Miguel Ventosa

Mass spectrometry and animal science: Protein identification strategies and particularities of farm animal species ☆

Proteomic approaches are gaining increasing importance in the context of all fields of animal and veterinary sciences, including physiology, productive characterization, and disease/parasite tolerance, among others. Proteomic studies mainly aim the proteome characterization of a certain organ, tissue, cell type or organism, either in a specific condition or comparing protein differential expression within two or more selected situations. Due to the high complexity of samples, usually total protein extracts, proteomics relies heavily on separation procedures, being 2D-electrophoresis and HPLC the most common, as well as on protein identification using mass spectrometry (MS) based methodologies. Despite the increasing importance of MS in the context of animal and veterinary science studies, the usefulness of such tools is still poorly perceived by the animal science community. This is primarily due to the limited knowledge on mass spectrometry by animal scientists. Additionally, confidence and success in protein identification is hindered by the lack of information in public databases for most of farm animal species and their pathogens, with the exception of cattle (Bos taurus), pig (Sus scrofa) and chicken (Gallus gallus). In this article, we will briefly summarize the main methodologies available for protein identification using mass spectrometry providing a case study of specific applications in the field of animal science. We will also address the difficulties inherent to protein identification using MS, with particular reference to experiments using animal species poorly described in public databases. Additionally, we will suggest strategies to increase the rate of successful identifications when working with farm animal species.

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.

Gene therapy approach to FAP: in vivo influence of T119M in TTR deposition in a transgenic V30M mouse model

Familial amyloidotic polyneuropathy (FAP) is a neurodegenerative disorder characterized by extracellular deposition of amyloid fibrils composed by mutated transthyretin (TTR) mainly in the peripheral nervous system. At present, liver transplantation is still the standard treatment to halt the progression of clinical symptoms in FAP, but new therapeutic strategies are emerging, including the use of TTR stabilizers. Here we propose to establish a new gene therapy approach using adeno-associated virus (AAV) vectors to deliver the trans-suppressor TTR T119M variant to the liver of transgenic TTR V30M mice at different ages. This TTR variant is known for its ability to stabilize the tetrameric protein. Analysis of the gastrointestinal tract of AAV-treated animals revealed a significant reduction in deposition of TTR non-fibrillar aggregates in as much as 34% in stomach and 30% in colon, as well as decreased levels of biomarkers associated with TTR deposition, namely the endoplasmic reticulum stress marker BiP and the extracellular matrix protein MMP-9. Moreover, we showed with different studies that our approach leads to an increase in tetrameric and more stable forms of TTR, in favor of destabilized monomers. Altogether our data suggest the possibility to use this gene therapy approach in a prophylactic manner to prevent FAP pathology.

Tissue remodeling after RNAi-mediated knockdown of TTR in a Familial Amyloidotic Polyneuropathy mouse model

Background Transthyretin (TTR) deposition in the peripheral nervous system (PNS) is the hallmark of Familial Amyloidotic Polyneuropathy (FAP). Mice expressing human TTR with the V30M mutation in a heterozygous heat shock factor 1 (Hsf-1) background show extensive TTR deposits in PNS and gastrointestinal tract, as well as extracellular matrix (ECM) remodeling, similar to those seen in human FAP patients. Currently, liver transplantation is the only available treatment to halt the progression of clinical symptoms in FAP. Due to the limitations of this procedure, it is of utmost importance to develop alternative therapeutic strategies. In this regard, an RNAi therapeutic targeting TTR for the treatment of FAP is currently in Phase 3 clinical development. An ongoing phase 2 clinical trial in FAP patients demonstrated promising results as a mean plasma TTR reduction of 80%, sustained for over nine months, led to a decrease in neuropathy progression compared to historical data.

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).

Tick-borne diseases in cattle: applications of proteomics and the development of new generation vaccines

Tick-borne diseases (TBDs) affect 80% of the world’s cattle population, severely hampering livestock production. Some of these diseases are caused by obligate intracellular tick-borne pathogens (TBPs) such as Theileria spp., Babesia spp., Anaplasma marginale and Ehrlichia ruminantium (1). Immunization strategies against TBDs are currently available (most of them being blood-derived or attenuated), but with variable efficacy (2); thus identification of new antigens is required to develop improved vaccines. Nowadays, new breakthroughs in vaccine research are increasingly reliant on novel ‘Omics’ approaches such as genomics, proteomics, transcriptomics, and metabolomics, to deepen our understanding of the key biological processes that lead to protective immunity (Figure 1). Despite the recent availability of TBPs genomes, the increased knowledge on their biology has proven to be difficult since they have complex life cycles (with different developmental forms) being able to infect and multiply within different types of cells. As limited genetic tools are currently available to complement existing genomic information, post-genomics strategies such as proteomics are nowadays being used more frequently.

The effect of colostrum intake on blood plasma proteomic profiles of newborn lambs

The consumption of colostrum by newborn ruminants plays a fundamental role in the acquisition of passive immunity. In fact, due to the characteristics of ruminant’s syndesmochorial placenta, the transfer of immunoglobulins from the dam to the fetus is very limited and not able to ensure the survival of the newborn (Castro et al., 2009).

Mass spectrometry for the veterinary and farm animal world

Proteomics approaches are gaining increasing importance in the context of all fields of animal and veterinary sciences, including physiology, productive characterization, and disease/parasite tolerance, among others. Proteomic studies mainly aim at the proteome characterization of a certain organ, tissue, cell type or organism, either in a specific condition or comparing protein differential expression within two or more selected situations. Due to the high complexity of samples, usually total protein extracts, proteomic studies rely heavily on protein identification and quantification using mass spectrometry (MS) based methodologies or with coupling to other methodologies, like liquid chromatography and gel electrophoresis. Despite the increasing importance of MS in the context of animal and veterinary sciences studies, the usefulness of such tools is still poorly perceived by the Animal Science community. This is primarily due to limited knowledge on Mass Spectrometry by animal scientists, which use nowadays still requires a high level of specialization. Additionally, confidence and success in protein identification is hindered by the lack of information in public databases for most of farm animal species and their pathogens, with the exception of cattle (Bos taurus), pig (Sus scrofa) and chicken (Gallusgallus). During this lecture, a description of the main MS methodologies available for proteome characterization and differential proteomic studies will be presented.