O. Etxebeste

Asexual spores are the main vehicle used by fungi to disperse to new niches. The Eurotiomycete Aspergillus nidulans is the main reference for the study of the genetic/molecular control of asexual development. In this species, Flb proteins control the expression of the master gene brlA, and thus, loss-of-function mutations in flb (upstream developmental activation [UDA]) genes block brlA transcription and, consequently, the production of conidiophores, the structures bearing asexual spores known as conidia. However, the aconidial phenotype of specific flb mutants, such as that of the Δ flbB strain, is reverted under salt-stress conditions. Previously, we generated a collection of second-site mutants of Δ flbB unable to conidiate on culture medium supplemented with NaH 2 PO 4 (0.65 M). Here, we identified a Gly347Stop mutation within flpA as responsible for the FLIP57 phenotype and characterized the role of the putative cyclin FlpA and the remaining putative components of the C-terminal domain kinase-1 (CTDK-1) complex in A. nidulans and Aspergillus fumigatus . FlpA, Stk47, and FlpB are necessary (i) for timely germination, (ii) in the transition from metulae to phialides (the cells generating conidia) during conidiophore development, and (iii) for the development of sexual structures (cleistothecia) in A. nidulans . The three proteins are nuclear, and the nucleoplasmic localization of Stk47 depends on the activity of FlpA, which correlates with the retention of Stk47 by FlpA in pull-down assays. Overall, this work links the putative CTDK-1 complex of aspergilli with growth and developmental control. Identification of a mutation in flpA as inhibitor of conidiation in A. nidulans and functional characterization of FlpA, Stk47 and FlpB as putative members of the C-terminal domain kinase complex CTDK-1 in A. nidulans and A. fumigatus . IMPORTANCE Aspergillus fumigatus has been included by the World Health Organization in the priority list of fungal pathogens because (i) it causes 90% of invasive aspergillosis cases, with a high mortality rate, and (ii) infections are becoming increasingly resistant to azole antifungals. A. nidulans is an opportunistic pathogen and a saprotroph which has served during the last 80 years as a reference system for filamentous fungi. Here, we characterized the role in morphogenesis and development of the putative transcriptional cyclin/kinase complex CTDK-1 in both aspergilli. The null mutants of the corresponding genes showed delayed germination, aberrant conidiophore development, and inhibition of cleistothecia production. While in higher eukaryotes this complex is formed only by a cyclin and a kinase, the fungal complex would incorporate a fungal-specific third component, FlpB, which would enable the interaction between the kinase (Stk47) and the cyclin (FlpA) and may be used as a target for a

Invasive aspergillosis is one of the most serious clinical invasive fungal infections, resulting in a high case fatality rate among immunocompromised patients. The disease is caused by saprophytic molds in the genus Aspergillus , including Aspergillus fumigatus , the most significant pathogenic species. The fungal cell wall, an essential structure mainly composed of glucan, chitin, galactomannan, and galactosaminogalactan, represents an important target for the development of antifungal drugs. UDP (uridine diphosphate)-glucose pyrophosphorylase (UGP) is a central enzyme in the metabolism of carbohydrates that catalyzes the biosynthesis of UDP-glucose, a key precursor of fungal cell wall polysaccharides. Here, we demonstrate that the function of UGP is vital for Aspergillus nidulans ( An UGP). To understand the molecular basis of An UGP function, we describe a cryoEM structure (global resolution of 3.5 Å for the locally refined subunit and 4 Å for the octameric complex) of a native An UGP. The structure reveals an octameric architecture with each subunit comprising an N-terminal α-helical domain, a central catalytic glycosyltransferase A-like (GT-A-like) domain, and a C-terminal (CT) left-handed β-helix oligomerization domain. An UGP displays unprecedented conformational variability between the CT oligomerization domain and the central GT-A-like catalytic domain. In combination with activity measurements and bioinformatics analysis, we unveil the molecular mechanism of substrate recognition and specificity for An UGP. Altogether, our study not only contributes to understanding the molecular mechanism of catalysis/regulation of an important class of enzymes but also provides the genetic, biochemical, and structural groundwork for the future exploitation of UGP as a potential antifungal target. IMPORTANCE Fungi cause diverse diseases in humans, ranging from allergic syndromes to life-threatening invasive diseases, together affecting more than a billion people worldwide. Increasing drug resistance in Aspergillus species represents an emerging global health threat, making the design of antifungals with novel mechanisms of action a worldwide priority. The cryoEM structure of UDP (uridine diphosphate)-glucose pyrophosphorylase (UGP) from the filamentous fungus Aspergillus nidulans reveals an octameric architecture displaying unprecedented conformational variability between the C-terminal oligomerization domain and the central glycosyltransferase A-like catalytic domain in the individual protomers. While the active site and oligomerization interfaces are more highly conserved, these dynamic interfaces include motifs restricted to specific clades of filamentous fungi. Functional study of these motifs could lead to the definition of new targets for antifungals inhibiting UGP activity and, thus, the architecture of the cell wall of filamentous fungal pathogens.