The differential interaction of Brucella and ochrobactrum with innate immunity reveals traits related to the evolution of stealthy pathogens Elías Barquero-Calvo, Raquel Conde-Alvarez, Carlos Chacón-Díaz, Lucía Quesada-Lobo, Anna Martirosyan, Caterina Guzmán-Verri, Maite Iriarte, Mateja Mancek-Keber, Roman Jerala, Jean Pierre Gorvel, et al. To cite this version: Elías Barquero-Calvo, Raquel Conde-Alvarez, Carlos Chacón-Díaz, Lucía Quesada-Lobo, Anna Mar- tirosyan, et al.. The differential interaction of Brucella and ochrobactrum with innate immunity reveals traits related to the evolution of stealthy pathogens. PLoS ONE, Public Library of Science, 2009, 4 (6), pp.e5893. 10.1371/journal.pone.0005893. hal-00431866 HAL Id: hal-00431866 https://hal.archives-ouvertes.fr/hal-00431866 Submitted on 27 Sep 2018 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Distributed under a Creative Commons Attribution| 4.0 International License The Differential Interaction of Brucella and Ochrobactrum with Innate Immunity Reveals Traits Related to the Evolution of Stealthy Pathogens Elı´as Barquero-Calvo1., Raquel Conde-Alvarez2., Carlos Chaco´ n-Dı´az1,3, Lucı´a Quesada-Lobo1, Anna Martirosyan4, Caterina Guzma´n-Verri1, Maite Iriarte2, Mateja Mancek-Keber5, Roman Jerala5, Jean Pierre Gorvel4, Ignacio Moriyo´ n2, Edgardo Moreno1*, Esteban Chaves-Olarte1,3* 1 Programa de Investigacio´n en Enfermedades Tropicales, Escuela de Medicina Veterinaria, Universidad Nacional, Heredia, Costa Rica, 2 Department of Microbiology, University of Navarra, Navarra, Spain, 3 Centro de Investigacio´n en Enfermedades Tropicales, Facultad de Microbiologı´a, Universidad de Costa Rica, San Jose´, Costa Rica, 4 Centre d’Immunologie de Marseille-Luminy, Aix Marseille Universite´, Faculte´ de Sciences de Luminy, INSERM U631, CNRS UMR6102, Marseille, France, 5 Department of Biotechnology, National Institute of Chemistry, Hajdrihova, Ljubljana, Slovenia Abstract Background: During evolution, innate immunity has been tuned to recognize pathogen-associated molecular patterns. However, some a-Proteobacteria are stealthy intracellular pathogens not readily detected by this system. Brucella members follow this strategy and are highly virulent, but other Brucellaceae like Ochrobactrum are rhizosphere inhabitants and only opportunistic pathogens. To gain insight into the emergence of the stealthy strategy, we compared these two phylogenetically close but biologically divergent bacteria. Methodology/Principal Findings: In contrast to Brucella abortus, Ochrobactrum anthropi did not replicate within professional and non-professional phagocytes and, whereas neutrophils had a limited action on B. abortus, they were essential to control O. anthropi infections. O. anthropi triggered proinflammatory responses markedly lower than Salmonella enterica but higher than B. abortus. In macrophages and dendritic cells, the corresponding lipopolysaccharides reproduced these grades of activation, and binding of O. anthropi lipopolysaccharide to the TLR4 co-receptor MD-2 and NF-kB induction laid between those of B. abortus and enteric bacteria lipopolysaccharides. These differences correlate with reported variations in lipopolysaccharide core sugars, sensitivity to bactericidal peptides and outer membrane permeability. Conclusions/Significance: The results suggest that Brucellaceae ancestors carried molecules not readily recognized by innate immunity, so that non-drastic variations led to the emergence of stealthy intracellular parasites. They also suggest that some critical envelope properties, like selective permeability, are profoundly altered upon modification of pathogen- associated molecular patterns, and that this represents a further adaptation to the host. It is proposed that this adaptive trend is relevant in other intracellular a-Proteobacteria like Bartonella, Rickettsia, Anaplasma, Ehrlichia and Wolbachia. Citation: Barquero-Calvo E, Conde-Alvarez R, Chaco´n-Dı´az C, Quesada-Lobo L, Martirosyan A, et al. (2009) The Differential Interaction of Brucella and Ochrobactrum with Innate Immunity Reveals Traits Related to the Evolution of Stealthy Pathogens. PLoS ONE 4(6): e5893. doi:10.1371/journal.pone.0005893 Editor: David M. Ojcius, University of California Merced, United States of America Received April 24, 2009; Accepted May 19, 2009; Published June 16, 2009 Copyright: ß 2009 Barquero-Calvo 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: This work was funded by grants from FIDA, Universidad Nacional de Costa Rica; FS-Conare UNA/UCR IFEG29 Costa Rica; NeTropica P00059 and F00013- 02; MICIT/CONICIT IFDG12; Fundacion CRUSA-CSIC; Centre National de la Recherche Scientifique, Institut National de la Sante et de la Recherche Medicale and the Ministry of Education in France; and Departamento de Microbiologia de la Universidad de Navarra AGL2008-04514-C03-01. Fellowship support for R.C-A. from the Ministerio de Ciencia y Tecnologia of Spain (BES-2005-10411) is gratefully 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: [email protected] (EM); [email protected] (ECO) . These authors contributed equally to this work. Introduction avoid immediate recognition by innate immunity, thus following a stealthy strategy of which Brucella can be considered as a model [3]. The class a-Proteobacteria includes microorganisms capable of In contrast, some bacteria close to Brucella are free living establishing close associations with eukaryotic cells in a wide range environmental microorganisms like Ochrobactrum, Daeguia, Crab- of lifestyles. Members of the genus Agrobacterium are pericellular to treella and Mycoplana. Although these four genera are included in plant cells and induce tumors, whereas Brucella, Bartonella, the Brucellaceae (http://www.bacterio.cict.fr/), only Ochrobactrum Phyllobacterium and Sinorhizobium are facultative extracellular- has been reported to display some degree of pathogenicity. intracellular bacteria that behave as pathogens or endosymbionts, Ochrobactrum anthropi, primarily a rhizosphere inhabitant, has been and Rickettsia, Anaplasma, Ehrlichia and Wolbachia are obligate isolated from immunocompromised individuals or patients intracellular pathogens of mammals and arthropods [1,2]. subjected to dialysis, catheterization, surgical interventions or Remarkably, animal pathogens of this group have the ability to transplantation [4,5,6,7,8,9,10,11] and often shows a broad PLoS ONE | www.plosone.org 1 June 2009 | Volume 4 | Issue 6 | e5893 Bacterial Stealthy Pathogens antibiotic resistance [12,13]. Ochrobactrum intermedium is another as the CFU numbers of intracellular B. abortus increased opportunistic member of the genus. Interestingly, Ochrobactrum is throughout the experiment, those of O. anthropi declined steadily the Brucellaceae member closest to Brucella according to several (Fig. 1B). In order to bypass the internalization deficiency and to molecular markers and genome comparisons [14]. determine if O. anthropi was able to multiply intracellularly, we used Owing to its close phylogenetic relatedness with the highly the Escherichia coli Cytotoxic Necrotizing Factor, a toxin that virulent Brucella [15], Ochrobactrum has received some attention. confers a phagocytic phenotype to HeLa cells [22]. This treatment Phenotypic analysis reveals that O. anthropi displays envelope increased the intracellular O. anthropi in one order of magnitude molecules known to be critical in Brucella virulence. They include (Fig. 1B) and most visualized bacteria were intracellular. phosphatidylcholine and a lipopolysaccharide (LPS) with a lipid A Nevertheless, O. anthropi was not able to achieve sustained carrying very long chain fatty acids (VLCFA). Also, like other replication, even though some survival was observed (Fig. 1B). Brucellaceae, O. anthropi free lipids contain acyl chains with a higher Then, we repeated the experiments in Raw 264.7 murine number of carbons than those found in typical Gram negative macrophages. Although at early times the intracellular numbers bacteria. Along with the VLCFAs of lipid A, these structural of O. anthropi paralleled those of B. abortus, the replication curves features are thought to be relevant in the construction of a firm coursed with opposite tendencies because, whereas B. abortus envelope which, in the case of Brucella, has been proposed to be replicated, O. anthropi did not (Fig. 1C). Noteworthy, the later important in pathogenicity [16,17]. Despite these similarities, bacteria remained cultivable 48 h after infection (Fig. 1C). there are differences in important outer membrane properties. Altogether, these results indicate that O. anthropi possesses a very Most notably,