Aspergillus fumigatus Hsp90 interacts with the main components of the cell wall integrity pathway and cooperates in heat shock and cell wall stress adaptation

Abstract

The initiation of Aspergillus fumigatus infection occurs via dormant conidia deposition into the airways. Therefore, conidial germination and subsequent hyphal extension and growth occur in a sustained heat shock (HS) environment promoted by the host. The cell wall integrity pathway (CWIP) and the essential eukaryotic chaperone Hsp90 are critical for fungi to survive HS. Although A. fumigatus is a thermophilic fungus, the mechanisms underpinning the HS response are not thoroughly described and important to define its role in pathogenesis, virulence and antifungal drug responses. Here, we investigate the contribution of the CWIP in A. fumigatus thermotolerance. We observed that the CWIP components PkcA, MpkA and RlmA are Hsp90 clients and that a PkcAG579R mutation abolishes this interaction. PkcAG579R also abolishes MpkA activation in the short‐term response to HS. Biochemical and biophysical analyses indicated that Hsp90 is a dimeric functional ATPase, which has a higher affinity for ADP than ATP and prevents MpkA aggregation in vitro. Our data suggest that the CWIP is constitutively required for A. fumigatus to cope with the temperature increase found in the mammalian lung environment, emphasising the importance of this pathway in supporting thermotolerance and cell wall integrity.

The Heat Shock Transcription Factor HsfA Is Essential for Thermotolerance and Regulates Cell Wall Integrity in Aspergillus fumigatus

“…However, it remained unclear how hsfA HSF 1 contributes to thermal adaptation and the disturbed cell wall homeostasis observed in the CWI pathway mutants. This is a follow-up study to our previous work (Rocha et al, 2020b), and our results show that cell wall ultrastructure is severely modified when cells are exposed to HS. We observe that hsfA is essential for thermotolerance, impacts the expression of genes related to the cell wall biosynthesis, and genetically interacts with the main players of the CWI and HOG pathway.…”

“…Also, Hsp90 potentiates the resistance to echinocandins in different fungal pathogens, including A. fumigatus, since depletion of this chaperone increases the susceptibility to these antifungals (Cowen, 2009;LaFayette et al, 2010;Lamoth et al, 2013Lamoth et al, , 2015Chatterjee and Tatu, 2017). We have previously demonstrated that three critical components of the A. fumigatus CWI pathway (PkcA, MpkA, and RlmA) are Hsp90 clients (Rocha et al, 2020b), suggesting an involvement of the Hsf1-Hsp90 regulatory function over the A. fumigatus CWI pathway. However, it remained unclear how hsfA HSF 1 contributes to thermal adaptation and the disturbed cell wall homeostasis observed in the CWI pathway mutants.…”

“…The CWI pathway, which comprises the apical kinase PkcA, the three-component mitogen-activated protein kinases (MAPKs) Bck1, Mkk2, and MpkA, and the transcription factor RlmA (Valiante et al, 2009;Rocha et al, 2015Rocha et al, , 2016, is one of the signaling cascades responsible for the regulation of cell wall biosynthesis and maintenance (Valiante et al, 2015). This signaling pathway also sustains the fungal cell wall viability during heat stress (Kamada et al, 1995;Fuchs and Mylonakis, 2009;Rocha et al, 2020b). However, despite the significance of the interplay between thermotolerance and CWI, little is known about the molecular events that reciprocally govern these two essential A. fumigatus virulence attributes.…”

Post-Translational Modifications Drive Success and Failure of Fungal–Host Interactions

“…PkcA is a critical kinase in the CWI pathway and necessary for fungal survival during exposure to antifungal drugs [78]. In the current study, mutations in the C1B domain of PkcA prevented interaction with Hsp90, abolishing the CWI pathway response and supporting the potential of PkcA as a novel antifungal target; however, similarity with the host PKCβII protein demonstrates cross-reactivity and blocking [77]. These findings determined that CWI proteins are Hsp90 substrates, and Hsp90 stabilizes the CWI proteins, regulating response to heat stress.…”

“…For example, in C. albicans, Hsp90 is phosphorylated by regulatory subunits of kinase CK2, impacting the stability and function of Hsp90 targets, including Hog1, which is necessary for stress response [75] (Figure 4B). Another example encompasses the role of CWI components (e.g., PkcA, MpkA, and RlmA), which interact with Hsp90 during heat shock and cell wall stress adaptation [77]. PkcA is a critical kinase in the CWI pathway and necessary for fungal survival during exposure to antifungal drugs [78].…”