Browsing by Author "Alves, Artur"
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- Detection of premature stop codons leading to truncated internalin A among food and clinical strains of Listeria monocytogenesPublication . Silva, Margarida Ferreira da; Ferreira, Vânia; Magalhães, Rui; Almeida, Gonçalo; Alves, Artur; Teixeira, PaulaListeria monocytogenes is a food-borne pathogen responsible for outbreaks and sporadic cases of listeriosis, a severe invasive disease. Internalin A (InlA) a protein encoded by inlA has a key role in the mechanism of pathogenesis in L. monocytogenes infection, specifically in the invasion of human intestinal epithelial cells. Studies on inlA have shown that mutations leading to premature stop codons (PMSCs) occur naturally and are associated with impaired virulence of L. monocytogenes strains. Increasing evidence suggests that inlA PMSCs mutations are frequent in strains from foods, but rare among clinical isolates. In this study, 22 L. monocytogenes strains collected in Portugal from the processing environment of a bakery industry (n = 1), different food products (n = 10) and human clinical cases (n = 11) were analysed for mutations in inlA and invasion efficiency in Caco-2 cells. Sequencing revealed previously reported mutations types leading to PMSCs in three food and one clinical strain presenting different molecular serotypes (i.e., IIa, IIb and IIc). The remaining 18 isolates did not show PMSCs in inlA. The four strains with PMSCs in inlA presented lower invasiveness efficiencies in Caco-2 cells (below 8.9%) when compared to the control strain (full-length InlA). In addition, one clinical isolate showed reduced invasion efficiency but no PMSCs in inlA. This isolate showed increased inlA transcript levels to that obtained for the laboratory control strain. Our data support the hypothesis that L. monocytogenes isolated from food have attenuated invasion due to the presence of inlA PMSCs. This information would be critically needed for adequate risk-assessments of the foodborne illness burden associated with L. monocytogenes strains.
- 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.
- 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.
- How temperature modulates the expression of pathogenesis-related molecules of the cross-kingdom pathogen Lasiodiplodia hormozganensisPublication . Félix, Carina; Meneses, Rodrigo; Gonçalves, Micael F. M.; Duarte, Ana S.; Jorrín-Novo, Jesus V.; van de Peer, Yves; Deforce, Dieter; Nieuwerburgh, Filip Van; Alves, Artur; Esteves, Ana C.Lasiodiplodia hormozganensis, initially recognized as a fungal plant pathogen, is recognized now acknowledged as a potential threat to humans. However, our understanding of the pathogenesis mechanisms of Lasiodiplodia species remains limited, and the impact of temperature on its pathogenicity is unclear. This study aims to elucidate the effects of temperature on the biology of L. hormozganensis, focusing on the expression of pathogenesis-related molecules and its ability to function as a cross-kingdom pathogen. We conducted experiments at two different temperatures, 25 and 37 °C, analyzing the proteome and transcriptome of L. hormozganensis. Using strain CBS339.90, initially identified as L. theobromae but confirmed through ITS and tef1-α sequence analysis to be L. hormozganensis, we aimed to understand the fungus's protein expression under varying temperature conditions. Results from the functional analysis of the secretome at 25 °C showed a noteworthy presence of proteins related to carbohydrate metabolism, catabolism, plant cell wall degradation, and pathogenesis. However, when grown at 37 °C, the fungus exhibited an increased production of stress response and pathogenesis-related proteins. Our findings identified various pathways crucial for pathogenesis in both plants and humans, suggesting that L. hormozganensis possesses the genetic foundation to infect both hosts. Specific pathogenesis-related proteins, including the phytotoxin snodprot1, aspartic protease aspergillopepsin, and virulence protein SSD1, were also identified. Concluding, we propose a possible mechanism of how L. hormozganensis adapts to different temperatures. The shift in temperature results in the expression of genes that favor human related pathogenesis molecules.
- Microbial consortia selection for the development of an innovative nature-based solution for air pollutants remediationPublication . Moreira, Irina S.; Novo, Beatriz; Monteiro, Pedro; Pinto, Glória; Castro, Paula M. L.; Alves, Artur; Tacão, Marta; Alves, MartaAir pollutants, like benzene, toluene, and xylene (BTX), pose significant health and environmental risks being associated with 6.7 million premature deaths annually. Despite plants natural air purification capabilities in green infrastructures, high pollution levels in cities can hinder urban greening when tolerance levels are exceeded. Therefore, this study aimed to develop plant-beneficial microbial consortia with the capacity to degrade air pollutants envisioning their application as plant inoculants to promote plants’ resilience and their efficiency in air purification.Leaves and rhizosphere samples from Tilia sp., Rhododendron sp. and Euonymus sp. were collected in Porto city (Portugal), in an area with high traffic-derived air pollution (NOx, benzene – as in Qualar database). Total pigments concentration showed low variability among plants genera (from 3.24 ± 0.45 to 3.57 ± 0.30 µmol g-1). The neutral to alkaline pH of leaf extracts and high relative water content results indicated good tolerance of the sampled plant genera to pollutants. To select BTX-degrading microbial consortia, leaves and rhizosphere samples were incubated with 300ppm of BTX as the only carbon source, far exceeding EU limits. BTX concentration was monitored through GC-FID. Microbial strains within BTX degrading consortia were identified through sequencing of microbial phylogenetic markers.While leaf samples showed no BTX degradation, rhizosphere samples from all three plant genera displayed significant degradation after 7 to 14 days. Remarkably, in a Rhododendron sp. sample, only 3.8% of the initial BTX concentration remained after 14 days, indicating efficient pollutant removal. Among the fungi genera detected were Penincillium sp., Umbelopsis sp, Fusarium sp. and Clonostachys sp. with species known for their role in plant growth promotion. The obtained microbial consortia have the potential to be used as plant inoculants to promote air remediation.
- Molecular techniques and target selection for the identification of Candida spp. in oral samplesPublication . Magalhães, Joana; Correia, Maria José; Silva, Raquel M.; Esteves, Ana Cristina; Alves, Artur; Duarte, Ana SofiaCandida species are the causative agent of oral candidiasis, with medical devices being platforms for yeast anchoring and tissue colonization. Identifying the infectious agent involved in candidiasis avoids an empirical prescription of antifungal drugs. The application of high-throughput technologies to the diagnosis of yeast pathogens has clear advantages in sensitivity, accuracy, and speed. Yet, conventional techniques for the identification of Candida isolates are still routine in clinical and research settings. Molecular approaches are the focus of intensive research, but conversion into clinic settings requires overcoming important challenges. Several molecular approaches can accurately identify Candida spp.: Polymerase Chain Reaction, Microarray, High-Resolution Melting Analysis, Multi-Locus Sequence Typing, Restriction Fragment Length Polymorphism, Loop-mediated Isothermal Amplification, Matrix Assisted Laser Desorption Ionization-mass spectrometry, and Next Generation Sequencing. This review examines the advantages and disadvantages of the current molecular methods used for Candida spp. Identification, with a special focus on oral candidiasis. Discussion regarding their application for the diagnosis of oral infections aims to identify the most rapid, affordable, accurate, and easy-to-perform molecular techniques to be used as a point-of-care testing method. Special emphasis is given to the difficulties that health care professionals need to overcome to provide an accurate diagnosis.
- 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.
- 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.
- P8 - Marine fungi exhibit antimicrobial activity against human oral pathogensPublication . Correia, Bruna L.; Devesas, Daniela; Noites, Rita; Gomes, Ana T. P. C.; Esteves, Ana Cristina; Alves, Artur; Duarte, Ana SofiaThe emergence of resistance to antibiotics and antimycotics has become a challenge in the treatment of infectious diseases, including infections of the oral cavity. Marine fungi are a source of novel biologically active compounds, namely in what concerns the development of antimicrobial and anticancer solutions. Our study aimed to test the antimicrobial activity and the cytotoxicity of the extracts of the two recent identified species of marine fungi, Penicillum lusitanum and Aspergillus affinis. Candida spp. and Enterococcus faecalis isolated from oral pathologies were included to evaluate the antimicrobial potential of the marine fungi by the disk diffusion assay. The cytotoxicity of the effective concentrations of the extract was tested using the Vero cell line (ECACC 88020401, African Green Monkey Kidney cells, GMK clone), according to the ISO 10993-5. The extracts of P. lusitanum and A. affinis were active against C. albicans and E. faecalis, respectively. Penicillum lusitanum active extracts are non-cytotoxic, in contrast to A. affinis extracts that showed high cytotoxic effects on Vero cells, for all concentrations tested. The results on the biological characterization of the P. lusitanumextract are promising and support the development of new disinfecting solutions that may be used during root canal therapy cleaning and shaping.
- 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.