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• W2055542034 abstract "Since the introduction of new anti-retroviral agents such as human immunodeficiency virus (HIV) protease inhibitors, oropharyngeal candidiasis is less often observed in acquired immune deficiency syndrome patients. Secretory aspartic proteases of Candida albicans, which have similarities to the HIV aspartic proteases, are pathogenicity factors that have been intensively investigated in recent years. The inhibitory effect of four different HIV aspartic protease inhibitors (ritonavir, saquinavir, indinavir, and nelfinavir), on the activity of different Candida albicans secretory aspartic proteases was demonstrated. These anti-retroviral agents were able to inhibit Candida albicans secretory aspartic proteases 1, 2, and 3 which are involved in Candida adherence. As a consequence of these results we used selected HIV protease inhibitors in an adherence assay of Candida cells to epithelial cells. Ritonavir and saquinavir inhibited adherence of Candida albicans under the chosen experimental conditions similarly to the in vitro results, whereas indinavir had no effect. This inhibition was shown to be concentration dependent. The specificity of these effects with respect to the secretory aspartic proteases was demonstrated by competitive binding experiments using purified recombinant secretory aspartic proteases. On the basis of these studies we conclude that lower rates of oropharyngeal candidiasis in individuals receiving potent anti-retroviral therapy could reflect not only an improvement in the immune system but also direct inhibition of Candida secretory aspartic proteases by HIV protease inhibitors. Since the introduction of new anti-retroviral agents such as human immunodeficiency virus (HIV) protease inhibitors, oropharyngeal candidiasis is less often observed in acquired immune deficiency syndrome patients. Secretory aspartic proteases of Candida albicans, which have similarities to the HIV aspartic proteases, are pathogenicity factors that have been intensively investigated in recent years. The inhibitory effect of four different HIV aspartic protease inhibitors (ritonavir, saquinavir, indinavir, and nelfinavir), on the activity of different Candida albicans secretory aspartic proteases was demonstrated. These anti-retroviral agents were able to inhibit Candida albicans secretory aspartic proteases 1, 2, and 3 which are involved in Candida adherence. As a consequence of these results we used selected HIV protease inhibitors in an adherence assay of Candida cells to epithelial cells. Ritonavir and saquinavir inhibited adherence of Candida albicans under the chosen experimental conditions similarly to the in vitro results, whereas indinavir had no effect. This inhibition was shown to be concentration dependent. The specificity of these effects with respect to the secretory aspartic proteases was demonstrated by competitive binding experiments using purified recombinant secretory aspartic proteases. On the basis of these studies we conclude that lower rates of oropharyngeal candidiasis in individuals receiving potent anti-retroviral therapy could reflect not only an improvement in the immune system but also direct inhibition of Candida secretory aspartic proteases by HIV protease inhibitors. oropharyngeal candidiasis secretory aspartic protease Oropharyngeal candidiasis (OC) is the most common fungal disease among patients suffering from human immunodeficiency virus (HIV) infection. Some 80–95% of HIV-infected patients will have at least one episode of OC during the course of their disease and two-thirds of these patients will have OC as their initial symptomatic illness. Candida albicans is responsible for the majority of these infections (for review seePenzak and Gubbins, 1998Penzak S.R. Gubbins P.O. Preventing and treating azole-resistant oropharyngeal candidiasis in HIV-infected patients.Am J Health Syst Pharm. 1998; 55: 279-283PubMed Google Scholar). Since the introduction of highly active anti-retroviral therapy using HIV aspartic protease inhibitors and nucleoside analogs, however, HIV-associated opportunistic infections such as OC due to C. albicans are less often observed even in the presence of viral resistance to treatment (Egger et al., 1997Egger M. Hirschel B. Francioli P. et al.Impact of new retroviral combination therapies in HIV infected patients in Switzerland—Prospective multicentre study.Br Med J. 1997; 315: 1194-1199Crossref PubMed Scopus (560) Google Scholar;Palella et al., 1998Palella F.J. Delaney K.M. Moorman A.C. Loveless M.O. Fuhrer J. Satten G.A. Aschman D.J. Holmberg S.D. Declining morbidity and mortality among patients with advanced human immunodefficiency virus infection.N Engl J Med. 1998; 338: 853-860Crossref PubMed Scopus (8262) Google Scholar;Ledergerber et al., 1999Ledergerber B. Egger M. Opravil M. et al.for the Swiss HIV Cohort Study: Highly active antiretroviral therapy: low rates of clinical disease progression despite high rates of virological failure.Lancet. 1999; 353: 863-868Abstract Full Text Full Text PDF PubMed Scopus (853) Google Scholar) Candida albicans is a dimorphic fungus that is both a commensal and opportunistic pathogen in humans (Odds, 1988Odds F.C. Candida and Candidosis. Ballière-Tindall, London1988Google Scholar). Adhesion of the fungus to host cells and tissues is the initial step leading to the establishment of infection and is thus considered to be essential to virulence (Cutler, 1991Cutler J.E. Putative virulence factors of Candida albicans.Annu Rev Microbiol. 1991; 45: 187-218Crossref PubMed Scopus (537) Google Scholar). The binding mechanisms are mediated by complementary molecules at both the surface of the fungus and the host. During recent years, C. albicans secreted aspartic proteases (Sap) have attracted a lot of attention as virulence factors, and have been demonstrated on the surface of fungal elements colonizing mucosa and penetrating tissues (Borg and Rüchel, 1988Borg M. Rüchel R. Expression of extracellular acid proteinase by proteolytic Candida spp during experimental infection of oral mucosa.Infect Immun. 1988; 56: 626-631Crossref PubMed Google Scholar). Evidence for the role of Sap in the adherence process has been derived from experiments with the specific inhibitor pepstatin A, which is able to block adherence of C. albicans cells (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). HIV aspartic proteases and Candida Sap are enzymes which belong to the same class of proteases (Rao et al., 1990Rao J.K.M. Erickson J.W. Wlodawer A. Structural and evolutionary relationships between retroviral and eucaryotic aspartic proteinases.Biochemistry. 1990; 30: 4663-4671Crossref Scopus (91) Google Scholar). Six closely related secreted C. albicans aspartic proteases Sap1–Sap6 have been successfully produced as recombinant proteins in the methylotrophic yeast Pichia pastoris and well characterized (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). The availability of purified recombinant Sap1–Sap6 allowed us to test the effect of four different HIV aspartic protease inhibitors, ritonavir, saquinavir, indinavir and nelfinavir on the C. albicans Sap activity. Additionally, we tested the influence of these HIV protease inhibitors on the adherence of various Candida strains to epithelial cells using a previously described assay (Borg-von Zepelin and Wagner, 1995Borg-von Zepelin M. Wagner T. Fluorescence assay for the detection of adherent Candida yeasts to target cells in microtest plates.Mycoses. 1995; 38: 339-347Crossref PubMed Scopus (25) Google Scholar). The results of the adherence assay were correlated to the direct effect of the HIV protease inhibitors on the different Sap leading to the hypothesis that the decrease in the frequency of oropharyngeal candidiasis might be assisted by inhibition of Sap involved in C. albicans adherence. The C. albicans wild-type strains SC5314, ATCC10231, 10261, 48867, CBS2730, DSM70014, and clinical isolates from the bacteriological department of the university clinics of Göttingen were used in this study. Forty-eight hours before use, the strains were plated on Sabouraud agar and incubated at room temperature. For the adherence assays, the yeasts were grown for 14 h in glucose broth at 35°C. At that time point the yeasts were at the end of the logarithmic growth phase. The different C. albicans Sap tested were expressed as recombinant proteins using the P. pastoris expression system and purified as previously described (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). Protein concentrations were measured by the method ofBradford, 1976Bradford M.M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem. 1976; 72: 248-254Crossref PubMed Scopus (205559) Google Scholar using a commercial kit (BioRad, Richmond, CA). The inhibitors ritonavir (ABT 538), saquinavir (Ro 31–8959), nelfinavir (AG 1343), and indinavir (L 735,524), obtained from commercial sources, were prepared as follows: Indinavir and saquinavir were dissolved in Aqua bidest. at concentrations of 20 mM and 2 mM, respectively. Nelfinavir and ritonavir were dissolved in methanol at concentrations of 20 mM and 40 mM, respectively. Pepstatin A was purchased from Sigma (Taufkirchen, Germany), and a stock solution was prepared in methanol at a concentration of 10-2 M. Each compound was added at different concentrations (compare Table 1) to 0.5 μg of enzyme in 0.45 ml of 50 mM sodium citrate buffer (pH 3.5–5.0 depending on the optimum pH of activity of the enzyme). The mixture was incubated for 10 min at 37°C, and assays were performed after addition of 50 μl of a 0.2% solution of resorufin-labeled casein. An appropriate control without inhibitor was assayed simultaneously. After a 60 min incubation at 37°C, the undigested substrate was precipitated with trichloroacetic acid (4% final concentration) and separated from the supernatant by centrifugation. The absorbance of the supernatant as a function of the Sap activity was measured in the alkaline range at 574 nm following addition of 30 μl 4 M NaOH.Table 1Sap enzyme activities in the presence of HIV aspartic protease inhibitors aPercent values of enzyme activity in the presence of inhibitors. A value of 100% corresponds to activity in the absence of inhibitor.InhibitorSap1Sap2Sap3Sap4Sap5Sap6Ritonavir 3.2 μM664686100100100 16 μM343365100100100 32 μM252749100100100Saquinavir 32 μM78978710010093 160 μM53918610010096 320 μM414678908777Nelfinavir 32 μM96818210010095 160 μM67676510010093 320 μM45174610010082Indinavir 32 μM1009178100100100 160 μM944658100100100 320 μM883916100100100a Percent values of enzyme activity in the presence of inhibitors. A value of 100% corresponds to activity in the absence of inhibitor. Open table in a new tab Candida cells at the end of the logarithmic growth phase were washed twice with cold phosphate-buffered saline (PBS). The number of Candida cells was determined photometrically by hemocytometry. The yeasts were then transferred to medium RPMI 1640 supplemented with 5% fetal bovine serum at a concentration of 2 × 106 cells per ml. The HIV protease inhibitors were added at a final concentration of 200 μM. For comparison, control tests without inhibitors containing the solvents only, were incubated in parallel. The cell concentrations in the presence and in the absence of HIV protease inhibitors were measured by hemocytometry after 2, 4, 6, 8, and 24 h of cultivation at 37°C (5% CO2, 95% humidity). Vero cells were cultivated in medium 1640 containing 5% fetal bovine serum in cell culture flasks. The preparation of the microtest plates for the standard adherence assay was performed as previously described (Borg-von Zepelin and Wagner, 1995Borg-von Zepelin M. Wagner T. Fluorescence assay for the detection of adherent Candida yeasts to target cells in microtest plates.Mycoses. 1995; 38: 339-347Crossref PubMed Scopus (25) Google Scholar). Briefly, the detached Vero cells were suspended in new cell culture medium at a concentration of 105 cells per ml. Each position of a flat-bottomed microtest plate was filled with 200 μl of this Vero cell suspension. Prior to testing adherence, the Vero cells were incubated in the microtest plates for 24 h at 37°C (5% CO2, 95% humidity). This assay was essentially performed as previously described (Borg-von Zepelin and Wagner, 1995Borg-von Zepelin M. Wagner T. Fluorescence assay for the detection of adherent Candida yeasts to target cells in microtest plates.Mycoses. 1995; 38: 339-347Crossref PubMed Scopus (25) Google Scholar). Candida cells at the end of the logarithmic growth phase were washed twice with cold PBS. The number of cells was determined photometrically at 630 nm on the basis of standard curves or by hemocytometry. The yeasts were cultivated in 0.1 M phosphate buffer + 50 mM glucose pH 6.0 or medium 1640 supplemented with 5% fetal bovine serum (adherence media) at a concentration of 2 × 106 cells per ml. The wells of the microtest plate containing Vero cells were infected with 200 μl of the Candida cell suspension. Yeasts and target cells were incubated for at least 1 h at 37°C. For the detection of adherent Candida cells, the dye Calcofluor white (stock solution 0.2 mg per ml) was directly applied to the adherence medium at each position (final concentration 20 μg per ml). Subsequently, the plates were incubated for 30 min at 37°C. All wells of the microtest plate were rinsed three times with PBS to remove nonadherent Candida cells. Finally, the amount of adherent fluorescent Candida cells was determined by an automatic fluorescence reader (CytoFluor II, Biosearch, Somerville, NJ) at extinction 360 nm and emission 460 nm. As a standard procedure, each test was performed at least four times in parallel on the same microtest plate in order to determine mean values and SD. Prior to the infection of Vero cells, differing concentrations of the HIV protease inhibitors were added to Candida yeast cells. Control tests without inhibitor, either containing 1% methanol as solvent for ritonavir or water as solvent for saquinavir and indinavir were performed in parallel on the same microtest plate. These controls without inhibitors were set to 100%. The results of the adherence assay with inhibitors were expressed as percentages of the corresponding control tests without inhibitors. The differences between the control tests without protease inhibitors and the Candida adherence in the presence of HIV protease inhibitors was analyzed statistically using the Student’s t test. In order to assay the concentration-dependence of selected inhibitors, saquinavir and ritonavir were first added to the adherence medium (0.1 M phosphate buffer + 50 mM glucose) beginning with a concentration of 200 μM. Inhibitors concentration was then 2-fold diluted in the same adherence medium in five steps. The adherence assay was performed as described above. In order to test the reversal of adherence inhibition caused by HIV inhibitors, ritonavir and the different Sap were mixed and preincubated in 50 mM citrate buffer, pH 4 for 15 min at 37°C. This mixture was then added to the Candida cells prior to the infection of the target cells. Vero cells were cultivated for 48 h in 24-well test plates (Nunc no. 143982) containing round glass coverslips (diameter 12 mm) in cell culture medium containing 5% fetal bovine serum. The target cells were then infected with Candida cells (2 × 106 cells per ml) in adherence medium in the presence or in the absence of aspartic protease inhibitors. Candida and Vero cells were incubated for at least 4 h at 37°C. Following coincubation, the wells were rinsed once with PBS. Freshly prepared paraformaldehyde (2% in PBS) was then applied and incubated for at least 1 h at room temperature. Immunofluorescence microscopy was performed using an anti-serum (α-Sap2) raised in New Zealand White rabbits reacting with Sap1, Sap2, and Sap3 (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). The fixed specimens were rinsed with PBS and subsequently incubated with PBS + 1% bovine serum albumin and 0.1% milk powder to reduce nonspecific binding for 1 h at room temperature. The anti-serum was applied (100-fold diluted in PBS + 1% bovine serum albumin + 0.1% milk powder) and incubated at 8°C for at least 12 h. After another series of rinses with PBS a second antibody fluorescein isothiocyanate-conjugate from goat was added (diluted 200-fold in the above-mentioned buffer). After 3 h incubation and five subsequent rinses in PBS, the tests were embedded in p-phenylenediamine according toJohnson et al., 1982Johnson G.D. Davidson R.S. McNamee K.C. Russel G. Goodwin D. Holborow E.J. Fading of immunofluorescence during microscopy: a study of the phenomenon and ist remedy.J Immunol Methods. 1982; 55: 231-242Crossref PubMed Scopus (642) Google Scholar. The binding of the conjugate was evaluated with a Zeiss fluorescence microscope using the filter combination, BP 450–490/FT 510/LP 520. Depending on the Sap isoenzyme produced, a yield of 20–80 μg of protein per ml of P. pastoris culture supernatant was obtained. Saquinavir, ritonavir, nelfinavir, and indinavir were tested at concentrations in the range of 3.2–320 μM (Table 1). Sap1, Sap2, and Sap3 were inhibited by all four inhibitors in a concentration-dependent manner. Ritonavir was by far the most effective inhibitor. Under our experimental conditions, a 50% inhibition was obtained with 3.2–32 μM of ritonavir, which are concentrations comparable with those achievable in patients (Hsu et al., 1997Hsu A. Granneman G.R. Witt G. et al.Multiple-dose pharmacokinetics of ritonavir in human immunodeficiency virus-infected subjects.Antimicrob Agents Chemother. 1997; 41: 898-905Crossref PubMed Google Scholar). Ten-fold higher concentrations of saquinavir, nelfinavir, and indinavir were necessary to obtain similar inhibition of Sap1, Sap2, and Sap3. In contrast, only a slight inhibition of activity was detected for Sap4, Sap5, and Sap6 with a concentration of up to 320 μM saquinavir. C. albicans SC5314 was used in an adherence assay to test the effect of ritonavir, which most effectively inhibited Sap activities in vitro, as well as saquinavir and indinavir. C. albicans adherence without inhibitor was arbitrarily set at 100%. Under the chosen experimental conditions, the addition of pepstatin A at a concentration of 100 μM was used as a positive control and revealed an adherence inhibition of 30%, which is in agreement with previously published results (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). Using phosphate buffer pH 6.0 as the adherence medium, the influence of the HIV protease inhibitors on the adherence of C. albicans SC 5314 was shown to differ (Figure 1). Saquinavir and ritonavir used at concentrations of 200 μM revealed a marked inhibition of the adherence. The addition of ritonavir and saquinavir reduced the adherence of C. albicans SC5314 to 30% and 50% of the controls, respectively, at the end of the adherence period. The differences in adherence between C. albicans SC5314 without protease inhibitors and in the presence of each inhibitor at a concentration of 200 μM was found to be highly significant (p < 0.001). In contrast, indinavir did not influence the adherence of C. albicans cells under the chosen experimental conditions (Figure 1). The inhibition measured in the presence of ritonavir and saquinavir was found to be concentration dependent (Figure 2). Whereas ritonavir still distinctly inhibited the adherence of C. albicans SC5314 up to a concentration of 25 μM, the end-point of adherence-inhibition caused by saquinavir was only clearly seen at a 4-fold higher concentration only (100 μM, Figure 2).Figure 2Concentration-dependent adherence of C. albicans SC5314 by the HIV-protease inhibitors ritonavir and saquinavir in 0.1 M phosphate buffer, pH 6.0 plus 50 mM glucose. Adherence values (A%) are a percentage of the results of the control adherence assays without HIV protease inhibitors, which are set to 100%. The mean ± maximal SD is depicted. The SD varied between 6% and 15%.View Large Image Figure ViewerDownload (PPT) The inhibition of adherence in the presence of HIV protease inhibitors was also visualized by immunofluorescence microscopy. Figure 3 demonstrates that in the presence of ritonavir, only few Candida cells are adherent. Compared with control tests without the HIV protease inhibitor it is additionally obvious that these few adherent Candida cells produce shorter germination tubes (Figure 3a,b). The presence of protease antigen on the fungal surfaces was demonstrated by the binding of the antibody α-Sap2, which recognizes the protease antigens of Sap1, Sap2, and Sap3 (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). There were no obvious differences between the Candida cells grown with or without the HIV protease inhibitor (Figure 3c,d). The HIV protease inhibitors did not affect Candida viability. Compared with Candida cells cultured without HIV protease inhibitors, neither differences in cell concentrations nor differences in growth kinetics were observed in the presence of the HIV protease inhibitors ritonavir and saquinavir at various time points of the cultivation (data not shown). The specificity of the adherence inhibition due to Candida Sap was concluded from competitive binding experiments of HIV protease inhibitors with recombinant Sap as shown in Figure 4. Ritonavir and Sap1, Sap2, and Sap3 were mixed and preincubated in 50 mM citrate buffer pH 4 for 15 min at 37°C. This mixture was then added to the Candida cells prior to the infection of the target cells. As controls, ritonavir was mixed with serum proteins as well as bovine serum albumin (5 and 10 μg per ml) under the same experimental conditions. The addition of the different Sap to ritonavir completely abolished the inhibition of Candida adherence, whereas unspecific serum proteins had no effect. The effects of the HIV-protease inhibitors saquinavir and ritonavir at a concentration of 200 μM were demonstrated with eight further C. albicans strains from different origins. As the control, the wild-type strain C. albicans SC5314 was tested in parallel. The comparison of these Candida strains in the presence and in the absence of the HIV protease inhibitors is shown in Figure 5. The addition of ritonavir reduced adherence in the same strains by 40–60% compared with the controls without inhibitor. For all strains, the addition of saquinavir was less effective with only a 20–40% reduction in adherence. Secretory proteases from C. albicans have attracted great interest as defined virulence factors. Nine genes encoding Sap have been cloned from C. albicans to date (Monod et al., 1994Monod M. Togni G. Hube B. Sanglard D. Multiplicity of genes encoding secreted aspartic proteinases in Candida species.Mol Microbiol. 1994; 13: 357-368Crossref PubMed Scopus (212) Google Scholar,Monod et al., 1998Monod M. Hube B. Hess D. Sanglard D. Differential regulation of SAP8 and SAP9 which encode two new members of the secreted aspartic proteinase family in Candida albicans.Microbiology. 1998; 144: 2731-2737Crossref PubMed Scopus (117) Google Scholar). Sap1, Sap2, and Sap3 as well as Sap4, Sap5, and Sap6 form 2 subgroups each containing closely related enzymes, whereas Sap7, Sap8, and Sap9 do not form a specific group. The fact that C. albicans inhabit diverse host niches leads to the question of whether different Sap are expressed by C. albicans as a reaction to specific adaptation requirements under different environmental conditions. The first studies concerned with such problems were carried out on the basis of gene expression. With the help of reverse transcription–polymerase chain reaction the expression of genes SAP1, SAP2, SAP3, and SAP6 was demonstrated in samples of experimental and clinical candidosis (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 expression of SAP1 and SAP2 was also shown by northern blot analysis using an experimental rat vaginitis model (De Bernardis et al., 1995De Bernardis F. Cassone A. Sturtervant J. Calderone R. Expression of Candida albicans SAP1 and SAP2 in experimental vaginitis.Infect Immun. 1995; 63: 1887-1892PubMed Google Scholar). In particular, Sap2 seemed to have a certain potential importance for the vaginal infection as was deduced from infection assays using protease-deficient mutant strains in the rat model (De Bernardis et al., 1999De Bernardis F. Arancia S. Morelli L. Hube B. Sanglard D. Schäfer W. Cassone A. Evidence that members of the secretory aspartyl proteinase gene family, in particular SAP2, are virulence factors for Candida vaginitis.J Infect Dis. 1999; 179: 201-208Crossref PubMed Scopus (151) Google Scholar). From these data it must be concluded that Sap1, Sap2, and Sap3 appear to be the Sap produced during mucosal adherence. In contrast, Sap4, Sap5, and Sap6 have been shown to be secreted in macrophages after phagocytosis of yeast cells (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). In these studies we have demonstrated that the Sap involved in adherence, Sap1, Sap2, and Sap3, are those inhibited by HIV protease inhibitors. We, therefore, tried selected anti-retroviral substances in a previously described adherence assay (Borg-von Zepelin and Wagner, 1995Borg-von Zepelin M. Wagner T. Fluorescence assay for the detection of adherent Candida yeasts to target cells in microtest plates.Mycoses. 1995; 38: 339-347Crossref PubMed Scopus (25) Google Scholar) to address possible inhibition of the adherence of C. albicans cells to the Vero target cells by HIV protease inhibitors. Epithelial cells were used as a model for mucosal infections instead of keratinocytic cells, e.g., HaCat line or keratinocytes obtained from human foreskins which could be used as a model for skin infections. The inhibition of adherence was not caused by diminishing the viability of the Candida cells (Diz et al., 1999Diz P. Ocampo A. Iglesias I. Otero I. Lack of ritonavir antifungal effect in vitro.Antimicrob Agents Chermother. 1999; 43: 997PubMed Google Scholar). Ritonavir, which is the most active inhibitor of Sap activity in vitro, diminished the adherence of C. albicans SC5314 to epithelial cells by up to 70% of the controls. A distinct reduction in Candida adherence was found in the range of 25 μM (corresponding to 15 μg per ml), which is a HIV protease inhibitor concentration regularly found as the peak serum level under the proposed therapeutic regimen for AIDS patients (Hsu et al., 1997Hsu A. Granneman G.R. Witt G. et al.Multiple-dose pharmacokinetics of ritonavir in human immunodeficiency virus-infected subjects.Antimicrob Agents Chemother. 1997; 41: 898-905Crossref PubMed Google Scholar). In our model unspecific serum proteins or bovine serum albumin had no effect on the activity of HIV inhibitors on Candida adherence. So far no data are available on substance concentrations in saliva or in mucosal cells so that both decreased bioavailability and accumulation of the drugs in the mucosal cells is possible. In accordance with the observed concentration-dependence of Candida adherence reduction, the specificity of the inhibitory effect of HIV protease inhibitors directed against the Candida Sap was demonstrated by competitive binding using recombinant Sap (Figure 4). In contrast to ritonavir and saquinavir, indinavir, which is the most water soluble substance among these substances, does not affect adherence of C. albicans to epithelial cells in our model. The fourth retroviral substance used, nelfinavir, seems to induce an augmentation of the C. albicans adherence under the chosen experimental conditions (phosphate buffer pH 6 + glucose). Using medium 1640 supplemented with 5% fetal bovine serum, however, nelfinavir revealed an adherence inhibition in the same range as saquinavir. Nelfinavir obviously must be bound to proteins before it is able to reduce adherence of Candida cells to epithelial target cells as could be demonstrated by further tests in phosphate buffer with and without supplementation of fetal bovine serum (data not shown). An unsolved problem remains the precise function of Candida Sap in the adherence process. Two hypotheses are possible: (i) the Candida Sap could act as ligands to surface proteins of the specific host cells, which does not necessarily require their enzymic activity (Cutler, 1991Cutler J.E. Putative virulence factors of Candida albicans.Annu Rev Microbiol. 1991; 45: 187-218Crossref PubMed Scopus (537) Google Scholar). (ii) The Candida cells use Sap as active enzymes to affect target structures of their host cells. This leads to a conformational change of the surface proteins or ligands of epithelial cells allowing a better adherence of the yeasts. There are at least a few indirect indications for such an hypothesis. The cleavage of angiotensinogen to angiotensin I by Candida proteases is optimal at pH 5.5. (Rüchel, 1983Rüchel R. On the renin-like activity of Candida proteinase and activation of blood coagulation in vitro.Zeitschr Bakteriol Hyg A. 1983; 255: 368-379PubMed Google Scholar). The conversion of prekallikrein to kallikrein catalyzed by Candida proteases is optimal between pH 5.2 and 5.5 (Kaminishi et al., 1990Kaminishi H. Tanaka M. Cho T. Maeda H. Hagihara Y. Activation of the plasma kallikrein-kinin system by Candida albicans proteinase.Infect Immunity. 1990; 58: 2139-2143PubMed Google Scholar) and the activation of blood clotting factor XII was shown to be optimal between pH 5.5 and pH 6.5 (Kaminishi et al., 1994Kaminishi H. Hamatake H. Cho T. et al.Activation of blood clotting factors by microbial proteinases.FEMS Microbiol Lett. 1994; 121: 327-332Crossref PubMed Scopus (56) Google Scholar). In fact, Sap1, Sap2, and Sap3 are weakly active at pH 6.0 at which the adherence assays were performed (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) and proteolytic activity could lead to some conformational changes in Candida ligands of epithelial cells. Indinavir which has a good anti-Candida effect in a rat vaginitis model (Cassone et al., 1998Cassone A. Adriani D. Tacconelli E. Cauda R. De Bernardis F. HIV protease inhibitors have a direct anti-Candida effect by inhibition of Candida aspartyl proteinase 12th World AIDS Conference. 1998Google Scholar) has almost no effect on the inhibition of adherence of C. albicans to Vero cells. This discrepancy in results could be explained by differences in Sap expression as a function of localization and time point of infection. Indeed, Sap2 which is strongly inhibited by indinavir (Table 1,Gruber et al., 1999Gruber A. Speth C. Lukasser-Vogl E. Zangerle R. Borg-von Zepelin M. Dierich M.P. Würzner R. Human immunodefiency virus type 1 protease inhibitor attenuates Candida albicans virulence properties in vitro.Immunopharmacology. 1999; 41: 227-234Crossref PubMed Scopus (55) Google Scholar) is the main acting proteinase in a rat vaginitis model (De Bernardis et al., 1999De Bernardis F. Arancia S. Morelli L. Hube B. Sanglard D. Schäfer W. Cassone A. Evidence that members of the secretory aspartyl proteinase gene family, in particular SAP2, are virulence factors for Candida vaginitis.J Infect Dis. 1999; 179: 201-208Crossref PubMed Scopus (151) Google Scholar). In contrast, Sap1 which appeared to be one of the most dominant proteinases in acute OC is more weakly inhibited by this HIV inhibitor (Table 1). The results of these studies would suggest that the dramatically lower rates of OC due to C. albicans in individuals receiving HIV aspartic protease inhibitors reflect not only an improvement in the immune system as measured by an increase of CD4 counts in patients (Hoegl et al., 1998Hoegl L. Thoma-Greber E. Rocken M. Korting H.C. Shift from persistent oral pseudomembranous to erythematous candidosis in a human immunodeficiency virus (HIV) -infected patient upon combination treatment with an HIV protease inhibitor.Mycoses. 1998; 41: 213-217Crossref PubMed Scopus (10) Google Scholar). At least some of these HIV protease inhibitors may display a specific anti-Sap activity leading to a reduced number of Candida cells on epithelial cells. Specific animal models might confirm these observations. Therefore, the development of specific aspartic protease inhibitors might be of interest for the treatment of mucosal candidiasis. We thank Professor Dr. H. Eiffert, Department of Bacteriology, University Clinics of Göttingen for providing some clinical isolates of Candida albicans, Barbara Léchenne for technical assistance and Mrs Cyrilla Maelicke for critical review of the manuscript and linguistic assistance. This work comprises parts of the medical thesis of I. Meyer." @default.
• W2055542034 created "2016-06-24" @default.
• W2055542034 creator A5047318148 @default.
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• W2055542034 title "HIV-Protease Inhibitors Reduce Cell Adherence of Candida Albicans Strains by Inhibition of Yeast Secreted Aspartic Proteases" @default.
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