title: Proteomic profiling of the outer membrane fraction of the obligate intracellular bacterial pathogen Ehrlichia ruminantium.
abstract: 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.
authors.affiliation: CIRAD, UMR CMAEE, Site de Duclos, Prise d'eau, F-97170, Petit-Bourg, Guadeloupe, France; INRA, UMR1309 CMAEE, F-34398, Montpellier, France; Université des Antilles et de la Guyane, 97159, Pointe-à-Pitre cedex, Guadeloupe, France.
authors.forename: Amal
authors.initials: A
authors.lastname: Moumène
authors.affiliation: Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901, Oeiras, Portugal; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157, Oeiras, Portugal.
authors.forename: Isabel
authors.initials: I
authors.lastname: Marcelino
authors.affiliation: Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901, Oeiras, Portugal; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157, Oeiras, Portugal.
authors.forename: Miguel
authors.initials: M
authors.lastname: Ventosa
authors.affiliation: Université des Antilles et de la Guyane, Institut de Biologie Paris Seine, UMR7138 UPMC-CNRS, Equipe Biologie de la Mangrove, UFR des Sciences Exactes et Naturelles, Département de Biologie, BP 592, 97159, Pointe-à-Pitre cedex, Guadeloupe, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Gros
authors.affiliation: INRA, UMR1309 CMAEE, F-34398, Montpellier, France.
authors.forename: Thierry
authors.initials: T
authors.lastname: Lefrançois
authors.affiliation: CIRAD, UMR CMAEE, Site de Duclos, Prise d'eau, F-97170, Petit-Bourg, Guadeloupe, France; INRA, UMR1309 CMAEE, F-34398, Montpellier, France.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Vachiéry
authors.affiliation: CIRAD, UMR CMAEE, Site de Duclos, Prise d'eau, F-97170, Petit-Bourg, Guadeloupe, France; INRA, UMR1309 CMAEE, F-34398, Montpellier, France.
authors.forename: Damien F
authors.initials: DF
authors.lastname: Meyer
authors.affiliation: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157, Oeiras, Portugal.
authors.forename: Ana V
authors.initials: AV
authors.lastname: Coelho
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D016995:Ehrlichia ruminantium / Q000737:chemistry / Q000378:metabolism*; D020543:Proteome / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001425:Bacterial Outer Membrane Proteins; D020543:Proteome