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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 ability to change between yeast and hyphal cells (dimorphism) is known to be a virulence property of the human pathogen Candida albicans. The pathogenesis of disseminated candidosis involves adhesion and penetration of hyphal cells from a colonized mucosal site to internal organs. Parenchymal organs, such as the liver and pancreas, are invaded by C. albicans wild-type hyphal cells between 4 and 24 h after intraperitoneal (i.p.) infection of mice. In contrast, a hypha-deficient mutant lacking the transcription factor Efg1 was not able to invade or damage these organs. To investigate whether this was due to the inability to undergo the dimorphic transition or due to the lack of hypha-associated factors, we investigated the role of secreted aspartic proteinases during tissue invasion and their association with the different morphologies of C. albicans. Wild-type cells expressed a distinct pattern of SAP genes during i.p. infections. Within the first 72 h after infection, SAP1, SAP2, SAP4, SAP5, SAP6, and SAP9 were the most commonly expressed proteinase genes. Sap1 to Sap3 antigens were found on yeast and hyphal cells, while Sap4 to Sap6 antigens were predominantly found on hyphal cells in close contact with host cells, in particular, eosinophilic leukocytes. Mutants lacking EFG1 had either noticeably reduced or higher expressed levels of SAP4 to SAP6 transcripts in vitro depending on the culture conditions. During infection, efg1 mutants had a strongly reduced ability to produce hyphae, which was associated with reduced levels of SAP4 to SAP6 transcripts. Mutants lacking SAP1 to SAP3 had invasive properties indistinguishable from those of wild-type cells. In contrast, a triple mutant lacking SAP4 to SAP6 showed strongly reduced invasiveness but still produced hyphal cells. When the tissue damage of liver and pancreas caused by single sap4, sap5, and sap6 and double sap4 and -6, sap5 and -6, and sap4 and -5 double mutants was compared to the damage caused by wild-type cells, all mutants which lacked functional SAP6 showed significantly reduced tissue damage. These data demonstrate that strains which produce hyphal cells but lack hypha-associated proteinases, particularly that encoded by SAP6, are less invasive. In addition, it can be concluded that the reduced virulence of hypha-deficient mutants is not only due to the inability to form hyphae but also due to modified expression of the SAP genes normally associated with the hyphal morphology.

Fungal infections constitute a serious clinical problem in the group of patients receiving total parenteral nutrition. The majority of species isolated from infections of the total parenteral nutrition patients belong to Candida genus. The most important factors of Candida spp. virulence are the phenomenon of “phenotypic switching,” adhesins, dimorphism of fungal cells and the secretion of hydrolytic enzymes such as proteinases and lipases, including aspartyl proteinases. We determined the proteolytic activity of yeast-like fungal strains cultured from the clinical materials of patients receiving total parenteral nutrition and detected genes encoding aspartyl proteinases in predominant species Candida glabrata—YPS2, YPS4, and YPS6, and Candida albicans—SAP1–3, SAP4, SAP5, and SAP6. C. albicans released proteinases on the various activity levels. All C. glabrata strains obtained from the clinical materials of examined and control groups exhibited secretion of the proteinases. All 13 isolates of C. albicans possessed genes SAP1–3. Gene SAP4 was detected in genome of 11 C. albicans strains, SAP5 in 6, and SAP6 in 11. Twenty-six among 31 of C. glabrata isolates contained YPS2 gene, 21 the YPS4 gene, and 28 the YPS6 gene. We observed that clinical isolates of C. albicans and C. glabrata differed in SAPs and YPSs gene profiles, respectively, and displayed differentiated proteolytic activity. We suppose that different sets of aspartyl proteinases genes as well as various proteinase-activity levels would have the influence on strains virulence.

In order to approximate and adhere to mucosal epithelial cells, Candida must traverse the overlying mucus layer. Interactions of Candida species with mucin and human buccal epithelial cells (BECs) were thus investigated in vitro. Binding of the Candida species to purified small intestinal mucin showed a close correlation with their hierarchy of virulence. Significant differences (P < 0.05) were found among three categories of Candida species adhering highly (C. dubliniensis, C. tropicalis, and C. albicans), moderately (C. parapsilosis and C. lusitaniae) or weakly (C. krusei and C. glabrata) to mucin. Adherence of C. albicans to BECs was quantitatively inhibited by graded concentrations of mucin. However, inhibition of adherence was reversed by pretreatment of mucin with pronase or C. albicans secretory aspartyl proteinase Sap2p but not with sodium periodate. Saturable concentration- and time-dependent binding of mucin to C. albicans was abrogated by pronase or Sap2p treatment of mucin but was unaffected by β-mercaptoethanol, sodium periodate, neuraminidase, lectins, or potentially inhibitory sugars. Probing of membrane blots of the mucin with C. albicans revealed binding of the yeast to the 66-kDa cleavage product of the 118-kDa C-terminal glycopeptide of mucin. Although no evidence was found for the participation of C. albicans cell surface mannoproteins in specific receptor-ligand binding to mucin, inhibition of binding by p-nitrophenol (1 mM) and tetramethylurea (0.36 M) revealed that hydrophobic interactions are involved in adherence of C. albicans to mucin. These results suggest that C. albicans may both adhere to and enzymatically degrade mucins by the action of Saps, and that both properties may act to modulate Candida populations in the oral cavity and gastrointestinal tract.

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.

The secreted aspartyl proteinases of Candida albicans (products of the SAP genes) are thought to contribute to virulence through their effects on Candida adherence, invasion, and pathogenicity. From a single strain of C. albicans (WO-1) which expresses a phenotypic switching system, three secreted aspartyl proteinases have been identified as determined by molecular weight and N-terminal sequence. Each of the three identified proteins represents the mature form of one of three distinct proteinase isoenzymes, two of which correspond to the recently cloned SAP1 and SAP2 genes (previously referred to as CAP, PEP, or PRA). A genomic library was screened under low-stringency hybridization conditions with a polymerase chain reaction fragment from SAP1. In addition to clones of SAP1 and SAP2, a clone containing SAP3, a novel third secreted proteinase gene, was identified and sequenced. The three aspartyl proteinase isoenzymes differ in primary sequence and pI, suggesting that they may play different roles in virulence and pathogenesis. All three of these proteinases are expressed in the same strain. However, the pattern of proteinase expression is correlated with the switch phenotype of the cell. Opaque cells of strain WO-1 express Sap1 and Sap3, while white cells of the same strain express Sap2. The differential expression of three Sap proteinases may contribute to virulence in C. albicans.

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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.

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Although the echinocandin caspofungin primarily inhibits the synthesis of cell wall 1,3-β-d-glucan, its fungicidal activity could also potentially perturb the expression of virulence factors involved in the ability of Candida albicans to cause infection. Expression of the C. albicans secretory aspartyl proteinase (SAP) and phospholipase B (PLB) virulence genes was determined by reverse transcription-PCR after the addition of caspofungin to cells grown for 15 h in Sabouraud dextrose broth. In cells that remained viable, expression of SAP1 to SAP3, SAP7 to SAP9, and PLB1 was unaltered after exposure to fungicidal concentrations (4 to 16 μg/ml) of caspofungin over a period of 7 h. However, expression of SAP5 increased steadily beginning 1 h after exposure to caspofungin. These results indicate that caspofungin is rapidly fungicidal against C. albicans, before any suppression of SAP or PLB1 gene expression can occur.

Candida albicans secreted aspartyl proteinases (Saps) are considered virulence-associated factors. Several members of the Sap family were claimed to play a significant role in the progression of candidiasis established by the hematogenous route. This assumption was based on the observed attenuated virulence of sap-null mutant strains. However, the exclusive contribution of SAP genes to their attenuated phenotype was not unequivocally confirmed, as the Ura status of these mutant strains could also have contributed to the attenuation. In this study, we have reassessed the importance of SAP1 to SAP6 in a murine model of hematogenously disseminated candidiasis using sap-null mutant strains not affected in their URA3 gene expression and compared their virulence phenotypes with those of Ura-blaster sap mutants. The median survival time of BALB/c mice intravenously infected with a mutant strain lacking SAP1 to SAP3 was equivalent to that of mice infected with wild-type strain SC5314, while those infected with mutant strains lacking SAP5 showed slightly extended survival times. Nevertheless, no differences could be observed between the wild type and a Δsap456 mutant in their abilities to invade mouse kidneys. Likewise, a deficiency in SAP4 to SAP6 had no noticeable impact on the immune response elicited in the spleens and kidneys of C. albicans-infected mice. These results contrast with the behavior of equivalent Ura-blaster mutants, which presented a significant reduction in virulence. Our results suggest that Sap1 to Sap6 do not play a significant role in C. albicans virulence in a murine model of hematogenously disseminated candidiasis and that, in this model, Sap1 to Sap3 are not necessary for successful C. albicans infection.

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.

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.

Background

Candida albicans infections are often associated with biofilm formation. Previous work demonstrated that the expression of HWP1 (hyphal wall protein) and of genes belonging to the ALS (agglutinin-like sequence), SAP (secreted aspartyl protease), PLB (phospholipase B) and LIP (lipase) gene families is associated with biofilm growth on mucosal surfaces. We investigated using real-time PCR whether genes encoding potential virulence factors are also highly expressed in biofilms associated with abiotic surfaces. For this, C. albicans biofilms were grown on silicone in microtiter plates (MTP) or in the Centres for Disease Control (CDC) reactor, on polyurethane in an in vivo subcutaneous catheter rat (SCR) model, and on mucosal surfaces in the reconstituted human epithelium (RHE) model.

Results

HWP1 and genes belonging to the ALS, SAP, PLB and LIP gene families were constitutively expressed in C. albicans biofilms. ALS1-5 were upregulated in all model systems, while ALS9 was mostly downregulated. ALS6 and HWP1 were overexpressed in all models except in the RHE and MTP, respectively. The expression levels of SAP1 were more pronounced in both in vitro models, while those of SAP2, SAP4 and SAP6 were higher in the in vivo model. Furthermore, SAP5 was highly upregulated in the in vivo and RHE models. For SAP9 and SAP10 similar gene expression levels were observed in all model systems. PLB genes were not considerably upregulated in biofilms, while LIP1-3, LIP5-7 and LIP9-10 were highly overexpressed in both in vitro models. Furthermore, an elevated lipase activity was detected in supernatans of biofilms grown in the MTP and RHE model.

Conclusions

Our findings show that HWP1 and most of the genes belonging to the ALS, SAP and LIP gene families are upregulated in C. albicans biofilms. Comparison of the fold expression between the various model systems revealed similar expression levels for some genes, while for others model-dependent expression levels were observed. This suggests that data obtained in one biofilm model cannot be extrapolated to other model systems. Therefore, the need to use multiple model systems when studying the expression of genes encoding potential virulence factors in C. albicans biofilms is highlighted.

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 α-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 Δsap4-6 triple mutant induced a significantly reduced activity of ALT in comparison to the reference strain. In contrast to the Δsap1, Δsap2, and Δsap3 mutants, the ALT induced by the Δsap4-6 mutant could not be further reduced by pepstatin A treatment, which indicates that Sap4-6 may contribute to virulence in this model.

Purpose

To correlate the morphogenic and molecular traits that affect fungal virulence in human corneas.

Methods

C. albicans wild-type strains SC5314 and VE175 were compared using in vitro growth kinetics, filamentation assays, and microarray analysis. Corneal virulence was assessed ex vivo by inoculating C. albicans onto superficially scarified human corneas that were processed after 1 and 3 days to measure hyphal penetration. For comparison, DSY459, a C. albicans homozygous deletion mutant deficient in secreted aspartyl proteinases (SAP) 4, 5, and 6, was evaluated.

Results

C. albicans strain SC5314 was highly filamentous in vitro and more invasive in human corneal explants while VE175 demonstrated limited filamentation and less corneal invasion. Among 6,655 C. albicans genes, 9.0% significantly (p<.05) differed by 2 fold or more between SC5314 and VE175. Genes involved in fungal filamentation that were upregulated in strain SC5314 compared to VE175 included SAP5, SAP6, and other hypha-associated genes. Compared to wild-type strains, DSY459 had intermediate filamentation and stromal penetration.

Conclusions

Fungal genes involved in filamentation likely contribute to virulence differences between wild-type strains of C. albicans. The corneal pathogenicity of C. albicans involves the morphogenic transformation of yeasts into hyphae.

The regulation of morphogenesis in the human fungal pathogen Candida albicans is under investigation to better understand how the switch between budding and hyphal growth is linked to virulence. Therefore, in this study we examined the ability of C. albicans to undergo a distinct type of morphogenesis to form large thick-walled chlamydospores whose role in infection is unclear, but they act as a resting form in other species. During chlamydospore morphogenesis, cells switch to filamentous growth and then develop elongated suspensor cells that give rise to chlamydospores. These filamentous cells were distinct from true hyphae in that they were wider and were not inhibited by the quorum-sensing factor farnesol. Instead, farnesol increased chlamydospore production, indicating that quorum sensing can also have a positive role. Nuclear division did not occur across the necks of chlamydospores, as it does in budding. Interestingly, nuclei divided within the suspensor cells, and then one daughter nucleus subsequently migrated into the chlamydospore. Septins were not detected near mitotic nuclei but were localized at chlamydospore necks. At later stages, septins localized throughout the chlamydospore plasma membrane and appeared to form long filamentous structures. Deletion of the CDC10 or CDC11 septins caused greater curvature of cells growing in a filamentous manner and morphological defects in suspensor cells and chlamydospores. These studies identify aspects of chlamydospore morphogenesis that are distinct from bud and hyphal morphogenesis.

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.

The yeast Candida albicans possesses a gene family that encodes secreted aspartic proteases (Saps), which are important for the virulence of this human fungal pathogen. Inhibitors of the Saps could therefore be used as novel antimycotic agents for the treatment of C. albicans infections. In the present study, we established a bioassay which allows testing of the activity of potential protease inhibitors against specific Sap isoenzymes by their ability to inhibit protease-dependent growth of C. albicans. In a medium containing bovine serum albumin (BSA) as the sole source of nitrogen, C. albicans specifically expresses the Sap2p isoenzyme, which degrades the BSA and thereby enables the fungus to grow. As the other SAP genes are not significantly expressed under these conditions, mutants lacking SAP2 are unable to utilize BSA as a nitrogen source and cannot grow in such a medium. To investigate whether forced expression of SAP genes other than SAP2 would also allow growth on BSA, we constructed a set of strains expressing each of the 10 SAP genes from a tetracycline-inducible promoter in a sap2Δ mutant background. Expression of Sap1p, Sap2p, Sap3p, Sap4p, Sap5p, Sap6p, Sap8p, and a C-terminally truncated, secreted Sap9p restored the growth of the sap2Δ mutant with different efficiencies. This set of strains was then used to test the activities of various aspartic protease inhibitors against specific Sap isoenzymes by monitoring growth on BSA in the presence of the inhibitors. While pepstatin blocked the activity of all of the Saps tested, the human immunodeficiency virus protease inhibitors ritonavir and saquinavir inhibited growth of the strains expressing Sap1p to Sap3p and Sap1p, respectively, but not that of strains expressing other Saps. Therefore, the strain set can be used to test the activity of new protease inhibitors against individual C. albicans Sap isoenzymes by their ability to block the growth of the pathogen.

The opportunistic fungal pathogen Candida albicans can cause superficial as well as systemic infections. Successful adaptation to the different host niches encountered during infection requires coordinated expression of various virulence traits, including the switch between yeast and hyphal growth forms and secretion of aspartic proteinases. Using an in vivo expression technology that is based on genetic recombination as a reporter of gene activation during experimental candidiasis in mice, we investigated whether two signal transduction pathways controlling hyphal growth, a mitogen-activated protein kinase cascade ending in the transcriptional activator Cph1p and a cyclic AMP-dependent regulatory pathway that involves the transcription factor Efg1p, also control expression of the SAP5 gene, which encodes one of the secreted aspartic proteinases and is induced by host signals soon after infection. Our results show that both transcriptional regulators are important for SAP5 activation in vivo. SAP5 expression was reduced in a cph1 mutant, although filamentous growth in infected tissue was not detectably impaired. SAP5 expression was also reduced, but not eliminated, in an efg1 null mutant, although this strain grew exclusively in the yeast form in infected tissue, demonstrating that in contrast to in vitro conditions, SAP5 activation during infection does not depend on growth of C. albicans in the hyphal form. In a cph1 efg1 double mutant, however, SAP5 expression in infected mice was almost completely eliminated, suggesting that the two signal transduction pathways are important for SAP5 expression in vivo. The avirulence of the cph1 efg1 mutant seemed to be caused not only by the inability to form hyphae but also by a loss of expression of additional virulence genes in the host.

Vaginal infections caused by the opportunistic yeast Candida albicans are a significant problem in women of child-bearing age. Several factors are recognized as playing a crucial role in the pathogenesis of superficial candidiasis; these factors include hyphal formation, phenotypic switching, and the expression of virulence factors, including a 10-member family of secreted aspartic proteinases. In the present investigation, we analyzed the secreted aspartic proteinase gene (SAP) expression profile of C. albicans that is elicited in the course of vaginal infection in mice and how this in vivo expression profile is associated with hyphal formation. We utilized two different genetic reporter systems that allowed us to observe SAP expression on a single-cell basis, a recombination-based in vivo expression technology and green fluorescent protein-expressing Candida reporter strains. Of the six SAP genes that were analyzed (SAP1 to SAP6), only SAP4 and SAP5 were detectably induced during infection in this model. Expression of both of these genes was associated with hyphal growth, although not all hyphal cells detectably expressed SAP4 and SAP5. SAP5 expression was induced soon after infection, whereas SAP4 was expressed at later times and in fewer cells compared with SAP5. These findings point to a link between morphogenetic development and expression of virulence genes during Candida vaginitis in mice, where host signals induce both hyphal formation and expression of SAP4 and SAP5, but temporal gene expression patterns are ultimately controlled by other factors.

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.

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.

In response to a wide variety of environmental stimuli, the opportunistic fungal pathogen Candida albicans exits the budding cycle, producing germ tubes and hyphae concomitant with expression of virulence genes, such as that encoding hyphal wall protein 1 (HWP1). Biochemical studies implicate cyclic AMP (cAMP) increases in promoting bud-hypha transitions, but genetic evidence relating genes that control cAMP levels to bud-hypha transitions has not been reported. Adenylate cyclase-associated proteins (CAPs) of nonpathogenic fungi interact with Ras and adenylate cyclase to increase cAMP levels under specific environmental conditions. To initiate studies on the relationship between cAMP signaling and bud-hypha transitions in C. albicans, we identified, cloned, characterized, and disrupted the C. albicans CAP1 gene. C. albicans strains with inactivated CAP1 budded in conditions that led to germ tube formation in isogenic strains with CAP1. The addition of 10 mM cAMP and dibutyryl cAMP promoted bud-hypha transitions and filamentous growth in the cap1/cap1 mutant in liquid and solid media, respectively, showing clearly that cAMP promotes hypha formation in C. albicans. Increases in cytoplasmic cAMP preceding germ tube emergence in strains having CAP1 were markedly diminished in the budding cap1/cap1 mutant. C. albicans strains with deletions of both alleles of CAP1 were avirulent in a mouse model of systemic candidiasis. The avirulence of a germ tube-deficient cap1/cap1 mutant coupled with the role of Cap1 in regulating cAMP levels shows that the Cap1-mediated cAMP signaling pathway is required for bud-hypha transitions, filamentous growth, and the pathogenesis of candidiasis.

Isolates of Candida albicans from the oral cavities of subjects at different stages of human immunodeficiency virus (HIV) infection or uninfected controls were examined for (i) production of aspartic proteinase(s), a putative virulence-associated factor(s); (ii) the presence in the fungal genome of two major genes (SAP1 and SAP2) of the aspartic proteinase family; and (iii) experimental pathogenicity in a murine model of systemic infection. It was found that the fungal isolates from symptomatic patients secreted, on average, up to eightfold more proteinase than the isolates from uninfected or HIV-infected but asymptomatic subjects. This differential property was stably expressed by the strains even after years of maintenance in stock cultures. Moreover, representative high-proteinase isolates were significantly more pathogenic for mice than low-proteinase isolates of C. albicans. The characters high proteinase and increased virulence were not associated with a single molecular type or category identifiable through DNA fingerprinting or pulsed-field electrophoretic karyotype, and both SAP1 and SAP2 genes were present in both categories of isolates, on the same respective chromosomes. In conclusion, our data suggest that during HIV infection more-virulent strains or biotypes of C. albicans which are identifiable by direct analysis of virulence determinants are selected. It also appears that the biotype switch to increased aspartic proteinase and virulence properties occurs before the HIV-infected subject enters the symptomatic stage and overt AIDS.

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.

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.