The Aspergillus fumigatus pkcAG579R Mutant Is Defective in the Activation of the Cell Wall Integrity Pathway but Is Dispensable for Virulence in a Neutropenic Mouse Infection Model

Abstract

Aspergillus fumigatus is an opportunistic human pathogen, which causes the life-threatening disease, invasive pulmonary aspergillosis. In fungi, cell wall homeostasis is controlled by the conserved Cell Wall Integrity (CWI) pathway. In A. fumigatus this signaling cascade is partially characterized, but the mechanisms by which it is activated are not fully elucidated. In this study we investigated the role of protein kinase C (PkcA) in this signaling cascade. Our results suggest that pkcA is an essential gene and is activated in response to cell wall stress. Subsequently, we constructed and analyzed a non-essential A. fumigatus pkcA G579R mutant, carrying a Gly579Arg substitution in the PkcA C1B regulatory domain. The pkcA G579R mutation has a reduced activation of the downstream Mitogen-Activated Protein Kinase, MpkA, resulting in the altered expression of genes encoding cell wall-related proteins, markers of endoplasmic reticulum stress and the unfolded protein response. Furthermore, PkcAG579R is involved in the formation of proper conidial architecture and protection to oxidative damage. The pkcA G579R mutant elicits increased production of TNF-α and phagocytosis but it has no impact on virulence in a murine model of invasive pulmonary aspergillosis. These results highlight the importance of PkcA to the CWI pathway but also indicated that additional regulatory circuits may be involved in the biosynthesis and/or reinforcement of the A. fumigatus cell wall during infection.

Transcriptomic and molecular genetic analysis of the cell wall salvage response ofAspergillus nigerto the absence of galactofuranose synthesis

“…1 × 10 7 conidia of A. niger N402 and ΔugmA strain were grown at 30 °C (200 rpm) in liquid CM during 18, 24 and 36 h or 24, 36 and 48 h for the wild‐type and Δ ugmA strains respectively. For protein extraction, 0.5 ml of lysis buffer added to the ground mycelia as described previously (Rocha et al, ). The supernatants were collected and the protein concentrations were determined according to the Lowry method modified by Hartree (Hartree, ).…”

Cell wall integrity signalling in human pathogenic fungi

“…fumigatus , AfPkcA, was recently studied for the first time (Rocha et al ., ). Attempts to delete the Af pkcA gene failed, which is in good agreement with several similar approaches in Aspergillus nidulans (Ichinomiya et al ., ; Ronen et al ., ; Teepe et al ., ; Rocha et al ., ). Rocha and colleagues therefore constructed an Af pkcA G579R mutant that carries a point mutation in the C1B regulatory domain of AfPkcA, a strategy that was previously successfully used to characterize the A .…”

“…nidulans PkcA homologue (Hill et al ., ; Teepe et al ., ). The Af pkcA G579R mutant is hypersensitive towards chitin‐binding agents, heat, oxidative stress, azole and echinocandin antifungals (Rocha et al ., ). Similar to CaPkc1, AfPkcA and AnidPkcA were shown to influence CWI signalling.…”

“…Similar to CaPkc1, AfPkcA and AnidPkcA were shown to influence CWI signalling. While overexpression of Anid pkcA increased the amount of phospho‐AnidMpkA, phosphorylation of AfMpkA was less inducible in the Af pkcA G579R mutant (Colabardini et al ., ; Rocha et al ., ). Surprisingly, despite increased susceptibility to cell wall perturbing conditions and increased internalization by macrophages under in vitro conditions, the virulence of the Af pkcA G579R mutant is not attenuated (Rocha et al ., ).…”

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

“…The A. fumigatus CWI pathway has already been characterized, and it is mainly composed of the PkcA-MpkA-Rlm1 module (36,(41)(42)(43)(44)(45). Recently, we demonstrated that the HOG MAPs SakA and MpkC are independently required to mount an adequate response to cell wall damage (29).…”