Secreted Aspartyl Proteinase Research Articles

Misexpression of the opaque-phase-specific gene PEP1 (SAP1) in the white phase of Candida albicans confers increased virulence in a mouse model of cutaneous infection.

Candida albicans WO-1 switches reversibly and at high frequency between a white and an opaque colony-forming phenotype that includes dramatic changes in cell morphology and physiology. A misexpression strategy has been used to investigate the role of the opaque-phase-specific gene PEP1 (SAP1), which encodes a secreted aspartyl proteinase, in the expression of the unique opaque-phase phenotype and phase-specific virulence in two animal models. The PEP1 (SAP1) open reading frame was inserted downstream of the promoter of the white-phase-specific gene WH11 in the transforming vector pCPW7, and the resulting transformants were demonstrated to misexpress PEP1 (SAP1) in the white phase. Misexpression did not confer any of the unique morphological characteristics of the opaque phase to cells in the white phase and had no effect on the switching process. However, misexpression conferred upon white-phase cells the increased capacity of opaque-phase cells to grow in medium in which protein was the sole nitrogen source. Misexpression of PEP1 (SAP1) had no effect on the virulence of white-phase cells in a systemic mouse model, in which white-phase cells were already more virulent than opaque-phase cells. Misexpression did, however, confer upon white-phase cells the dramatic increase in colonization of skin in a cutaneous mouse model that was exhibited by opaque-phase cells. Misexpression of PEP1 (SAP1) conferred upon white-phase cells two dissociable opaque-phase characteristics: increased adhesion and the capacity to cavitate skin. The addition of pepstatin A to the cutaneous model inhibited the latter, but not the former, suggesting that the latter is effected by released enzyme, while the former is effected by cell-associated enzyme.

Germ tubes and proteinase activity contribute to virulence of Candida albicans in murine peritonitis.

Peritonitis with Candida albicans is an important complication of bowel perforation and continuous ambulatory peritoneal dialysis. To define potential virulence factors, we investigated 50 strains of C. albicans in a murine peritonitis model. There was considerable variation in their virulence in this model when virulence was measured as release of organ-specific enzymes into the plasma of infected mice. Alanine aminotransferase (ALT) and alpha-amylase (AM) were used as parameters for damage of the liver and pancreas, respectively. The activities of ALT and AM in the plasma correlated with invasion into the organs measured in histologic sections and the median germ tube length induced with serum in vitro. When the activity of proteinases was inhibited in vivo with pepstatin A, there was a significant reduction of ALT and AM activities. This indicates that proteinases contributed to virulence in this model. Using strains of C. albicans with disruption of secreted aspartyl proteinase gene SAP1, SAP2, SAP3, or SAP4 through SAP6 (collectively referred to as SAP4-6), we showed that only a Deltasap4-6 triple mutant induced a significantly reduced activity of ALT in comparison to the reference strain. In contrast to the Deltasap1, Deltasap2, and Deltasap3 mutants, the ALT induced by the Deltasap4-6 mutant could not be further reduced by pepstatin A treatment, which indicates that Sap4-6 may contribute to virulence in this model.

Biotyping and virulence properties of skin isolates of Candida parapsilosis.

The biotype and virulence of skin isolates of Candida parapsilosis were compared with blood isolates of the same fungus. Morphotype, resistotype, and electrophoretic karyotype determinations did not reveal any special cluster with a unique or dominant pathogenic feature among all of the isolates, regardless of their source. However, all cutaneous isolates had uniformly elevated secretory aspartyl-protease (Sap) activity, more than four times higher than the enzyme activity of the blood isolates. They were also highly vaginopathic in a rat vaginitis model, being significantly more virulent than blood isolates in this infection model. In contrast, skin isolates were nonpathogenic in systemic infection of cyclophosphamide-immunodepressed mice, while some blood isolates were, in this model, highly pathogenic (median survival time, 2 days, with internal organ invasion at autopsy). Finally, skin isolates did not differ, as a whole, from blood isolates in their adherence to plastic. This property was associated with a morphotype, as defined by a colony with continuous fringe, which was present among both skin and blood isolates. While confirming the genetic heterogenicity of C. parapsilosis, our data strongly suggest that the potential of this fungus to cause mucosal disease is associated with Sap production and is substantially distinct from that of systemic invasion.

Dissimilar Attenuation of Candida albicans Virulence Properties by Human Immunodeficiency Virus Type 1 Protease Inhibitors

The secreted aspartyl proteinase (Sap) of Candida albicans, which is believed to represent an important virulence factor of this opportunistic yeast, and the human immunodeficiency virus type 1 (HIV 1) protease, which is obligatory for the production of infectious virions, both belong to the same family of aspartyl proteinases. We have previously shown that the HIV -1 protease inhibitor Indinavir directly inhibits secretion and proteinase activity of Sap in a dosedependent manner. Furthermore, at very high concentrations, viability of C. albicans is markedly reduced by Indinavir, indicating that HIV-1 protease inhibitors may possess antifungal activity. We thus proposed that these drugs may add to the resolution of mucosal candidiasis in HIV-1 infected subjects. We have now compared three different HIV-1 protease inhibitors. The rank order of Sap inhibition, already significant at 0.1 mg/ml for all protease inhibitors, was Ritonavir > Indinavir > Saquinavir. However, the cross-reactivity of Ritonavir to pepsin was also more pronounced compared with the other two. Indinavir did not affect Candida viability at concentrations up to 1 mg/ml, in line with our previous study. In contrast, at this concentration Saquinavir was even fungicidal as assessed by three different viability assays (colony formation assay, MTT assay, propidium iodide staining) whereas Ritonavir significantly affected the mitochondrial activity only (MTT assay). No influence on Candida viability was observed for any of the three at concentrations of 0.1 mg/ml or lower. It remains to be examined whether HIV-1 protease inhibitors or derivatives thereof may be suitable for in vivo therapy of subjects suffering from mucosal candidiasis resistant to current antimycotics.

Inhibition of growth and secreted aspartyl proteinase production in Candida albicans by lysozyme.

Lysozyme (muramidase) is a non-specific, antimicrobial protein ubiquitous in human mucosal secretions such as saliva. Although its antibacterial and antifungal activities are well recognised, there are no data on the specific concentrations necessary to affect the growth of Candida albicans or about the effect of lysozyme on the production of secreted aspartyl proteinase (Sap), a putative virulence factor of C. albicans. Five Sap-producing isolates of C. albicans were cultured in YCB-BSA medium with various concentrations of lysozyme to examine its effect on yeast cell growth, ultrastructural cellular topography and extracellular and intracellular Sap concentration and activity. Lysozyme was candidacidal at high concentrations and decreased significantly the extracellular Sap concentration at sublethal doses, accompanied by intracellular accumulation of the enzyme. At low concentrations of lysozyme (c. 10 microg/ml), Sap activity decreased more than two-fold and Sap concentration decreased five-fold without any appreciable effect on cell growth or viability. Ultrastructural investigations showed ballooned cells and cells with invaginations (especially present near bud scars), indicating that cell-wall components may be possible targets for this enzyme. All concentrations of lysozyme tested were well within physiologically attainable levels. These data suggest that lysozyme has, at least, a bimodal action on C. albicans, killing the organism at higher concentrations and modulating Sap metabolism at lower concentrations.

The expression of secreted aspartyl proteinases of Candida species in human whole saliva.

The expression of secreted aspartyl proteinases (Saps) by clinical isolates of Candida albicans, C. tropicalis and C. parapsilosis in human saliva supplemented with glucose and in a proteinase-inducing medium (YCB-BSA), was investigated. Also, yeast growth, pH fluctuation and total protein concentration of the saliva cultures during incubation were measured. Sap expression was assessed by evaluating the enzyme activity as well as the antigen concentration. Saps were expressed well in human saliva supplemented with glucose by all three Candida species, although greater expressions was found in YCB-BSA medium. C. albicans isolates were significantly more proteolytic than the non-albicans isolates. In general, for all three species, the rate of yeast growth, pH fluctuation and percentage reduction of total salivary protein concentration concurred with the degree of expression of Saps. These data strongly suggest that Saps of C. albicans, C. tropicalis and C. parapsilosis may play an active role in the progression of oral candidoses, particularly with regard to the abundance of low pH microenvironments in the oral cavity, which are regularly replenished with dietary carbohydrates.

In vitro and in vivo anticandidal activity of human immunodeficiency virus protease inhibitors.

Highly active antiretroviral therapy that includes human immunodeficiency virus (HIV) aspartyl protease inhibitors (PIs) causes a decline in the incidence of some opportunistic infections in AIDS, and this decline is currently attributed to the restoration of specific immunity. The effect of two PIs (indinavir and ritonavir) on the enzymatic activity of a secretory aspartyl protease (Sap) of Candida albicans (a major agent of mucosal disease in HIV-infected subjects) and on growth and experimental pathogenicity of this fungus was evaluated. Both PIs strongly (>/=90%) and dose dependently (0.1-10 microM) inhibited Sap activity and production. They also significantly reduced Candida growth in a nitrogen-limited, Sap expression-dependent growth medium and exerted a therapeutic effect in an experimental model of vaginal candidiasis, with an efficacy comparable to that of fluconazole. Thus, besides the expected immunorestoration, patients receiving PI therapy may benefit from a direct anticandidal activity of these drugs.

High aspartyl proteinase production and vaginitis in human immunodeficiency virus-infected women.

Vaginal isolates of Candida albicans from human immunodeficiency virus-positive (HIV+) and HIV- women with or without candidal vaginitis were examined for secretory aspartyl proteinase (Sap) production in vitro and in vivo and for the possible correlation of Sap production with pathology and antimycotic susceptibility in vitro. HIV+ women with candidal vaginitis were infected by strains of C. albicans showing significantly higher levels of Sap, a virulence enzyme, than strains isolated from HIV+, C. albicans carrier subjects and HIV- subjects with vaginitis. The greater production of Sap in vitro was paralleled by greater amounts of Sap in the vaginal fluids of infected subjects. In an estrogen-dependent, rat vaginitis model, a strain of C. albicans producing a high level of Sap that was isolated from an HIV+ woman with vaginitis was more pathogenic than a strain of C. albicans that was isolated primarily from an HIV-, Candida carrier. In the same model, pepstatin A, a strong Sap inhibitor, exerted a strong curative effect on experimental vaginitis. No correlation was found between Sap production and antimycotic susceptibility, as most of the isolates were fully susceptible to fluconazole, itraconazole, and other antimycotics, regardless of their source (subjects infected with strains producing high or low levels of Sap, subjects with vaginitis or carrier subjects, or subjects with or without HIV). Thus, high Sap production is associated with virulence of C. albicans but not with fungal resistance to fluconazole in HIV-infected subjects, and Sap is a potentially new therapeutic target in candidal vaginitis.

In vivo analysis of secreted aspartyl proteinase expression in human oral candidiasis.

Secreted aspartyl proteinases are putative virulence factors in Candida infections. Candida albicans possesses at least nine members of a SAP gene family, all of which have been sequenced. Although the expression of the SAP genes has been extensively characterized under laboratory growth conditions, no studies have analyzed in detail the in vivo expression of these proteinases in human oral colonization and infection. We have developed a reliable and sensitive procedure to detect C. albicans mRNA from whole saliva of patients with oral C. albicans infection and those with asymptomatic Candida carriage. The reverse transcription-PCR protocol was used to determine which of the SAP1 to SAP7 genes are expressed by C. albicans during colonization and infection of the oral cavity. SAP2 and the SAP4 to SAP6 subfamily were the predominant proteinase genes expressed in the oral cavities of both Candida carriers and patients with oral candidiasis; SAP4, SAP5, or SAP6 mRNA was detected in all subjects. SAP1 and SAP3 transcripts were observed only in patients with oral candidiasis. SAP7 mRNA expression, which has never been demonstrated under laboratory conditions, was detected in several of the patient samples. All seven SAP genes were simultaneously expressed in some patients with oral candidiasis. This is the first detailed study showing that the SAP gene family is expressed by C. albicans during colonization and infection in humans and that C. albicans infection is associated with the differential expression of individual SAP genes which may be involved in the pathogenesis of oral candidiasis.

Human immunodeficiency virus type 1 protease inhibitor attenuates Candida albicans virulence properties in vitro.

The putative virulence factor secreted aspartyl proteinase (SAP) of Candida albicans and the human immunodeficiency virus type 1 (HIV-1) protease both belong to the aspartyl proteinase family. The present study demonstrates that the HIV-1 protease inhibitor Indinavir is a weak but specific inhibitor of SAP. In addition, Indinavir reduces the amount of cell bound as well as released SAP antigen from C. albicans. Furthermore, viability and growth of C. albicans are markedly reduced by Indinavir. These findings indicate that HIV-1 protease inhibitors may possess antifungal activity and we speculate that in vivo SAP inhibition may add to the resolution of mucosal candidiasis in HIV-1 infected subjects.

In Vivo Expression and Localization of Candida albicans Secreted Aspartyl Proteinases during Oral Candidiasis in HIV-Infected Patients

Isoforms of aspartyl proteinase (Sap), which are encoded by at least nine related SAP genes, have been implicated to be a major virulence factor of the opportunistic yeast Candida albicans in experimental infections. Although it is generally assumed that proteinases are important for infections, detailed information on the pathogenetic role of Saps is still lacking. The same applies to the question whether the genes and corresponding isoforms of the enzyme are expressed during oral infection. For in vivo investigations, parts of the lesional oral epithelium were collected from three HIV-infected patients with oropharyngeal candidiasis. Immunoelectron microscopy was performed (pre- and post-embedding gold labeling with silver enhancement) using an anti-Sap murine monoclonal antibody directed against the gene products Sap1-3. It was possible to demonstrate expression of Sap antigens in each of the three samples of human oral candidiasis. This suggests that at least one of the genes SAP1-3 was expressed at the time of sample collection. Furthermore, a possible role of the enzymes during the interaction of yeast cells and mucosal cells is suggested: the majority of Sap antigens is secreted by those C. albicans cells that adhere directly to the epithelial surface. Sap immunoreactivity can be detected in particular at the site of close contact between C. albicans and epithelial cells, suggesting a pathogenetic role of the Saps in host-fungal interaction. Thus, inhibition of the enzyme might prove to be an important alternative in the prevention and treatment of candidiasis.

Evidence that members of the secretory aspartyl proteinase gene family, in particular SAP2, are virulence factors for Candida vaginitis.

Virulence of Candida albicans strains with targeted disruption of secretory aspartyl proteinase genes (SAP1 to SAP6) was assessed in an estrogen-dependent rat vaginitis model. Null sap1 to sap3 but not sap4 to sap6 mutants lost most of the virulence of their parental strain SC5314. In particular, the sap2 mutant was almost avirulent in this model. Reinsertion of the SAP2 gene into this latter mutant led to the to recovery of the vaginopathic potential. The vaginal fluids of the animals infected by the wild type strain or by the sap1 or sap3 mutants expressed a pepstatin-sensitive proteinase activity in vitro. No traces of this activity were found in the vaginal fluid of rats challenged by the sap2 mutant. All strains were capable of developing true hyphae during infection. Thus, members of SAP family, in particular SAP2, play a clear pathogenic role in vaginitis and may constitute a novel target for chemoimmunotherapy of this infection.

Overexpression of Candida albicans secretory aspartyl proteinase 2 and its expression in Saccharomyces cerevisiae do not augment virulence in mice.

To elucidate the implications of secreted aspartyl proteinase (Sap)2p in the pathogenesis of Candida infections, the SAP2 gene was expressed in Saccharomyces cerevisiae and overexpressed in Candida albicans. The coding region of SAP2, including its signal sequence and propeptide, was amplified by PCR and cloned downstream of the S. cerevisiae or C. albicans ADH1 promoter. Plasmid expression of SAP2 in S. cerevisiae showed that the signal peptide was functional. Integrative transformation of S. cerevisiae and C. albicans was accomplished by homologous recombination within the URA3 locus for S. cerevisiae and the SAP2 locus for C. albicans. Negative control transformants carried plasmids either without the SAP2 insert or with mutated sap2. S. cerevisiae and C. albicans transformants showed similar growth rates to their parental strains or negative controls, when grown in medium containing amino acids. However, in medium with BSA as sole nitrogen source, constitutive expression of SAP2 enabled S. cerevisiae to grow and increased the growth rate of C. albicans. In both media, only S. cerevisiae transformants harbouring SAP2 secreted the enzyme, as confirmed by proteinase activity assays and immunoblotting. When C. albicans was grown in amino acids medium, the enzyme was detected exclusively in transformants constitutively expressing SAP2. However, in BSA medium these strains secreted enzyme earlier and secreted higher amounts of enzyme and total proteinase activity. In pathogenicity studies in intact mice, expression of Sap2p as a sole putative virulence factor did not cause S. cerevisiae to become virulent and constitutive overexpression of SAP2 did not augment virulence of C. albicans in experimental oral or systemic infection.

Secreted aspartyl proteinases and interactions of Candida albicans with human endothelial cells.

The endothelial cell interactions of homozygous null mutants of Candida albicans that were deficient in secreted aspartyl proteinase 1 (Sap1), Sap2, or Sap3 were investigated. Only Sap2 was found to contribute to the ability of C. albicans to damage endothelial cells and stimulate them to express E-selectin. None of the Saps studied appears to play a role in C. albicans adherence to endothelial cells.

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 delta 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.

Candida dubliniensis: phylogeny and putative virulence factors.

Candida dubliniensis is a recently identified species which is implicated in oral candidosis in HIV-infected and AIDS patients. The species shares many phenotypic characteristics with, and is phylogenetically closely related to, Candida albicans. In this study the phylogenetic relationship between these two species was investigated and a comparison of putative virulence factors was performed. Four isolates of C. dubliniensis from different clinical sources were chosen for comparison with two reference C. albicans strains. First, the distinct phylogenetic position of C. dubliniensis was further established by the comparison of the sequence of its small rRNA subunit with representative Candida species. The C. dubliniensis isolates formed true unconstricted hyphae under most induction conditions tested but failed to produce true hyphae when induced using N-acetylglucosamine. Oral C. dubliniensis isolates were more adherent to human buccal epithelial cells than the reference C. albicans isolates when grown in glucose and equally adherent when grown in galactose. The C. dubliniensis isolates were sensitive to fluconazole, itraconazole, ketoconazole and amphotericin B. Homologues of seven tested C. albicans secretory aspartyl proteinase (SAP) genes were detected in C. dubliniensis by Southern analysis. In vivo virulence assays using a systemic mouse model suggest that C. dubliniensis is marginally less virulent than C. albicans. These data further confirm the distinct phenotypic and genotypic nature of C. dubliniensis and suggest that this species may be particularly adapted to colonization of the oral cavity.

KEX2 Influences Candida albicans Proteinase Secretion and Hyphal Formation

Candida albicans possesses at least seven differentially expressed genes that encode virulence-related secretory aspartyl proteinases (Saps). Sap DNA sequences predict post-translational processing at lysine-arginine residues in the preproteins, reminiscent of the maturation of Saccharomyces cerevisiae alpha-factor, where a prepropolypeptide is converted into a biologically active pheromone by Kex2, a subtilisin-like proprotein convertase. To investigate involvement of a C. albicans KEX2 homologue in Sap activation, a genetic selection was performed based on KEX2 function. A kex2 strain of S. cerevisiae was transformed with a C. albicans genomic DNA library and screened for the production of active alpha-factor. Positive clones were assayed for killer toxin activity, another Kex2-dependent phenotype. Plasmids that rescued both defects contained a sequence encoding a protein homologous to S. cerevisiae Kex2. Both alleles of the C. albicans KEX2 were inactivated by successive mutations. Null mutants continued to secrete active Sap2; however, the enzyme was abnormally processed and secreted at reduced levels. Unexpectedly, null mutants were incapable of forming hyphae, instead differentiating into aberrantly shaped cells. The ability to normally process Sap2 and form hyphae was restored upon transformation of null mutants with a KEX2-containing plasmid.

A triple deletion of the secreted aspartyl proteinase genes SAP4, SAP5, and SAP6 of Candida albicans causes attenuated virulence.

Secreted aspartyl proteinases (Saps) from Candida albicans are encoded by a multigene family with at least nine members (SAP1 to SAP9) and are considered putative virulence factors important for the pathogenicity of this human pathogen. The role of Sap isoenzymes in the virulence of C. albicans has not yet been clearly established, and therefore, using recent progress in the genetics of this yeast, we have constructed a panel of isogenic yeasts, each with a disruption of one or several SAP genes. We focused on the construction of a C. albicans strain in which three related SAP genes (SAP4, SAP5, and SAP6) were disrupted. Growth of the delta sap4,5,6 triple homozygous null mutant DSY459 in complex medium was not affected, whereas, interestingly, growth in a medium containing protein as the sole nitrogen source was severely impaired compared to the growth of the wild-type parent strain SC5314. Since the presence of Sap2 is required for optimal growth on such medium, this suggests that Sap4, Sap5, or Sap6 plays an important role for the process of induction of SAP2. When guinea pigs and mice were injected intravenously with DSY459, their survival time was significantly longer than that of control animals infected with the wild-type SC5314. Attenuated virulence of DSY459 was followed by a significant reduction of yeast cells in infected organs. These data suggest that the group of Sap4, Sap5, and Sap6 isoenzymes is important for the normal progression of systemic infection by C. albicans in animals.

Disruption of each of the secreted aspartyl proteinase genes SAP1, SAP2, and SAP3 of Candida albicans attenuates virulence.

Secreted aspartyl proteinases (Saps), encoded by a gene family with at least nine members (SAP1 to SAP9), are one of the most discussed virulence factors produced by the human pathogen Candida albicans. In order to study the role of each Sap isoenzyme in pathogenicity, we have constructed strains which harbor mutations at selected SAP genes. SAP1, SAP2, and SAP3, which are regulated differentially in vitro, were mutated by targeted gene disruption. The growth rates of all homozygous null mutants were similar to those of the isogenic wild-type parental strain (SC5314) in complex and defined media. In medium with protein as the sole source of nitrogen, sap1 and sap3 mutants grew with reduced growth rates but reached optical densities similar to those measured for SC5314. In contrast, sap2 null mutants tended to clump, grew poorly in this medium, and produced the lowest proteolytic activity. Addition of ammonium ions reversed such growth defects. These results support the view that Sap2 is the dominant isoenzyme. When sap1, sap2, and sap3 mutants were injected intravenously in guinea pigs and mice, the animals had increased survival rates compared to those of control animals infected with SC5314. However, reduction of proteolytic activity in vitro did not correlate directly with the extent of attenuation of virulence observed for all Sap-deficient mutants. These data suggest that SAP1, SAP2, and SAP3 all contribute to the overall virulence of C. albicans and presumably all play important roles during disseminated infections.

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 pronounced 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.