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Exploring Cryptococcus neoformans CYP51 and Its Cognate Reductase as a Drug Target
Yasmeen N Ruma1, Mikhail V Keniya1,2, Brian C Monk1
1Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago, Dunedin 9016, New Zealand.
Abstract:
Cryptococcus remains a leading cause of invasive fungal infections in immunocompromised people. Resistance to azole drugs has imposed a further challenge to the effective treatment of such infections. In this study, the functional expression of full-length hexahistidine-tagged Cryptococcus neoformans CYP51 (CnCYP51-6×His), with or without its cognate hexahistidine-tagged NADPH-cytochrome P450 reductase (CnCPR-6×His), in a Saccharomyces cerevisiae host system has been used to characterise these enzymes. The heterologous expression of CnCYP51-6×His complemented deletion of the host CYP51 and conferred increased susceptibility to both short-tailed and long-tailed azole drugs. In addition, co-expression of CnCPR-6×His decreased susceptibility 2- to 4-fold for short-tailed but not long-tailed azoles. Type 2 binding of azoles to CnCYP51-6×His and assay of NADPH cytochrome P450 reductase activity confirmed that the heterologously expressed CnCYP51 and CnCPR are functional. The constructs have potential as screening tools and use in structure-directed antifungal discovery.
Insights
Cryptococcus neoformans CYP51 (CnCYP51) and NADPH-cytochrome P450 reductase (CnCPR) were functionally expressed in yeast. This system aids in understanding azole drug resistance in fungal infections.
Area of Science:
- Medical Mycology
- Molecular Biology
- Drug Discovery
Background:
- Cryptococcus is a major cause of fungal infections in immunocompromised individuals.
- Azole drug resistance complicates treatment of Cryptococcus infections.
Purpose of the Study:
- To characterize the functional expression of Cryptococcus neoformans CYP51 (CnCYP51) and its reductase (CnCPR) in a Saccharomyces cerevisiae host system.
- To assess the enzymes' role in azole drug susceptibility.
Main Methods:
- Heterologous expression of hexahistidine-tagged CnCYP51 and CnCPR in Saccharomyces cerevisiae.
- Complementation of host CYP51 deletion and azole drug susceptibility assays.
- Analysis of azole binding and enzyme activity.
Main Results:
- Heterologous expression of CnCYP51 restored growth in yeast lacking CYP51 and increased susceptibility to azole drugs.
- Co-expression of CnCPR modulated azole susceptibility, decreasing it for short-tailed azoles.
- Enzyme activity and azole binding confirmed the functionality of expressed CnCYP51 and CnCPR.
Conclusions:
- The functional expression system for CnCYP51 and CnCPR provides a valuable tool for studying azole drug interactions.
- These constructs can be utilized for screening antifungal agents and structure-directed drug discovery against Cryptococcus.
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