Analysing the role of the GEF Rom2 in cell wall integrity and significance of functional septa for echinocandin tolerance in the opportunistic pathogenic mold Aspergillus fumigatus

Aspergillus fumigatus is a major opportunistic, filamentous fungal pathogen causing invasive aspergillosis (IA), a fatal systemic infection in immunocompromised patients with significant mortality rate. The fungal cell is protected by a rigid but highly dynamic cellular structure, the cell wall that forms the first level of defence against environmental stress. The cell wall being an essential and unique structure of the fungus has always been an ideal drug target. The major antifungal drugs used currently either target the fungal cell membrane or cell wall. However, due to the poor efficacy of current antifungal therapy, the CWI (cell wall integrity) pathway has emerged as the focus of research in recent years to discover potential molecular drug targets for designing antifungal therapy with novel mode of action. This signaling cascade is dedicated to monitoring and maintaining functional integrity of the cell wall, remodelling its structure in response to cell surface stress. This MAPK (mitogen activated protein kinase) cascade is highly coordinated to transduce the stress signals to the nucleus and consequently trigger necessary gene expression to counteract the stress. In this study, we explored the pivotal role of guanine nucleotide exchange factor (GEF), Rom2 in cell wall stress response and antifungal drug susceptibility. The findings of this work reveal that the Rom2 GEF is essential for viability of the pathogen. Additionally, characterization of a conditional rom2 mutant functionally links it to the previously identified CWI sensors, namely, Wsc1, Wsc3 and MidA in A. fumigatus. The conditional mutant shows severe growth defects under repressive conditions such as hyper-susceptibility to heat, Calcofluor white and Congo red, similar to the ∆midA mutant. Additionally, similar to the ∆wsc1, the rom2 mutant cultured under repressive conditions is increasingly susceptible to the actively used antifungal and inhibitor of cell wall β-1,3-glucan synthesis, echinocandin such as caspofungin. Furthermore, the Rom2 shows a sub-cellular localization similar to the Rho1 GTPase to hyphal tips and also physically interacts with the GTPase. Thus, these relevant findings establish the integral role of Rom2 as an intermediate relay molecule acting between the cell surface sensors and Rho1 GTPase as well as the downstream MAPK module. This study also reports a novel mechanism imparting echinocandin tolerance to the pathogen. This work explores two possibilities that may explain the fungistatic nature of echinocandins against Aspergillus: one either owing to incomplete inhibiton of β-1,3-glucan synthesis or that the cell wall β-1,3-glucan is not essential for A. fumigatus viability. In order to evaluate the role of the β-1,3-glucan synthase subunit, Fks1 in viability, growth and antifungal response of the mold, a conditional fks1 mutant was generated. Downregulation of fks1 expression results in characteristic growth behaviour which phenocopies the effect of wild type treated with echinocandins. The mutant cultured under repressive growth conditions also displays significant decrease in cell surface β-1,3-glucan and enhanced galactomannan shedding, marked with a compensatory increase in chitin content. Importantly, the growth of the conditional fks1 mutant is not completely abolished in presence of echinocandin and an fks1 deletion mutant is surprisingly viable. These results strongly reflect that β-1,3-glucan is not essential in A. fumigatus, and thereby justifies the limited activity of β-1,3-glucan synthesis inhibitor echinocandin on the mold. The novel findings of the work also suggest that presence of septa is an essential means of survival for A. fumigatus upon echinocandin treatment. Compounds inhibiting septum formation exhibit significant synergism with the echinocandin caspofungin. Thus, the present study identifies and proposes that septum inhibition is a promising strategy for enhancing echinocandin fungicidal potency and improving existing antifungal therapy.