Isabel Marcelino

Metabolic changes during cell growth inhibition by p27 overexpression

The overexpression of p27, a cyclin-dependent kinase (CDK) inhibitor, has been shown to effectively inhibit cell growth at the G1-phase of different cell lines, potentiating a valid genetic strategy for cell proliferation control. In order to characterize the energy requirements after p27 overexpression in CHO cells expressing SEAP (secreted form of the human alkaline phosphatase enzyme), key metabolic parameters were evaluated. Cell growth inhibition led to a significant increase in cell size concomitant with a 2-fold increase in cell protein content. The simultaneous increase of the intracellular proteolytic activity with protein content suggests higher protein synthesis. A general 2-fold increase in oxygen, glutamine and glucose consumption rates, coupled with an increase in lactate and ammonia production was observed. p27 overexpression led to a significant increase in the intracellular pool of AMP (8.5-fold), ADP (6-fold) and, more uncommonly, ATP (4.5-fold). Nevertheless, cells were able to maintain the equilibrium among the three adenine nucleotides since both the ATP/ADP ratio and the energy charge values remained similar to those observed with non-growth inhibited cells. This work shows that the observed 4-fold increase in SEAP specific productivity after cell growth inhibition by p27, occurred concomitantly with a higher expenditure of cell energy. This characterization of cell metabolism becomes important in demonstrating the applicability of growth inhibition systems.

Efficiency of inactivated vaccines against heartwater in Burkina Faso: impact of Ehrlichia ruminantium genetic diversity.

In order to identify the appropriate strains to use in vaccination trials against heartwater in Burkina Faso, the protective effect of Gardel and Welgevonden strains was assessed against local strains on sheep vaccinated by infection-and-treatment method: Gardel protected significantly against Burkina Faso strains tested (survival rate 59% for immunised sheep vs 13% for control sheep) while Welgevonden did not (survival rate 45% for immunised sheep vs 25% for control sheep). The efficacy of the ISA50 inactivated vaccine, produced under industrial process, was evaluated in sheep during field challenges in two successive years. During year 1, there was a limited protective effect of the Gardel vaccine with 65% of survival rate for the vaccinated group compared to 49% for the control group (N=153, p=0.053). During year 2, the vaccine containing Gardel and a local strain gave an increased protective effect compared to the first trial: 72% of the vaccinated animals survived compared to 47% of the naïve animals (N=173, p<0.001). There was an important genetic diversity of strains in the field with detection of 11 different map1 genotypes in brains from control and vaccinated sheep post mortem. Map1 genotyping of strains detected in brains from control sheep showed that genotype distribution varied according to time and study areas, which could explain the difference in efficacy of the vaccine.

Global gene expression profiling of Ehrlichia ruminantium at different stages of development

Ehrlichia ruminantium (ER), the causative agent of heartwater on ruminants, is an obligate intracellular bacterium transmitted by ticks of the genus Amblyomma. Previous studies have shown that early stages of development may be critical for Ehrlichia pathogenicity. To gain insights into the biology of intracellular ER, we determined the genome-wide transcriptional profile of ER replicating inside bovine aortic endothelial cells using DNA microarrays. At intermediate and late stages of infection (reticulate and elementary bodies, respectively), a total of 54 genes were differentially expressed. Among them, we measured by q-RTPCR the overexpression of 11 of 14 genes. A number of genes involved in metabolism, nutrient exchange, and defense mechanisms, including those involved in resistance to oxidative stress, were significantly induced in ER reticulate bodies. This is consistent with the oxidative stress condition and nutrient starvation that seem to occur in Ehrlichia-containing vacuoles. During the lysis stage of development, when ER is infectious, we showed the overexpression of a transcription factor, dksA, which is also known to induce virulence in other pathogens such as Salmonella typhimurium. Our results suggest a possible role of these genes in promoting ER development and pathogenicity.

Portuguese winemaking residues as a potential source of natural anti-adenoviral agents

Abstract To date there are no licensed systemic or topical treatments in Europe or the USA for adenovirus infections. In the present paper, we evaluate the effect of a polyphenol-based grape extract (NE) obtained from Portuguese white-winemaking by-products, and Resveratrol in pure form, on adenovirus type 5 infection. For this purpose, recombinant adenovirus vectors (Ad-5) and a human-derived cell line (293) were used as models. The NE and Resveratrol at the used concentrations do not induce cell cytotoxicity or direct virucidal activity; however, they reduce 4.5 and 6.5 log (TCID50/ml) on total infectious Ad-5 production, respectively. The capacity of Ad-5 replication upon removal of NE and Resveratrol after 24 h post infection was also evaluated. In contrast to Resveratrol, the highest evaluated NE concentration inhibits irreversibly the Ad-5 replication. These results provide useful information for the use of NE and Resveratrol as potential sources of promising natural antiviral agents on Ad-5 infection.

Proteomic analyses of Ehrlichia ruminantium highlight differential expression of MAP1-family proteins

The Rickettsiales Ehrlichia ruminantium (ER) is the causative agent of heartwater, a fatal tick-borne disease of livestock in sub-Saharan Africa and in the Caribbean, posing strong economical constraints to livestock production. In an attempt to identify the most prominent proteins expressed by this bacterium, especially those encoded by the major antigenic protein 1 (map1) multigene family, a proteome map of ER cultivated in endothelial cells was constructed by using two dimensional gel electrophoresis combined with mass spectrometry. Among the sixty-four spots detected, we could identify only four proteins from the MAP1-family; the other proteins detected were mainly related to energy, amino acid and general metabolism (26%), to protein turnover, chaperones and survival (21%) and to information processes (14%) or classified as hypothetical proteins (23%). Additional studies on MAP1-family protein using immunochemical labeling also revealed that these proteins are differentially expressed along the bacterium life cycle, presenting different structural organization. Interestingly, when infectious elementary bodies (EBs) are released from host cells, MAP1 appears to be organized in SDS and heat-resistant dimers and trimers stabilized by disulfide bridges. Overall, the results presented herein not only reveal the first partial proteome map of ER but provide new insights on the expression ER MAP1-family proteins in host endothelial cells.

Understanding Anaplasmataceae pathogenesis using "Omics" approaches.

This paper examines how “Omics” approaches improve our understanding of Anaplasmataceae pathogenesis, through a global and integrative strategy to identify genes and proteins involved in biochemical pathways key for pathogen-host-vector interactions. The Anaplasmataceae family comprises obligate intracellular bacteria mainly transmitted by arthropods. These bacteria are responsible for major human and animal endemic and emerging infectious diseases with important economic and public health impacts. In order to improve disease control strategies, it is essential to better understand their pathogenesis. Our work focused on four Anaplasmataceae, which cause important animal, human and zoonotic diseases: Anaplasma marginale, A. phagocytophilum, Ehrlichia chaffeensis, and E. ruminantium. Wolbachia spp. an endosymbiont of arthropods was also included in this review as a model of a non-pathogenic Anaplasmataceae. A gap analysis on “Omics” approaches on Anaplasmataceae was performed, which highlighted a lack of studies on the genes and proteins involved in the infection of hosts and vectors. Furthermore, most of the studies have been done on the pathogen itself, mainly on infectious free-living forms and rarely on intracellular forms. In order to perform a transcriptomic analysis of the intracellular stage of development, researchers developed methods to enrich bacterial transcripts from infected cells. These methods are described in this paper. Bacterial genes encoding outer membrane proteins, post-translational modifications, eukaryotic repeated motif proteins, proteins involved in osmotic and oxidative stress and hypothetical proteins have been identified to play a key role in Anaplasmataceae pathogenesis. Further investigations on the function of these outer membrane proteins and hypothetical proteins will be essential to confirm their role in the pathogenesis. Our work underlines the need for further studies in this domain and on host and vector responses to infection.

Proteomic profiling of the outer membrane fraction of the obligate intracellular bacterial pathogen #Ehrlichia ruminantium#

The outer membrane proteins (OMPs) of Gram-negative bacteria play a crucial role in virulence and pathogenesis. Identification of these proteins represents an important goal for bacterial proteomics, because it aids in vaccine development. Here, we have developed such an approach for Ehrlichia ruminantium, the obligate intracellular bacterium that causes heartwater. A preliminary whole proteome analysis of elementary bodies, the extracellular infectious form of the bacterium, had been performed previously, but information is limited about OMPs in this organism and about their role in the protective immune response. Identification of OMPs is also essential for understanding Ehrlichia’s OM architecture, and how the bacterium interacts with the host cell environment. First, we developed an OMP extraction method using the ionic detergent sarkosyl, which enriched the OM fraction. Second, proteins were separated via one-dimensional electrophoresis, and digested peptides were analyzed via nano-liquid chromatographic separation coupled with mass spectrometry (LC-MALDI-TOF/TOF). Of 46 unique proteins identified in the OM fraction, 18 (39%) were OMPs, including 8 proteins involved in cell structure and biogenesis, 4 in transport/virulence, 1 porin, and 5 proteins of unknown function. These experimental data were compared to the predicted subcellular localization of the entire E. ruminantium proteome, using three different algorithms. This work represents the most complete proteome characterization of the OM fraction in Ehrlichia spp. The study indicates that suitable subcellular fractionation experiments combined with straightforward computational analysis approaches are powerful for determining the predominant subcellular localization of the experimentally observed proteins. We identified proteins potentially involved in E. ruminantium pathogenesis, which are good novel targets for candidate vaccines. Thus, combining bioinformatics and proteomics, we discovered new OMPs for E. ruminantium that are valuable data for those investigating new vaccines against this organism. In summary, we provide both pioneering data and novel insights into the pathogenesis of this obligate intracellular bacterium.

Comparative Proteomic Profiling of Ehrlichia ruminantium Pathogenic Strain and Its High-Passaged Attenuated Strain Reveals Virulence and Attenuation-Associated Proteins.

The obligate intracellular bacterium Ehrlichia ruminantium (ER) causes heartwater, a fatal tick-borne disease in livestock. In the field, ER strains present different levels of virulence, limiting vaccine efficacy, for which the molecular basis remains unknown. Moreover, there are no genetic tools currently available for ER manipulation, thus limiting the knowledge of the genes/proteins that are essential for ER pathogenesis and biology. As such, to identify proteins and/or mechanisms involved in ER virulence, we performed the first exhaustive comparative proteomic analysis between a virulent strain (ERGvir) and its high-passaged attenuated strain (ERGatt). Despite their different behaviors in vivo and in vitro, our results from 1DE-nanoLC-MS/MS showed that ERGvir and ERGatt share 80% of their proteins; this core proteome includes chaperones, proteins involved in metabolism, protein-DNA-RNA biosynthesis and processing, and bacterial effectors. Conventional 2DE revealed that 85% of the identified proteins are proteoforms, suggesting that post-translational modifications (namely glycosylation) are important in ER biology. Strain-specific proteins were also identified: while ERGatt has an increased number and overexpression of proteins involved in cell division, metabolism, transport and protein processing, ERGvir shows an overexpression of proteins and proteoforms (DIGE experiments) involved in pathogenesis such as Lpd, AnkA, VirB9 and B10, providing molecular evidence for its increased virulence in vivo and in vitro. Overall, our work reveals that ERGvir and ERGatt proteomes are streamlined to fulfill their biological function (maximum virulence for ERGvir and replicative capacity for ERGatt), and we provide both pioneering data and novel insights into the pathogenesis of this obligate intracellular bacterium.

A user-friendly and scalable process to prepare a ready-to-use inactivated vaccine: the example of heartwater in ruminants under tropical conditions.

The use of cheap and thermoresistant vaccines in poor tropical countries for the control of animal diseases is a key issue. Our work aimed at designing and validating a process for the large-scale production of a ready-to-use inactivated vaccine for ruminants. Our model was heartwater caused by the obligate intracellular bacterium Ehrlichia ruminantium (ER). The conventional inactivated vaccine against heartwater (based on whole bacteria inactivated with sodium azide) is prepared immediately before injection, using a syringe-extrusion method with Montanide ISA50. This is a fastidious time-consuming process and it limits the number of vaccine doses available. To overcome these issues, we tested three different techniques (syringe, vortex and homogenizer) and three Montanide ISA adjuvants (50, 70 and 70M). High-speed homogenizer was the optimal method to emulsify ER antigens with both ISA70 and 70M adjuvants. The emulsions displayed a good homogeneity (particle size below 1 μm and low phase separation), conductivity below 10 μS/cm and low antigen degradation at 4 °C for up to 1 year. The efficacy of the different formulations was then evaluated during vaccination trials on goats. The inactivated ER antigens emulsified with ISA70 and ISA70M in a homogenizer resulted in 80% and 100% survival rates, respectively. A cold-chain rupture assay using ISA70M+ER was performed to mimic possible field conditions exposing the vaccine at 37 °C for 4 days before delivery. Surprisingly, the animal survival rate was still high (80%). We also observed that the MAP-1B antibody response was very similar between animals vaccinated with ISA70+ER and ISA70M+ER emulsions, suggesting a more homogenous antigen distribution and presentation in these emulsions. Our work demonstrated that the combination of ISA70 or ISA70M and homogenizer is optimal for the production of an effective ready-to-use inactivated vaccine against heartwater, which could easily be produced on an industrial scale.

Automatic prediction of PTMs in Ehrlichia ruminantium – creating new datasets for Quickmod analyses

Ehrlichia ruminantium (ER) is an obligate intracellular bacterium, from the order Rickettsiales, which causes Heartwater, a fatal tick-borne disease in ruminants. This disease is a major limitation to livestock production in sub-Saharan Africa and in some Caribbean islands. Recent studies showed that key proteins such as the Major Antigenic Protein 1 (MAP1) are glycosylated (Postigo et al., 2008) and that about 25 % of ER proteome account for isoforms, indicating the importance of post-translational modifications (PTMs) in the ER infection process (Marcelino et al., 2012).