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Characterization of pneumocystis major surface glycoprotein gene (msg) promoter activity in Saccharomyces cerevisiae
Geetha Kutty1, Robert Shroff, Joseph A Kovacs
1Critical Care Medicine Department, NIH Clinical Center, NIH, Bethesda, Maryland, USA.
Abstract:
Major surface glycoprotein (Msg), the most abundant cell surface protein of Pneumocystis, plays an important role in the interaction of this opportunistic pathogen with host cells, and its potential for antigenic variation may facilitate evasion of host immune responses. In the present study, we have identified and characterized the promoter region of msg in 3 species of Pneumocystis: P. carinii, P. jirovecii, and P. murina. Because Pneumocystis cannot be cultured, promoter activity was measured in Saccharomyces cerevisiae, a related fungus, using a yeast vector modified to utilize the gene coding for Renilla luciferase as a reporter gene. The 5'-flanking sequences of msg from all three Pneumocystis species showed considerable promoter activity, with increases in luciferase activity up to 15- to 44-fold above baseline. Progressive deletions helped define an ∼13-bp sequence in each Pneumocystis species that appears to be critical for promoter activity. Electrophoretic mobility shift analysis using P. carinii-specific msg promoter sequences demonstrated binding of nuclear proteins of S. cerevisiae. The 144-bp 5'-flanking region of P. murina msg showed 72% identity to that of P. carinii. The 5'-flanking region of P. jirovecii msg showed 58 and 61% identity to those of P. murina and P. carinii, respectively. The msg promoter is a good candidate for inclusion in a construct designed for genetic manipulation of Pneumocystis species.
Insights
Researchers characterized the promoter region of the major surface glycoprotein (Msg) in Pneumocystis species. This critical region is essential for gene expression and may enable genetic manipulation of this opportunistic pathogen.
Area of Science:
- Mycology
- Molecular Biology
- Immunology
Background:
- The major surface glycoprotein (Msg) is a key cell surface protein in Pneumocystis, crucial for host cell interactions and immune evasion.
- Antigenic variation in Msg may contribute to Pneumocystis's ability to evade host immune responses.
Purpose of the Study:
- To identify and characterize the promoter region of the msg gene in three species of Pneumocystis: P. carinii, P. jirovecii, and P. murina.
- To assess the promoter activity and identify critical sequences for gene regulation.
Main Methods:
- Reporter gene assay using Renilla luciferase in Saccharomyces cerevisiae due to the inability to culture Pneumocystis.
- Progressive deletion analysis to pinpoint essential promoter elements.
- Electrophoretic mobility shift assays (EMSA) to investigate protein binding to the promoter sequences.
Main Results:
- The 5'-flanking sequences of msg from all three Pneumocystis species exhibited significant promoter activity (15- to 44-fold increase in luciferase activity).
- A conserved approximately 13-bp sequence was identified as critical for promoter activity across the studied species.
- EMSA confirmed the binding of Saccharomyces cerevisiae nuclear proteins to the P. carinii msg promoter.
- Sequence identity analysis revealed high conservation between P. murina and P. carinii msg promoters (72%) and moderate identity with P. jirovecii (58-61%).
Conclusions:
- The msg promoter demonstrates significant activity and contains a conserved critical element essential for its function.
- The identified msg promoter is a promising candidate for developing constructs for genetic manipulation of Pneumocystis species, aiding future research and therapeutic development.
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