The Aspergillus fumigatus CrzA Transcription Factor Activates Chitin Synthase Gene Expression during the Caspofungin Paradoxical Effect

Abstract

Aspergillus fumigatus is an opportunistic fungal pathogen that causes invasive aspergillosis (IA), a life-threatening disease in immunocompromised humans. The echinocandin caspofungin, adopted as a second-line therapy in combating IA, is a β-1,3-glucan synthase inhibitor, which, when used in high concentrations, reverts the anticipated A. fumigatus growth inhibition, a phenomenon called the “caspofungin paradoxical effect” (CPE). The CPE has been widely associated with increased chitin content in the cell wall due to a compensatory upregulation of chitin synthase-encoding genes. Here, we demonstrate that the CPE is dependent on the cell wall integrity (CWI) mitogen-activated protein kinase MpkAMPK1 and its associated transcription factor (TF) RlmARLM1, which regulate chitin synthase gene expression in response to different concentrations of caspofungin. Furthermore, the calcium- and calcineurin-dependent TF CrzA binds to and regulates the expression of specific chitin synthase genes during the CPE. These results suggest that the regulation of cell wall biosynthetic genes occurs by several cellular signaling pathways. In addition, CrzA is also involved in cell wall organization in the absence of caspofungin. Differences in the CPE were also observed between two A. fumigatus clinical isolates, which led to the identification of a novel basic leucine zipper TF, termed ZipD. This TF functions in the calcium-calcineurin pathway and is involved in the regulation of cell wall biosynthesis genes. This study therefore unraveled additional mechanisms and novel factors governing the CPE response, which ultimately could aid in developing more effective antifungal therapies.

The Aspergillus fumigatus Phosphoproteome Reveals Roles of High-Osmolarity Glycerol Mitogen-Activated Protein Kinases in Promoting Cell Wall Damage and Caspofungin Tolerance

“…According to FunCat annotation enrichment and the interaction network analyses of this novel phosphoproteome, the proteins phosphorylated in response to CR were involved in a variety of important biological processes, including signal transduction (osmosensing and response, Ca 2ϩ -mediated signal transduction, and MAPKKK cascade), stress response, protein kinases, actin cytoskeleton, and budding cell polarity and filament formation. These results emphasize the importance of osmotic stress, calcium metabolism, and MAPKs in the A. fumigatus CWI pathway, as previously demonstrated (29,34,(78)(79)(80)(81)(82).…”

Aspergillus fumigatus calcium-responsive transcription factors regulate cell wall architecture promoting stress tolerance, virulence and caspofungin resistance

“…Furthermore, calcineurin-CrzA pathway promotes antifungal resistance, since combining calcium or calcineurin inhibitors with azoles or echinocandins restores drug-mediated growth inhibition for some antifungal resistant fungal strains [16,[19][20][21][22][23][24][25]. In A. fumigatus CrzA mediates cellular tolerance to increased concentrations of calcium and manganese, controls the transcription of several calcium transporters and channels, and is important for virulence [26][27][28][29][30][31][32].…”

Echinocandins for the Treatment of Invasive Aspergillosis: from Laboratory to Bedside

“…However, this pathway does not seem to play a role in PE via overexpression of chitin synthases. On the contrary, mpkA had a repressive effect on these genes but was also shown to positively impact the expression of other cell wall components, such as ␤-1,3-glucan or ␣-1,3-glucans (22,32). Indeed, Loiko and Wagener (33) demonstrated that the key adaptive mechanism in PE does not seem to be related to the increased expression of chitin synthases but rather to the recovery of ␤-1,3-glucan synthase activity.…”

“…In addition to the calcineurin pathway, a role of the cell wall integrity (CWI) pathway was recently highlighted. Deletions of the CWI MAPK gene mpkA and its downstream transcription factor rlmA resulted in loss of the PE (22). However, this pathway does not seem to play a role in PE via overexpression of chitin synthases.…”

“…Activated calcineurin then dephosphorylates the transcription factor CrzA that moves to the nucleus and binds to specific promoter regions (calcineurin-dependent reporter elements) of the chitin synthase-encoding genes (chsA, chsC, chsG, and csmB) (22). Both calcineurin and CrzA are necessary to increase expression of chitin synthases and chitin content of the cell wall after caspofungin exposure (14,22,23). The cnaA and crzA deletion mutants also exhibit decreased ␤-1,3-glucan in the cell wall and lack the PE in response to caspofungin (23).…”