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Expression analysis of PCSTE3, a putative pheromone receptor from the lung pathogenic fungus Pneumocystis carinii
Pawan K Vohra1, John G Park, Bharati Sanyal
1Thoracic Diseases Research Unit, Division of Pulmonary, Critical Care and Internal Medicine, Department of Medicine, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.
Abstract:
The fungal pathogen Pneumocystis carinii remains the most prevalent opportunistic infection in patients infected with HIV. Fungal pheromone receptors are seven transmembrane domain G-protein-coupled receptors which are expressed on specific mating types, and have ligand-binding extracellular domains for specific pheromones from cells of the opposite mating type. We have cloned and characterized PCSTE3 from P. carinii, which encodes a seven transmembrane domain protein orthologous to the Saccharomyces cerevisiae pheromone receptor Ste3. We detect PCSTE3 by indirect immunofluorescence using antibodies designed to extracellular domains of the receptor in yeast expressing the protein. Using a downstream Fus1-lacZ reporter gene, we determined that PCSTE3 does not recognize a- or alpha-factor pheromones as ligands for the receptor. We isolated P. carinii life cycle stages and examined PCSTE3 expression by immunofluorescence microscopy and flow cytometry, and found PCSTE3 expression exclusively on a population of trophic forms. PCSTE3 receptor expression was not found on cysts.
Insights
Pneumocystis carinii Ste3 (PCSTE3) is a fungal pheromone receptor. PCSTE3 is expressed on trophic forms but does not bind known yeast pheromones, suggesting unique P. carinii signaling.
Area of Science:
- Mycology
- Molecular Biology
- Immunology
Background:
- Pneumocystis carinii is a major opportunistic pathogen in HIV-infected individuals.
- Fungal pheromone receptors are G-protein-coupled receptors involved in mating.
- Understanding P. carinii signaling is crucial for developing targeted therapies.
Purpose of the Study:
- To clone and characterize the P. carinii Ste3 ortholog, PCSTE3.
- To investigate the ligand specificity and expression pattern of PCSTE3.
Main Methods:
- Cloning and expression of PCSTE3 in yeast.
- Indirect immunofluorescence using specific antibodies.
- Reporter gene assays (Fus1-lacZ) to detect pheromone binding.
- Immunofluorescence microscopy and flow cytometry to determine PCSTE3 localization in P. carinii life cycle stages.
Main Results:
- PCSTE3 encodes a seven-transmembrane domain protein, orthologous to Saccharomyces cerevisiae Ste3.
- PCSTE3 does not recognize a-factor or alpha-factor pheromones.
- PCSTE3 is exclusively expressed on trophic forms of P. carinii, not on cysts.
Conclusions:
- PCSTE3 represents a novel fungal pheromone receptor in P. carinii.
- Its expression pattern suggests a role in trophic form biology.
- PCSTE3's lack of response to known yeast pheromones indicates distinct ligand interactions in P. carinii.
