Candida albicans proteinases

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is a continuing challenger for the medical mycology field. Attributes that contribute ... and better agents that target fundamental biological processes and or pathogenic determinants ..... defined protein-free media34-35. The expression of SAP8 ...

Braz J Oral Sci. January-March 2006 - Vol. 5 - Number 16

Candida albicans proteinases Rita de Cássia Mardegan1 Mary Ann Foglio2 Reginaldo Bruno Gonçalves3 José Francisco Höfling4 1

DDS, MSc, Graduate Student at the University of Campinas – Piracicaba Dental School Department of Oral Diagnosis, São Paulo, Brazil 2 DDS, PhD, Professor - Research Center for Chemistry, Biology and Agriculture (CPQBA) Department of Phytochemical, University of Campinas, São Paulo, Brazil 3 DDS, PhD, Professor at the University of Campinas - Piracicaba Dental SchoolDepartment of Oral Diagnosis, São Paulo, Brazil,. 4 BS, MS, PhD, Professor at the University of Campinas - Piracicaba Dental School Department of Oral Diagnosis, São Paulo, Brazil.

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

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

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: [email protected]

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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 determinants 2. 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 action 8. 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 processes 3 . These more direct virulence functions may include digesting or distorting host cell membranes to facilitate adhesion and tissue invasion,

Candida albicans proteinases.

which has been demonstrated in plants9 and insects 10, 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 fumigatus 12 , Cryptococcus neoformans13, and C. albicans14-17. 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. dubliniensis 18 C. tropicalis and C. parapsilosis 19 . 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 probe22-23, 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

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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. albicans 29 , C. dubliniensis 18 , C. tropicalis 30 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 nitrogen34-35 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

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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 mucin39-40 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 cells 43 . Wu and Samaranayake 44 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 vimetin6,28,31. C albicans proteinases may also evade host defenses by directly degrading

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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 strains23,47. 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 media34-35. 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

Candida albicans proteinases.

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 carriers52-53. 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 candidiasis 54. All reports showed that C. albicans isolates from symptomatic patients with vaginal candidiasis were significantly more proteolytic than isolates from asymptomatic vaginal carriers55-56. 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

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greater levels of tissue colonization in the liver, kidneys, and spleen 58. 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 fungus 14. 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 Odds 59 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 60 . The discovery that C. albicans possessed a multitude of proteinase genes that were differentially expresses under a variety of environmental conditions in vitro43-44 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

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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 determined64-65. 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

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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 directly67-68. A total of four HIV Pis, namely Ritonavir, Saquinavir, Indinavir and Nelfinavir, have been investigated for their ability to inhibit Candida proteinase activity69-72. 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 candidiasis 75. The identification of novel Candida Sap inhibitors, either

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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 species80-81, numerous xanthones from Tovomita krokovvi82, the serratene triterpenes from Lycopodium cernuum 83, 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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References 1. 2. 3.

4. 5.

6. 7.

8. 9.

10.

11.

12.

13. 14.

15.

16. 17. 18.

19.

20.

21.

22.

23.

24.

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Sweet SP. Selection and pathogenicity of Candida albicans in HIV infection. Oral Dis. 1997; 3: s88-s95. Finlay BB, Falkow S. Common themes in microbial pathogenicity. Microbiol Rev. 1989; 53: 210-30. Naglik JR, Challacombe SJ, Hube B. Candida albicans secreted aspartyl proteinases in virulence and pathogenesis. Microbiol Mol Biol Rev. 2003; 67: 400-28. Calderone RA, Fonzi WA. Virulence factors of Candida albicans. Trends Microbiol. 2001; 9: 327-35. McKerrow JH, Sun E, Rosenthal PJ, Bouvier J. The proteases and patogenicity of parasitic protozoa. Annu Rev Microbiol. 1993; 47: 821-53. Ogrydziak DM. Yeast extracellular proteases. Crit Rev Biotechnol. 1993; 13: 1-55. Cunningham E L. Agard D A. Disabling the folding catalyst is the last critical step in alpha-lytic protease folding. Protein Sci. 2004; 13: 325-31. Barrett AJ, Rawlings ND. Types and families of endopeptidases. Biochem Soc Trans. 1991; 19: 707-15. Choi GH, Pawlyk DM, Rae B, Shapira R, Nuss DL. Molecular analysis and overexpression of the gene encoding endothiapepsin, an aspartic protease from Cryphonectria parasitica. Gene. 1993; 125: 135-41. Smithson SL, Paterson IC, Bailey AM, Screen SE, Hunt BA, Cobb BD, et al. Cloning and characterization of gene encoding a cuticle-degrading protease from the insect pathogenic fungus Metarhizium anisopliae. Gene. 1995; 166: 161-5. Salyers A, Witt D. Virulence factors that promote colonization. In: Salyers A., Witt D (cd), Bacterial pathogenesis: a molecular approach. Washington: ASM Press; 1994. p. 30-46. Jaton-Ogay K, Paris S, Huerre M, Guadroni M, Falchetto R, Togni G, et al. Cloning and disruption of the gene encoding an extracellular metalloprotease of aspergillus fumigatus. Mol Microbiol. 1994; 14: 917-28. Brueske CH. Proteolytic activity of a clinical isolate of Cryptococcus neoformans. J Clin Microbiol. 1986; 23: 631-3. Monod M, Borg-Von Zepelin M. Secreted proteinases and other virulence mechanisms of Candida albicans. Chem Immunol; 2002. 81: 114-28. De Bernardis F, Sullivan PA, Cassone A. Aspartyl proteinases of Candida albicans and their role in pathogenicity. Med Mycol. 2001. 39: 303-13. Hube B, Naglik J. Candida albicans proteinases: resolving the mystery of a gene family. Microbiology. 2001; 147: 1997-2005. Ghannoum MA. Potential role of phospholipases in virulence and fungal pathogenesis. Clin Microbiol Rev. 2000; 13: 122-43. Gilfillan GD, Sullivan DJ, Haunes K, Parkinson T, Coleman DC, Gow NA. Candida dubliniensis: phylogeny and putative virulence factors. Microbiology. 1998; 144: 829-38. Monod M, Togni G, Hube B, Sangland D. Multiplicity of genes encoding secreted aspartic proteinases in Candida species. Mol Microbiol. 1994; 13: 357-68. Hube B, Turver CJ, Odds FC, Eiffert H, Boulnois GJ, Köchel H, et al. Sequence of the Candida albicans gene encoding the secretory aspartate proteinase. J Med Vet Mycol. 1991; 29: 129-32. Wright RJ, Carne A, Hieber AD, Lamont IL, Emerson GW, Sullivan PA. A second gene for a secreted aspartate proteinase in Candida albicans. J Bacteriol. 1992; 174: 7848-53. Monod M, Togni G, Hube B, Sanglard D. Multiplicity of genes encoding secreted aspartic proteinase. Mol Microbiol. 1994; 13: 357-68. White TC, Miyasaki SH, Agabian N. Three distint secreted aspartic ptoteinase in Candida albicans. J. Bacteriol. 1993; 174: 6126-33. Miyasaki SH, White TC, Agabian N. A fourth secreted aspartic

25.

26.

27.

28. 29.

30.

31.

32. 33.

34.

35.

36. 37.

38. 39.

40.

41. 42.

43.

44.

proteinase gene (SAP4) and a CARE2 repetitive element are located upstream of the SAP1 gene in Candida albicans. J Bacteriol. 1994; 176: 1702-10. Newport G, Agabian N. KEX2 influences Candida albicans proteinase secretion and hyphal formation. J Biol Chem. 1997; 272: 28954-61. Togni G, Sanglard D, Quadroni M, Foundling SI, Monod M. Acid proteinase secreted by Candida tropicalis: functional analysis of preproregion cleavages in C. tropicalis and Saccharomyces cerevisiae. Microbiology. 1996; 142: 493-503. Koelsch G, Tang J, Loy JA, Monod M, Jackson K, Foundling SI, et al. Enzymic characteristics of secreted aspartic proteases of Candida albicans. Biochim Biophys Acta. 2000; 1480: 117-31. Hube B. Candida albicans secreted aspartic proteinases. Curr Top Med Mycol. 1996; 7: 55-69. Magee BB, Hube B, Wright RJ, Sullivan PJ, Magee PT. The genes encoding the secreted aspartyl proteinases of Candida albicans constitute a family with at least three members. Infect Immun. 1993; 618: 3240-3. Zaugg C, Borg-Von Zepelin M, Reichard U, sanglard D, Monod D. Secreted aspartic proteinase family of Candida tropicalis. Infect Immun. 2001; 69: 405-12. Ray TL, Payne CD, Ruchel R, Ritter B, Schaffrinski M. Comparative production and rapid purification of Candida acid proteinase from protein-supplemented cultures. Infect Immun. 1990; 273: 391-403. Macdonald F. Secretion of inducible proteinase by pathogenic Candida species. Sabouraudia. 1984; 22: 79-82. Rüchel R. Proteinase. In: Bennett JE, Hay RJ, Peterson PK, editors. New strategies in fungal disease. Edinburgh: Churchill Livingstone; 1992. p.17-31. Hube B, Monod M, Schofield DA, Brown AJP, Gow NAR. Expression of seven members of the gene family encoding secretory aspartyl proteinases in Candida albicans. Mol Microbiol. 1994; 14: 87-99. White TC, Agabian N. Candida albicans secreted aspartic proteinases: isoenzyme pattern is determined by cell type, and levels are determined by environmental factors. J Bacteriol. 1995; 177: 5215-21. Staib F. serum-proteins as nitrogen source for yeastlike fungi. Sabouraudia. 1965; 4: 187-93. Remold H, Fasold H, Staib F. Purification and characterization of a proteolytic enzyme from Candida albicans. Biochim Biophys Acta. 1968; 167: 399-406. Odds FC. Candida species and virulence. ASM News. 1994; 60: 313-8. Colina A, Aumont RF, Deslauriers N, Belhumeur P, de Repentigny L. Evidence for degradation of gastrointestinal mucin by Cândida albicans secretory aspartyl proteinase. Infect Immun. 1996; 64: 4514-9. de Repentigny L, Aumont F, Bernard K, Belhumeur P. Characterization of binding of Candida albicans to small intestinal mucin and its role in adherence to mucosal epithelial cells. Infect Immun. 2000; 68: 3172-9. Rüchel R. Cleavage of immunoglobulins by pathogenic yeast of the genus Candida. Microbial Sci. 1986; 3: 316-9. Kilian M, Mestecky J, Russell MW. Defense mechanisms involving Fc-dependent functions of immunoglobulin A and their subversion by bacterial immunoglobulin A proteases. Microbiol Rev. 1988; 52: 296-303. Hube B, Monod M, Schofield DA, Brow AJ, Gow NA. Expression of seven members of the gene family encoding secretory aspartic proteinase in Candida albicans. Mol Microbiol. 1994; 14: 87-99 Vudhichamnong K, Walker DM, Ryley HC. The effect of secretory immunoglobulin A on the in vitro adherence of the yeast candida albicans to human epithelial cells. Arch Oral Biol. 1982; 27: 617-21.

Braz J Oral Sci. 5(16):944-952

45. White TC, Agabian N. Candida albicans secreted aspartyl proteinases: isoenzyme pattern is determined by cell type, and levels are determined by environmental factors. J Bacteriol. 1995; 177: 5215-21. 46. Wu T, Samaranayake LP. The expression of secreted aspartyl proteinases of Candida species in human whole saliva. J Med Microbiol. 1999; 48: 711-20. 47. Germaine GR, Tellefson LM. Effect of pH and human saliva on protease production by Candida albicans. Infect Immun. 1981; 31: 323-6. 48. Kaminishi H, Hamatake H, Cho T, Tamaki T, Suenaga N, Hisamatsu M, et al. Degradation of humoral host defense by Cândida albicans proteinase. Infect Immun. 1995; 63: 984-8. 49. Morrow B, Srikantha T, Soll,DS. Transcription of the gene for a pepstinogen, PEP1, is regulated by while-opaque switching in Candida albicans. Mol Cell. Biol. 1992; 12; 2997-3005. 50. Monod M, Hube B, Hess D, Sangland D. Differential regulation of SAP8 and SAP9 which encode two new members of the secreted aspartic proteinase family in Candida albicans. Microbiology. 1998; 144: 2731-7. 51. Schaller M, Schäfer W, Korting HC, Hube B. Differential expression of secreted aspartyl proteinases in a model of human oral candidosis and in patient samples from the oral cavity. Mol Microbiol. 1998; 29: 605-15. 52. Schaller M, Korting HC, Schafer W, Bastert J, Chen W, Hube B. Secreted aspartic proteinase (Sap) activity contributes to tissue damage in a model of human oral candidosis. Mol Microbiol. 1999; 34: 169-80. 53. Straib P, Kretschman M, Nichterlein T, Holf H, Morschhauser J. Differential activation of a Candida albicans virulence gene family during infection. Proc. Natl Acad Sci USA. 2000; 23; 97: 6102-7. 54. De Bernardis F, Boccanera M, Rainaldi L, Guerra CE, Quinti I, Cassone A. The secretion of aspartyl proteinase, a virulence enzyme, by isolates of Candida albicans from the oral cavity of HIV-infected subjects. Eur J Epidemiol. 1992; 8: 362-7. 55. Wu T, Samaranayake LP, Cao BY, Wang J. In vitro proteinase production by oral Candida albicans isolates from individuals with and without HIV infection and its attenuation by antimycotic agents. J Med Microbiol. 1996; 44: 311-6. 56. Ollert MW, Wend C, Gorlich M, McMullan-Vogel CG, Borg-von Zepelin M, Vogel CW, et al. Increased expression of Candida albicans secretory proteinase, a putative virulence factor, in isolates from human immunodeficiency virus-positive patients. J Clin Microbiol. 1995; 33: 2543-9. 57. Agatensi L, Franchi F, Montello F, Bevilacqua RL, Ceddia T, De Bernardis F, et al. Vaginopathic and proteolytic Candida species in outpatients attending a gynaccology clinic. J Clin Pathol. 1991; 44: 826-30. 58. Soll DR. Higt-frequency switching in Candida albicans. Clin microbial rev. 1992; 5: 183-203. 59. Cassone A, De Bernardis F, Mondello F, Ceddia T, Agatensi L. Evidence for a correlation between proteinase secretion and vulvovaginal candidosis. J Inf Dis. 1987; 156: 777-83. 60. De Bernardis F, Mondello F, Scaraveli G, Pachi A, Girolamo A, Agatensi L, et al. Higt aspartyl proteinase production and vaginitis in human immunodeficiency vírus-infected women. J Clin Microbiol. 1999; 37: 1376-80. 61. Kondoh Y, Shimizu K, Tanaka K. Proteinase production and pathogenicity of Candida albicans. II . Virulence for mice of C. albicans strains of different proteinase activity. Microbiol Immunol. 1987; 31: 1061-9. 62. Macdonald F, Odds FC. Virulence for mice of a proteinase secreting strain of Candida albicans and a proteinase-deficient mutant. J Gen Microbiol. 1983; 129: 431-8. 63. Borg M, Ruchel R. Demonstration of fungal proteinase during

Candida albicans proteinases

64.

65.

66.

67.

68.

69.

70.

71.

72.

73.

74.

75.

76.

77.

78.

phagocytosis of Candida albicans and Candida tropicalis. J Med Vet Mycol. 1990; 28: 3-14. Naglik JR, Newport G, White TC, Fernandes-Naglik LL, Grenspan JS, Grenspan D, et al. In vivo analysis of secreted aspartyl proteinase expression in human oral candidiasis. Infect Immun. 1999; 67: 2482-90. Naglik JR, Rodgers CA, Shirlaw PJ, Dobbie JL, Fernandes-Naglik LL, Greenspan D, et al. Differential expression of Candida albicans secreted aspartyl proteinase and phospholipase B genes in humans correlates with oral and vaginal infections. J Infect Dis. 2003; 188: 465-75. Schaller M, Januschkert E, Schackert C, Woerle B, Korting HC. Different isoforms of secreted aspartyl proteinases (Sap) are expressed by Candida albicans during oral and cutaneous candidosis in vivo. J Med Microbiol. 2001; 50: 743-7. Adab-Zapatero C, Goldman CR, Muchmore SW, Hutchins C, Stewart K, Navaza J, et al. Struture of a secreted aspartic protease from C. albicans complexed with a potent inhibitor: implications for the design of antifungal agent. Protein Sci. 1996; 5: 640-52. Cutfield SM, Dodson EJ, Anderson BF, Noody PC, Marshall CJ, Sullivan PA, et al. The crystal structure of a major secreted aspartic proteinase from Candida albicans in complexes with two inhibitors. Structure. 1995; 3: 1261-71. Morschhauser J, Virkola R, Korhonen TK, Hacker J. Degradation of human subendothelial extracellular matrix by proteinase secreting Candida albicans. FEMS Microbiol. 1997; 153: 34955. Cauda R, Tacconelli M, Tumbarello M, Morace G, De Bernardis F, Torosantucci A, et al. Role of protease inhibitors in preventing recurrent oral candidosis im patients with HIV infection: a prospective case-control study. J Acquir Immun Defic Syndr. 1999; 21: 20-5. Gruber A, Speth C, Lukasser-Vogl E, Zangerle R, Borg von Zepelin M, Dierich MP, et al. Human immunodeficiency virus type 1 protease inhibitor attenuates Candida albicans virulence properties in vitro. Immunopharmacology. 1999; 41: 227-34. Borg-Von Zeppelin M, Meyer I, Thomssen R, Wurzner R, Sanglard D, Telenti A, et al. HIV-protease inhibitors reduce cell adherence of Candida albicans strains by inhibition of yeast secreted aspartic proteases. J Investig Dermatol. 1999; 113: 747-51. Cassone A, De Bernardis F, Torosantucci A, Tacconelli M, Tumbarello M, Cauda R. In vitro and in vivo anticandidal activity of human immunodeficiency vírus protease inhibitors. J Infect Dis. 1999; 180: 448-53. Korting HC, Schaller M, Eder G, Hamm G, Bohmer U, Hube B. Effect of the human immunodeficiency virus (HIV) proteinase inhibitors saquinavir and indinavir on in vitro activities of secreted aspartil proteinases of Candida albicans isolates from HIVinfected patients. 1999; Antimicrob Agents Chemother. 1999; 43: 2038-42. Pichova I, Pavlickova L, Dostal J, Dolejsi E, HruskovaHeidingsfeldova O, Weber J, et al. Secreted aspartic proteases of Candida albicans, Candida tropicalis, Candida parapsilosis and Candida lusitaneae. Inhibition with peptidomimetic inhibitors. Eur J Biochem. 2001; 268: 2669-77. Gruber A, Berlit J, Speth C, Lass-Florl C, Kofler G, Nagl M, et al. Dissimilar attenuation of Candida albicans virulence properties by human immunodeficienty virus type 1 protease inhibitors. Immunobiology. 1999; 201: 133-44. Hoegl L, Thoma-Gleber E, Rocken M, Korting HC. HIV proteases inhibitors influence the prevalence of oral candidosis in HIVinfected patients: a 2-years study. Mycoses. 1998; 41: 321-5. Cassone A, Tacconelli M, De Bernardis F, Tumbarello M, Torosantucci A, Chiani R, et al. Antiretroviral therapy with protease inhibitors hás na early, immune reconstitution-

951

Braz J Oral Sci. 5(16):944-952

79.

80.

81.

82.

83.

84.

85.

86.

952

independent beneficial effect on Cândida virulence and oral candidiasis im humam immunodeficiency virus-infected subjects. J Infect Dis. 2002; 185: 188-95. Edison AM, Manning-Zweerink M. Comparison of the extracellular proteinase activity produced by a low-virulence mutant of Candida albicans and wild-type parent. Infect Immun. 1988; 56: 1388-90. Fallon K, Bausch K, Noonan J, Huguenel E, Tamburine P. Role of aspartic proteases in disseminated Candida albicans infection in mice. Infect Immun. 1997; 65: 551-6. Rüchel R, Ritter B, Schaffrinski M. Modulation of experimental systemic murine candidosis by intravenouns pepstatin. Zentbl Bakteriol . 1990; 273: 391-403. Zotter C, Haustein UF, Schonborn C, Grimmecke HD, Wand H. Effect of pepstatin A on candida albicans infection in the mouse. Dermatol Monatsschrift. 1990; 176: 189-98. Sato T, Nagai K, Shibazaki M, Abe K, Takebayashi Y, Lumanau B, et al. Novel aspartyl protease inhibitors, YF-0200R-A and B. J. Antibiot. (Tokyo) 1994; 47: 566-70. Sato T, Shibazaki M, Yamaguchi H, Abe K, Matsumoto H, Shimizu M. Novel Cândida albicans aspartyl protease inhibitor. II. A new pepstatin-ahpatinin group inhibitor, YF-044P-D. J antibiotic. 1994; 47: 588-90. Zhang Z, El Sohly HN, Jacob MR, Pasco DS, Walker LA, Clark AM. Natural products inhibiting Candida albicans secreted aspartic proteases from Tovomita krukovii. Plant Med. 2002; 68: 49-54. Zhang Z, El Sohly HN, Jacob MR, Pasco DS, Walker LA, Clark AM. Natural products inhibiting Candida albicans secreted aspartic proteases from Lycopodium cernuum. J Nat Prod. 2002; 65: 979-85.

Candida albicans proteinases