Abstract

The yeast exocyst is a multiprotein complex comprised of eight subunits (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84) which orchestrates trafficking of exocytic vesicles to specific docking sites on the plasma membrane during polarized secretion. To study SEC6 function in Candida albicans, we generated a conditional mutant strain in which SEC6 was placed under the control of a tetracycline-regulated promoter. In the repressed state, the tetR-SEC6 mutant strain (denoted tSEC6) was viable for up to 27 h; thus, all phenotypic analyses were performed at 24 h or earlier. Strain tSEC6 under repressing conditions had readily apparent defects in cytokinesis and endocytosis and accumulated both post-Golgi apparatus secretory vesicles and structures suggestive of late endosomes. Strain tSEC6 was markedly defective in secretion of aspartyl proteases and lipases as well as filamentation under repressing conditions. Lack of SEC6 expression resulted in markedly reduced lateral hyphal branching, which requires the establishment of a new axis of polarized secretion. Aberrant localization of chitin at the septum and increased resistance to zymolyase activity were observed, suggesting that C. albicans Sec6 plays an important role in mediating trafficking and delivery of cell wall components. The tSEC6 mutant was also markedly defective in macrophage killing, indicating a role of SEC6 in C. albicans virulence. Taken together, these studies indicate that the late secretory protein Sec6 is required for polarized secretion, hyphal morphogenesis, and the pathogenesis of C. albicans.

Highlights

  • Section of Infectious Diseases, New Mexico VA Healthcare System, Albuquerque, New Mexico, USAa; Division of Infectious Diseases, University of New Mexico Health Science Center, Albuquerque, New Mexico, USAb; School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Aberdeen, United Kingdomc

  • We found that C. albicans Sec6 plays multiple roles in vegetative growth, cell wall biosynthesis, and virulence of this fungus

  • Microscopy of cells stained with calcofluor white revealed an increased concentration of chitin at the bud neck in C. albicans tSEC6 strains under restrictive conditions

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Summary

Introduction

The ability of strain tSEC6 to grow on medium containing cell wall stressors was tested on agar plates in the presence or absence of DOX and compared to that of controls. Expression of SEC6 in tSEC6 and the control strain, THE1-CIp10, in the absence or presence of DOX was analyzed by RT-PCR (see Fig. S1 in the supplemental mateadded to a concentration of 20 cells/ml, poured into individual petri rial).

Results
Conclusion

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