Transcriptome and Biochemical Analysis Reveals That Suppression of GPI-Anchor Synthesis Leads to Autophagy and Possible Necroptosis in Aspergillus fumigatus

Jianghong Yan,Haomiao Ouyang,Hua Lu,Wan Zhao,Ting Du,Thomas Hartmann,Xuejun Jiang,Yang Lü,Lei Sun,Cheng Jin,Maureen J Donlin

doi:10.1371/journal.pone.0059013

Abstract

Previously, it has been shown that GPI proteins are required for cell wall synthesis and organization in Aspergillus fumigatus, a human opportunistic pathogen causing life-threatening invasive aspergillosis (IA) in immunocompromised patients. Blocking GPI anchor synthesis leads to severe phenotypes such as cell wall defects, increased cell death, and attenuated virulence. However, the mechanism by which these phenotypes are induced is unclear. To gain insight into global effects of GPI anchoring in A. fumigatus, in this study a conditional expression mutant was constructed and a genome wide transcriptome analysis was carried out. Our results suggested that suppression of GPI anchor synthesis mainly led to activation of phosphatidylinositol (PtdIns) signaling and ER stress. Biochemical and morphological evidence showed that autophagy was induced in response to suppression of the GPI anchor synthesis, and also an increased necroptosis was observed. Based on our results, we propose that activation of PtdIns3K and increased cytosolic Ca2+, which was induced by both ER stress and PtdIns signaling, acted as the main effectors to induce autophagy and possible necroptosis.

Highlights

Aspergillus fumigatus is an opportunistic pathogen causing lifethreatening invasive aspergillosis (IA) in immunocompromised patients [1,2]
Construction of the conditional afpig-a mutant A conditional expression mutant was constructed by replacing the native promoter of the afpig-a gene with PalcA, a strictly regulated promoter that can be induced by ethanol, glycerol or threonine and repressed completely on YEPD medium [22,32]
PCR analysis confirmed that a 1217-bp fragment of pyr-4 and a 2023-bp fragment of PalcA-afpig-a were amplified from the genomic DNA of the mutant, while no such fragments were amplified from the WT DNA (Fig.1A)

Summary

Introduction

Aspergillus fumigatus is an opportunistic pathogen causing lifethreatening invasive aspergillosis (IA) in immunocompromised patients [1,2]. The crude mortality from IA is 60–90% [3]. This high mortality is due to a poor understanding of A. fumigatus and the low efficiency of drug therapies available. A deeper understanding of A. fumigatus at the molecular level would help to develop new drugs or new strategies to treat IA. As the fungal cell wall directly contacts the host cells and provides physical protection against an adverse environment, it has long been treated as an ideal target for drug development. The cell wall of A. fumigatus is mainly composed of a covalently connected polysaccharide skeleton (glucans and chitin) that is interlaced and coated with glycoproteins [4,5,6]. Some cell surface proteins are modified at their C-terminus by the addition of a glycosylphosphatidylinositol (GPI) anchor and transported to the plasma membrane and cell wall, where they are directly or indirectly involved in cell wall organization [7,8,9,10,11,12,13,14]

Methods

Results

Conclusion