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.