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Cloning and heterologous expression of the Candida albicans gene PMI 1 encoding phosphomannose isomerase
D J Smith1, A E Proudfoot, M Detiani
1Glaxo Institute for Molecular Biology, Geneva, Switzerland.
Abstract:
Using a DNA fragment derived from the Saccharomyces cerevisiae phosphomannose isomerase (PMI) structural gene as a probe against a random ordered array library of genomic DNA from the pathogenic fungus Candida albicans, we have cloned the C. albicans PMI 1 gene. This gene, which is unique in the C. albicans genome, can functionally complement PMI-deficient mutants of both S. cerevisiae and Escherichia coli. The DNA sequence of the PMI 1 gene predicts a protein with 64.1% identity to PMI from S. cerevisiae. Sequential gene disruption of PMI 1 produces a strain with an auxotrophic requirement for D-mannose. The heterologous expression of the PMI 1 gene at levels up to 45% of total cell protein in E. coli leads to partitioning of the enzyme between the soluble and particulate fractions. The protein produced in the soluble fraction is indistinguishable in kinetic properties from the material isolated from C. albicans cells.
Insights
Researchers cloned the Candida albicans phosphomannose isomerase (PMI) 1 gene, demonstrating its unique function in complementing deficient mutants. Gene disruption confirmed its essential role in D-mannose metabolism.
Area of Science:
- Molecular Biology
- Mycology
- Genetics
Background:
- Phosphomannose isomerase (PMI) is crucial for D-mannose metabolism in various organisms.
- Understanding fungal gene function is vital for developing targeted antifungal strategies against pathogens like Candida albicans.
Purpose of the Study:
- To clone and characterize the phosphomannose isomerase (PMI) 1 gene from the pathogenic fungus Candida albicans.
- To investigate the functional complementation and essentiality of the C. albicans PMI 1 gene.
Main Methods:
- Cloning of the C. albicans PMI 1 gene using a Saccharomyces cerevisiae PMI gene probe.
- Functional complementation assays in PMI-deficient mutants of S. cerevisiae and Escherichia coli.
- DNA sequencing and sequence comparison with S. cerevisiae PMI.
- Sequential gene disruption of PMI 1 in C. albicans.
- Heterologous expression of the C. albicans PMI 1 gene in E. coli.
Main Results:
- The unique C. albicans PMI 1 gene was successfully cloned and shown to functionally complement PMI-deficient mutants.
- The deduced protein sequence exhibits 64.1% identity to S. cerevisiae PMI.
- Disruption of the PMI 1 gene resulted in an auxotrophic requirement for D-mannose in C. albicans.
- Heterologous expression in E. coli produced soluble PMI with indistinguishable kinetic properties from the native enzyme.
Conclusions:
- The cloned C. albicans PMI 1 gene is essential for D-mannose metabolism.
- The C. albicans PMI enzyme is functionally conserved and shares significant sequence homology with its S. cerevisiae counterpart.
- This study provides a foundation for further investigation into PMI's role in C. albicans virulence and potential as an antifungal target.