Margarete Borg-von Zepelin

Secreted aspartic proteinase family of Candida tropicalis.

ABSTRACT Medically important yeasts of the genus Candida secrete aspartic proteinases (Saps), which are of particular interest as virulence factors. Like Candida albicans, Candida tropicalis secretes in vitro one dominant Sap (Sapt1p) in a medium containing bovine serum albumin (BSA) as the sole source of nitrogen. Using the gene SAPT1 as a probe and under low-stringency hybridization conditions, three new closely related gene sequences, SAPT2 to SAPT4, encoding secreted proteinases were cloned from a C. tropicalis λEMBL3 genomic library. All bands identified by Southern blotting ofEcoRI-digested C. tropicalis genomic DNA withSAPT1 could be assigned to a specific SAP gene. Therefore, the SAPT gene family of C. tropicalis is likely to contain only four members. Interestingly, the SAPT2 and SAPT3 gene products, Sapt2p and Sapt3p, which have not yet been detected in C. tropicaliscultures in vitro, were produced as active recombinant enzymes with the methylotrophic yeast Pichia pastoris as an expression system. As expected, reverse transcriptase PCR experiments revealed a strong SAPT1 signal with RNA extracted from cells grown in BSA medium. However, a weak signal was obtained with all otherSAPT genes under several conditions tested, showing that these SAPT genes could be expressed at a basic level. Together, these experiments suggest that the gene products Sapt2p, Sapt3p, and Sapt4p could be produced under conditions yet to be described in vitro or during infection.

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

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

Human Immunodeficiency Virus Type 1 gp160 and gp41 Binding to Candida albicans Selectively Enhances Candidal Virulence In Vitro

Previously, it has been shown that human immunodeficiency virus (HIV)-1 envelope proteins gp160 and gp41 bind to Candida albicans. Whether this interaction affects candidal virulence in vitro was investigated. HIV-1 gp160 or gp120 treatment of C. albicans significantly altered neither growth nor phospholipase activity of the fungus. However, treatment of C. albicans with gp160, but not with gp120, led to an elevation of free and cell-bound aspartate proteinase. In addition, culture supernatants obtained from C. albicans treated with gp160 or gp41, but not with gp120, showed a strong increase in proteinase activity. Finally, C. albicans viable yeast cells treated with gp160 or gp41 and serum were phagocytosed by polymorphonuclear leukocytes to a lesser extent than was C. albicans treated with gp120 and serum or serum alone. These findings suggest that the interaction between HIV-1 gp160 and C. albicans may promote the virulence of C. albicans in HIV-1-positive patients.

Changes in the spectrum of fungal isolates: results from clinical specimens gathered in 1987/88 compared with those in 1991/92 in the University Hospital Gottingen, Germany

Summary. In the University Hospital in Gottingen, the spectra of fungal species in clinical specimens of respiratory secretions, bronchial secretions and urine were compared over periods of 15 months (10/87 to 12/88 and 1/91 to 3/92) before and after the introduction of fluconazole. The following changes could be demonstrated:

Adherence of different Candida dubliniensis isolates in the presence of fluconazole.

Background The recently described yeast species Candida dubliniensis is closely related to C. albicans and has been recovered predominantly from the oral cavities of HIV-infected individuals and AIDS patients who are often receiving fluconazole as prophylactic or therapeutic treatment for oropharyngeal candidiasis. Like C. albicans, C. dubliniensis secretes aspartic poteinases which in C. albicans have been shown to be involved in adherence. Objective To explain the increasing prevalence of C. dubliniensis in AIDS patients and to investigate the virulence factors of this yeast. Methods An in vitro assay was developed to compare the adherence to epithelial cells of C. dubliniensis from HIV-patients with that of C. albicans. Results All C. albicans isolates adhered better than the 22 C. dubliniensis isolates. In the presence of fluconazole, the C. dubliniensis isolates tested showed increased adherence as compared with controls without fluconazole. In contrast, all C. albicans isolates decreased in adherence to epithelial cells in the presence of fluconazole independently of their in vitro susceptibility to this drug. Proteinase antigens are present on the surface of C. dubliniensis cells adherent to epithelial target cells. In the presence of fluconazole this proteinase antigen was more strongly expressed. Conclusion Increased adherence of C. dubliniensis strains in the presence of fluconazole could explain its high recovery rate from HIV-positive patients in recent years. The induction of proteinase secretion in the presence of fluconazole found for most of the C. dubliniensis isolates could be one of the factors involved in adherence.

Detection of characteristic metabolites of Aspergillus fumigatus and Candida species using ion mobility spectrometry - metabolic profiling by volatile organic compounds

Volatile metabolites of Aspergillus fumigatus and Candida species can be detected by gas chromatography/mass spectrometry (GC/MS). A multi‐capillary column – ion mobility spectrometer (MCC‐IMS) was used in this study to assess volatile organic compounds (VOCs) in the headspace above A. fumigatus and the four Candida species Candida albicans, Candida parapsilosis, Candida glabrata and Candida tropicalis in an innovative approach, validated for A. fumigatus and C. albicans by GC/MS analyses. For the detection of VOCs, a special stainless steel measurement chamber for the microbial cultures was used. The gas outlet was either attached to MCC‐IMS or to adsorption tubes (Tenax GR) for GC/MS measurements. Isoamyl alcohol, cyclohexanone, 3‐octanone and phenethylalcohol can be described as discriminating substances by means of GC/MS. With MCC‐IMS, the results for 3‐octanone and phenethylalcohol are concordant and additionally to GC/MS, ethanol and two further compounds (p_0642_1/p_683_1 and p_705_3) can be described. Isoamyl alcohol and cyclohexanone were not properly detectable with MCC‐IMS. The major advantage of the MCC‐IMS system is the feasibility of rapid analysis of complex gas mixtures without pre‐concentration or preparation of samples and regardless of water vapour content in an online setup. Discrimination of fungi on genus level of the investigated germs by volatile metabolic profile and therefore detection of VOC is feasible. However, a further discrimination on species level for Candida species was not possible.

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

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

Impact of N-Chlorotaurine on Viability and Production of Secreted Aspartyl Proteinases of Candida spp.

ABSTRACT N-Chlorotaurine, an endogenous long-lived oxidant, demonstrated fungicidal activity against Candida spp. and a postantifungal effect. Secreted aspartyl proteinases, important fungal virulence factors, proved to be a first target of impact. These results provide support for the topical application of N-chlorotaurine as an antimicrobial agent in yeast infections.

Fluorescence assay for the detection of adherent Candida yeasts to target cells in microtest plates.

Summary. We describe an assay based on photometric analysis for the measurement of adherence of Candida species to epithelial target cells (Vero cell line). Adherent Candida cells were detected by staining the cells with the fluorescent dye Calcofluor white (CFW), which binds to chitin and glucan in the yeasts. The tests were performed on microtest plates, which were analysed automatically by fluorescence plate readers. The assay is based on the following steps: (i) coating of the microtest plates with target cells (e.g. Vero cells); (ii) infection with Candida: (iii) staining of Candida with CFW; (iv) rinsing to remove non‐adherent Candida cells and unbound dye; (v) detection of adherent fluorescent Candida cells. The test was able to detect 4times104 cells ml‐1. The standard deviation was ±8%. Day‐to‐day variation was ± 10% at most. The adherence of strains of different Candida species was assayed by a standard procedure. The results confirmed the order of adherence, with C. albicans ranking first, followed by C. tropicalis, C. parapsilosis and C. glabrata.

Effect of Micafungin (FK463) on Candida albicans Adherence to Epithelial Cells

Background: Adherence is considered a major virulence trait of Candida albicans. FK463 is a new investigational intravenous antifungal of the ‘candin family’ with potent in vitro and in vivo activity against Candida spp. Objective: The aim of the present study was to investigate the effect of Micafungin (FK463) on Candida adherence to epithelial cells of azole-sensitive and azole-resistant C. albicans isolates. Methods: An in vitro assay using microtest plate technology and fluorescence measurement was developed to compare the adherence of C. albicans SC5314 and of paired C. albicans isolates to epithelial cells in the presence and in the absence of FK463. Results: FK463 showed a marked inhibitory effect on the adherence of C. albicans SC5314. The addition of FK463 reduced the adherence of C. albicans SC5314 to 90% of the value of control without drug. A dose-dependent adherence inhibition was observed with FK463 in the range of 10–0.015 µg ml–1. The comparison of paired C. albicans isolates, either a fluconazole-susceptible and a fluconazole-resistant isolate of one patient, revealed no significant difference in the adherence behavior between azole-susceptible and azole-resistant. Conclusion: Micafungin (FK463) has the capacity to reduce adherence of C. albicans azole-susceptible and azole-resistant strains to epithelial cells.