In filamentous fungi the conserved MAPK pheromone response, filamentous growth, osmotic stress response and cell wall integrity (CWI) pathways have been shown to influence numerous virulence traits including invasive growth, biofilm formation, mycotoxin production and antifungal tolerance. A. fumigatus has four MAPKs: (i) MpkA, the central regulator of CWI pathway also plays a role in oxidative stress, (ii) MpkB is the putative homologue of filamentous growth/pheromone response pathway, still uncharacterized and (iii) MpkC and SakA, homologues of the Saccharomyces cerevisiae Hog1, constitute the main regulator of the high osmolarity glycerol response (HOG) pathway. The MpkC protein sequence is very similar to that of SakA. SakA and MpkC have also been shown to play a role in caspofungin adaptation and carbon source utilization, respectively. Our laboratories have been investigating A. fumigatus MAPKs and their importance for the establishment of virulence/pathogenicity and mediation of drug resistance.
We have used a series of procedures, such as, construction of deletion and GFP mutants, extensive phenotypic assays, immunoblot analysis, and virulence studies by using a murine model of pulmonary aspergillosis, lung histopathology and fungal burden, aiming to characterize the biological functions of A. fumigatus SakA and MpkC.
Here, we investigated which stress responses were influenced by the MpkC and SakA mitogen-activated protein (MAP) kinases of the high-osmolarity glycerol (HOG) pathway in the fungal pathogen Aspergillus fumigatus. The ΔsakAand the double ΔmpkC ΔsakA mutants were more sensitive to osmotic and oxidative stresses, and to cell wall damaging agents. Both MpkC::GFP and SakA::GFP translocated to the nucleus upon osmotic stress and cell wall damage, with SakA::GFP showing a quicker response to both stresses. The phosphorylation state of MpkA was determined post exposure to high concentrations of Congo Red and Sorbitol. In the wild-type strain, MpkA phosphorylation levels progressively increased in both treatments. In contrast, the ΔsakA mutant had reduced MpkA phosphorylation, and surprisingly, the double ΔmpkC ΔsakA had no detectable MpkA phosphorylation. A. fumigatus ΔsakA and ΔmpkC were virulent in mouse survival experiments, but they had a 40 % reduction in fungal burden. In contrast, the ΔmpkC ΔsakA double mutant showed highly attenuated virulence, with approximately 50 % mice surviving and a 75 % reduction in fungal burden. We propose that both CWI and HOG pathways collaborate, and that MpkC could act by modulating SakA activity upon exposure to several types of stress and during cell wall biosynthesis.
In summary, we have identified an interaction between MpkC and SakA to counteract osmotic, oxidative, high temperature stresses, and also to regulate cell wall biosynthesis. Furthermore, this interaction is essential for virulence and macrophage recognition. It remains to be investigated how MpkC and SakA are affecting MpkA phosphorylation and the organization of the cell wall. Since most of the phenotypes observed for ΔmpkC were milder than for ΔsakA mutant, we propose that both the CWI and HOG pathways collaborate, and that MpkC could act by modulating SakA activity upon exposure to several different types of stress and during cell wall biosynthesis.