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• W2033383118 abstract "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. 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. secreted aspartyl proteinase (gene) secreted aspartyl proteinase (protein) The opportunistic yeast Candida albicans, the major cause of cutaneous and mucosal candidiasis, possesses a panel of putative virulence factors that are thought to enable the fungus to invade host tissue (Homma et al., 1992Homma M. Kanabe T. Hiroji C. Tanak K. Detection of intracellular forms of secretory aspartic proteinase in.Candida Albicans. J Gen Microbiol. 1992; 138: 627-633Crossref PubMed Scopus (18) Google Scholar;Hoegl et al., 1996Hoegl L. Ollert M.W. Korting H.C. The role of Candida albicans secreted aspartic proteinase in the development of candidoses.J Mol Med. 1996; 74: 135-142Crossref PubMed Scopus (52) Google Scholar). Among these virulence attributes, many studies have focused on secreted aspartyl proteinases (Saps). Sap enzymatic activities have received considerable attention in several in vitro (Borg-von Zepelin & Rüchel 1988;De Bernardis et al., 1992De Bernardis F. Boccanera M. Rainaldi L. Guerra C.E. Quinti I. Cassone A. The secretion of aspartyl proteinase, a virulence enzyme, by isolates of Candida albicans from the oral cavity of HIV-infected subjects.Eur J Epidemiol. 1992; 8: 362-367Crossref PubMed Scopus (33) Google Scholar;Homma et al., 1992Homma M. Kanabe T. Hiroji C. Tanak K. Detection of intracellular forms of secretory aspartic proteinase in.Candida Albicans. J Gen Microbiol. 1992; 138: 627-633Crossref PubMed Scopus (18) Google Scholar;Ollert et al., 1993Ollert M.W. Söhnchen R. Korting H.C. Ollert U. Bräutigam S. Bräutigam W. Mechanisms of adherence of Candida albicans to cultured human epidermal keratinocytes.Infect Immun. 1993; 61: 4560-4568Crossref PubMed Google Scholar) and animal studies (MacDonald and Odds, 1983MacDonald F. Odds F.C. Virulence for mice of a proteinase-secreting strain of Candida albicans and a proteinase-deficient mutant.J Gen Microbiol. 1983; 129: 431-438PubMed Google Scholar;Kwon-Chung et al., 1985Kwon-Chung K.J. Lehman D. Good C. Magee P.T. Genetic evidence for role of extracellular proteinase in virulence of.Candida Albicans. Infect Immun. 1985; 49: 571-575PubMed Google Scholar;Ghannoum and Abu Elteen, 1986Ghannoum M. Abu Elteen K. Correlative relationship between proteinase production, adherence and pathogenicity of various strains of Candida albicans..J Med Vet Mycol. 1986; 24: 407-413Crossref PubMed Scopus (102) Google Scholar;Ray and Payne, 1988Ray T.L. Payne C.D. Scanning electron microscopy of epidermal adherence and cavitation in murine candidiasis: a role for Candida acid proteinase.Infect Immun. 1988; 56: 1942-1949Crossref PubMed Google Scholar;Fallon et al., 1997Fallon K. Bausch K. Noonan J. Huguenel E. Tamburini P. Role of aspartic proteases in disseminated Candida albicans infection in mice.Infect Immun. 1997; 65: 551-556Crossref PubMed Google Scholar). In these studies various biologic functions have been attributed to the Saps. For example, it has been suggested that Saps may enhance attachment to mucosal surfaces (Borg-von Zepelin and Rüchel, 1988Borg-von Zepelin M. Rüchel R. Expression of extracellular acid proteinase by proteolytic Candida spp during experimental infection of oral mucosa.Infect Immun. 1988; 56: 626-631PubMed Google Scholar), help to escape phagocytosis by granulocytes and macrophages (MacDonald and Odds, 1983MacDonald F. Odds F.C. Virulence for mice of a proteinase-secreting strain of Candida albicans and a proteinase-deficient mutant.J Gen Microbiol. 1983; 129: 431-438PubMed Google Scholar;Ghannoum and Abu Elteen, 1986Ghannoum M. Abu Elteen K. Correlative relationship between proteinase production, adherence and pathogenicity of various strains of Candida albicans..J Med Vet Mycol. 1986; 24: 407-413Crossref PubMed Scopus (102) Google Scholar;Borg-von Zepelin et al., 1998Borg-von Zepelin M. Beggah S. Boggian K. Sanglard D. Monod M. The expression of the secreted aspartyl proteinases Sap4 to Sap6 from Candida albicans in murine macrophages.Mol Microbiol. 1998; 28: 543-554Crossref PubMed Scopus (157) Google Scholar), or hydrolyze immunoglobulins such as secretory IgA (Hoegl et al., 1996Hoegl L. Ollert M.W. Korting H.C. The role of Candida albicans secreted aspartic proteinase in the development of candidoses.J Mol Med. 1996; 74: 135-142Crossref PubMed Scopus (52) Google Scholar;Hube, 1996Hube B. Candida albicans secreted aspartyl proteinases.Curr Top Med Mycol. 1996; 7: 55-69PubMed Google Scholar). In previous electronmicroscopic investigations of the interaction of C. albicans cells with human keratinocytes, the influence of keratinolytic enzymes on the initiation of disease was postulated (Borg-von Zepelin and Rüchel, 1988Borg-von Zepelin M. Rüchel R. Expression of extracellular acid proteinase by proteolytic Candida spp during experimental infection of oral mucosa.Infect Immun. 1988; 56: 626-631PubMed Google Scholar). Using surface electron microscopy it has been shown that the addition of the aspartyl proteinase inhibitor pepstatin A reduced the number of Candida elements adhering to epithelia in experimental infections (Borg-von Zepelin and Rüchel, 1988Borg-von Zepelin M. Rüchel R. Expression of extracellular acid proteinase by proteolytic Candida spp during experimental infection of oral mucosa.Infect Immun. 1988; 56: 626-631PubMed Google Scholar), scanning electron microscopic investigations also demonstrated that inhibition of Saps by pepstatin reduces a cavitation process of C. albicans on mouse corneocyte surfaces (Ray and Payne, 1988Ray T.L. Payne C.D. Scanning electron microscopy of epidermal adherence and cavitation in murine candidiasis: a role for Candida acid proteinase.Infect Immun. 1988; 56: 1942-1949Crossref PubMed Google Scholar). Antibodies against Saps in systemic candidiasis as indirect evidence for expression in man were demonstrated (Rüchel, 1983Rüchel R. On the role of proteinases from Candida albicans in the pathogenesis of acronecrosis.Zbl Bakt Hyg Orig A. 1983; 255: 524-536Google Scholar). Until now at least nine different SAP genes have been identified (Monod et al., 1998Monod M. Togni G. Hube B. Heβ D. Sanglard D. Cloning, sequencing and expression of two new members of the secreted aspartyl proteinase family of.Candida Albicans. Microbiology. 1998; 144: 2731-2737Google Scholar). Northern analysis of the SAP gene family showed a differential regulation of these genes in response to environmental factors and cell morphology (Hube et al., 1994Hube B. Monod M. Schofield D.A. Brown A.J.P. Gow N.A.R. Expression of seven members of the gene family encoding secretory aspartyl proteinase in.Candida Albicans. Mol Microbiol. 1994; 14: 87-99Crossref PubMed Scopus (323) Google Scholar;White and Agabian, 1995White T.C. Agabian N. Candida albicans secreted aspartyl proteinases: isoenzyme pattern is determined by cell type, levels are determined by environmental factors.J Bacteriol. 1995; 177: 5215-5221Crossref PubMed Google Scholar;Hube, 1996Hube B. Candida albicans secreted aspartyl proteinases.Curr Top Med Mycol. 1996; 7: 55-69PubMed Google Scholar;Monod et al., 1998Monod M. Togni G. Hube B. Heβ D. Sanglard D. Cloning, sequencing and expression of two new members of the secreted aspartyl proteinase family of.Candida Albicans. Microbiology. 1998; 144: 2731-2737Google Scholar). This suggests that distinct SAP genes may play different roles during the infection process (Hube et al., 1994Hube B. Monod M. Schofield D.A. Brown A.J.P. Gow N.A.R. Expression of seven members of the gene family encoding secretory aspartyl proteinase in.Candida Albicans. Mol Microbiol. 1994; 14: 87-99Crossref PubMed Scopus (323) Google Scholar). Western blot analysis of C. albicans cell culture supernatants experiments showed that Sap isoenzymes were predominantly regulated on the transcriptional level (White and Agabian, 1995White T.C. Agabian N. Candida albicans secreted aspartyl proteinases: isoenzyme pattern is determined by cell type, levels are determined by environmental factors.J Bacteriol. 1995; 177: 5215-5221Crossref PubMed Google Scholar). The expression of SAP1 and SAP2 has been demonstrated by northern blot analysis by means of an experimental rat vaginitis model (De Bernardis et al., 1995De Bernardis F. Cassone A. Sturvetant J. Calderone R. Expression of Candida albicans SAP1 and SAP2 in experimental vaginitis.Infect Immun. 1995; 63: 1887-1892PubMed Google Scholar). Animal experiments with SAP null mutants indirectly showed that SAP1, 2, 3 and at least one of the closely related SAP4–6 genes are expressed during disseminated infections, and presumably contribute to the overall virulence of C. albicans (Hube et al., 1997Hube B. Sanglard D. Odds F.C. Hess D. Monod M. Schäfer W. Brown A.J.P. Gow N.A.R. Gene disruption of each of the secreted aspartyl proteinase genes SAP1, SAP2 and SAP3 in Candida albicans attenuates virulence.Infect Immun. 1997; 65: 3529-3538PubMed Google Scholar;Sanglard et al., 1997Sanglard D. Hube B. Monod M. Odds F.C. Gow N.A.R. A triple deletion in SAP4, SAP5 and SAP6 secretory aspartyl proteinase genes of Candida albicans causes attenuated virulence.Infect Immun. 1997; 65: 3539-3546Crossref PubMed Google Scholar). The regulation of SAP mRNA levels was recently investigated in an in vitro model of oral candidiasis based on reconstituted human epithelium. In these experimental infections, the expression of SAP1, SAP3, and SAP6 correlated chronologically with severe histologic alterations of the epithelium. Furthermore, immunoelectron microscopical investigations of the infected tissue demonstrated Sap antigen within the C. albicans and the epithelial cells, suggesting an important role of Sap isoenzymes during mucosal infections (Schaller et al., 1998Schaller M. Schäfer W. Korting H.C. Hube B. Differential expression of secreted aspartyl proteinases in a model of human oral candidosis and in patient samples from the oral cavity.Mol Microbiol. 1998; 29: 605-615Crossref PubMed Scopus (183) Google Scholar). A similar gene expression pattern (SAP1, SAP2, SAP3, SAP6) was also detected by reverse transcriptase-polymerase chain reaction in clinical samples from patients suffering from oral candidiasis (Schaller et al., 1998Schaller M. Schäfer W. Korting H.C. Hube B. Differential expression of secreted aspartyl proteinases in a model of human oral candidosis and in patient samples from the oral cavity.Mol Microbiol. 1998; 29: 605-615Crossref PubMed Scopus (183) Google Scholar). In this report we demonstrate the localization and expression of Sap antigen during human oropharyngeal candidiasis for the first time, using a monoclonal antibody directed against the secreted isoforms of Sap1–3. The localization of the enzymes in vivo was studied by pre- and post-embedding immunoelectron microscopy. Samples of pseudomembrane were removed from the tongue of three volunteer male HIV-infected patients (A, B, C). They were 37, 56, and 44 y old and had been suffering from pseudomembraneous oropharyngeal candidiasis for at least 1 y. In spite of continuous treatment with systemic anti-mycotic drugs at the time the specimens were obtained, no improvement of the oropharyngeal candidiasis had been achieved. Parts of the clinical material of all three patients were used for microbiologic culture, biochemical characterization, and immunoelectron microscopical investigations. The specimens obtained were inoculated on Kimmig’s Agar (Merck, Darmstadt, Germany) and incubated for 72 h at 37°C. Biochemical identification of C. albicans was based on the use of the Ready-Made System ATB 32 C (API System, bio Mérieux, La Balme-les-Grottes, Montalieu Vercieu, France). Post-embedding immunogold labeling was carried out for intracellular detection of antigen. Scrapings of the tongue were fixed in Karnovsky solution for 30 min at room temperature and embedded in glycide ether. Sections of 80–100 nm thickness were mounted on nickel grids. The grids were rinsed on drops of dH2O for 10 min, and floated on drops of phosphate-buffered saline (PBS), containing 5% (vol/vol) normal goat serum, for 2 × 10 min. Grids were then incubated with the anti-Sap 1–3 monoclonal mouse IgG antibody FX 7–10 (Ollert et al., 1995Ollert M.W. Wende C. Görlich M. McMullan-Vogel M.G. Borg-von Zepelin M. Vogel C.-W. Korting H.C. Increased expression of Candida albicans secretory proteinase, a virulence factor, in isolates from human immunodeficiency virus- positive patients.J Clin Microbiol. 1995; 33: 2543-2549Crossref PubMed Google Scholar;Schaller et al., 1998Schaller M. Schäfer W. Korting H.C. Hube B. Differential expression of secreted aspartyl proteinases in a model of human oral candidosis and in patient samples from the oral cavity.Mol Microbiol. 1998; 29: 605-615Crossref PubMed Scopus (183) Google Scholar) diluted in PBS supplemented with 0.1% bovine serum albumin and 0.05% Tween 20 (PBS-BT) at a ratio of 1:50 for 14 h at 4°C. As demonstrated by enzyme-linked immunosorbent assay, FX 7–10 was shown to react strongly with Sap1, Sap2, and Sap3, but not with Sap4, Sap5, and Sap6. Cross-reactivity with pepsin and cathepsin D was not detected (Borg-von Zepelin, personal communication). The exact experimental system for testing the specificity of the antibody has been described previously (Borg-von Zepelin and Grüness, 1993Borg-von Zepelin M. Grüness V. Characterization of two monoclonal antibodies against secretory proteinase against Candida tropicalis DSM 4328.J Med Vet Mycol. 1993; 31: 1-15Crossref PubMed Scopus (12) Google Scholar). Grids were washed repeatedly with PBS-BT and incubated with the 10 nm gold-conjugated goat anti-mouse IgG (Auroprobe EM Immunogold reagents, Amersham, Buckinghamshire, U.K.) diluted 1:25 in TBS (0.02 M Tris hydrochloride acid buffer, 0.15 M NaCl, 0.015 M Na-azide, 0.1% bovine serum albumin, 0.05% Tween 20, adjusted to pH 8.2) for 1 h at room temperature. In control samples the anti-Sap1–3 monoclonal antibody was omitted. After several washing steps with TBS-BT, grids were fixed with 2% glutaraldehyde and washed again in dH2O. They were stained with 0.5% uranyl acetate for 10 min, and 2.7% lead citrate for 5 min (Ultrastainer, Leica, Bensheim, Germany) at 20°C. Grids were examined with a Zeiss EM 902 transmission electron microscope (Zeiss, Oberkochen, Germany) operating at 80 kV, at magnifications between ×3000 and ×85,000. Pre-embedding immunogold labeling was carried out for studying antigen distribution on the cell surfaces. The unfixed scrapings from the tongue were preincubated with 5% normal goat serum (Amersham) in PBS for 20 min at room temperature and then incubated with the anti-Sap1–3 monoclonal mouse IgG antibody FX 7–10 (Ollert et al., 1995Ollert M.W. Wende C. Görlich M. McMullan-Vogel M.G. Borg-von Zepelin M. Vogel C.-W. Korting H.C. Increased expression of Candida albicans secretory proteinase, a virulence factor, in isolates from human immunodeficiency virus- positive patients.J Clin Microbiol. 1995; 33: 2543-2549Crossref PubMed Google Scholar;Schaller et al., 1998Schaller M. Schäfer W. Korting H.C. Hube B. Differential expression of secreted aspartyl proteinases in a model of human oral candidosis and in patient samples from the oral cavity.Mol Microbiol. 1998; 29: 605-615Crossref PubMed Scopus (183) Google Scholar) diluted in PBS supplemented with 0.1% bovine serum albumin and 0.05% Tween 20 (PBS-BT) at a ratio of 1:10 for 12 h at 4°C. After washing with PBS, tissue samples were incubated with 5 nm or the 10 nm gold-conjugated goat anti-mouse IgG (Auroprobe EM Immunogold reagents, Amersham), diluted 1:5 in TBS for 3 h at room temperature. In control samples the anti-Sap1–3 monoclonal antibody was omitted. Tissue samples were washed with TBS, supplemented with 1% bovine serum albumin, fixed in Karnovsky solution for 1 h at room temperature, and repeatedly washed with PBS. For higher detection efficiency silver enhancement (IntenSE M Silver Enhancement System, Amersham) was performed in a subset of the samples, which were labeled with 5 nm gold particles. The other subset was post-fixed in Dalton solution for 30 min at room temperature. Both subsets were dehydrated in graded series of ethanol, and embedded in glycide ether. Sections of 60–90 nm thickness were mounted on uncoated copper grids and stained with 0.5% uranyl acetate for 10 min and 2.7% lead citrate for 5 min (Ultrastainer, Leica, Germany) at 20°C, and examined with the Zeiss EM 902 transmission electron microscope operating at 80 kV, at magnifications between ×3000 and ×85,000. All three samples investigated were from HIV-infected patients because higher proteolytic activity in strains isolated from those patients was demonstrated (Ollert et al., 1995Ollert M.W. Wende C. Görlich M. McMullan-Vogel M.G. Borg-von Zepelin M. Vogel C.-W. Korting H.C. Increased expression of Candida albicans secretory proteinase, a virulence factor, in isolates from human immunodeficiency virus- positive patients.J Clin Microbiol. 1995; 33: 2543-2549Crossref PubMed Google Scholar). Candida albicans was identified microbiologically in all three patients. In all patient samples bacteria (not shown) and different morphologic types of C. albicans cells (Figure 1, Figure 2,Figure 3) could be observed on the ultrastructural level. The diameters of the C. albicans cells ranged from 1.5 μm to 3 μm. Often yeast elements were attached to keratinocytes (Figure 2), hyphal cells were penetrating parakeratotic keratinocytes or corneocytes (Figure 3) and were also found within these cells (Figure 1). Figure 1 and Figure 2 demonstrate Sap immunoreactivity after post-embedding immunogold labeling, whereas Sap immunoreactivity after pre-embedding immunogold labeling is shown in Figure 3. Two different labeling methods were performed in all samples in order to detect intra- and extracellularly localized Sap antigens with a monoclonal antibody directed against Sap1–3. Five nanometer gold particles were used to improve sensitivity. Silver enhancement was performed to intensify the labeling of the 5 nm gold particles so that detection of the immunoreactivity could occur more easily. Sap immunoreactivity was seen in all three cases of oral infections and findings were similar in each patient. Sap antigens were detected within and in close vicinity to different morphologic types of C. albicans cells, e.g., within yeast cells attached to keratinocytes or with hyphal elements penetrating parakeratotic keratinocytes or corneocytes. For detection of Sap immunoreactivity within the C. albicans cells or the keratinocytes, post-embedding labeling with 10 nm gold particles showed two different patterns of expression. In a subset of cells, intensive gold labeling, representing the enzymes, could be detected in a regular distribution within the cytoplasm of the Candida cells, whereas only a few gold particles were seen within the cell wall and the epithelial cells near by (Figure 1). In another subset the majority of Sap immunoreactivity was identified in the outer area of the Candida cells, mainly concentrated at the site of direct contact between Candida and the host cell. Gold labeling was detected especially in close proximity to the invaginations lying between the plasma membrane and the cell wall, and within the adjoining epithelial cells (Figure 2). It is tempting to speculate that the intracellular distribution of Sap1–3 antigen in the cytoplasm of Candida cells might represent a first step during the infection process of a particular cell. Further steps comprised localization of the Saps in the space between invaginations of the cytoplasmic membrane and the Candida cell wall and, after secretion, within the epithelial cells. The main concentration of intracellular gold labeling was often detected at the site of direct interaction of the Candida and the host cells, indicating a role of Saps in this location of infection. We did not find large pools of internal Sap antigen. In contrast the greatest part of total gold labeling was located outside the Candida cells, supporting the view that Saps are secreted immediately after translation (Homma et al., 1993Homma M. Chibana H. Tanaka K. Induction of extracellular proteinase in.Candida Albicans. J Gen Microbiol. 1993; 139: 1187-1193Crossref PubMed Scopus (27) Google Scholar). Membrane bound vesicles were seen within the Candida cells at the inner side of the cell wall. Vesicle transport through the C. albicans cell wall was obvious. The interior of the vesicles found was not labeled. In contrast to the secretion of phospholipase within vesicles through the cell wall (Pugh and Cawson, 1975Pugh D. Cawson R.A. The cytochemical localization of phospholipase A in Candida albicans..Sabouraudia. 1975; 13: 110-115Crossref PubMed Scopus (50) Google Scholar), such a pathway may not be relevant in the case of secretion of Sap1–3 in vivo. Release of enzymes into the periplasmatic and extracellular space in yeast cells is directed by the secretory pathway system. Transport of proteins occurs from the endoplasmatic reticulum via the Golgi apparatus and secretory vesicles to the plasma membrane. Secretion to the cell surface is achieved by fusion of the vesicles with the plasma membrane. The fusion event results in invaginations of the plasma membrane at the inner side of the cell wall (Reid, 1991Reid G.A. Protein targeting in yeasts.J Gen Microbiol. 1991; 137: 1765-1772Crossref PubMed Scopus (8) Google Scholar). This later stage of protein transport was frequently observed in our samples, as demonstrated by gold labeling of the invagination in Figure 2, indicating that Saps are released into the periplasmatic space.Figure 2Post-embedding immunogold labeling with 10 nm gold particles in a sample of C. albicans from oral candidiasis of patient B. Deposition of Saps within the invaginations of the cytoplasmic membrane (arrow), the cell wall of a C. albicans cell (double arrow), and the epithelial cell (EC). Note the vesicles (arrowheads) within the Candida cell and at the outer surface of the cell wall. Scale bar: 0.5 μm.View Large Image Figure ViewerDownload (PPT)Figure 3Pre-embedding immunogold labeling with 5 nm gold particles and silver enhancement in a sample of C. albicans from oral candidiasis of patient A. Antibody deposition, indicating the presence of Saps at the site of penetration of an epithelial cell representing a possible microniche (arrows). Note the proteolysis of the epithelial outer cell membrane and the keratin (stars) at the site of penetration. CM, intact cell membrane;arrowheads, exocytosis of vesicles from the Candida cell. Silver enhanced labeling is sometimes associated with the presence of vesicles (arrows and arrowheads). Scale bar: 0.5 μm.View Large Image Figure ViewerDownload (PPT) Minute inspection of Candida cells directly attached to epithelial cells gave evidence of Sap antigens at the site of penetration, and clearly showed destruction of the outer cell membrane and the keratin of the parakeratotic epithelial cells (Figure 3). At a lower degree Sap immunoreactivity was also seen on the outer surface of the epithelial cells in the close vicinity, but without alterations of their outer membranes (not shown). Sap immunoreactivity was also associated with the presence of vesicles, but was never seen within the vesicles (Figure 3). Exocytosis of these vesicles from C. albicans cells was clearly demonstrated (Figure 3). In control experiments under identical conditions, yet without addition of monoclonal antibody, no gold marker could be detected. The substrate specifity of C. albicans Sap2 and probably most other Sap isoenzymes is very broad. Host proteins, such as keratin, collagen, and mucin, were considered to be possible targets for Saps during infection (Borg-von Zepelin and Rüchel, 1988Borg-von Zepelin M. Rüchel R. Expression of extracellular acid proteinase by proteolytic Candida spp during experimental infection of oral mucosa.Infect Immun. 1988; 56: 626-631PubMed Google Scholar;Colina et al., 1996Colina A.R. Aumont F. Deslauriers N. Belhumeur P. De Repenigny L. Evidence for degradation of gastrointestinal mucin by Candida albicans secretory aspartyl proteinase.Infect Immun. 1996; 64: 4514-4519PubMed Google Scholar;Hube, 1996Hube B. Candida albicans secreted aspartyl proteinases.Curr Top Med Mycol. 1996; 7: 55-69PubMed Google Scholar). According to our results, degradation of parakeratotic keratinocytes or corneocytes is obviously demonstrated in the presence of Sap immunoreactivity at the site of hyphal penetration (Figure 3). A part of the clinical material from one patient (patient C) was previously used to show a distinct SAP gene expression (Schaller et al., 1998Schaller M. Schäfer W. Korting H.C. Hube B. Differential expression of secreted aspartyl proteinases in a model of human oral candidosis and in patient samples from the oral cavity.Mol Microbiol. 1998; 29: 605-615Crossref PubMed Scopus (183) Google Scholar). In this sample SAP1, SAP2, SAP3, and SAP6 transcripts were detected, suggesting a role of these genes during oral candidiasis (Schaller et al., 1998Schaller M. Schäfer W. Korting H.C. Hube B. Differential expression of secreted aspartyl proteinases in a model of human oral candidosis and in patient samples from the oral cavity.Mol Microbiol. 1998; 29: 605-615Crossref PubMed Scopus (183) Google Scholar). The in vivo detection of SAP gene expression and Sap immunoreactivity in the same clinical sample of patient C indicates the presence of secretory aspartyl proteinases in human oral candidiasis in HIV-infected patients for the first time. This also includes the expression of SAP2 during human oral infections. Previously, this fact was questionable, because an acidic pH range was necessary for the induction and activity of Sap2 in several in vitro studies (Hube, 1996Hube B. Candida albicans secreted aspartyl proteinases.Curr Top Med Mycol. 1996; 7: 55-69PubMed Google Scholar); however, Sap2 may act in acidic microniches within the normally neutral milieu of the oral cavity (Rüchel et al., 1991Rüchel R. Zimmermann F. Böning-Stutzer B. Helmchen U. Candidiasis visualized by proteinase-directed immunofluorescence.Virchows Arch a Pathol Anat. 1991; 419: 199-202Crossref Scopus (39) Google Scholar). Such a possible microniche with an acidic pH range might be demonstrated in Figure 3. The protein keratin is an important differentiation product of the human epidermis and represents the main component of ortho- or parakeratotic corneocytes. During this differentiation process, cells of stratified squamous epithelia become filled mainly with fibrous and amorphous proteins and enveloped by a thickened and resistant plasma membrane. Keratinolytic enzymes are necessary for the ability of C. albicans to invade corneocytes. In 1984, production of a keratinolytic proteinase was demonstrated when C. albicans was cultivated in a medium containing human stratum corneum as a nitrogen source (Negi et al., 1984Negi M. Tsuboi R. Matsui T. Ogawa H. Isolation and characterization of proteinase from Candida albicans: substrate specificity.J Invest Dermatol. 1984; 83: 32-36Abstract Full Text PDF PubMed Scopus (94) Google Scholar). It was suggested that this proteinase may play an important role in superficial infection by affecting the human corneocytes (Negi et al., 1984Negi M. Tsuboi R. Matsui T. Ogawa H. Isolation and characterization of proteinase from Candida albicans: substrate specificity.J Invest Dermatol. 1984; 83: 32-36Abstract Full Text PDF PubMed Scopus (94) Google Scholar). The presence of Sap immunoreactivity directly at the site of keratinolytic degradation of the host during the infection process, suggests an important role of this virulence factor for the development of oral candidiasis in vivo. This provides a rationale for the development of Sap inhibitors as anti-mycotics for clinical use (Abad-Zapatero et al., 1996Abad-Zapatero C. Goldman R. Muchmore S.W. et al.Structure of secreted aspartic protease from C. albicans complexed with a potent inhibitor: implication for the design of antifungal agents.Protein Science. 1996; 5: 640-652Crossref PubMed Scopus (105) Google Scholar). The authors thank E. Januschke (Ludwig-Maximilians-University, Munich, Germany) for excellent technical assistance and M. Roecken (University of Munich, Germany) for critical reading of the manuscript." @default.
• W2033383118 created "2016-06-24" @default.
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• W2033383118 date "1999-03-01" @default.
• W2033383118 modified "2023-10-16" @default.
• W2033383118 title "In Vivo Expression and Localization of Candida albicans Secreted Aspartyl Proteinases during Oral Candidiasis in HIV-Infected Patients" @default.
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