Biosensor-based screening and characterization of HIV-1 inhibitor interactions with Sap 1, Sap 2, and Sap 3 from Candida albicans

A surface plasmon resonance (SPR) biosensor-based strategy for identification and characterization of compounds has been devised as a tool for the discovery of specific drugs for treatment of Candida albicans infections. Three secreted aspartic proteases (Saps 1-3) from C. albicans were used as parallel targets. The stepwise procedure involved screening of 104 HIV-1 pro-tease inhibitors at a single concentration for binding to the targets. Twenty-four compounds that appeared to interact with the targets were identified in the screen. False positives and compounds with low affinities or very fast dissociation rates could be removed after a series of additional measurements of these compounds at 3 different concentrations. Kinetic characterization was performed with 13 compounds, giving information about the interaction mechanism and interaction kinetic parameters (k(on), k(off), and K(D)). The pH dependence of the interaction and the inhibitory effect of a final small set of compounds were also evaluated. The strategy resulted in the identification of ritonavir as the compound generally exhibiting the highest affinity for the Candida enzymes. It had similar interaction kinetic characteristics for Sap 1 and Sap 2 but a lower affinity for Sap 3 due to a slower association rate. Several additional compounds with high affinity and/or slow dissociation rates for the targets were identified, revealing 2 other structural scaffolds for Sap inhibitors. In addition, important differences in the specificity for these types of compounds by the Saps were identified. The stepwise biosensor-based strategy was consequently efficient for identification and characterization of new lead compounds for 3 important drug targets.     

Enzymic characteristics of secreted aspartic proteases of Candida albicans

Candida yeasts are rarely infectious, but frequently cause life-threatening systemic infections in patients immunocompromised by AIDS or by immunosuppressive therapeutics. The secreted aspartic proteases (Saps) are known virulence factors of pernicious Candida species. The most virulent, Candida albicans, possesses at least nine SAP genes, some of which are specifically expressed from cells with morphologies associated with virulence. Only one of these proteases, Sap2, has been previously purified from yeast in sufficient quantities for enzymic studies. The other enzymes are present in low amounts in yeast culture and are difficult to purify. As a consequence, enzyme properties, including the substrate specificities, of all Saps are poorly studied. Therefore, four Saps that are known to be expressed in C. albicans, Sap1, Sap2, Sap3 and Sap6, were produced in Escherichia coli as recombinant zymogens and purified in large quantities. These proenzymes were autoactivated and purified as active proteases. The enzymic properties including the substrate specificities at the P(1) and P(1)' sites were determined using a competitive hydrolysis method employing synthetic substrate mixtures. All four Saps cleave peptide bonds between larger hydrophobic amino acids, but these somewhat broad specificities differ in detail among the four enzymes at both sites. At the P(1) site, Sap1, Sap2 and Sap6 prefer Phe while Sap3 prefers Leu. Positively charged amino acids are also accommodated, especially by Sap2 and Sap3. The specificities at P(1)' are broader than at P(1) for all four enzymes. Sap6 prefers Ala, whereas other Saps prefer Tyr. Acidic side chains are also accommodated at this site. Analysis of substrates with a hydrophobic amino acid in P(1)' reveals that all the Saps possess a unique preference for Ala at this site. The observed differences of residue preferences among Saps may be utilized for the design of specific substrates and inhibitors.     

The crystal structure of the secreted aspartic proteinase 3 from Candida albicans and its complex with pepstatin A

The family of secreted aspartic proteinases (Sap) encoded by 10 SAP genes is an important virulence factor during Candida albicans (C. albicans) infections. Antagonists to Saps could be envisioned to help prevent or treat candidosis in immunocompromised patients. The knowledge of several Sap structures is crucial for inhibitor design; only the structure of Sap2 is known. We report the 1.9 and 2.2 A resolution X-ray crystal structures of Sap3 in a stable complex with pepstatin A and in the absence of an inhibitor, shedding further light on the enzyme inhibitor binding. Inhibitor binding causes active site closure by the movement of a flap segment. Comparison of the structures of Sap3 and Sap2 identifies elements responsible for the specificity of each isoenzyme.     

Secreted aspartic proteases of Candida albicans, Candida tropicalis, Candida parapsilosis and Candida lusitaniae. Inhibition with peptidomimetic inhibitors.

The frequency of Candida infections has increased in recent years and it has been accompanied by a significant rise in morbidity and mortality. The secretion of aspartic proteases by Candida spp. was demonstrated to be one of the virulence determinants. Candida albicans is classified as the major human pathogen in the genus Candida. However, other species of this genus have been found to cause an increasing number of candidiases. We isolated secreted aspartic proteases (Saps) of C. albicans (Sap2p), C. tropicalis (Sapt1p), C. parapsilosis (Sapp1p), and C. lusitaniae (Saplp) from culture media. All the isolated proteases were N-terminally sequenced. Their specific proteolytic activities and sensitivity to series of peptidomimetic inhibitors modified in the type of scissile bond replacement as well as in the N- and C-termini were analyzed. The most divergent substrate specificity was observed for the Sap of C. tropicalis. The specificity of Sap of C. lusitaniae is most closely related to that of Sap of C. parapsilosis. We designed and prepared an inhibitor containing phenylstatine isoster that was equipotent towards all four proteases within the range of 10-10-10-9 M. The HIV-1 protease inhibitors ritonavir, saquinavir, indinavir, and nelfinavir were also tested for the inhibition of four Saps. Only ritonavir and saquinavir inhibited Sap2p, Sapt1p, Sapp1p, and Saplp in micromolar concentrations.     

Secreted aspartic proteases as virulence factors of Candida species

Candida infections have emerged as a significant medical problem during the last few decades. Among the different virulence traits of C. albicans, secreted proteolytic activity has been intensively investigated. Pathogenesis of the various forms of candidiasis was shown to be associated with the differential and temporal regulation of the expression of genes coding for secreted aspartic proteases (Sap). These enzymes act as cytolysins in macrophages after phagocytosis of Candida, are present in tissue penetration and are also involved in adherence to epithelial cells. Since the introduction of new antiretroviral therapeutics such as HIV protease inhibitors, oropharyngeal candidiasis is less often observed in AIDS patients. Different HIV aspartic protease inhibitors were able to inhibit the C. albicans Saps involved in adherence. The lower rates of oropharyngeal candidiasis observed in individuals receiving antiretroviral combination therapy could reflect not only an improvement in the immune system but also direct inhibition of Candida Saps by HIV protease inhibitors. Therefore, the development of specific aspartic protease inhibitors might be of interest for the inhibition of candidiasis.     

Structure-based specificity mapping of secreted aspartic proteases of Candida parapsilosis, Candida albicans, and Candida tropicalis using peptidomimetic inhibitors and homology modeling

Secreted aspartic proteases (Saps) of pathogenic Candida spp. represent a specific target for antifungal drug development. We synthesized a series of peptidomimetic inhibitors with different isosteric groups and modifications at individual positions and tested them with purified Saps from C. albicans (Sap2p), C. tropicalis (Sapt1p), and C. parapsilosis (Sapp1p). The kinetic parameters indicated that all three proteases prefer binding of inhibitors containing bulky hydrophobic residues between positions P3 and P3'. The most divergent specificity was found for Sapp1p. The sequence alignment of Sap2p, Sapt1p, and Sapp1p, and homology modeling of Sapp1p with the crystal structure of Sapt1p and the complex of Sap2p with a peptidomimetic inhibitor showed that the overall folds of Sap2p, Sapt1p, and Sapp1p are similar. However, the N- and C-terminal loops formed by disulfide bonds between residues 47-53 and 258-292 are significantly shorter in Sapp1p, and a unique insertion following Tyr 129 in Sapp1p results in the formation of a loop that can interact with inhibitor residues. These Sapp1p structural differences might lead to its altered susceptibility to inhibition.     

The expression of the secreted aspartyl proteinases Sap4 to Sap6 from Candida albicans in murine macrophages

Medically important yeasts of the genus Candida secrete aspartyl proteinases (Sap), which are of particular interest as virulence factors. Six closely related gene sequences, SAP1 to SAP6 , for secreted proteinases are present in Candida albicans . The methylotrophic yeast Pichia pastoris was chosen as an expression system for preparing substantial amounts of each Sap isoenzyme. Interestingly, Sap4, Sap5 and Sap6, which have not yet been detected in C . albicans cultures in vitro , were produced as active recombinant enzymes. Different Sap polyclonal antibodies were raised in rabbits and tested before further application by enzyme-linked immunosorbent assay (ELISA) against each recombinant Sap. Two antisera recognized only Sap4 to Sap6. Using these antisera, together with sap null mutants obtained by targeted mutagenesis, we could demonstrate a high production of Sap4, Sap5 and Sap6 by C . albicans cells after phagocytosis by murine peritoneal macrophages. Furthermore, a Δ sap4,5,6 null mutant was killed 53% more effectively after contact with macrophages than the wild-type strain. These results support a role for Sap4 to Sap6 in pathogenicity.     

<Emphasis Type="Italic">Candida albicans VPS4</Emphasis> is Required for Secretion of Aspartyl Proteases and In Vivo Virulence

Candida albicans secretes aspartyl proteases (Saps) during infection. Although Saps are secretory proteins, little is known about the intracellular trafficking and secretion of these proteins. We previously cloned and analyzed the C. albicans pre-vacuolar protein sorting gene VPS4, and demonstrated that extracellular Sap2p is absent in the culture supernatants of the vps4delta null mutant. We therefore investigated the role of the C. albicans pre-vacuolar secretion pathway in the trafficking of Sap4-6p and in vivo virulence. The C. albicans vps4delta mutant failed to produce extracellular Sap4-6p. Next, when tested in a mouse model of disseminated candidiasis, the vps4delta mutant was greatly attenuated in virulence. Histopathological analysis indicated that infection with the vps4delta mutant did not cause renal microabscess formation, in contrast to the wild-type strain. Our results imply that VPS4 is required for extracellular secretion of Sap4-6p, and that C. albicans requires an intact pre-vacuolar secretory pathway for wild-type virulence in vivo.     

Proteolytic cleavage of covalently linked cell wall proteins by Candida albicans Sap9 and Sap10

The cell wall of the human-pathogenic fungus Candida albicans is a robust but also dynamic structure which mediates adaptation to changing environmental conditions during infection. Sap9 and Sap10 are cell surface-associated proteases which function in C. albicans cell wall integrity and interaction with human epithelial cells and neutrophils. In this study, we have analyzed the enzymatic properties of Sap9 and Sap10 and investigated whether these proteases cleave proteins on the fungal cell surface. We show that Sap9 and Sap10, in contrast to other aspartic proteases, exhibit a near-neutral pH optimum of proteolytic activity and prefer the processing of peptides containing basic or dibasic residues. However, both proteases also cleaved at nonbasic sites, and not all tested peptides with dibasic residues were processed. By digesting isolated cell walls with Sap9 or Sap10, we identified the covalently linked cell wall proteins (CWPs) Cht2, Ywp1, Als2, Rhd3, Rbt5, Ecm33, and Pga4 as in vitro protease substrates. Proteolytic cleavage of the chitinase Cht2 and the glucan-cross-linking protein Pir1 by Sap9 was verified using hemagglutinin (HA) epitope-tagged versions of both proteins. Deletion of the SAP9 and SAP10 genes resulted in a reduction of cell-associated chitinase activity similar to that upon deletion of CHT2, suggesting a direct influence of Sap9 and Sap10 on Cht2 function. In contrast, cell surface changes elicited by SAP9 and SAP10 deletion had no major impact on the phagocytosis and killing of C. albicans by human macrophages. We propose that Sap9 and Sap10 influence distinct cell wall functions by proteolytic cleavage of covalently linked cell wall proteins.     

Candida albicans secreted aspartyl proteinases: isoenzyme pattern is determined by cell type, and levels are determined by environmental factors.

For the pathogenic yeast Candida albicans, secreted aspartyl proteinase (Sap) activity has been correlated with virulence. A family consisting of at least eight SAP genes can be drawn upon to produce Sap enzymatic activity. In this study, the levels of Sap1, Sap2, and Sap3 isoenzymes were monitored under a variety of growth conditions for several strains, including strain WO-1, which alternates between two switch phenotypes, white (W) and opaque (O). When cultured under proteinase-inducing conditions, most strains and W cells produce Sap2, while O cells produce Sap1, Sap2, and Sap3. Both W and O cells of strain WO-1 produce Saps in enriched and defined media that do not induce Saps from other strains. The specific Sap isoenzyme that is produced is determined by the cell type, while the level of Sap production is determined by environmental factors. The levels and temporal regulation of the SAP mRNAs as determined by Northern (RNA) analysis were consistent with Sap protein levels and with previous results. S1 analysis showed that SAP6 is the predominant SAP gene transcribed during hyphal induction at neutral pH. These studies define the culture conditions which control the levels of SAP mRNAs and Sap proteins, and they indicate that both the yeast/hyphal transition and phenotypic switching can determine which of the Sap isoenzymes is produced.     

HIV aspartyl protease inhibitors as promising compounds against Candida albicans André Luis Souza dos Santos

Cells of Candida albicans (C. albicans) can invade humans and may lead to mucosal and skin infections or to deep-seated mycoses of almost all inner organs, especially in immunocompromised patients. In this context, both the host immune status and the ability of C. albicans to modulate the expression of its virulence factors are relevant aspects that drive the candidal susceptibility or resistance; in this last case, culminating in the establishment of successful infection known as candidiasis. C. albicans possesses a potent armamentarium consisting of several virulence molecules that help the fungal cells to escape of the host immune responses. There is no doubt that the secretion of aspartyl-type proteases, designated as Saps, are one of the major virulence attributes produced by C. albicans cells, since these hydrolytic enzymes participate in a wide range of fungal physiological processes as well as in different facets of the fungal-host interactions. For these reasons, Saps clearly hold promise as new potential drug targets. Corroborating this hypothesis, the introduction of new anti-human immunodeficiency virus drugs of the aspartyl protease inhibitor-type (HIV PIs) have emerged as new agents for the inhibition of Saps. The introduction of HIV PIs has revolutionized the treatment of HIV disease, reducing opportunistic infections, especially candidiasis. The attenuation of candidal infections in HIV-infected individuals might not solely have resulted from improved immunological status, but also as a result of direct inhibition of C. albicans Saps. In this article, we review updates on the beneficial effects of HIV PIs against the human fungal pathogen C. albicans, focusing on the effects of these compounds on Sap activity, growth behavior, morphological architecture, cellular differentiation, fungal adhesion to animal cells and abiotic materials, modulation of virulence factors, experimental candidiasis infection, and their synergistic actions with classical antifungal agents.     

Candida albicans secreted aspartyl proteinases in virulence and pathogenesis.

Candida albicans is the most common fungal pathogen of humans and has developed an extensive repertoire of putative virulence mechanisms that allows successful colonization and infection of the host under suitable predisposing conditions. Extracellular proteolytic activity plays a central role in Candida pathogenicity and is produced by a family of 10 secreted aspartyl proteinases (Sap proteins). Although the consequences of proteinase secretion during human infections is not precisely known, in vitro, animal, and human studies have implicated the proteinases in C. albicans virulence in one of the following seven ways: (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. Sap proteins fulfill a number of specialized functions during the infective process, which include the simple role of digesting molecules for nutrient acquisition, digesting or distorting host cell membranes to facilitate adhesion and tissue invasion, and digesting cells and molecules of the host immune system to avoid or resist antimicrobial attack by the host. We have critically discussed the data relevant to each of these seven criteria, with specific emphasis on how this proteinase family could contribute to Candida virulence and pathogenesis.     

Anti-fungal therapy at the HAART of viral therapy

HIV-positive patients receiving combination therapy (highly active anti-retroviral treatment, HAART) suffer significantly fewer oral infections with the opportunistic fungal pathogen Candida albicans than non-HAART-treated patients. One component of HAART is an inhibitor of the HIV proteinase, the enzyme required for correct processing of retroviral precursor proteins. It would appear that HIV proteinase inhibitors also have a direct effect on one of the key virulence factors of C. albicans, the secreted aspartic proteinases (Saps). This suggests that the reduction in C. albicans infections in HIV-positive patients might not be solely the result of improved immunological status but could also be caused by the HAART treatment directly inhibiting Candida proteinases.     

Comprehensive characterization of secreted aspartic proteases encoded by a virulence gene family in Candida albicans

Candida albicans is a commensal organism, but causes life-threatening infections in immunocompromised patients. Certain factors such as yeast-hyphae transition and hydrolytic enzymes are suggested as virulence attributes of C. albicans. Among them, 10 types of secreted aspartic protease (SAP) genes have received particular attention as a major virulence gene family. However, their full functional repertoire, including its biochemical properties, remains to be elucidated. Hence, we purified all Sap isozymes using Pichia pastoris and comprehensively determined and compared their biochemical properties. While optimum pH of Sap7 was 6.5 and that of Sap8 was 2.5, presence of other Sap isozymes functioning within a broad range of optimum pH could allow C. albicans to survive and cause infections in various tissues. The substrate specificities of Sap isozymes were analysed by using FRETS-25Xaa libraries. Sap7 and Sap10 showed high substrate specificity, while other Sap isozymes had broad substrate specificities. Principal component analysis revealed that the 10 Sap isozymes were clustered into 3 distinct groups in terms of their substrate specificities. Interestingly, Sap4-6, which are coproduced in the hyphal form, were clustered as the same group, indicating that they may target similar host proteins. These results will lead to further understanding of C. albicans pathogenicity.     

HIV-1 and its transmembrane protein gp41 bind to different Candida species modulating adhesion

Oral candidiasis in HIV-1-infected individuals is widely believed to be triggered by the acquired T-lymphocyte immunodeficiency. Recently, binding of the HIV-1 envelope protein gp160 and its subunit gp41, and also of the whole virus itself, to Candida albicans has been shown. The present study shows that, in addition to C. albicans, HIV-1 gp41 also binds to yeast and hyphal forms of Candida dubliniensis, a species which is closely related to C. albicans, and to Candida tropicalis but not to Candida krusei, Candida glabrata or Saccharomyces cerevisiae. The previous finding that gp41 binding to C. albicans augments fungal virulence in vitro is supported by the observation that the yeast showed an enhanced adhesion to HIV-infected H9 cells in comparison to uninfected cells. In line with these results soluble gp41 itself reduced binding of C. albicans to both endothelial and epithelial cell lines, confirming a dominant role of the gp41 binding moiety on the surface of Candida for adhesion. Surface-associated secreted aspartic proteinases (Saps) play an important role in candidial adhesion, but are not likely to be involved in the interaction as gp41 binding to the C. albicans parental wild-type strain was comparable to that of three different isogenic Sap deletion mutants. Furthermore, gp41 binding to the yeast killer toxin-susceptible C. albicans strain 10S was not inhibitable by an anti-YKT receptor antibody. In conclusion, HIV-1 interacts with different clinically important Candida spp., and may thereby affect the outcome of the respective fungal infection.     

Distribution of secreted aspartyl proteinases using a polymerase chain reaction assay withSAP specific primers inCandida albicans isolates

Secreted aspartyl proteinase (Sap) distribution among different C. albicans isolates was determined using SAP-specific primers in polymerase chain reaction (PCR) assay. SAP1, SAP2, and SAP3 were detected in 13 of 40 (32.5%), SAP4 in 38/40 (95%), SAP5 were detected in 30/40 (75%), SAP6 in 23/40 (57.5%) of C. albicans strains isolated from blood cultures. SAP1-SAP3 were detected in 37 of 40 (92.5%), SAP4 were detected in 3/40 (7.5%), SAP5 in 3/40 (7.5%), SAP6 in 5/40 (12.5%) of C. albicans strains isolated from vaginal swab cultures. Sap1, Sap2 and Sap3 isoenzymes were found to be related to the vaginopathic potential of C. albicans; Sap4, Sap5 and Sap6 isoenzymes were found to be correlated with systemic infections.