address this, Stewart et al. (227) undertook a comparative structural study with the sequences of SAP1 SAP6 by molecular modeling. Although the structures of Sap1 to Sap6 and the electrostatic charge of their active sites were generally similar, sufficient differences existed to allow for different substrate specificities, with the difference between Sap1 to Sap3 and Sap4 to Sap6 being clearly evident. Furthermore, a potentially significant trend in the total electrostatic charge of the Sap1 to Sap6 enzymes was observed; the six enzymes had an overall net charge of -8, -21, -22, -5, +2, and +2, respectively. It is somewhat puzzling that the charge of the non-active-site regions of Sap1 to Sap6 varies so much, but it might contribute in part to our understanding of the different pH optima and range of pH activities of the Sap enzymes (ranging from pH 2.0 to 7.0). One more interesting observation resulting from the molecular modeling of Sap1 to Sap6 was the clear difference in the carboxy end-terminal extension between SAP1 to SAP3 and SAP4 to SAP6 at amino acid positions 323 to 324 and 335 (SAP1 to SAP3, NE and A, SAP4 to SAP6 = RK and Q, E) (227). While speculative, this carboxy-terminal extension appears to resemble an "attachment" anchor (C. Abad-Zapatero, personal communication). If true, this may support the hypothesis that the C. albicans proteinases may target specific cell proteins or tissue compartments during the infective process.

Discussion. Although proteinases other than Sap2 (specifically Sap1 and Sap3 to Sap6) have recently been purified (16, 210, 246), it remains to be determined whether they have the same broad substrate specificities as Sap2. Furthermore, purified proteins of Sap7, Sap8, Sap9, and Sap10 have not been isolated or biochemically characterized, and thus the proteolytic properties of these proteinases remain totally unknown. On the one hand, it might seem unlikely that the different members of the Sap family have the same broad substrate specificities as Sap2, since it would seem unnecessary for C. albicans to possess a family of 10 proteins which are differentially expressed under a variety of environmental conditions and in different tissues (see "SAP gene expression during Candida infections" below) simply to digest the same substrates. On the other hand, C. albicans may require a family of extracellular proteolytic enzymes, each optimized to certain environmental conditions or different local pH values and/or particular tissues, to help the fungus colonize and infect multiple sites of the body. However, there are clearly differences in the substrate specificities of the Sap proteins; for example, sequence similarities of C. albicans Sap9 and Sap10 to the S. cerevisiae yapsins, including potential C-terminal consensus sequences for GPI anchors, suggest that Sap9 and Sap10 may have different specificities and functions from the other C. albicans Sap proteins (A. Albrecht, I. Pichova, M. Monod, and B. Hube, unpublished data).

In all likelihood, there is probably considerable overlap in the substrate specificities of many of the Sap members. Since the pH activity of the individual hydrolytic enzymes range between pH 2.0 and 7.0 (75, 182, 210, 247), this would allow for the concomitant expression of a number of similar SAP genes at environments with different pH values. In addition, there are differences in the promoter sequences of the different SAP genes, which indicates that their expression might be controlled by different SAP-specific transcriptional regulators and possibly suggests that the SAP genes might have evolved to possess distinct functions. Moreover, the coordinated regulation of the SAP genes with other virulence factors, including hyphal formation and phenotypic switching, would permit several proteinases to act in unison to carry out a series of tasks to not only digest a complex mixture of target proteins but also to provide C. albicans with a biological advantage to specifically enhance the pathogenic ability of the fungus (97). With these considerations in mind, it is entirely plausible that C. albicans has adapted to certain niche sites by expressing a combination of SAP genes (and other virulence genes), which are called upon as and when required.

(i) Sap proteins facilitate C. albicans adherence to many host tissues and cell types.

(ii) Hypha formation and SAP4 to SAP6 expression are coordinately regulated, but the signaling pathways remain to be elucidated.

(iii) SAP1 appears to be regulated by phenotypic switching, but the contribution of switching to C. albicans virulence in vivo is not yet clear.

Many of the early proteinase studies focused on the influence of culture conditions on Sap expression and proteolytic activity in vitro (reviewed in reference 94). 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 hyphal state and can undergo phenotypic switching (214). 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 including adherence was investigated.

Sap production and C. albicans adherence. Adhesion of Candida to host tissues allows the fungus to attain a foothold and to colonize a specific niche environment. Under suitable predisposing conditions when the host is compromised, this colonized site provides the base for candidal proliferation, invasion, and, in some instances, dissemination. Adherence of C. albicans to host cells is a complex, multifactorial process involving several types of candidal adhesins on a morphogenically changing cell surface (reviewed in the references in Table 1), and one mechanism through which Candida adherence might be promoted is via the production of proteinases.

One of the first early studies to link proteinase production to adherence in C. 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 (74). A more recent report correlated proteinase production with increased adherence to buccal epithelial cells and death of mice; the higher-Sap-producing strains showed greater levels of tissue colonization in the liver, kidneys, and spleen (2). However, the majority of studies linking Sap production with C. albicans adherence have been performed using the proteinase inhibitor pepstatin, which inhibits Sap2 (and probably Sap1 and Sap3) very efficiently (168). Borg and Rüchel (12) demonstrated a marked reduction in C. albicans adhesion and invasion of human mucosa by pepstatin, and a similar reduction of C. albicans adherence using pepstatin was also shown with human epidermal cells (60, 165). Pepstatin could also inhibit the development of cavitations after yeast cells adhered to epidermal corneocytes (173). Some years earlier, Klotz et al. (114) observed that yeast cells formed cavitations and burrowed rapidly into vascular endothelium in vitro by a mechanism independent of germ tube formation. However, at that time the burrowing was not associated with proteinase production, but the results of the work by Ray and Payne (173) clearly implicated proteinases in the process.

The actual Sap proteins involved in adherence and cavitation (and possibly subsequent penetration) of host tissues were not studied, but a recent report by Kvaal et al. (121) indicated that Sap1 might be involved. Using a gene misexpression strategy in the switching strain WO-1, in which white-phase cells misexpressed the opaque-specific gene SAP1, the authors demonstrated in a cutaneous mouse model that SAP1 conferred two opaque-specific characteristics upon white cells: increased adhesion and the capacity to cavitate skin (237). Interestingly, the addition of pepstatin inhibited cavitation but not the enhanced adhesion (which confirmed the data of Ray and Payne [173]), suggesting that cavitation was the consequence of secreted Sap1 enzyme, while increased adhesion was the result of other cell-associated factors. Other, more recent studies using HIV aspartyl proteinase inhibitors have also implicated Sap1 to Sap3 in C. albicans adherence; however, these studies are explained in full in "Modulation of C. albicans virulence by aspartyl proteinase inhibitors" (below).

(i) How do Sap proteins contribute to adherence? These pepstatin studies demonstrating the inhibition of C. albicans adherence clearly indicate that the Sap family plays some kind of role in C. albicans adherence. Although the precise mechanisms by which Sap proteins contribute to the adherence process are not clear, two hypotheses are currently favored. In the first, C. albicans proteinases 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 cells (i.e., epithelial cells), which may alter surface hydrophobicity or lead to conformational changes, thus allowing better adhesion of the fungus (145). If the Sap proteins can indeed function directly as C. albicans adhesins, this will add to the growing number of virulence properties already possessed by the Sap family (i.e., tissue damage, invasion, and evasion of host defenses) and establish the proteinases as one of the most versatile and multifunctional virulence gene families possessed by C. albicans.

Sap production and yeast-to-hypha transition. Research efforts by many investigators in different laboratories have concentrated on the study of C. albicans morphogenesis, as well as the identification and characterization of cell wall components that are growth phase (yeast and hypha) specific and associated with virulence. The foundation of these studies is based on two factors: (i) the common acceptance that the hyphal form is related to the invasive properties of C. albicans and (ii) the importance of morphogenesis as a biological phenomenon.

The ability of C. albicans to transform into hyphae may be considered a pathogenic determinant in the initial processes of superficial tissue invasion, whereby hyphae may promote the adherence and penetration of C. albicans to host tissues. In culture medium, the main proteinases associated with hyphal formation are the SAP4 to SAP6 subfamily (96, 246), and pH and hypha induction alone are sufficient for the induction of SAP4 to SAP6 (it should be noted that SAP4 transcripts were not detected in several experiments).

(i) Coordinate regulation of hypha-formation and SAP4 to SAP6 expression. Although hypha formation and SAP4 to SAP6 expression were linked, more direct proof was required to determine whether the two phenomena were coordinately regulated. Sequence analysis of the promoter regions of SAP4 to SAP6 revealed the presence of consensus sequences [CATTC(A/C)] for the TEA/ATTS transcription factor Tec1 (206). C. albicans mutant strains lacking TEC1 failed to produce hyphal cells in vitro and were not able to express SAP4 to SAP6, suggesting the existence of joint or coordinated regulatory pathways for hypha production and proteinase expression. The concept of coordinated pathways was supported by the observation that SAP4 to SAP6 expression increased in a hyperfilamentous strain lacking CPP1 (a mitogen-activated protein kinase phosphatase) (205) and had a modified expression pattern in a strain lacking EFG1 (a key transcriptional regulator of dimorphism), which has a strongly reduced ability to form hyphae (228). Moreover, in a murine systemic intraperitoneal model, the EFG1-deficient mutant had a strongly reduced ability to produce hyphae, which was associated with reduced expression of SAP4 to SAP6 and an inability to invade or damage parenchymal organs including the liver and pancreas (65). Interestingly, a triple null C. albicans mutant lacking SAP4 to SAP6 showed strongly reduced invasiveness but still produced hyphal cells. Finally, SAP5 activation during in vivo infection was shown not to depend on growth of C. albicans in the hyphal form; however, the two major hyphal signaling pathways in C. albicans (defined by Cph1 and Efg1) were required for SAP5 expression (224). Together, these studies indicate that not only are hypha formation and proteinase production coordinately regulated but also C. albicans hyphal cells require the support of hydrolytic enzymes (specifically SAP4 to SAP6) in order to be fully invasive in vivo.

The observation that certain transcriptional factors which regulate the yeast-to-hypha transition also regulate proteinase expression has recently been addressed using C. albicans DNA microarrays. Transcriptional profiling of C. albicans mutants lacking factors that regulate the dimorphic transition has helped to elucidate signaling pathways and to clarify the coordinated regulation between morphology and proteinase production. The reader is guided to "Functional genomics and Candida" (below) for more details.

Sap production and phenotypic switching. The selection of phenotypically altered strains may be enhanced in C. albicans by a phenomenon known as high-frequency phenotypic switching, whereby Candida cells randomly switch their phenotype, especially in response to stress (217, 218). While many prokaryotic and eukaryotic microorganisms can switch between alternate phenotypes under different environmental conditions (207), C. albicans appears to have an enhanced ability for chromosomal rearrangement and genetic reorganization. Unlike switching in other microbial pathogens, switching in C. albicans is pleiotropic, affecting several morphological and physiological parameters and a number of virulence traits (214), all of which may allow the fungus to adapt to different host environments during the course of an infection. Recent work suggests that phenotypic switching is based on heritable changes in chromatin structure and supports the notion that acetylation of histones plays a selective role in regulating the switching process (113, 220).

(i) Which SAP genes are regulated by phenotypic switching? In a C. albicans strain named WO-1, the discovery and characterization of the white (W)-to-opaque (O) transition indicated that switching could affect a variety of cellular characteristics, including proteinase production (214). As a result, a correlation between Sap secretion and switching was subsequently described in C. albicans strains WO-1 (151) and 3153A (149). In both strains, transcripts of SAP1 were abundant in specific switching-regulated forms and Sap1 was primarily responsible for the higher extracellular proteolytic activity observed in these switching states. As a result, SAP1 was the first cloned switching-regulated gene detected in C. albicans (151), a year after the gene was first isolated by Hube et al. (101).

Sequence analysis of the 5'-untranslated regions of SAP1 from different C. albicans strains indicated that during switching, SAP1 expression was regulated by activation or deactivation of phase-specific trans-acting factors, which in turn were regulated by a "master switch" event (215). Since SAP1 was shown to be switching regulated, it was not surprising that the expression of this gene was not dependent on the presence of exogenous protein (96, 151, 246). This is in contrast to SAP2 (not switching regulated), which is expressed in both the white and opaque phenotypes of C. albicans strain WO-1 but only in the presence of exogenous protein (96, 246).

SAP3 expression may also be regulated by phenotypic switching (150, 247), but its regulation is different from that of SAP1 and SAP2 in that SAP3 is detected in C. albicans strains when SAP2 is expressed (96, 210, 246). Another SAP gene that is differentially expressed during switching is SAP8, since transcripts were detected in the opaque but not the white phenotype (99). However, since SAP8 was shown to be up-regulated at 25°C compared with 37°C (146) and since the opaque phenotype is stable only at 25°C (149, 151, 219), this suggested that SAP8 expression in opaque cells may be temperature regulated rather than switching regulated.

At present, in C. albicans strain WO-1, SAP1 is the only proteinase that is strictly regulated by phenotypic switching. However, phenotypic switching is a very complicated process, which is by no means fully understood. In fact, very little is known about this phenomenon outside of C. albicans strain WO-1. Other clinical or laboratory strains may have switching processes divergent from or even unrelated to that of WO-1, each affecting SAP gene expression and other virulence genes in distinctive ways. Therefore, it cannot yet be concluded which proteinases are regulated by switching in vivo or what contribution this phenomenon makes, in terms of SAP gene expression, to the virulence of C. albicans.

Discussion. 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. At the most basic level, one could conclude that yeast cells predominantly express one set of SAP genes (SAP1 to SAP3), hyphae predominantly express another (SAP4 to SAP6), and phenotypically switched cells predominantly express yet another (SAP1 and SAP3). Although this might be attractive, it is almost certainly too simplistic, since these conclusions have usually been drawn from the use of one strain of C. albicans grown under laboratory-controlled conditions. In vivo, the environmental milieu and immune selective pressures may affect SAP gene expression and phenotypic switching in individual yeast and hyphal cells in a unique fashion, which cannot be tested or controlled for in the laboratory. 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 regulation.

(i) Sap proteins are produced in vivo during mucosal and systemic infections.

(ii) Proteinases are localized to the cell wall during C. albicans infections.

(iii) C. albicans proteinases are immunogenic and elicit mucosal and systemic antibody responses.

(iv) The inhibitory and protective effects of Sap antibodies against Candida infections remain unclear and the protective B- and T-cell epitopes of the Sap family are unknown.

Sap protein production during C. albicans infections. Several studies have provided strong evidence demonstrating the production of Sap protein in vivo. Early work using murine models of disseminated candidiasis revealed the presence of Sap proteins on the surface of C. albicans cells in murine kidneys (116, 132). Using indirect-immunofluroescence microscopy, the presence of Sap proteins was also detected within the cell wall of yeast and hyphal cells in all organs of immunocompromised patients who had succumbed to systemic C. albicans infections, including the mucosa, central nervous system, lungs, heart, liver, pancreas, and kidneys (190). With regard to mucosal infections, elevated levels of Sap proteins were observed in vaginal fluids of candidiasis patients compared with Candida carrier subjects as determined by enzyme-linked immunosorbent assay and immunoblotting using Sap2 polyclonal antibodies (43), indicating a link between Sap production in vivo and infection.

The presence of Sap protein has also been demonstrated during phagocytosis of C. albicans by leukocytes. Macdonald and Odds (134) 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 surface (13). Recently, the Sap4 to Sap6 family have been implicated in the evasion of phagocytosis by C. albicans, since the expression of Sap4 to Sap6 but not Sap1 to Sap3 was upregulated on yeast and germ tubes after phagocytosis by murine peritoneal macrophages (16). In the same study, C. albicans mutants lacking SAP4 to SAP6 were significantly more susceptible to phagocytosis than were wild-type cells. These results strongly indicate that by preventing macrophage killing, Sap4 to Sap6 play at least one significant role in evading host immune defenses.

Sap localization to the cell wall. More recent studies using immunogold-labeling techniques demonstrated that Sap proteins are localized to the cell wall during C. albicans infections. In a rat vaginitis model, Sap1 to Sap3 were present in the yeast cell wall during early stages of infection, a pattern that correlated with the in vitro localization of Sap (229). Three studies using polyclonal antibodies raised against Sap1 to Sap6 demonstrated the presence of Sap1 to Sap3 on the surface of both yeast and hyphal cells, while Sap4 to Sap6 antigens were found predominantly on hyphal cells (65, 119, 202). Also, in biopsy specimens of oral epithelial lesions collected from three HIV-infected patients with oropharyngeal candidiasis, most Sap was secreted at the locations where C. albicans directly adhered to epithelial cells or at sites in close contact between C. albicans and epithelial cells (199). However, it is important to recognize that the Sap1 to Sap3 and Sap4 to Sap6 antibodies used were not able to differentiate between the individual Sap proteins within each of the two subfamilies investigated. Therefore, the identities of the individual proteinases that localize to the cell wall or are detected in vivo during experimental infections could not be determined. Antibodies specific for individual Sap proteins do not yet exist, but the development of such antibodies would be a valuable addition to existing molecular techniques in determining the localization patterns of the individual proteinases as well as the expression patterns of the proteinases during different stages and types of C. albicans infections.

Antibodies against Sap induced by C. albicans infections. (i) Antibodies produced during systemic infection. Numerous studies have described the presence of Sap protein during C. albicans infections; however, few studies have described antibody responses to the C. albicans proteinases in human patients. Macdonald and Odds (133) were the first to detect proteinase-specific IgG antibodies in sera of patients with disseminated candidiasis at significantly higher levels than those found in healthy individuals. These findings were later confirmed by Ray and Payne (172) and Rüchel et al. (186, 187), further demonstrating the production of proteinases during systemic candidiasis. However, in the latter study, sera of a fifth of the patients suffering from candidiasis did not produce high titers of antibodies against purified Sap2, probably reflecting the inability of many high-risk patients to mount a normal immune response (185).

(ii) Antibodies produced during mucosal infection. The above studies investigated anti-Sap IgG responses during systemic infections, but few studies have investigated the IgA response, and in particular secretory IgA, to Sap proteins during mucosal Candida infections, such as those in the oral cavity and vaginal lumen. This would clearly be more relevant than IgG responses, since IgA is the predominant antibody present at mucosal surfaces and is known to prevent the attachment of C. albicans to the mucosal epithelium (243).

Two reports have recently addressed this issue. Using a time-resolved immunofluorometric assay, total levels of IgA against Sap1, Sap2, and Sap6 were found to be higher in saliva from HIV-positive patients with oral and oropharyngeal candidiasis than from HIV-positive patients without oral candidiasis or HIV-negative healthy controls (57, 142). The authors concluded that during oral infection, HIV-positive patients have an increased mucosal antibody response specifically directed against C. albicans virulence antigens, in this case the proteinases (57). However, this study did not include an HIV-negative patient group with oral candidiasis, so the observed increase in the level of salivary IgA against the proteinases may be related to the HIV status of the individual as well as to candidiasis. Interestingly, over the 1-year period, variations in Candida colonization levels in the oral cavity and episodes of oropharyngeal candidiasis correlated with variations in salivary anti-Sap6 IgA antibody levels (142). This may indicate a direct relationship whereby as C. albicans numbers proliferate during mucosal infections, more Sap6 is produced, resulting in the induction of a corresponding mucosal IgA antibody response. However, since it is highly likely that the polyclonal antibodies induced by certain Sap proteins during human mucosal infections cross-react with other Sap proteins (especially within homologous subfamilies), more work is clearly needed before this hypothesis can be substantiated.

Functional anti-Sap antibodies. Antibodies have many functions in many diseases, but the role of antibody immunity in protection against mucosal and systemic candidiasis is unclear. Indeed, the majority of patients with mucosal Candida infections have normal or even elevated levels of both serum and mucosal anti-Candida antibodies (37, 112, 137, 233). This indicates that although patients are able to produce high levels of antibodies in response to Candida infection, these high antibody titers are not able to clear candidal infection. However, secretory IgA antibodies are able to bind to Candida and reduce the adherence of Candida to epithelial cells, theoretically preventing or maintaining low levels of Candida colonization (126, 243).

(i) Systemic infections. In patients with systemic candidiasis, there is some evidence for a role of anti-C. albicans antibodies in recovery from infection. Development of antibodies against C. albicans heat shock protein 90 (HSP90) appear to be protective against human and animal Candida infections; higher titers of antibodies against a 47-kDa breakdown product of C. albicans HSP90 were present in patients recovering from candidemia than in the early stages of infection (108, 139), and antibodies against this 47-kDa product were protective in a murine model of systemic candi