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Functional Analysis of the Exocyst Subunit Sec15 in Candida albicans
Alba A Chavez-Dozal1, Stella M Bernardo1, Hallie S Rane2
1Section of Infectious Diseases, New Mexico VA Healthcare System, Albuquerque, New Mexico, USA Division of Infectious Diseases, University of New Mexico Health Science Center, Albuquerque, New Mexico, USA.
Eukaryotic Cell
|October 11, 2015
Summary
The exocyst subunits Sec15 and Sec6 in Candida albicans have distinct roles in cell wall integrity and polarized secretion. Sec15 is crucial for hyphal growth and secretion, while Sec6 regulates branching.
Area of Science:
- Microbiology
- Cell Biology
- Mycology
Background:
- The exocyst complex is essential for polarized secretion in eukaryotic cells.
- Previous studies in Candida albicans identified roles for Sec6 in cell wall integrity, secretion, and filamentation, with its absence leading to reduced hyphal branching.
- The function of other exocyst subunits, like Sec15, in C. albicans filamentation and secretion remained less understood.
Purpose of the Study:
- To investigate the specific roles of the exocyst subunit Sec15 in polarized secretion and filamentation in Candida albicans.
- To compare the functions of Sec15 with the previously studied Sec6 subunit within the exocyst complex.
- To elucidate the impact of Sec15 dysfunction on key cellular structures involved in polarized growth.
Main Methods:
- Generation of a conditional mutant strain (tetR-SEC15) in C. albicans, where SEC15 expression is regulated by a tetracycline-inducible promoter.
- Assessment of cell viability, secretion of Sap and lipase, sensitivity to cell wall degrading enzymes (Zymolyase, chitinase), and hyphal morphology under repressed conditions.
- Microscopic analysis of the localization of Spitzenkörper (Mlc1-GFP), polarisomes (Spa2-GFP), and exocysts (Exo70-GFP) in tetR-SEC15 and tetR-SEC6 mutants during filamentation.
Main Results:
- Depletion of Sec15 in C. albicans led to cell death within 5 hours and severe defects in Sap and lipase secretion.
- The tetR-SEC15 mutant exhibited increased sensitivity to Zymolyase and chitinase, indicating compromised cell wall integrity.
- In contrast to the reduced branching in tetR-SEC6 mutants, tetR-SEC15 mutants displayed a hyperbranching phenotype.
- Mislocalization of Spitzenkörper, polarisomes, and exocysts was observed in tetR-SEC15 mutants, with sequential recovery of these structures upon removal of doxycycline.
- Sec6 localization remained normal in the tetR-SEC6 mutant.
Conclusions:
- The exocyst subunits Sec15 and Sec6 play distinct and non-redundant roles in mediating polarized secretion and filamentation in Candida albicans.
- Sec15 is critical for maintaining cell wall integrity, proper secretion, and regulating hyphal branching patterns.
- Proper localization of the Spitzenkörper, polarisome, and exocyst complex is dependent on Sec15 function during filamentation.
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