The significance of regulatory domains C1A and C1B in PkcA activation in Aspergillus fumigatus and their Impact on cell wall stress response

• Grant number: 24/10777-7
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: October 01, 2024
• End date: September 30, 2025
• Field of knowledge: Biological Sciences - Microbiology - Biology and Physiology of Microorganisms
• Principal Investigator: Iran Malavazi
• Grantee: Giulia Ayane Sato
• Host Institution: Centro de Ciências Biológicas e da Saúde (CCBS). Universidade Federal de São Carlos (UFSCAR). São Carlos , SP, Brazil

Abstract

The filamentous fungus Aspergillus fumigatus stands out for its ability to cause diseases in immunocompromised individuals, being the species responsible for the highest incidence rate of infections in humans. In the at-risk population, various diseases can occur after contact with the fungal conidia, including invasive pulmonary aspergillosis (IA), the most serious form of infection. Adaptive mechanisms are necessary for this fungus to survive within the host, among which the maintenance of cell wall integrity (CWI) is highlighted. The CWI pathway begins with the detection of external signals through membrane receptors (mechanosensors). Subsequently, Rho-GTPases assist in activating protein kinase C, named PkcA, in A. fumigatus. PkcA is a key protein in the pathway, as it activates the downstream MAPK signaling cascade, culminating in the activation of the transcription factor RlmA. Despite the importance of PkcA in responding to cell wall damage, there are still gaps regarding how this apical kinase is activated at the beginning of the signal transduction mediated by the CWI pathway. In mammalian PKCs, two conserved in tandem cysteine-rich domains named C1A and C1B are responsible for binding to phosphatidylserine and the second messenger, diacylglycerol (DAG). This event allows the translocation of the protein to the plasma membrane and its consequent activation. However, current literature, especially in budding yeast and sequence alignments, suggests that fungal PKCs cannot bind to DAG. Therefore, the activation mechanism of PkcA would be independent of secondary messengers. To elucidate how PkcA from A. fumigatus is activated and the importance of the C1 domains in response to cell wall stress, this study aims to perform the guided deletion of the C1A and C1B domains, using the CRISPR-Cas9 methodology, as well as conducting phenotypic and virulence assays in an invertebrate model, among others. These studies focused on the C1 domains could provide novel insights about the activation of this central protein of the CWI pathway and the importance of these domains. In addition, they are fundamental to the characterization of PkcA and the functioning of the pathway. Additionally, they may pave the way for future biochemical studies to identify whether these domains have any binding function to DAG and/or phosphatidylserine. Moreover, the results could be used in studies on discovering new pharmacological targets and optimizing the treatment of a wide spectrum of fungal diseases, whose epidemiology has become increasingly alarming in recent decades.