Alternaria species are cosmopolitan fungi darkly pigmented by melanin that infect numerous plant species causing economically important agricultural spoilage of various food crops. Alternaria spp. also infect animals, being described as entomopathogenic fungi but also infecting warm-blooded animals, including humans. Their clinical importance in human health, as infection agents, lay in the growing number of immunocompromised patients. Moreover, Alternaria spp. are considered some of the most abundant and potent sources of airborne sensitizer allergens causing allergic respiratory diseases, as severe asthma. Among the numerous strategies deployed by Alternaria spp. to attack their hosts, the production of toxins, carrying critical concerns to public health as food contaminant, and the production of hydrolytic enzymes such as proteases, can be highlighted. Alternaria proteases also trigger allergic symptoms in individuals with fungal sensitization, acting as allergens and facilitating antigen access to the host subepithelium. Here, we review the current knowledge about the mechanisms of Alternaria pathogenesis in plants and animals, the strategies used by Alternaria to cope with the host defenses, and the involvement Alternaria allergens and mechanisms of sensitization.

Endodontic biofilms eradication from the infected root canal system remains as the primary focus in endodontic field. In this study it was assessed the efficacy of antimicrobial Photodynamic Therapy (aPDT) with the Zn(II)chlorin e6 methyl ester (Zn(II)e6Me) activated by red light against monospecies and mixed biofilms of Enterococcus faecalis and Candida albicans. The results were compared with the ones obtained with Rose Bengal (RB), Toluidine Blue-O (TBO), the synthetic tetracationic porphyrin (TMPyP) as well as classical endodontic irrigants (3% NaOCl, 17% EDTA and 2% CHX). The antimicrobial efficacy of aPDT towards monospecies and mixed biofilms was quantified resorting to safranin red method. The changes of biofilm organization and of cellular ultrastructure were evaluated through several microscopy techniques (light, laser confocal and transmission electron microscopy). Zn(II)e6Me once activated with light for 60 or 90 s was able to remove around 60% of the biofilm’s biomass. It was more efficient than TBO and RB and showed similar efficiency to TMPyP and classical irrigants, CHX and EDTA. As desirable in a PS, Zn(II)e6Me in the dark showed smaller activity than TMPyP. Only NaOCl revealed higher efficiency, with 70-90% of the biofilm’s biomass removal. The organization of biofilms and the normal microbial cell ultrastructure were extensively damaged by the presence of Zn(II)e6Me. aPDT with Zn(II)e6Me showed to be an efficient antimicrobial strategy deserving further studies leading to a future clinical usage in endodontic disinfection.

Coffee is one of the most popular and consumed products in the world, generating tons of solid waste known as spent coffee grounds (SCG), containing several bioactive compounds. Here, the antifungal activity of ethanolic SCG extract from caffeinated and decaffeinated coffee capsules was evaluated against yeasts and filamentous fungi. These extracts had antifungal activity against Candida krusei, Candida parapsilosis, Trichophyton mentagrophytes, and Trichophyton rubrum, all skin fungal agents. Moreover, SCG had fungicidal activity against T. mentagrophytes and T. rubrum. To understand the underlying mechanisms of the antifungal activity, fungal cell membrane and cell wall components were quantified. SCG caused a significant reduction of the ergosterol, chitin, and β-(1,3)-glucan content of C. parapsilosis, revealing the synthesis of this membrane component and cell wall components as possible targets of these extracts. These extracts were cytotoxic for the tumoral cell lines tested but not for the non-tumoral PLP2 cell line. The analysis of the phenolic compounds of these extracts revealed the presence of caffeoylquinic acid, feruloylquinic acid, and caffeoylshikimic acid derivatives. Overall, this confirmed the antifungal activity of spent coffee grounds, presenting a potential increase in the sustainability of the life cycle of coffee grounds, as a source for the development of novel antifungal formulations, especially for skin or mucosal fungal infections.

The importance of Alternaria sp. fungi to human health ranges from being etiological agents of serious infections with poor prognosis in immunosuppressed individuals to their association with respiratory allergic diseases. The present work focuses on Alternaria infectoria, as a model organism of the genus, and was designed to unravel melanin production in the response to antifungals. After the characterization of the pigment produced by A. infectoria, we studied the dynamics of dihydroxynaphthalene (DHN)-melanin production during growth, the degree of melanization in response to antifungals, and how melanization affected the susceptibility to several classes of therapeutic drugs. We demonstrate that A. infectoria increased melanin deposition in the cell walls in response to nikkomycin Z, caspofungin and itraconazole but not in response to fluconazole or amphotericin B. These results indicated that A. infectoria activates DHN-melanin synthesis in response to certain fungal drugs, possibly as a protective mechanism against antifungal drugs. Inhibition of DHN-melanin synthesis by pyroquilon resulted in a lower minimum effective concentration (MEC) of caspofungin, with enhanced morphological changes (increased balloon size) characterized by thinner and less organized A. infectoria cell walls. In summary, A. infectoria synthesizes melanin in response to certain antifungal drugs and its susceptibility is influenced by melanization suggesting a therapeutic potential to drug combinations that affect melanin synthesis.

Acknowledgements TG and CF are funded by FEDER funds through the Operational Programme Competitiveness Factors – COMPETE and national funds by FCT – Foundation for Science and Technology under the strategic project UID/NEU/04539/2013. C.F. is a recipient of a postdoctoral fellowship from FCT-Fundação para a Ciência e Tecnologia (SFRH/BPD/63733/2009). NG is funded by The Wellcome Trust (080088, 086827, 075470, 099215 & 097377), the FungiBrain Marie Curie Network and the Medical Research Council (UK).Peer reviewedPostprin

The pathway for the synthesis of di-myo-inositol-phosphate (DIP) was recently elucidated on the basis of the detection of the relevant activities in cell extracts ofArchaeoglobus fulgidusand structural characterization of products by nuclear magnetic resonance (NMR) (N. Borges, L. G. Gonçalves, M. V. Rodrigues, F. Siopa, R. Ventura, C. Maycock, P. Lamosa, and H. Santos, J. Bacteriol.188:8128-8135, 2006). Here, a genomic approach was used to identify the genes involved in the synthesis of DIP. Cloning and expression inEscherichia coliof the putative genes for CTP:l-myo-inositol-1-phosphate cytidylyltransferase and DIPP (di-myo-inositol-1,3′-phosphate-1′-phosphate, a phosphorylated form of DIP) synthase from several (hyper)thermophiles (A. fulgidus,Pyrococcus furiosus,Thermococcus kodakaraensis,Aquifex aeolicus, andRubrobacter xylanophilus) confirmed the presence of those activities in the gene products. The DIPP synthase activity was part of a bifunctional enzyme that catalyzed the condensation of CTP andl-myo-inositol-1-phosphate into CDP-l-myo-inositol, as well as the synthesis of DIPP from CDP-l-myo-inositol andl-myo-inositol-1-phosphate. The cytidylyltransferase was absolutely specific for CTP andl-myo-inositol-1-P; the DIPP synthase domain used onlyl-myo-inositol-1-phosphate as an alcohol acceptor, but CDP-glycerol, as well as CDP-l-myo-inositol and CDP-d-myo-inositol, were recognized as alcohol donors. Genome analysis showed homologous genes in all organisms known to accumulate DIP and for which genome sequences were available. In most cases, the two activities (l-myo-inositol-1-P cytidylyltransferase and DIPP synthase) were fused in a single gene product, but separate genes were predicted inAeropyrum pernix,Thermotoga maritima, andHyperthermus butylicus. Additionally, usingl-myo-inositol-1-phosphate labeled on C-1 with carbon 13, the stereochemical configuration of all the metabolites involved in DIP synthesis was established by NMR analysis. The two inositol moieties in DIP had different stereochemical configurations, in contradiction of previous reports. The use of the designation di-myo-inositol-1,3′-phosphate is recommended to facilitate tracing individual carbon atoms through metabolic pathways.