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Heterologous expression of Candida albicans Pma1p in Saccharomyces cerevisiae
Mikhail V Keniya1, Richard D Cannon, ÂnBình Nguyễn
1The Sir John Walsh Research Institute, School of Dentistry, University of Otago, Dunedin, New Zealand. mikhail.keniya@otago.ac.nz
Abstract:
Candida albicans is a major cause of opportunistic and life-threatening systemic fungal infections, especially in the immunocompromised. The plasma membrane proton-pumping ATPase (Pma1p) is an essential enzyme that generates the electrochemical gradient required for cell growth. We expressed C. albicans Pma1p (CaPma1p) in Saccharomyces cerevisiae to facilitate screening for inhibitors. Replacement of S. cerevisiae PMA1 with C. albicans PMA1 gave clones expressing CaPma1p that grew slowly at low pH. CaPma1p was expressed at significantly lower levels and had lower specific activity than the native Pma1p. It also conferred pH sensitivity, hygromycin B resistance, and low levels of glucose-dependent proton pumping. Recombination between CaPMA1 and the homologous nonessential ScPMA2 resulted in chimeric suppressor mutants that expressed functional CaPma1p with improved H(+) -ATPase activity and growth rates at low pH. Molecular models of suppressor mutants identified specific amino acids (between 531 and 595 in CaPma1p) that may affect regulation of the activity of Pma1p oligomers in S. cerevisiae. A modified CaPma1p chimeric construct containing only 5 amino acids from ScPma2p enabled the expression of a fully functional enzyme for drug screens and structural resolution.
Insights
Researchers engineered Candida albicans Pma1p (CaPma1p) in yeast for drug screening. Chimeric CaPma1p, modified with yeast genes, showed improved function, enabling antifungal drug discovery.
Area of Science:
- Biochemistry
- Mycology
- Molecular Biology
Background:
- Candida albicans causes severe opportunistic infections, particularly in immunocompromised individuals.
- The essential plasma membrane proton-pumping ATPase (Pma1p) is crucial for fungal cell growth and survival.
- Targeting CaPma1p offers a potential strategy for developing new antifungal therapies.
Purpose of the Study:
- To express and characterize Candida albicans Pma1p (CaPma1p) in Saccharomyces cerevisiae for antifungal drug screening.
- To identify modifications that restore CaPma1p function and enable its use in drug discovery.
- To investigate the structural basis for CaPma1p regulation and activity.
Main Methods:
- Expressing C. albicans PMA1 in S. cerevisiae, replacing the native gene.
- Generating chimeric CaPma1p constructs through recombination with S. cerevisiae PMA2.
- Analyzing CaPma1p expression, activity, pH sensitivity, and drug resistance.
- Utilizing molecular modeling to identify key amino acid residues.
Main Results:
- Direct expression of CaPma1p resulted in slow growth and reduced enzyme activity.
- Chimeric CaPma1p variants exhibited improved H(+)-ATPase activity and growth at low pH.
- Specific amino acid regions (531–595) were implicated in CaPma1p regulation.
- A modified chimeric CaPma1p with 5 amino acids from ScPma2p was fully functional.
Conclusions:
- Engineered CaPma1p can be functionally expressed in yeast, serving as a valuable tool for antifungal drug discovery.
- Modifications involving homologous recombination can restore the activity of essential fungal enzymes.
- Understanding CaPma1p structure-function relationships is key to developing targeted antifungal agents.
