Deletion of the sec4 Homolog srgA from Aspergillus fumigatus Is Associated with an Impaired Stress Response, Attenuated Virulence and Phenotypic Heterogeneity

Margaret V Powers-Fletcher,Karthik Krishnan,David S Askew,Xizhi Feng,Robert A Cramer

doi:10.1371/journal.pone.0066741

Margaret V Powers-Fletcher, Karthik Krishnan + Show 3 more

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https://doi.org/10.1371/journal.pone.0066741

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Journal: PLoS ONE	Publication Date: Jun 13, 2013
Citations: 24	License type: CC BY 4.0

Affiliation: University of Cincinnati Medical Center

Abstract

Small GTPases of the Rab family are master regulators of membrane trafficking, responsible for coordinating the sorting, packaging and delivery of membrane-bound vesicles to specific sites within eukaryotic cells. The contribution of these proteins to the biology of the human pathogenic fungus Aspergillus fumigatus has not been explored. In this study, we characterized the srgA gene, encoding a Rab GTPase closely related to Sec4. We found that a GFP-SrgA fusion protein accumulated preferentially at hyphal tips and mature condiophores. The radial growth of a ΔsrgA mutant was impaired on both rich and minimal medium, consistent with a role for SrgA in filamentous growth. In addition, the ΔsrgA mutant revealed dysmorphic conidiophores that produced conidia with heterogeneous morphology. The ΔsrgA mutant was hypersensitive to brefeldin A-mediated inhibition of vesicular trafficking and showed increased temperature sensitivity relative to wild type A. fumigatus. However, the most striking phenotype of this mutant was its phenotypic heterogeneity. Individual colonies isolated from the original ΔsrgA mutant showed variable morphology with colony sectoring. In addition, each isolate of the ΔsrgA mutant displayed divergent phenotypes with respect to thermotolerance, in vitro stress response and virulence in a Galleria mellonella infection model. Taken together, these results indicate that SrgA contributes to the asexual development and filamentous growth of A. fumigatus. However, the discordant phenotypes observed among individual isolates of the ΔsrgA mutant suggest that the absence of srgA exerts selective pressure for the acquisition of compensatory changes, such as second-site suppressor mutations.

Highlights

Filamentous fungi elongate and branch by apical extension, a mode of growth that involves the establishment of a stable axis of polarity, followed by the maintenance of growth in the same direction [1]
Identification of the Sec4 Homolog secretionrelated GTPase A (SrgA) in A. fumigatus SrgA was previously identified in A. niger as one of five different secretion-related GTPases thought to be involved in mediating different stages of vesicle transport [17]
The most striking finding was that srgA deletion was associated with phenotypic heterogeneity, which was manifested by distinct colony morphologies and variable responses to both in vitro and in vivo stress conditions

Summary

Introduction

Filamentous fungi elongate and branch by apical extension, a mode of growth that involves the establishment of a stable axis of polarity, followed by the maintenance of growth in the same direction [1]. The ability to sustain polarization requires a constant stream of new cell wall and plasma membrane material to the hyphal apex [2] This is accomplished by packaging components required for membrane and cell wall biogenesis into membraneenclosed vesicles of the secretory system and delivering them to the growing tip cell [3]. The Spitzenkorper is an apical cluster of vesicles and cytoskeletal components that assists in this process by providing a vesicle supply center for the rapid delivery of enzymes into and across the apical cell membrane [7] This contrasts the budding yeast Saccharomyces cerevisiae, where the continual delivery of vesicles across the entire cell surface promotes spherical rather than polarized growth [8]

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