Secreted Aspartyl Proteinase Genes Research Articles

Upregulation of secreted aspartyl proteinase genes of fluconazole-sensitive Candida albicans isolates.

Candida albicans infection development depends on several factors associated with this etiological agent, including secreted aspartyl protease (Sap) production. Sap expression commonly occurs under selective pressure caused by the presence of antifungals. Fluconazole is the main antifungal drug used for treatment or prophylaxis. This study investigated the influence of inhibitory and sub-inhibitory fluconazole concentrations on Sap activity and their gene transcription for three clinical C. albicans isolates. Two isolates presented significant increases in Sap activity and transcription of SAP 1-8 genes in the presence of 1 MIC80 of fluconazole compared to the absence of the antifungal agent. The results suggest that the increase in Sap activity occurs due to an upregulation of the SAP gene transcription influenced by fluconazole. This suggests the importance of all SAP genes in the progression of bloodstream infections compared to primary tissue infection. However, this phenomenon does not occur everywhere, and it is multifactorial. This may be related to the selective pressure effect on transcription modulators. Although preliminary, these results open a new perspective for the study of virulence factors.

Genotypic Patterns of Secreted Aspartyl Proteinase Gene in Various Candida Species Isolated from Antenatal Women with Vulvovaginal Candidiasis

Genotypic Patterns of Secreted Aspartyl Proteinase Gene in Various Candida Species Isolated from Antenatal Women with Vulvovaginal Candidiasis

Farnesol, a Fungal Quorum-Sensing Molecule Triggers Candida Albicans Morphological Changes by Downregulating the Expression of Different Secreted Aspartyl Proteinase Genes

The aim of this study was to determine the effect of exogenous farnesol in yeast-to-hyphae morphogenesis, and Saps (2, 4, 5 and 6) mRNA expressions by a Candida strain that does not produce endogenous farnesol. C. albicans was cultured in the absence and presence of farnesol at various concentrations (10, 100, and 300 µM), in proteinase induction medium, and then used to determine yeast-to- hyphae changes, Candida ultrastructure and to determine Saps 2, 4, 5 and 6 expressions using q-TR-PCR and ELISA (for Sap2). Data demonstrated that farnesol greatly reduced the yeast-to-hyphae morphogenesis of a Candida strain that does not produce endogenous farnesol. Farnesol induced several ultrastructural alterations, including changes in the cell-wall shape, a visible disconnection between the cell wall and cytoplasm with an electron-lucent zone between them, and the presence of electron-dense vacuoles. Tested on gene expressions, farnesol was able to significantly (p < 0.01) decrease Sap2 secretion and mRNA expression. Farnesol downregulated also Sap4-6 mRNA expression. These results demonstrated for the first time that farnesol modules Candida morphogenesis through a downregulation of Saps 2, 4, 5 and 6 expressions. Overall these data point to the potential use of farnesol as an antifungal molecule

An in vitro evaluation of Candida tropicalis infectivity using human cell monolayers

The aim of this study was to investigate the interaction of Candida tropicalis with three different human cell lines: TCC-SUP (epithelial cells from urinary bladder), HeLa (epithelial cells from cervical carcinoma) and Caco-2 (epithelial cells from colorectal adenocarcinoma). In particular we sought to assess the degree of cell damage and activity reduction induced by C. tropicalis adhesion and the role of secreted aspartyl proteinase (SAP) gene expression in this process. Two C. tropicalis strains were used: the reference strain ATCC 750 and a clinical isolate from urine (U69). The ability of C. tropicalis to adhere to a confluent layer of human cells was determined using an adaptation of the crystal violet staining method; cell damage and cell activity inhibition induced by the adhesion of C. tropicalis were assessed by measuring lactate dehydrogenase and tetrazolium salt (MTS) reduction, respectively. C. tropicalis SAP gene expression was determined by real-time PCR. Both C. tropicalis strains were able to adhere to the different human cells, although in a strain- and cell-line-dependent manner. Concerning the cellular response to C. tropicalis, the highest inhibition of cell activity was obtained for Caco-2, followed by TCC-SUP and HeLa cells. The highest percentage of cell damage (around 14 %) was observed for TCC-SUP cells in contact with the U69 isolate and for Caco-2 in contact with the reference strain. Real-time PCR analysis revealed a wide range of expression profiles of SAP genes for both C. tropicalis strains in contact with the different types of epithelial cells. SAPT3 was the gene expressed at the highest level for both C. tropicalis strains in contact with the three human epithelial cell lines. The results highlight that the response of human cells to C. tropicalis adhesion, as well as production of SAPs, is dependent on both the strain and the epithelial cell line.

The role of secreted aspartyl proteinases in Candida tropicalis invasion and damage of oral mucosa

Candida virulence attributes include the ability to colonize and invade host tissues, and the secretion of hydrolytic enzymes. Although Candida albicans is regarded as the principal fungi causing infections in humans, other species, particularly Candida tropicalis, are increasingly being recognized as human pathogens. Relatively little is known, however, about the virulence attributes associated with C. tropicalis. The present study aimed to investigate epithelial infection by C. tropicalis using a reconstituted human oral epithelium (RHOE) together with confocal laser scanning microscopy and real-time PCR. A comparison of clinical strains was made in terms of tissue colonization, invasion and C. tropicalis secreted aspartyl proteinase (SAPT) gene expression. All C. tropicalis strains were able to colonize RHOE in a strain-dependent manner. After 12 h of infection, C. tropicalis was found to be highly invasive, with extensive tissue damage occurring after 24 h. Real-time PCR of C. tropicalis SAPT1-4 genes showed that expression was strain-dependent, with SAPT2-4 transcripts being frequently detected and SAPT1 rarely detected. Tissue invasion and damage was not inhibited by the presence of pepstatin A. Accordingly, and given that an increase in infection time was not accompanied with an increase in SAPT gene expression, it can be suggested that the proteinases are not involved in invasion and damage of RHOE by C. tropicalis. In summary, C. tropicalis can be considered as highly invasive with the ability to induce significant tissue damage. These features, however, do not appear to be related to specific SAPT gene expression.

Differential expression ofCandida dubliniensis-secreted aspartyl proteinase genes (CdSAP1–4) under different physiological conditions and during infection of a keratinocyte culture

The in vitro and keratinocyte (HaCAT cells) culture expression of four putative genes coding for secreted aspartyl proteases of Candida dubliniensis-CdSAP1, CdSAP2, CdSAP3, and CdSAP4 (CdSAP1-4) - is reported for the first time. In addition, CdSAP7, 8, 9, and 10, orthologous genes of Candida albicans, were recognized in C. dubliniensis genome. There are no orthologs of C. albicans SAP5 and 6 in C. dubliniensis. The expression of CdSAP1 and 2 was independent of the morphological stage of C. dubliniensis; they are expressed at both pH 4 and pH 7, and were induced with albumin as nitrogen source. CdSAP3 expression was regulated by the pH, and was related to the infection process of keratinocytes. Expression of CdSAP4 predominated during the mycelial phase and the initial stage of keratinocyte infection. During infection of the HaCaT cell line, only genes CdSAP3-4 were expressed, and keratinocytes were affected in their number and shape by the infection with C. dubliniensis; however, this effect decreased in the presence of pepstatin A (aspartyl protease inhibitor). Pepstatin A was not able to inhibit keratinocyte damage. Based on the aforementioned, we suggest that the Saps from C. dubliniensis could be considered a virulence factor just as those from C. albicans, and participants in the nitrogen metabolism of the yeast for nutrient acquisition.

Quantitative expression of the Candida albicans secreted aspartyl proteinase gene family in human oral and vaginal candidiasis

A quantitative real-time RT-PCR system was established to identify which secreted aspartyl proteinase (SAP) genes are most highly expressed and potentially contribute to Candida albicans infection of human epithelium in vitro and in vivo. C. albicans SC5314 SAP1-10 gene expression was monitored in organotypic reconstituted human epithelium (RHE) models, monolayers of oral epithelial cells, and patients with oral (n=17) or vaginal (n=17) candidiasis. SAP gene expression was also analysed in Deltasap1-3, Deltasap4-6, Deltaefg1 and Deltaefg1/cph1 mutants to determine whether compensatory SAP gene regulation occurs in the absence of distinct proteinase gene subfamilies. In monolayers, RHE models and patient samples SAP9 was consistently the most highly expressed gene in wild-type cells. SAP5 was the only gene significantly upregulated as infection progressed in both RHE models and was also highly expressed in patient samples. Interestingly, the SAP4-6 subfamily was generally more highly expressed in oral monolayers than in RHE models. SAP1 and SAP2 expression was largely unchanged in all model systems, and SAP3, SAP7 and SAP8 were expressed at low levels throughout. In Deltasap1-3, expression was compensated for by increased expression of SAP5, and in Deltasap4-6, expression was compensated for by SAP2: both were observed only in the oral RHE. Both Deltasap1-3 and Deltasap4-6 mutants caused RHE tissue damage comparable to the wild-type. However, addition of pepstatin A reduced tissue damage, indicating a role for the Sap family as a whole in inducing epithelial damage. With the hypha-deficient mutants, RHE tissue damage was significantly reduced in both Deltaefg1/cph1 and Deltaefg1, but SAP5 expression was only dramatically reduced in Deltaefg1/cph1 despite the absence of hyphal growth in both mutants. This indicates that hypha formation is the predominant cause of tissue damage, and that SAP5 expression can be hypha-independent and is not solely controlled by the Efg1 pathway but also by the Cph1 pathway. This is believed to be the first study to fully quantify SAP gene expression levels during human mucosal infections; the results suggest that SAP5 and SAP9 are the most highly expressed proteinase genes in vivo. However, the overall contribution of the Sap1-3 and Sap4-6 subfamilies individually in inducing epithelial damage in the RHE models appears to be low.

Characterization of Candida albicans infection of an in vitro oral epithelial model using confocal laser scanning microscopy

Oral candidosis presents as several distinct forms and one of these, chronic hyperplastic candidosis, is distinguished by penetration of the epithelium by Candida. The aim of this study was to use confocal laser scanning microscopy to examine invasion of the oral epithelium by Candida albicans from different oral conditions and to determine whether inherent strain differences exist that could relate to infection type. Reverse transcription-polymerase chain reaction was also used to detect products from virulence gene families. C. albicans (n = 19) was used to infect reconstituted human oral epithelium, which was incubated for 12 h. One half of the reconstituted human oral epithelium was then fixed and stained with concanavalin A-Alexa 594, pan-cytokeratin antibody-Alexa 488 and Hoechst nucleic acid dye. RNA was extracted from the remaining tissue for reverse transcription-polymerase chain reaction targeting secreted aspartyl proteinase, phospholipase and agglutinin-like sequence genes of C. albicans. Confocal laser scanning microscopy revealed strain-dependent tissue invasion, with differences evident in surface colonization, C. albicans morphology and the extent and pattern of tissue penetration. Hyphae were seen to directly penetrate epithelial cells and migrate between keratinocytes with yeast budding also evident in the reconstituted human oral epithelium. A relationship between 'high tissue invasion' and expression of secreted aspartyl proteinase genes 4-6 was noted. Interestingly, four of the five 'high invaders' originated from chronic hyperplastic candidosis. Confocal laser scanning microscopy permitted high resolution analysis of reconstituted human oral epithelium invasion by C. albicans and identified strain differences in the invasion process. Association between extensive hyphal morphology, direct epithelial penetration and high surface colonization were made with the 'highly invasive' strains.

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.

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.

Molecular targets for the development of AIDS therapeutics : C. Debouck, Department of Molecular Genetics, SmitKline Beecham Pharmaceuticals, King of Prussia, PA 19406, USA

Candida virulence attributes include the ability to colonize and invade host tissues, and the secretion of hydrolytic enzymes. Although Candida albicans is regarded as the principal fungi causing infections in humans, other species, particularly Candida tropicalis, are increasingly being recognized as human pathogens. Relatively little is known, however, about the virulence attributes associated with C. tropicalis. The present study aimed to investigate epithelial infection by C. tropicalis using a reconstituted human oral epithelium (RHOE) together with confocal laser scanning microscopy and real-time PCR. A comparison of clinical strains was made in terms of tissue colonization, invasion and C. tropicalis secreted aspartyl proteinase (SAPT) gene expression. All C. tropicalis strains were able to colonize RHOE in a strain-dependent manner. After 12 h of infection, C. tropicalis was found to be highly invasive, with extensive tissue damage occurring after 24 h. Real-time PCR of C. tropicalis SAPT1-4 genes showed that expression was strain-dependent, with SAPT2-4 transcripts being frequently detected and SAPT1 rarely detected. Tissue invasion and damage was not inhibited by the presence of pepstatin A. Accordingly, and given that an increase in infection time was not accompanied with an increase in SAPT gene expression, it can be suggested that the proteinases are not involved in invasion and damage of RHOE by C. tropicalis. In summary, C. tropicalis can be considered as highly invasive with the ability to induce significant tissue damage. These features, however, do not appear to be related to specific SAPT gene expression.