The Aspergillus fumigatus pkcA G579R Mutant Is Defective in the Activation of the Cell Wall Integrity Pathway but Is Dispensable for Virulence in a Neutropenic Mouse Infection Model

Marina Campos Rocha; Krissia Franco de Godoy; Patrícia Alves de Castro; Juliana Issa Hori; Vinícius Leite Pedro Bom; Neil Andrew Brown; Anderson Ferreira da Cunha; Gustavo Henrique Goldman; Iran Malavazi

doi:10.1371/journal.pone.0135195

The Aspergillus fumigatus pkcA G579R Mutant Is Defective in the Activation of the Cell Wall Integrity Pathway but Is Dispensable for Virulence in a Neutropenic Mouse Infection Model

PLoS One. 2015 Aug 21;10(8):e0135195. doi: 10.1371/journal.pone.0135195. eCollection 2015.

Authors

Marina Campos Rocha¹, Krissia Franco de Godoy¹, Patrícia Alves de Castro², Juliana Issa Hori³, Vinícius Leite Pedro Bom², Neil Andrew Brown⁴, Anderson Ferreira da Cunha¹, Gustavo Henrique Goldman⁵, Iran Malavazi¹

Affiliations

¹ Departamento de Genética e Evolução, Centro de Ciências Biológicas e da Saúde, Universidade Federal de São Carlos, São Carlos, São Paulo, Brazil.
² Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil.
³ Departamento de Farmacologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil.
⁴ Department of Plant Biology and Crop Science, Rothamsted Research, Harpenden, Herts, United Kingdom.
⁵ Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil; Laboratório Nacional de Ciência e Tecnologia do Bioetanol, Campinas, São Paulo, Brazil.

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 pkcAG579R mutant, carrying a Gly579Arg substitution in the PkcA C1B regulatory domain. The pkcAG579R 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 pkcAG579R 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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Aspergillus fumigatus / genetics*
Aspergillus fumigatus / metabolism
Aspergillus fumigatus / pathogenicity
Cell Wall / chemistry
Cell Wall / metabolism
Disease Models, Animal
Endoplasmic Reticulum Stress / genetics
Female
Fungal Proteins / chemistry
Fungal Proteins / genetics*
Fungal Proteins / metabolism
Gene Expression Regulation, Fungal*
Genetic Engineering
Humans
Invasive Pulmonary Aspergillosis / microbiology*
Invasive Pulmonary Aspergillosis / mortality
Invasive Pulmonary Aspergillosis / pathology
Mice
Mice, Inbred BALB C
Mitogen-Activated Protein Kinases / genetics
Mitogen-Activated Protein Kinases / metabolism
Mutation
Neutropenia / microbiology*
Neutropenia / mortality
Neutropenia / pathology
Phagocytosis
Protein Kinase C-alpha / chemistry
Protein Kinase C-alpha / genetics*
Protein Kinase C-alpha / metabolism
Protein Structure, Tertiary
Signal Transduction
Spores, Fungal / chemistry
Spores, Fungal / metabolism
Survival Analysis
Tumor Necrosis Factor-alpha / biosynthesis
Unfolded Protein Response / genetics
Virulence

Substances

Fungal Proteins
Tumor Necrosis Factor-alpha
Protein Kinase C-alpha
Mitogen-Activated Protein Kinases

Grants and funding

This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo—FAPESP (http://www.fapesp.br/en/), grant number: 2009/53546-5 to IM; Conselho Nacional de Desenvolvimento Científico e Tecnológico—CNPq (http://www.cnpq.br), grant number 485478/2011-0) to IM.