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- Toxicity of recombinant necrosis and ethylene-inducing proteins (NLPs) from Neofusicoccum parvumPublication . Pour, Forough Nazar; Cobos, Rebeca; Coque, Juan José Rubio; Serôdio, João; Alves, Artur; Félix, Carina; Ferreira, Vanessa; Esteves, Ana Cristina; Duarte, Ana SofiaNeofusicoccum parvum is a fungal pathogen associated with a wide range of plant hosts. Despite being widely studied, the molecular mechanism of infection of N. parvum is still far from being understood. Analysis of N. parvum genome lead to the identification of six putative genes encoding necrosis and ethylene-inducing proteins (NLPs). The sequence of NLPs genes (NprvNep 1-6) were analyzed and four of the six NLP genes were successfully cloned, expressed in E. coli and purified by affinity chromatography. Pure recombinant proteins were characterized according to their phytotoxic and cytotoxic effects to tomato leaves and to mammalian Vero cells, respectively. These assays revealed that all NprvNeps tested are cytotoxic to Vero cells and also induce cell death in tomato leaves. NprvNep2 was the most toxic to Vero cells, followed by NprvNep1 and 3. NprvNep4 induced weaker, but, nevertheless, still significant toxic effects to Vero cells. A similar trend of toxicity was observed in tomato leaves: the most toxic was NprvNep 2 and the least toxic NprvNep 4. This study describes for the first time an overview of the NLP gene family of N. parvum and provides additional insights into its pathogenicity mechanism.
- An analysis of protein patterns present in the saliva of diabetic patients using pairwise relationship and hierarchical clusteringPublication . Soares, Airton; Esteves, Eduardo; Rosa, Nuno; Esteves, Ana Cristina; Lins, Anthony; Bastos-Filho, Carmelo J. A.Molecular diagnosis is based on the quantification of RNA, proteins, or metabolites whose concentration can be correlated to clinical situations. Usually, these molecules are not suitable for early diagnosis or to follow clinical evolution. Large-scale diagnosis using these types of molecules depends on cheap and preferably noninvasive strategies for screening. Saliva has been studied as a noninvasive, easily obtainable diagnosis fluid, and the presence of serum proteins in it enhances its use as a systemic health status monitoring tool. With a recently described automated capillary electrophoresis-based strategy that allows us to obtain a salivary total protein profile, it is possible to quantify and analyze patterns that may indicate disease presence or absence. The data of 19 persons with diabetes and 58 healthy donors obtained by capillary electrophoresis were transformed, treated, and grouped so that the structured values could be used to study individuals’ health state. After Pairwise Relationships and Hierarchical Clustering analysis were observed that amplitudes of protein peaks present in the saliva of these individuals could be used as differentiating parameters between healthy and unhealthy people. It indicates that these characteristics can serve as input for a future computational intelligence algorithm that will aid in the stratification of individuals that manifest changes in salivary proteins.
- Secondary metabolites produced by macrophomina phaseolina isolated from eucalyptus globulusPublication . Salvatore, Maria Michela; Félix, Carina; Lima, Fernanda; Ferreira, Vanessa; Naviglio, Daniele; Salvatore, Francesco; Duarte, Ana Sofia; Alves, Artur; Andolfi, Anna; Esteves, Ana CristinaIn the course of investigations on the role of secondary metabolites in plant‐microbe interactions, the production of secondary metabolites by Macrophomina phaseolina isolates from Eucalyptus globulus, was studied. This fungus is responsible for several plant diseases which affect crop productivity and industry. Although secondary metabolites may play a role in disease development, there are very few reports on M. phaseolina metabolomics and, as far as we know, isolates from eucalypts have not been investigated for secondary metabolites production. In the present paper, metabolites typical of fungi, from the family Botryosphaeriaceae, were identified for the first time as products of M. phaseolina. Furthermore, the isolate under examination was grown in the presence and absence of host stem tissue, and metabolite profiles were compared. Five products are reported for the first time in this species and azelaic acid was exclusively produced in the presence of eucalypt stem. Finally, phytotoxicity and cytotoxicity tests of culture filtrates and crude organic extracts were also performed. Key Contribution: Lipophilic metabolites produced by M. phaseolina might play a role in the plant‐fungus interactions responsible for serious diseases of E. globulus.
- Effect of γ-aminobutyric acid (GABA) on the metabolome of two strains of Lasiodiplodia theobromae isolated from grapevinePublication . Salvatore, Maria Michela; Félix, Carna; Lima, Fernanda; Ferreira, Vanessa; Duarte, Ana Sofia; Salvatore, Francesco; Alves, Artur; Esteves, Ana Cristina; Andolfi, AnnaThe effect of γ-aminobutyric acid (GABA) on the metabolome of two strains of Lasiodiplodia theobromae isolated from grapevine that hold a different degree of virulence to the host plant (LA-SOL3 (more virulent), LA-SV1 (less virulent)) was investigated. The culture filtrates and crude extracts from the two strains grown in the presence and absence of 10 mM of GABA were tested for phytotoxicity on tomato plant cuttings and leaves, respectively. Considering the opportunistic nature of this fungus for humans, crude extracts were also tested for cytotoxicity on mammalian cell lines. We found that culture filtrates and crude extracts have a decreased toxicity in the presence of GABA. Metabolomic analysis, conducted on both strains at both growth conditions, revealed the production of several compounds, such as indole-3-carboxylic acid (ICA, which is the main compound produced by L. theobromae), 3-indolecarboxyaldehyde, (3R,4S)-botryodiplodin, (R)-mellein. Finally, data demonstrate that GABA both induces a decrease in the amount of ICA, and a diversification of the metabolites produced by L. theobromae.
- Neptunomyces aureus gen. et sp. nov. (Didymosphaeriaceae, Pleosporales) isolated from algae in Ria de Aveiro, PortugalPublication . Gonçalves, Micael F. M.; Vicente, Tânia F. L.; Esteves, Ana C.; Alves, ArturA collection of fungi was isolated from macroalgae of the genera Gracilaria, Enteromorpha and Ulva in the estuary Ria de Aveiro in Portugal. These isolates were characterized through a multilocus phylogeny based on ITS region of the ribosomal DNA, beta-tubulin (tub2) and translation elongation factor 1 alpha (tef1-a) sequences, in conjunction with morphological and physiological data. These analyses showed that the isolates represented an unknown fungus for which a new genus, Neptunomyces gen. nov. and a new species, Neptunomyces aureus sp. nov. are proposed. Phylogenetic analyses supported the affiliation of this new taxon to the family Didymosphaeriaceae. Copyright Micael F.M. Gonçalves et al.
- SalivaPrint as a non-invasive diagnostic toolPublication . Esteves, Eduardo; Cruz, Igor; Esteves, Ana Cristina; Barros, Marlene; Rosa, NunoCurrently, the molecular diagnosis is based on the quantification of RNA, proteins and metabolites because they present changes in their quantity related to clinical situations. The same molecules are not generally suitable for early diagnosis or to follow clinical evolution, making necessary strategies to evaluate the complete molecular scenario. There are already experimental strategies that allow the determination of total protein profiles from saliva samples (the SalivaPrint). The goal of this work is to identify a profile of saliva proteins (similar to a fingerprint) and, using computational methods, identify how this profiles changes with age and gender. So far it has been possible to collect 79 samples as well as the metadata associated with each sample using an electronic questionnaire developed by us. A total protein profile was obtained and their association with gender was verified using statistical methods. Currently we are developing the Python scripts for automatic data acquiring and normalization. Total protein profiles annotation on a database (SalivaPrintDB) and their integration with the factors that affects them using machine learning strategies can empower the use of the approach proposed on this work as a tool for monitoring the individual's health status.
- A multi-omics analysis of the grapevine pathogen Lasiodiplodia theobromae reveals that temperature affects the expression of virulence- and pathogenicity-related genesPublication . Félix, Carina; Meneses, Rodrigo; Gonçalves, Micael F. M.; Tilleman, Laurentijn; Duarte, Ana S.; Jorrín-Novo, Jesus V.; Peer, Yves Van de; Deforce, Dieter; Nieuwerburgh, Filip Van; Esteves, Ana C.; Alves, ArturLasiodiplodia theobromae (Botryosphaeriaceae, Ascomycota) is a plant pathogen and human opportunist whose pathogenicity is modulated by temperature. The molecular effects of temperature on L. theobromae are mostly unknown, so we used a multi-omics approach to understand how temperature affects the molecular mechanisms of pathogenicity. The genome of L. theobromae LA-SOL3 was sequenced (Illumina MiSeq) and annotated. Furthermore, the transcriptome (Illumina TruSeq) and proteome (Orbitrap LC-MS/MS) of LA-SOL3 grown at 25 °C and 37 °C were analysed. Proteins related to pathogenicity (plant cell wall degradation, toxin synthesis, mitogen-activated kinases pathway and proteins involved in the velvet complex) were more abundant when the fungus grew at 25 °C. At 37 °C, proteins related to pathogenicity were less abundant than at 25 °C, while proteins related to cell wall organisation were more abundant. On the other hand, virulence factors involved in human pathogenesis, such as the SSD1 virulence protein, were expressed only at 37 °C. Taken together, our results showed that this species presents a typical phytopathogenic molecular profile that is compatible with a hemibiotrophic lifestyle. We showed that L. theobromae is equipped with the pathogenesis toolbox that enables it to infect not only plants but also animals.
- Dual RNA sequencing of vitis vinifera during lasiodiplodia theobromae infection unveils host–pathogen interactionsPublication . Gonçalves, Micael F. M.; Nunes, Rui B.; Tilleman, Laurentijn; Peer, Yves Van De; Deforce, Dieter; Nieuwerburgh, Filip Van; Esteves, Ana C.; Alves, ArturLasiodiplodia theobromae is one of the most aggressive agents of the grapevine trunk disease Botryosphaeria dieback. Through a dual RNA-sequencing approach, this study aimed to give a broader perspective on the infection strategy deployed by L. theobromae, while understanding grapevine response. Approximately 0.05% and 90% of the reads were mapped to the genomes of L. theobromae and Vitis vinifera, respectively. Over 2500 genes were significantly differentially expressed in infected plants after 10 dpi, many of which are involved in the inducible defense mechanisms of grapevines. Gene expression analysis showed changes in the fungal metabolism of phenolic compounds, carbohydrate metabolism, transmembrane transport, and toxin synthesis. These functions are related to the pathogenicity mechanisms involved in plant cell wall degradation and fungal defense against antimicrobial substances produced by the host. Genes encoding for the degradation of plant phenylpropanoid precursors were up-regulated, suggesting that the fungus could evade the host defense response using the phenylpropanoid pathway. The up-regulation of many distinct components of the phenylpropanoid pathway in plants supports this hypothesis. Moreover, genes related to phytoalexin biosynthesis, hormone metabolism, cell wall modification enzymes, and pathogenesis-related proteins seem to be involved in the host responses observed. This study provides additional insights into the molecular mechanisms of L. theobromae and V. vinifera interactions.
- Three new species of Neocamarosporium isolated from saline environments: N. aestuarinum sp. nov., N. endophyticum sp. nov. and N. halimiones sp. nov.Publication . Gonçalves, M. F. M.; Aleixo, A.; Vicente, T. F. L.; Esteves, A. C.; Alves, A.Neocamarosporium species are typically halotolerant, being commonly found in saline environments like saline water, hypersaline soils and especially in association with halophytes. Several isolates were obtained from saline water, dead leaves of the seaweed Zostera noltii and live tissues of the halophyte Halimione portulacoides. Phylogenetic analysis based on ITS sequence data placed these isolates into three clades within the genus Neocamarosporium distinct from the currently known species. Isolates from each clade showed clear differences in conidial morphology. Three new species N. aestuarinum sp. nov., N. endophyticum sp. nov. and N. halimiones sp. nov. are described and illustrated. Our results show that the salt marsh plant H. portulacoides harbours a high diversity of Neocamarosporium species.
- Unveiling biological activities of marine fungi: the effect of sea saltPublication . Gonçalves, Micael F. M.; Paço, Ana; Escada, Luís F.; Albuquerque, Manuela S. F.; Pinto, Carlos A.; Saraiva, Jorge A.; Duarte, Ana Sofia; Rocha-Santos, Teresa A. P.; Esteves, Ana Cristina; Alves, ArturThere is an urgent need for new substances to overcome current challenges in the health sciences. Marine fungi are known producers of numerous compounds, but the manipulation of growth conditions for optimal compound production can be laborious and time-consuming. In Portugal, despite its very long coastline, there are only a few studies on marine fungi. From a collection of Portuguese marine fungi, we screened for antimicrobial, antioxidant, enzymatic, and cytotoxic activities. Mycelia aqueous extracts, obtained by high pressure-assisted extraction, and methanolic extracts of culture media showed high antioxidant, antimicrobial, and cytotoxic activities. The mycelium extracts of Cladosporium rubrum showed higher antioxidant potential compared to extracts from other fungi. Mycelia and culture media extracts of Aspergillus affinis and Penicillium lusitanum inhibited the growth of Staphylococcus aureus, Kocuria rhizophila, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, including multiresistant strains. Penicillium lusitanum and Trichoderma aestuarinum inhibited the growth of clinical strains of Candida albicans, C. glabrata, C. parapsilosis, and C. tropicalis. All extracts from culture media were cytotoxic to Vero cells. Sea salt induced alterations in the mycelium's chemical composition, leading to different activity profiles.