Summary

In view of the increasing threat of Candida infections, particularly to immuno-comprom-ised patients (immuno-suppressive therapy, AIDS), it is imperative to look for novel drugs with new targets. The experimental rationale is to develop a screening system on a suitable yeast background for effectors which block an early regulatory step in the acquired mechanism of the pathogen’s drug defence machinery. The novel and innovative aspect of this approach is searching for inhibitors of the synthesis of regulators and of their newly identified target MDR genes in favour of regaining the efficiency of already existing antifungals.

Problem

Candida albicans accounts for more than 50% of fungal infections in immuno-compromised individuals and thus, becomes a severe concern to clinicians. The search for novel drugs has become urgent as the common Candida strains acquire resistance to antifungals (multidrug resistance, MDR) and thus, severely hamper successful therapy. In this context leading experts in the field have joined their efforts to combat MDR of C. albicans on the following complementary areas: 1) systematic analysis of the functional domains of the transcription regulators and cell surface glycoproteins involved in MDR. 2) Search for new targets of MDR transcription regulators by using micro-array DNA chip technology. 3) Development of a screening system, in a suitable yeast background, for a block in an early regulatory step (including entry of the drug) in the acquired mechanism of MDR.

Aim

The main objectives of the project are to understand the mechanisms underlying MDR in order to define new antifungal targets and to develop a yeast based test system suitable for screen of negative effectors of the regulation of MDR components. The basic idea of this approach is to block an early regulatory step in the acquired mechanism of MDR of a pathogenic yeast. This will be monitored by testing the expression of a gene, able to inhibit growth, set under control of the promoter of a specific drug exporter, e.g. Pdr5p, Cdr1p, or of an enzyme related to protein glycosylation such as Pmt1p. This strain will only grow under conditions when the regulatory step is inhibited. The novel and innovative aspect of this approach in combating MDR of human pathogens is to block the synthesis of regulators of MDR genes in favour of regaining the efficiency of already existing antifungals. This approach emphasises the inhibition of regulatory components of the MDR defence machinery rather than looking for new antifungals, to which organism will develop resistance in due course of time.

Potential effectors (regulatory inhibitors) will allow the growth of constructed yeast strains in selective media. This positive growth selection marker is of significant advantage since it excludes false positive signals by general metabolic inhibitors.

Expected results

• Establishment of functional domains of MDR gene products (transcriptional regulators, drug transporters)
• Revealing of the subset of genes up-regulated by drug treatment
• Identification of targets of MDR regulatory genes by DNA array analysis
• Establishment of the role of cell surface glycosylation in MDR
• Identification of glycosylation target genes of C. albicans
• Construction of C. albicans mutant strains lacking or over-expressing pmt genes
• Establishment of a yeast based screening system for inhibitors and novel components of the MDR regulatory network in C. albicans
• The screening system will also have potential to reveal so far unknown cofactors of MDR transcription regulators of drug entry/export machinery, including cell surface glycosylation.

Potential applications

Elaboration of novel strategies to combat multidrug resistance of animal and human pathogens has a strong relevance to the quality of life and health aspects of the European population. Development of novel approaches to combat these infections is, therefore, of fundamental interest for the population in Europe and elsewhere. The participating industrial partner in collaboration with participating clinical partners will use the obtained results for further clinical testing of Candida infection treatment.

The yeast based screening system has the potential to reveal new components of the MDR regulatory cascade. Eventually, it may be exploited to search for pharmaceuticals related to other diseases (tumour chemotherapy), thus offering a promising alternative to animal tests.

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Last update:Oktober 23, 2002 webmaster