Elsa Alverca

IDENTIFICATION OF BACTERIA ASSOCIATED WITH DINOFLAGELLATES (DINOPHYCEAE) ALEXANDRIUM SPP. USING TYRAMIDE SIGNAL AMPLIFICATION–FLUORESCENT IN SITU HYBRIDIZATION AND CONFOCAL MICROSCOPY1

In the marine environment, phytoplankton and bacterioplankton can be physically associated. Such association has recently been hypothesized to be involved in the toxicity of the dinoflagellate genus Alexandrium. However, the methods, which have been used so far to identify, localize, and quantify bacteria associated with phytoplankton, are either destructive, time consuming, or lack precision. In the present study we combined tyramide signal amplification–fluorescent in situ hybridization (TSA‐FISH) with confocal microscopy to determine the physical association of dinoflagellate cells with bacteria. Dinoflagellate attached microflora was successfully identified with TSA‐FISH, whereas FISH using monolabeled probes failed to detect bacteria, because of the dinoflagellate autofluorescence. Bacteria attached to entire dinoflagellates were further localized and distinguished from those attached to empty theca, by using calcofluor and DAPI, two fluorochromes that stain dinoflagellate theca and DNA, respectively. The contribution of specific bacterial taxa of attached microflora was assessed by double hybridization. Endocytoplasmic and endonuclear bacteria were successfully identified in the nonthecate dinoflagellate Gyrodinium instriatum. In contrast, intracellular bacteria were not observed in either toxic or nontoxic strains of Alexandrium spp. Finally, the method was successfully tested on natural phytoplankton assemblages, suggesting that this combination of techniques could prove a useful tool for the simultaneous identification, localization, and quantification of bacteria physically associated with dinoflagellates and more generally with phytoplankton.

Morphological and ultrastructural effects of microcystin-LR from Microcystis aeruginosa extract on a kidney cell line.

The aim of this study was to examine the toxic effects of a microcystin-LR (MCLR)-containing cyanobacteria extract on the subcellular organization of a kidney cell line (Vero-E6). Cells were exposed to different MCLR concentrations (1.3-150 microM) for 24, 48 and 72h and two cytotoxicity assays were performed. This information was combined with the analysis of lysosomal, mitochondrial and cytoskeleton integrity and with an ultrastructural study. Biochemical and microscopic data revealed a good agreement and demonstrated that cellular response to MCLR is dependent on the dose/exposure time. Cell viability decayed markedly after 24h of exposure to toxin concentrations greater than 30 microM. Furthermore, it was demonstrated that lysosome destabilization precedes mitochondria dysfunction. The ultrastructural analysis showed that mild toxin incubation conditions induce endoplasmic reticulum (ER) vacuolization and assembly of large autophagic vacuoles, suggesting that autophagy is an early cellular response to the toxin. After exposure to higher MCLR doses, the number of apoptotic cells increased, as identified by microscopic observations and confirmed with TUNEL assay. Additionally, drastic exposure conditions induced the increase of necrotic cells. These results suggest that the ER is the primary microcystin target in Vero cells and that autophagy, apoptosis and necrosis are induced in a dose- and time-dependent manner.

In situ identification and localization of bacteria associated with Gyrodinium instriatum (Gymnodiniales, Dinophyceae) by electron and confocal microscopy

The presence of intracellular bacteria in the dinoflagellate Gyrodinium instriatum Freudenthal & Lee has previously been described but the bacterial flora associated with this species has not been characterized. In this study, new results of transmission electron microscopy (TEM) and in situ hybridization using several bacterial group-specific oligonucleotide probes are presented. The long-term association of endocytoplasmic and endonuclear bacteria with G. instriatum has been confirmed. All endonuclear and most of the endocytoplasmic bacteria labelled were identified as belonging to the betaproteobacteria. Large clusters of Cytophaga-Flavobacterium-Bacteroides (CFB) were labelled and observed in the cytoplasm of the dinoflagellate cells, but were absent from the nucleus. Gammaproteobacteria were only observed outside the dinoflagellates. No alphaproteobacteria were detected either free-living or intracellular. Empirical observation of intracellular CFB reflected a degradation process of moribund dinoflagellate cells, whereas the systematic colonization of dinoflagellate nucleoplasm by betaproteobacteria suggested a true symbiotic relationship. Natural colonization may have occurred, perpetuated by vertical transmission of intracellular bacteria to the dinoflagellate daughter cells, via a pool of bacteria sequestered within the nucleus. Dividing bacteria were observed in the nucleus and equilibrium may be maintained by release of endonuclear bacteria to the cytoplasm through nuclear envelope constrictions.

Bacterium-dinoflagellate interactions: investigative microscopy of Alexandrium spp. (Gonyaulacales, Dinophyceae)

Abstract The association of bacteria with dinoflagellates has been a neglected field of study, which has gained prominence in recent years because of the possible role of bacteria in toxin synthesis. A number of dinoflagellates undergo sexual reproduction, passing through various life-cycle stages in addition to the vegetative form. The presence of bacteria within dinoflagellates has been well established, but their presence throughout the dinoflagellate life-cycle has not been investigated. Using cultures of Alexandrium (A. tamarense, A. fundyense), we investigated the association of bacteria with various vegetative growth phases (lag, log, stationary) and sexual life-cycle stages (planozygote, planomeiocyte, hypnozygote), using scanning electron microscopy, transmission electron microscopy (TEM) and epifluorescence microscopy. Bacteria were found to be associated with the surfaces of vegetative cells, planozygotes, hypnozygotes and planomeiocytes. TEM showed bacteria to be present within all vegetative growth phases, as well as in the sexual planozygote, cyst and planomeiocyte. The presence of intra-cellular bacteria in vegetative cells was also confirmed using DAPI staining combined with epifluorescence microscopy, and lipopolysaccharide staining combined with TEM.

Involvement of endoplasmic reticulum and autophagy in microcystin-LR toxicity in Vero-E6 and HepG2 cell lines

This work investigates the involvement of the endoplasmic reticulum (ER) and autophagy in microcystin-LR (MCLR) toxicity in Vero-E6 and HepG2 cell lines. Additionally, morphological alterations induced by MCLR in lysosomes and mitochondria were studied. Cytotoxicity evaluation showed that pure MCLR and MCLR from LMECYA110 extract induce concentration dependent viability decays after 24h exposure. HepG2 cells showed an increased sensitivity to MCLR than Vero cells, with lower cytotoxic thresholds and EC(50) values. Conversely, LC3B immunofluorescence showed that autophagy is triggered in both cell lines as a survival response to low MCLR concentrations. Furthermore, MCLR induced a MCLR concentration-dependent decrease of GRP94 expression in HepG2 cells while in Vero cells no alteration was observed. This suggests the involvement of the ER in HepG2 apoptosis elicited by MCLR, while in Vero cells ER destructuration could be a consequence of cytoskeleton inflicted damages. Additionally, in both cell lines, lysosomal destabilization preceded mitochondrial impairment which occurred at high toxin concentrations. Although not an early cellular target of MCLR, mitochondria appears to serve as central mediators of different signaling pathways elicited by the organelles involved in MCLR toxicity. As a result, kidney and hepatic cell lines exhibit cell type and dose-dependent mechanisms to overcome MCLR toxicity.

Integrated approach to the in vivo genotoxic effects of a titanium dioxide nanomaterial using LacZ plasmid-based transgenic mice

Titanium dioxide (TiO2) nanomaterials (NMs) are widely used in a diversity of products including cosmetics, pharmaceuticals, food, and inks, despite uncertainties surrounding the potential health risks that they pose to humans and the environment. Previous studies on the genotoxicity of TiO2 have reported discrepant or inconclusive findings in both in vitro and in vivo systems. This study explores the in vivo genotoxic potential of a well‐characterized uncoated TiO2 NM with an average diameter of 22 nm (NM‐102, from JRC repository) using several genotoxicity endpoints in the LacZ plasmid‐based transgenic mouse model. Mice were exposed by intravenous injection to two daily doses of NM‐102: 10 and 15 mg/kg of body weight/day. Micronuclei were analyzed in peripheral blood reticulocytes 42 hr after the last treatment. DNA strand breaks (comet assay) and gene mutations were determined in the spleens and livers of the same animals 28 days after the last treatment. Histopathological and cytological analyses were also performed in liver samples. Genotoxic effects were not detected in mice exposed to the nanosized TiO2 under the experimental conditions used, despite a moderate inflammatory response that was observed in the liver. Considering the biopersistence of TiO2 in mouse liver and the moderate inflammatory response, the possibility of a secondary genotoxic effect at higher doses and in conditions that result in a stronger inflammatory response, for example, within a longer time window, should be investigated further. Environ. Mol. Mutagen. 55:500–509, 2014.

Exploring the Contribution of Mycobacteria Characteristics in Their Interaction with Human Macrophages

Tuberculosis (TB) is a major health problem. The emergence of multidrug resistant (MDR) Mycobacterium tuberculosis (Mtb) isolates confounds treatment strategies. In Portugal, cases of MDR-TB are reported annually with an increased incidence noted in Lisbon. The majority of these MDR-TB cases are due to closely related mycobacteria known collectively as the Lisboa family and Q1 cluster. Genetic determinants linked to drug resistance have been exhaustively studied resulting in the identification of family and cluster specific mutations. Nevertheless, little is known about other factors involved in development of mycobacteria drug resistance. Here, we complement genetic analysis with the study of morphological and structural features of the Lisboa family and Q1 cluster isolates by using scanning and transmission electron microscopy. This analysis allowed the identification of structural differences, such as cell envelope thickness, between Mtb clinical isolates that are correlated with antibiotic resistance. The infection of human monocyte derived macrophages allowed us to document the relative selective advantage of the Lisboa family isolates over other circulating Mtb isolates.