Oxystress and human fungal pathogens: clinical and applied aspects

This WP will focus on applied aspects of oxystress; we are exploring whether hypoxia or ROS influence the interaction of antifungal drugs (polyenes, candines, azoles) against fungi, the production of fungal metabolites and/or biomarkers and whether host cells are affected in fighting against fungi. Influences on drug/microbe combinations have major therapeutical implications (1) because the release of biomarkers is the basis for early sensitive and specific diagnostic approaches in lifethreatening fungal infections (2); the release of cytokines and interaction of macrophages and neutrophils is crucial in preventing germination and hyphal elongation (3, 4). Each topic needs to be investigated in relation to oxygen availability (normoxia/hypoxia) to ensure a clinical- therapeutical implication. Several experimental models will be used in this work package: 1. Assaying antifungal sensitivity. We will investigate the in vitro activity of the most clinically important drugs against C. albicans, A. fumigatus and zygomycetes (R. oryzae, Mucor spec.) under normoxia and hypoxia. Antifungal drug resistance is usually quantified using the minimum inhibitory concentration (MIC); the assessment of in vitro activity of echinocandins against molds is complicated by the fact that under normoxia MICs often exceed safely achievable plasma concentrations and face the phenomenon of trailing. Preliminary data showed that end point reading for anidulafungin, caspofungin and micafungin under hypoxic conditions (1% oxygen) was superior (4). The underlying reasons are not known but should be clarified within this project. We will test for cross resistance and evaluate resistance of several genotypically well defined resistant strains under oxystress. Furthermore, we will analyse antifungal resistance of oxystress adapted strains/mutants established in WP3. 2. Biomarker release. Fungi secrete a wide variety of molecules from enzymes to carbohydrates, also in the infected human host. The detection of circulating fungal antigens in serum for Aspergillus galactomannan (GM) or ß-D-glucan has become an accepted diagnostic strategy. However, sensitivity and specificity varies extremely and the reasons are only partially clear; therefore, we will check whether normoxia and hypoxia influence the physiological kinetics of GM and ß-glucan unshielding/release. 3. Novel antifungals based on oxystress (collaboration with PFIZER). New antifungal drugs are required to treat the increasing numbers of patients suffering from serious fungal infections. Existing antifungal drugs are active on only a limited number of molecular targets. Therefore, new agents acting on novel targets such as molecules/reactions occuring during hypoxia/oxystress adaptation can be developed. It is highly likely that results obtained in WPs1-3 will establish novel antifungal targets that will be evaluated for antifungal screening in collaboration with PFIZER. In addition, synergisms of established and yet experimental antifungals (supplied by PFIZER) will be investigated under hypoxia, CO2 and/or ROS. 4. Virulence tests in animal infection models. Established murine models of systemic infection will be used to assess the virulence of C. albicans mutants lacking suspected oxystress regulators. Likewise, for A. fumigatus a murine infection model for invasive pulmonary aspergillosis will be employed, in which immunosuppressed mice are infected intranasally with spores of different mutant strains to monitor the contribution of the deleted genes on virulence; furthermore, growth of the mutants within the lung tissue will be examined by histopathology. 5. In vitro immune responses. Several in vitro killing assays will be performed in addition to molecular- based strategies (WP1-3) under oxystress: the conidiocidal assay to evaluate the phagocytes` efficiency in killing phagocytosed conidia, the germination inhibition assay to evaluate whether phagocytes are able to inhibit ingested conidia in their growth, the phagocytosis assay to assess the efficiency of phagocytic cells to engulf conidia of a given fungus. The MTT assay will be used to quantify hyphal damage. (1) Antimicrob Agents Chemother 2008, 52:3504-11; (2) Lancet 2004, 10:1467-74; (3) Clin Microbiol Rev 2009, 4:535-51; (4) Antimicrob Agents Chemother 2008, 5:1873-5.

Invasive fungal infections have increased over the last decades due to the growing number of patients at risk. These are mainly immunocompromised patients undergoing treatment against haematological malignancies and those who have received solid organ transplantation. Candida and Aspergillus species are the most frequent source of human fungal infections, but in recent years the epidemiology has changes and more and more atypical pathogens, such as Mucorales have been isolated from patient material. Given the complexity of the patient risk factors and the increasing variety of fungal pathogens, clinical outcome is still poor and it is essential to advance diagnosis and to optimize therapeutic strategies. Therefore, a detailed understanding of the development of fungal disease that implies fungal growth in host environment and the reactions of the immune system is necessary. In the frame of this project we studied by which means low oxygen conditions as they exist in the infected tissue might influence fungal growth, the secretion of so called biomarkers and the efficacy of antifungal drugs.Biomarkers are fungal components that are secreted in the surrounding tissue and are used for diagnosis, as they can be detected by commercially available test kits. In brief, our study showed, that hypoxia increased the release of galactomannan and ?-(1,3)-glucan only at early growth stages indicating only a marginal influence of hypoxic conditions on biomarkers important for diagnosis. In order to optimize treatment regimes, data from in vitro assays are used to check for susceptibility of fungal isolates to antifungal drugs, and also to determine efficient concentrations. Such test do usually do not take conditions within the host into account. Therefore, we tested the antifungal susceptibility of the main fungal pathogens under low oxygen conditions. Summarizing, changes of minimal inhibitory concentrations due to hypoxia largely depend on the method of choice (Etest or microbroth dilution), the species and the ability to grow at hypoxia. Second, a hypoxic environment makes visual determination of inhibitory concentration easier. The antifungal activity of the tested agents was not significantly altered in hypoxia for Aspergillus spp. and Mucorales, except for A. terreus, for which activity changed from fungicidal to fungistatic. For azoles, which target sterol biosynthesis, less fungal damage in hypoxic growth conditions was detected for A. fumigatus. Sterol pattern did not change due to hypoxia alone, but when fungi were treated with azoles, changes in amount of ergosterol, eburicol and lanosterol were detected, indicating lower efficacy of azoles in hypoxia.