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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.
- 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.
- Genotypic and phenotypic traits of blaCTX-M-carrying Escherichia coli strains from an UV-C-treated wastewater effluentPublication . Tavares, Rafael D. S.; Tacão, Marta; Figueiredo, Ana S.; Duarte, Ana S.; Esposito, Fernanda; Lincopan, Nilton; Manaia, Célia M.; Henriques, IsabelWastewater treatment plants (WWTPs) are relevant sources of antibiotic resistance into aquatic environments. Disinfection of WWTPs’ effluents (e.g. by UV-C irradiation) may attenuate this problem, though some clinically relevant bacteria have been shown to survive disinfection. In this study we characterized 25 CTX-M-producing Escherichia coli strains isolated from a WWTP’s UV-C-irradiated effluent, aiming to identify putative human health hazards associated with such effluents. Molecular typing indicated that the strains belong to the phylogroups A, B2 and C and clustered into 9 multilocus sequence types (STs), namely B2:ST131 (n ¼ 7), A:ST58 (n ¼ 1), A:ST155 (n ¼ 4), C:ST410 (n ¼ 2), A:ST453 (n ¼ 2), A:ST617 (n ¼ 2), A:ST744 (n ¼ 1), A:ST1284 (n ¼ 3) and a putative novel ST (n ¼ 3). PCR-screening identified 9 of the 20 antibiotic resistance genes investigated [i.e. sul1, sul2, sul3, tet(A), tet(B), blaOXA-1-like, aacA4, aacA4-cr and qnrS1]. The more prevalent were sul1, sul2 (n ¼ 15 isolates) and tet(A) (n ¼ 14 isolates). Plasmid restriction analysis indicated diverse plasmid content among strains (14 distinct profiles) and mating assays yielded cefotaxime-resistant transconjugants for 8 strains. Two of the transconjugants displayed a multi-drug resistance (MDR) phenotype. All strains were classified as cytotoxic to Vero cells (9 significantly more cytotoxic than the positive control) and 10 of 21 strains were invasive towards this cell line (including all B2:ST131 strains). The 10 strains tested against G. mellonella larvae exhibited a virulent behaviour. Twenty-four and 7 of the 25 strains produced siderophores and haemolysins, respectively. Approximately 66% of the strains formed biofilms. Genome analysis of 6 selected strains identified several virulence genes encoding toxins, siderophores, and colonizing, adhesion and invasion factors. Freshwater microcosms assays showed that after 28 days of incubation 3 out of 6 strains were still detected by cultivation and 4 strains by qPCR. Resistance phenotypes of these strains remained unaltered. Overall, we confirmed WWTP’s UV-C-treated outflow as a source of MDR and/or virulent E. coli strains, some probably capable of persisting in freshwater, and that carry conjugative antibiotic resistance plasmids. Hence, disinfected wastewater may still represent a risk for human health. More detailed evaluation of strains isolated from wastewater effluents is urgent, to design treatments that can mitigate the release of such bacteria.
- 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.
- 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.