Abstract
Aspergillus fumigatus, a saprophytic filamentous fungus, is a serious opportunistic pathogen of mammals and it is the primary causal agent of invasive aspergillosis (IA). Mitogen activated protein Kinases (MAPKs) are important components involved in diverse cellular processes in eukaryotes. A. fumigatus MpkC and SakA, the homologs of the Saccharomyces cerevisiae Hog1 are important to adaptations to oxidative and osmotic stresses, heat shock, cell wall damage, macrophage recognition, and full virulence. We performed protein pull-down experiments aiming to identify interaction partners of SakA and MpkC by mass spectrometry analysis. In presence of osmotic stress with sorbitol, 118, and 213 proteins were detected as possible protein interactors of SakA and MpkC, respectively. Under cell wall stress caused by congo red, 420 and 299 proteins were detected interacting with SakA and MpkC, respectively. Interestingly, a group of 78 and 256 proteins were common to both interactome analysis. Co-immunoprecipitation (Co-IP) experiments showed that SakA::GFP is physically associated with MpkC:3xHA upon osmotic and cell wall stresses. We also validated the association between SakA:GFP and the cell wall integrity MAPK MpkA:3xHA and the phosphatase PtcB:3xHA, under cell wall stress. We further characterized A. fumigatus PakA, the homolog of the S. cerevisiae sexual developmental serine/threonine kinase Ste20, as a component of the SakA/MpkC MAPK pathway. The ΔpakA strain is more sensitive to cell wall damaging agents as congo red, calcofluor white, and caspofungin. Together, our data supporting the hypothesis that SakA and MpkC are part of an osmotic and general signal pathways involved in regulation of the response to the cell wall damage, oxidative stress, drug resistance, and establishment of infection. This manuscript describes an important biological resource to understand SakA and MpkC protein interactions. Further investigation of the biological roles played by these protein interactors will provide more opportunities to understand and combat IA.
Highlights
Protein-protein interactions (PPI) are essential for the accomplishment of many biological functions (Bork et al, 2004)
The full group of proteins that co-purified with SakA and MpkC during osmotic and CR stresses are listed in Supplementary Tables S1, S2, respectively
In fungi the signaling through the Mitogen activated protein Kinases (MAPKs) cascades result in altered gene expression that regulates many processes including pheromone response, filamentous growth, biosynthesis of cell wall components, the HOG pathway, the establishment of virulence, and mediation of drug resistance (Bahn et al, 2005; Monge et al, 2006; Román et al, 2007; Valiante et al, 2008, 2009, 2015a,b; de Castro et al, 2014; Altwasser et al, 2015; Bom et al, 2015; Winkelströter et al, 2015a,b)
Summary
Protein-protein interactions (PPI) are essential for the accomplishment of many biological functions (Bork et al, 2004). Interactome analyses can increase the understanding of the biological interactions and cellular processes within an organism. Recent high-throughput studies have obtained a great amount of PPI data, including model organisms such as Saccharomyces cerevisiae (Lam et al, 2015) and Aspergillus nidulans (Bayram et al, 2012; Jaimes-Arroyo et al, 2015), as well as plant pathogens Fusarium graminearum (Zhao et al, 2009), Magnaporthe grisea (He et al, 2008), and Phomopsis longicolla (Li et al, 2018). MAPK pathways control the response to multiple stresses including, oxidative, osmotic, heat shock, reactive oxygen species, nutrient limitation, and high concentrations of heavy metals (Rispail et al, 2009; Hamel et al, 2012; Turra et al, 2014)
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