Investigating the upstream mechanisms of Aspergillus fumigatus protein kinase C activation as a strategy for the inhibition of the cell wall integrity pathway and the search for new antifungal approaches

Abstract

The cell wall is essential for fungal cell survival, exerting protection against osmotic damage. In addition, it is also a component of the mammalian host immune system recognition during the infection caused by fungal pathogens. Understanding the mechanisms whereby fungal cell builds and remodels the cell wall under stress conditions is relevant given the high morbidity and mortality rates caused by these pathogens, including the mold Aspergillus fumigatus. Our laboratory is focused on investigating one of the signal transduction pathways responsible for cell wall synthesis in fungi, i. e., the cell wall integrity pathway (CWI). Our data have indicated that CWI functioning begins with the activation of apical protein kinase C (PkcA), an important event for the subsequent activation of downstream MAPK cascade and the main CWI-associated transcription factor, RlmA. Rho-GTPases are essential components for activating PKCs because they connect extracellular signals to the pathway stimulation through physical interaction with PKC. However, direct evidence of Rho-GTPases mediated PkcA activation is not available in A. fumigatus, possibly due to the redundancy of these proteins and the absence of information about hierarchy and the importance of Rho-GTPases. Consequently, which domain(s) of PkcA (C1 and/or HR1) are involved in binding with Rho-GTPases is unknown in A. fumigatus or any other filamentous fungus. PKCs such as PkcA have a complex and multimodular activation mechanism, and the conversion of the inactive form of the enzyme to the active enzyme may depend on secondary messengers such as DAG (diacylglycerol), depending on the enzyme class. However, the binding of DAG in fungal PKCs is still controversial, and conclusions about the inability of these fungal enzymes to bind DAG in the C1B regulatory domain have been suggested only from predictions of PkcA primary sequence alignments and without experimental evidence. The lack of knowledge about the mechanisms of activation of this critical signaling pathway in fungi is the central premise of this project and the experiments associated with it. Thus, the goals of this proposal are: (i) to explore the relevance of the regulatory domains C1 and HR1 of PkcA in binding with Rho-GTPases; (ii) to clarify the dependence or not of PkcA on secondary messengers such as DAG for its activation; (iii) validate a possible new RlmA binding motif as output via CWI. Together, the results will generate unprecedented information about the initial events of CWI activation in A. fumigatus and, consequently, about the biology of filamentous fungi. Identifying associated mechanisms and proteins involved in this signaling pathway may provide opportunities for the development of new approaches in the search for active molecules. (AU)