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Research Project
Applied Molecular Biosciences Unit
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The Effects of Tetrabromobisphenol A (TBBPA) on the Mussel Mytilus galloprovincialis: A Multi-Biomarker Approach
Publication . Copeto, Sandra; Ganço, Sara; Ferreira, Inês João; Silva, Marco; Motta, Carla; Diniz, Mário
Tetrabromobisphenol A (TBBPA) is a fire-retardant containing bromine, produced in large quantities worldwide and extensively used in several industrial products. This compound was identified as a potential contaminant of the environment, causing toxicity to organisms. However, its toxicity remains poorly understood in marine bivalves. The first objective of this work was to evaluate the impact of TBBPA on mussels (Mytilus galloprovincialis) exposed for 28 days to various concentrations of TBBPA (0, 1, 10, and 100 µg·L−1), by assessing stress biomarkers’ responses (Glutathione S-transferase, superoxide dismutase, catalase, lipid peroxidation, total antioxidant capacity, total ubiquitin, caspase-3 and acetylcholinesterase). The results showed that lower concentrations (1 and 10 µg·L−1) were efficiently detoxified, as suggested by GST activities, which were supported by the responses of the other biomarkers. The most pronounced effects were observed in animals exposed to the highest concentration of TBBPA (100 µg·L−1), suggesting oxidative stress. Additionally, significant strong correlations were found between total antioxidant capacity and some biomarkers (superoxide dismutase and lipid peroxidation), showing that processes involved in oxidative stress fighting are working to avoid cell injury. In brief, mussels’ defense mechanisms were capable of dealing with exposure to the lower concentrations tested. Despite this, the risk of consuming shellfish or other fishery products contaminated with TBBPA should be a cause for concern.
The Type III Secretion Effector CteG Mediates Host Cell Lytic Exit of Chlamydia trachomatis
Publication . Pereira, Inês Serrano; Pais, Sara Vilela; Borges, Vítor; Borrego, Maria José; Gomes, João Paulo; Mota, Luís Jaime
Chlamydia trachomatis is an obligate intracellular bacterium causing ocular and urogenital infections in humans that are a significant burden worldwide. The completion of its characteristic infectious cycle relies on the manipulation of several host cell processes by numerous chlamydial type III secretion effector proteins. We previously identified the C. trachomatis CteG effector and showed it localizes at the host cell plasma membrane at late stages of infection. Here, we showed that, from 48 h post-infection, mammalian cells infected by wild-type C. trachomatis contained more infectious chlamydiae in the culture supernatant than cells infected by a CteG-deficient strain. This phenotype was CteG-dependent as it could be complemented in cells infected by the CteG-deficient strain carrying a plasmid encoding CteG. Furthermore, we detected a CteG-dependent defect on host cell cytotoxicity, indicating that CteG mediates chlamydial lytic exit. Previous studies showed that Pgp4, a global regulator of transcription encoded in the C. trachomatis virulence plasmid, also mediates chlamydial lytic exit. However, by using C. trachomatis strains encoding or lacking Pgp4, we showed that production and localization of CteG are not regulated by Pgp4. A C. trachomatis strain lacking both CteG and Pgp4 was as defective in promoting host cell cytotoxicity as mutant strains lacking only CteG or Pgp4. Furthermore, CteG overproduction in a plasmid suppressed the host cell cytotoxic defect of CteG- and Pgp4-deficient chlamydiae. Overall, we revealed the first chlamydial type III secretion effector involved in host cell lytic exit. Our data indicates that CteG and Pgp4 participate in a single cascade of events, but involving multiple layers of regulation, leading to lysis of host cells and release of the infectious chlamydiae.
A Search for Novel Legionella pneumophila Effector Proteins Reveals a Strain Specific Nucleotropic Effector
Publication . Monteiro, Inês P.; Sousa, Sofia; Borges, Vítor; Gonçalves, Paulo; Gomes, João Paulo; Mota, Luís Jaime; Franco, Irina S.
Legionella pneumophila is an accidental human pathogen that causes the potentially fatal Legionnaires' disease, a severe type of pneumonia. The main virulence mechanism of L. pneumophila is a Type 4B Secretion System (T4SS) named Icm/Dot that transports effector proteins into the host cell cytosol. The concerted action of effectors on several host cell processes leads to the formation of an intracellular Legionella-containing vacuole that is replication competent and avoids phagolysosomal degradation. To date over 300 Icm/Dot substrates have been identified. In this study, we searched the genome of a L. pneumophila strain (Pt/VFX2014) responsible for the second largest L. pneumophila outbreak worldwide (in Vila Franca de Xira, Portugal, in 2014) for genes encoding potential novel Icm/Dot substrates. This strain Pt/VFX2014 belongs to serogroup 1 but phylogenetically segregates from all other serogroup 1 strains previously sequenced, displaying a unique mosaic genetic backbone. The ability of the selected putative effectors to be delivered into host cells by the T4SS was confirmed using the TEM-1 β-lactamase reporter assay. Two previously unknown Icm/Dot effectors were identified, VFX05045 and VFX10045, whose homologs Lpp1450 and Lpp3070 in clinical strain L. pneumophila Paris were also confirmed as T4SS substrates. After delivery into the host cell cytosol, homologs VFX05045/Lpp1450 remained diffused in the cell, similarly to Lpp3070. In contrast, VFX10045 localized to the host cell nucleus. To understand how VFX10045 and Lpp3070 (94% of identity at amino acid level) are directed to distinct sites, we carried out a comprehensive site-directed mutagenesis followed by analyses of the subcellular localization of the mutant proteins. This led to the delineation of region in the C-terminal part (residues 380 to 534) of the 583 amino acid-long VFX10045 as necessary and sufficient for nuclear targeting and highlighted the fundamental function of the VFX10045-specific R440 and I441 residues in this process. These studies revealed a strain-specific nucleotropism for new effector VFX10045/Lpp3070, which anticipates distinct functions between these homologs.
The Impact of Perfluorooctanoic Acid (PFOA) on the Mussel Mytilus galloprovincialis: A Multi-Biomarker Evaluation
Publication . Copeto, Sandra; Ganço, Sara; Ferreira, Inês João; Sanchez, Didier; Nunes, Maria João; Motta, Carla; Silva, Marco; Diniz, Mário
Perfluorooctanoic acid (PFOA) has been widely studied due to its environmental persistence and bioaccumulation potential, raising concerns about its effects on aquatic life. This research evaluates the impact of PFOA on the antioxidant defenses and stress response systems of the mussel Mytilus galloprovincialis. Mussels were exposed to three concentrations of PFOA (1, 10, and 100 µg·L−1) over 28 days. Several biomarkers, including glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), lipid peroxidation (LPO), total antioxidant capacity (TAC), vitellogenin (VTG), ubiquitin (UBI), and caspase-3 (CASP) were analyzed. The results suggest stress responses, particularly in animals exposed to higher concentrations, as shown by GST and SOD activities which increased according to PFOA concentrations. Additionally, oxidative stress markers such as MDA and CAT showed variable responses depending on the exposure concentration tested. This study underscores the need for further investigation into the effects of PFOA on mollusks but also the need to unveil gender-specific responses in aquatic organisms exposed to this contaminant. The concentrations of PFOA used in our research are lower than those examined in previous studies, providing crucial insights into the impacts of even minimal exposure levels. It highlights the potential of M. galloprovincialis as a bioindicator in environmental monitoring programs, providing crucial insights for environmental management and policymaking regarding regulating and monitoring PFOA in marine settings. Consequently, in a country where seafood consumption is the second largest in Europe, implementing environmental policies and regulatory measures to manage and monitor PFOA levels in marine environments is crucial.
Assessment of the Genotoxic Hazard of Estuarine Sediments Using an Integrative Approach With LacZ Plasmid‐Based Transgenic Mice
Publication . Pinto, Miguel; Sacadura, Joana; Costa, Pedro M.; Caeiro, Sandra; Louro, Henriqueta; Silva, Maria J.
Under the influence of multiple anthropogenic pressures, from industrial to agricultural activities, estuaries have long been regarded as particularly sensitive ecosystems to contamination. The present study aimed at investigating the genotoxic potential of a contaminated sediment sample from an urban and industrial area of the Sado Estuary, by combining the analysis of multiple endpoints in the LacZ plasmid‐based transgenic mouse model exposed for 28 days to contaminated estuarine sediment extracts through drinking water. The DNA and chromosome damaging effects were monitored in peripheral blood at 7‐day intervals using the standard and enzyme‐modified Comet assay, as well as the micronucleus assays in peripheral blood cells. After euthanasia, DNA damage was analyzed in several mouse tissues, and LacZ mutant frequencies were determined in the liver. Livers were also surveyed for histopathological analysis. A time‐dependent increase in micronuclei frequency was seen at all tested doses, in spite of no induction of DNA damage in any organ or mutation induction in the liver of exposed mice. The liver from mice exposed to sediment extracts did not reveal major alterations besides evidence of inflammation. Overall, the integration of the endpoints analyzed in the mice is suggestive of potential chronic, rather than acute, adverse effects in vivo, and points to the need for further research in the resident human population in the area. This experimental design can be used to assess the genotoxicity of complex environmental mixtures, understand how they work, and reduce costs and resources while speeding up data collection and interpretation.
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Funding agency
Fundação para a Ciência e a Tecnologia
Funding programme
6817 - DCRRNI ID
Funding Award Number
UIDP/04378/2020