Iriarte1*. Abstract Brucella abortus Ornithine Lipids Are Dispensable Outer Membrane Components Devoid of a Marked Pathogen- Associated Molecular Pattern Leyre Palacios-Chaves1, Raquel Conde-A´ lvarez1,2, Yolanda Gil-Ramı´rez1, Amaia Zu´ n˜ iga-Ripa1, Elı´as Barquero-Calvo 4 , Carlos Chaco´ n-Dı´az3,4, Esteban Chaves-Olarte3,4, Vilma Arce-Gorvel5, Jean-Pierre Gorvel5, Edgardo Moreno3,6, Marı´a-Jesu´ s de Miguel7, Marı´a-Jesu´ s Grillo´ 8, Ignacio Moriyo´ n1., Maite 1 Departamento de Microbiolog´ıa y Parasitolog´ıa, Universidad de Navarra, Pamplona, Spain, 2 Focal Area Infection Biology, Biozentrum of the University of Basel, Basel, Switzerland, 3 Centro de Investigacio´ n en Enfermedades Tropicales, Facultad de Microbiolog´ıa, Universidad de Costa Rica, San Jose´, Costa Rica, 4 Programa de Investigacio´ n en Enfermedades Tropicales, Escuela de Medicina Veterinaria, Universidad Nacional, Heredia, Costa Rica, 5 Centre d’Immunologie de Marseille-Luminy, Aix Marseille Universite´, Faculte´ de Sciences de Luminy, Marseille, INSERM U631, CNRS UMR6102, Marseille, France, 6 Instituto Clodomiro Picado, Facultad de Microbiolog´ıa, Universidad de Costa Rica, San Pedro, Costa Rica, 7 Centro de Investigacio´ n y Tecnolog´ıa Agroalimentaria (CITA), Unidad de Sanidad Animal, Gobierno de Arago´ n, Zaragoza, Spain, 8 Instituto de Agrobiotecnolog´ıa, CSIC-UPNA-Gobierno de Navarra, Pamplona, Spain The brucellae are a-Proteobacteria facultative intracellular parasites that cause an important zoonosis. These bacteria escape early detection by innate immunity, an ability associated to the absence of marked pathogen-associated molecular patterns in the cell envelope lipopolysaccharide, lipoproteins and flagellin. We show here that, in contrast to the outer membrane ornithine lipids (OL) of other Gram negative bacteria, Brucella abortus OL lack a marked pathogen-associated molecular pattern activity. We identified two OL genes (olsB and olsA) and by generating the corresponding mutants found that olsB deficient B. abortus did not synthesize OL or their lyso-OL precursors. Liposomes constructed with B. abortus OL did not trigger IL-6 or TNF-a release by macrophages whereas those constructed with Bordetella pertussis OL and the olsB mutant lipids as carriers were highly active. The OL deficiency in the olsB mutant did not promote proinflammatory responses or generated attenuation in mice. In addition, OL deficiency did not increase sensitivity to polymyxins, normal serum or complement consumption, or alter the permeability to antibiotics and dyes. Taken together, these observations indicate that OL have become dispensable in the extant brucellae and are consistent within the trend observed in a-Proteobacteria animal pathogens to reduce and eventually eliminate the envelope components susceptible of recognition by innate immunity. Citation: Palacios-Chaves L, Conde-A´ lvarez R, Gil-Ram´ırez Y, Zu´ n˜ iga-Ripa A, Barquero-Calvo E, et al. (2011) Brucella abortus Ornithine Lipids Are Dispensable Outer Membrane Components Devoid of a Marked Pathogen-Associated Molecular Pattern. PLoS ONE 6(1): e16030. doi:10.1371/journal.pone.0016030 Editor: David M. Ojcius, University of California Merced, United States of America Received October 28, 2010; Accepted December 3, 2010; Published January 7, 2011 Copyright: 2011 Palacios-Chaves et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Research at the laboratories of the authors is supported by the Ministerio de Ciencia y Tecnolog´ıa of Spain (AGL2008-04514-C03); FIDA, Universidad Nacional de Costa Rica; FS-Conare UNA/UCR IFEG29 Costa Rica; NeTropica P00059 and F00013-02; MICIT/CONICIT IFDG12; Fundacio´ n CRUSA-CSIC (CR2008-0006); Centre National de la Recherche Scientifique, Institut National de la Sante´ et de la Recherche Me´dicale and the Ministry of Education in France. Fellowship support for L.P.-C., Y.G.-R. and A.Z.-R from the Ministerio de Ciencia y Tecnolog´ıa of Spain, Gobierno de Navarra and Friends of the University of Navarra is also acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: miriart@unav.es . These authors contributed equally to this work. Introduction The members of the genus Brucella are a-2 Proteobacteria that cause brucellosis, an important disease affecting livestock and wild life as well as human beings. These bacteria trigger only low proinflammatory responses in the initial stages of infection and, accordingly, they follow a stealthy behavior that allows them to reach sheltered intracellular niches before effective immunity activation. The outer membranes (OM) of brucellae are of critical importance in this strategy. Whereas most gram-negative have OM molecules bearing the pathogen-associated molecular pat- terns (PAMP) recognized by innate immunity, at least the Brucella OM lipopolysaccharide (LPS), lipoproteins and flagellin display a reduced PAMP [1,2]. Moreover, smooth (S) brucellae such as B. abortus and B. melitensis have OMs that are unusually resistant to the disrupting action of bactericidal peptides and complement. Thus, periplasmic and internal PAMP-bearing molecules like peptido- glycan or DNA are not readily accessible to pathogen recognition receptors [1,3–8]. The Brucella LPS is clearly implicated in these properties and there is evidence that other lipid molecules also contribute. Brucella OMs contain large amounts of phosphatidyl- choline (PC) and blockage of the synthesis of PC with the subsequent replacement by phosphatidylethanolamine (PE) gen- erates attenuation [9,10]. Ornithine lipids (OLs) are present in relatively large amounts in Brucella [11] and, although they have interesting properties in other bacteria, have not been investigated. PLoS ONE | www.plosone.org 1 January 2011 | Volume 6 | Issue 1 | e16030