A fourth secreted aspartyl proteinase gene (SAP4) and a CARE2 repetitive element are located upstream of the SAP1 gene in Candida albicans.

Candida albicans secreted aspartyl proteinases (Sap), products of the SAP genes, which are presumed to act as virulence factors. In the C. albicans strain WO-1, the ability to secrete Sap1 is regulated with switch phenotype, another putative virulence factor. KpnI restriction fragment length polymorphisms differentiate between several distinct SAP1 alleles in laboratory and clinical strains. Both SAP1 alleles from strain WO-1 along with their 5'- and 3'-flanking regions were cloned and sequenced, as were both alleles from another strain, SS. The 5'-flanking regions were remarkably similar in all four of the sequenced alleles over approximately 1,500 nucleotides. S1 analysis revealed that both alleles of WO-1 are transcribed. Characterization of the one allele from strain WO-1 identified a 284-nucleotide insertion flanked by 8-bp direct repeats that shows homology to the CARE2 repetitive element and that is not present in the other alleles. Characterization of the SAP1 alleles also identified a fourth SAP gene (SAP4) that includes an extended leader sequence. SAP4 is positioned upstream, in tandem to SAP1, in all strains tested and may encode another closely related secreted aspartyl proteinase.     

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.     

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.     

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.     

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.     

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.     

A highly immunogenic recombinant and truncated protein of the secreted aspartic proteases family (rSap2t) of Candida albicans as a mucosal anticandidal vaccine

Sap2 (secreted aspartyl proteinase2) is a member of the Sap family of Candida albicans, a human opportunistic pathogen, which acts as a virulence factor in experimental animal models of mucosal candidiasis. The C. albicans SAP2 was subcloned into vector pDS56-RBSII-6xhis, under the control of an inducible promoter to produce a truncated 6xhis-tagged, enzymatically inactive Sap2, lacking the N-terminus 76 amino acids (rSap2t). This recombinant protein was purified to homogeneity by one-step nickel-chelate affinity chromatography and used to immunize intravaginally oophorectomized estradiol-treated rats. These animals raised local anti-rSap2t immunoglobulin G (IgG) and IgA antibodies and were protected from the challenge of a highly vaginopathic strain of the fungus. Protection was possibly due to the specific antibodies as suggested by the passive transfer of immune vaginal fluid and the protective effects of passive vaccination with anti-rSap2t IgM and IgG monoclonal antibodies. Hence, this new recombinant proteinase constitutes a novel tool to investigate mechanisms of anti-Candida protection at the vaginal level and as vaccination against vaginal candidiasis, a common, frequently recurrent and sometimes antimycotic-refractory infection in women.     

Candida albicans proteinases and host/pathogen interactions

Candida infections are common, debilitating and often recurring fungal diseases and a problem of significant clinical importance. Candida albicans, the most virulent of the Candida spp., can cause severe mucosal and life-threatening systemic infections in immunocompromised hosts. Attributes that contribute to C. albicans virulence include adhesion, hyphal formation, phenotypic switching and extracellular hydrolytic enzyme production. The extracellular hydrolytic enzymes, especially the secreted aspartyl proteinases (Saps), are one of few gene products that have been shown to directly contribute to C. albicans pathogenicity. Because C. albicans is able to colonize and infect almost every tissue in the human host, it may be crucial for the fungus to possess a number of similar but independently regulated and functionally distinct secreted proteinases to provide sufficient flexibility in order to survive and promote infection at different niche sites. The aim of this review is to explore the functional roles of the C. albicans proteinases and how they may contribute to the host/pathogen interaction in vivo.     

X-ray structures of Sap1 and Sap5: structural comparison of the secreted aspartic proteinases from Candida albicans

Proteolytic activity is an important virulence factor for Candida albicans (C. albicans). It is attributed to the family of the secreted aspartic proteinases (Saps) from C. albicans with a minimum of 10 members. Saps show controlled expression and regulation for the individual stages of the infection process. Distinct isoenzymes can be responsible for adherence and tissue damage of local infections, while others cause systemic diseases. Earlier, only the structures of Sap2 and Sap3 were known. In our research, we have now succeeded in solving the X-ray crystal structures of the apoenzyme of Sap1 and Sap5 in complex with pepstatin A at 2.05 and 2.5 A resolution, respectively. With the structure of Sap1, we have completed the set of structures of isoenzyme subgroup Sap1-3. Of subgroup Sap4-6, the structure of the enzyme Sap5 is the first structure that has been described up to now. This facilitates comparison of structural details as well as inhibitor binding modes among the different subgroup members. Structural analysis reveals a highly conserved overall secondary structure of Sap1-3 and Sap5. However, Sap5 clearly differs from Sap1-3 by its electrostatic overall charge as well as through structural conformation of its entrance to the active site cleft. Design of inhibitors specific for Sap5 should concentrate on the S4 and S3 pockets, which significantly differ from Sap1-3 in size and electrostatic charge. Both Sap1 and Sap5 seem to play a major part in superficial Candida infections. Determination of the isoenzymes' structures can contribute to the development of new Sap-specific inhibitors for the treatment of superficial infections with a structure-based drug design program.     

Ultrastructural localization of the secretory aspartyl proteinase in Candida albicans cell wall in vitro and in experimentally infected rat vagina

Detection and ultrastructural localization of aspartyl proteinase (Sap) in Candida albicans experimentally infecting rat vagina were studied. Two Sap-positive (Sap+) and one Sap-negative (Sap-) strains of the fungus, endowed with high and low experimental vaginopathic potential, respectively, were used. Both Sap+ strains produced consistent Sap levels in the rat vagina, while the Sap- strain did not produce any measurable Sap. Electron microscopy of thin sections of chemically-fixed vaginal scrapings showed clear evidence of hyphae of proteolitic strains of C. albicans invading the keratinized epithelial cell layer of the vagina. The fungal cells exhibited a pronounced fibrillar layer on the cell wall with a marked intermixing of fungal and vaginal materials especially pronunced at the hyphal tip. Post-embedding immunogold techniques with the use of anti-Sap polyclonal and the specifically generated monoclonal antibody GF1 showed that Sap was essentially localized in the cell wall of C. albicans early during infection, in a cytological pattern mirroring Sap localization in C. albicans cells grown in Sap-inductive media in vitro. In summary, the data offer a new biochemical and ultrastructural evidence that Sap is actively secreted during experimental rat vaginitis by C. albicans. Cell wall localization of Sap is probably inherent to this active secretion process. This revised version was published online in August 2006 with corrections to the Cover Date.     

Amphotericin B resistance leads to enhanced proteinase and phospholipase activity and reduced germ tube formation in Candida albicans

Resistance to amphotericin B is an emerging phenomenon in Candida albicans. Amphotericin B-resistant strain of C. albicans was developed under laboratory conditions and the stability of acquired resistance was confirmed in vitro as well as in vivo. This AMB-resistant strain exhibited reduced germ tube formation as compared to parent strain of C. albicans (ATCC10231). Enzymatic activity of virulence factors like secreted aspartyl proteinase and phospholipase were found to be significantly high in AMB-R as compared to parent strain whereas ergosterol content of AMB-R was drastically reduced. The behavior of AMB-R strain is an interesting phenomenon and opens up a wide area of research regarding pathways and mechanisms.     

cDNA cloning of an aspartic proteinase secreted by Candida albicans.

cDNA of an aspartic proteinase secreted by Candida albicans No. 114 was isolated using the polymerase chain reaction (PCR). The primary structure of the enzyme was deduced from the nucleotide sequence of the cDNA and compared with the structures of Saccharomyces cerevisiae proteinase A and vacuolar aspartyl proteinase of C. albicans. The mature aspartic proteinase consisted of 341 amino acid residues, and was 17.6 and 15.3% identical with the proteinase A and the aspartyl proteinase, respectively. Two active aspartic acid sites and the amino acids near those sites were conserved in the aspartic proteinase. We also showed that there is another gene of aspartic proteinase than that of strain ATCC10231 reported by Hube et al (J. Med. Vet. Mycol. 29 (1991)) in the same C. albicans genome, both in that strain and in No. 114.     

<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.