Functional Coupling between the Unfolded Protein Response and Endoplasmic Reticulum/Golgi Ca ²⁺ -ATPases Promotes Stress Tolerance, Cell Wall Biosynthesis, and Virulence of Aspergillus fumigatus

Abstract

Many species of pathogenic fungi deploy the unfolded protein response (UPR) to expand the folding capacity of the endoplasmic reticulum (ER) in proportion to the demand for virulence-related proteins that traffic through the secretory pathway. Although Ca2+ plays a pivotal role in ER function, the mechanism by which transcriptional upregulation of the protein folding machinery is coordinated with Ca2+ homeostasis is incompletely understood. In this study, we investigated the link between the UPR and genes encoding P-type Ca2+-ATPases in the human-pathogenic mold Aspergillus fumigatus. We demonstrate that acute ER stress increases transcription of the srcA gene, encoding a member of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) family, as well as that of pmrA, encoding a secretory pathway Ca2+-ATPase (SPCA) in the Golgi membrane. Loss of the UPR transcription factor HacA prevented the induction of srcA and pmrA transcription during ER stress, defining these ER/Golgi Ca2+ pumps as novel downstream targets of this pathway. While deletion of srcA alone caused no major deficiencies, a ΔsrcA/ΔpmrA mutant displayed a severe polarity defect, was hypersensitive to ER stress, and showed attenuated virulence. In addition, cell wall analyses revealed a striking reduction in mannose levels in the absence of both Ca2+ pumps. The ΔhacA mutant was hypersensitive to agents that block calcineurin-dependent signaling, consistent with a functional coupling between the UPR and Ca2+ homeostasis. Together, these findings demonstrate that the UPR integrates the need for increased levels of chaperone and folding enzymes with an influx of Ca2+ into the secretory pathway to support fungal growth, stress adaptation, and pathogenicity. IMPORTANCE The UPR is an intracellular signal transduction pathway that maintains homeostasis of the ER. The pathway is also tightly linked to the expression of virulence-related traits in diverse species of human-pathogenic and plant-pathogenic fungal species, including the predominant mold pathogen infecting humans, Aspergillus fumigatus. Despite advances in the understanding of UPR signaling, the linkages and networks that are governed by this pathway are not well defined. In this study, we revealed that the UPR is a major driving force for stimulating Ca2+ influx at the ER and Golgi membranes and that the coupling between the UPR and Ca2+ import is important for virulence, cell wall biosynthesis, and resistance to antifungal compounds that inhibit Ca2+ signaling.

A Human IRE1 Inhibitor Blocks the Unfolded Protein Response in the Pathogenic Fungus Aspergillus fumigatus and Suggests Noncanonical Functions within the Pathway

“…Dithiothreitol (DTT) is the most widely used approach to experimentally induce ER stress because it reduces the disulfide bridges that stabilize many proteins in the secretory pathway, resulting in a rapid increase in the level of unfolded proteins that activate the IRE1/IreA sensor ( 32 ). Consistent with previous reports ( 33 ), DTT treatment of A. fumigatus hyphae induced accumulation of hacA i mRNA, reflecting activation of the IreA RNase domain ( Fig. 2A ).…”

“…The compound also blocked the ability of DTT to induce the expression of srcA and pmrA ( Fig. S2A ), encoding ER/Golgi P-type Ca 2+ ATPases that we have recently identified as targets of the UPR in A. fumigatus ( 33 ). Interestingly, we found that a Δ srcA /Δ pmrA double deletion mutant was hypersensitive to the effects of 4μ8C relative to its parental strain ( Fig.…”

“…S2B ). Since the absence of these Ca 2+ ATPases exacerbates ER stress ( 33 ), the increased sensitivity of this mutant to 4μ8C is likely to be due to the ability of the compound to prevent the canonical UPR from mounting an adaptive response to low Ca 2+ levels in the secretory pathway caused by the absence of these Ca 2+ pumps. We conclude that 4μ8C is able to enter the fungus and reach the IreA sensor, thereby inhibiting the RNase activity that is required for hacA u mRNA processing and downstream activation of UPR target genes.…”

Calcofluor white hypersensitive proteins contribute to stress tolerance and pathogenicity in entomopathogenic fungus, Metarhizium acridum

“…Pathogenic fungal lifestyle is largely supported by the ER, which enables secretion of cell wall components and virulence-related proteins. 41,42 Our results show that the cell wall structure and components were drastically affected in the absence of Macwh1 or Macwh43. Thus, the roles of MaCwh1 and MaCwh43 in fungal tolerance to CFW were less likely to be associated with the domains in CWH43 than with the secretion and remodeling of cell wall components by ER.…”

“…Consistent with an ER localization of Cwh43 in S. cerevisiae and S. pombe , 24,40 both MaCwh1 and MaCwh43 were localized at the ER. Pathogenic fungal lifestyle is largely supported by the ER, which enables secretion of cell wall components and virulence‐related proteins 41,42 . Our results show that the cell wall structure and components were drastically affected in the absence of Macwh1 or Macwh43 .…”