Identification and functional characterization of the putative members of the CTDK-1 kinase complex as regulators of growth and development in Aspergillus nidulans and Aspergillus fumigatus.

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 ΔflbB strain, is reverted under salt-stress conditions. Previously, we generated a collection of second-site mutants of ΔflbB unable to conidiate on culture medium supplemented with NaH2PO4 (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.IMPORTANCEAspergillus 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 antifungals.

Study of antifungal agent caspofungin adsorption to laboratory materials.

Treatment of invasive fungal infections with Caspofungin is used as the first-line antifungal agents. The minimum inhibitory concentration value is a test which indicates the degree of sensitivity of a strain regarding a drug. However, no value of minimum inhibitory concentration for caspofungin is available because very variable value is obtained. In this work, we study the link with the adsorption phenomenon of CSF previously described in literature and the lack of minimum inhibitory concentration value. A systematic study of the impact of different parameters on CSF adsorption is reported. The effect of the nature of container material, the aqueous solution pH and the organic solvent proportion was studied. In addition, the possibility of using a coating agent to minimize the adsorption was assayed and evaluated. Results obtained showed the importance of the material used during the manipulation of CSF. The use of acidic pH aqueous solution or the addition of acetonitrile or methanol proportions (50 % and 70 %, respectively) were found efficient to avoid adsorption of CSF on glassware material, which is the relevant strategy for analytical samples of caspofungin. The treatment of HPLC glass vials and 96-well plates with N-(2-aminoethyl)-3-aminopropyltrimethoxysilane reduced the adsorption. The significant adsorption observed in this work especially with plastic materials, questions the results obtained before in different assays and explained the absence of MIC value.

Melanin and pyomelanin in Aspergillus fumigatus: from its genetics to host interaction.

Aspergillus fumigatus is a worldwide-distributed saprophytic fungus and the major cause of invasive aspergillosis. This fungus can produce two types of melanin-dihydroxynaphthalene melanin (DHN-melanin) and pyomelanin. These pigments are considered important resistance mechanisms to stress, as well as virulence factors. The aim of this review is to present the current knowledge of the genetic basis and metabolic pathways of melanin production, their activation, function, and interaction with the host immune system. The DHN-melanin pathway is encoded in a cluster that includes six genes (abr1, abr2, ayg1, arp1, arp2, and pksP/alb1 genes) whose encoded proteins seem to be the origin of the pigment in endosomes. These vesicles are secreted and the pigment is subsequently located in the wall of the conidium beneath the rodlet layer. Unlike DHN-melanin, pyomelanin does not have its own biosynthetic pathway but is related to the activation of the L-tyrosine/L-phenylalanine degradation pathway that includes a cluster of six genes (hppD, hmgX, hmgA, fahA, maiA, and hmgR). Its production is due to the polymerization of homogentisic acid and is linked to conidial germination. Despite the knowledge gained in recent years, further studies will be necessary to confirm the pathways that produce these pigments and their role in the virulence mechanisms of A. fumigatus.