Lopez-Goñi, I. (Ignacio)
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- Evaluation of the relatedness of Brucella spp. and Ochrobactrum anthropi and description of Ochrobactrum intermedium sp. nov., a new species with a closer relationship to Brucella spp(Society for General Microbiology, 1998) Lopez-Goñi, I. (Ignacio); Moriyon, I. (Ignacio); Leiva, J. (José); Diaz, R. (Ramón); Romero, C. (Conchi); Velasco, J. (Julián)The relatedness of Brucella spp. and Ochrobactrum anthropi was studied by protein profiling, Western blot, immunoelectrophoresis and 16S rRNA analysis. Whole-cell and soluble proteins of brucellae and O. anthropi showed serological cross-reactivities quantitatively and qualitatively more intense than those existing with similar extracts of Agrobacterium spp. Numerical analysis of Western blot profiles of whole-cell extracts showed that O. anthropi LMG 3301 was closer to Brucella spp. than to O. anthropi LMG 3331T, a result not obtained by protein profiling. These differences were not observed by Western blot with soluble fractions, and immunoelectrophoretic analyses suggested that this was due to destruction of conformational epitopes in Western blot procedures with the subsequent simplification of antigenic profile. Analysis of the 16S rRNA sequences of strains previously used in the species definition confirmed that strain LMG 3301, and also LMG 3306, were closer to the brucellae, and that LMG 3331T was in a separate cluster. The LMG 3301 and the LMG 3331T clusters could also be separated by their different colistin sensitivity and by PCR with 16S rRNA Brucella primers, and both methods showed strains of both clusters among clinical isolates classified as O. anthropi by conventional tests. These results and those of previous DNA-DNA hybridization studies [Holmes, B., Popoff, M., Kiredjian, M. & Kersters, K. (1988). Int J Syst Bacteriol 38, 406-416] show that the LMG 3301 cluster and related clinical isolates should be given a new species status for which the name Ochrobactrum intermedium sp. nov. is proposed (type strain is LMG 3301T=NCTC 12171T = CNS 2-75T).
- Improved method for purification of bacterial DNA from bovine milk for detection of Brucella spp. by PCR(American Society for Microbiology, 1999) Lopez-Goñi, I. (Ignacio); Romero, C. (Conchi)Different methods of extraction of bacterial DNA from bovine milk to improve the direct detection of Brucella by PCR were evaluated. We found that the use of a lysis buffer with high concentrations of Tris, EDTA, and NaCl, high concentrations of sodium dodecyl sulfate and proteinase K, and high temperatures of incubation was necessary for the efficient extraction of Brucella DNA. The limit of detection by PCR was 5 to 50 Brucella CFU/ml of milk.
- Spontaneous excision of the O-polysaccharide wbkA glycosyltranferase gene is a cause of dissociation of smooth to rough Brucella colonies(American Society for Microbiology, 2012) Lopez-Goñi, I. (Ignacio); Mancilla, M. (Marcos); Moriyon, I. (Ignacio); Marin, C.M. (C. M.); Zarraga, A.M. (Ana María); Blasco, J.M. (J. M.)The brucellae are Gram-negative pathogens that cause brucellosis, a zoonosis of worldwide importance. The genus Brucella includes smooth and rough species that differ in that they carry smooth and rough lipopolysaccharides, respectively. Brucella abortus, B. melitensis, and B. suis are typical smooth species. However, these smooth brucellae dissociate into rough mutants devoid of the lipopolysaccharide O-polysaccharide, a major antigen and a virulence determinant encoded in regions wbo (included in genomic island-2) and wbk. We demonstrate here the occurrence of spontaneous recombination events in those three Brucella species leading to the deletion of a 5.5-kb fragment carrying the wbkA glycosyltranferase gene and to the appearance of rough mutants. Analysis of the recombination intermediates suggested homologous recombination between the ISBm1 insertion sequences flanking wbkA as the mechanism generating the deletion. Excision of wbkA was reduced but not abrogated in a recA-deficient mutant, showing the existence of both RecA-dependent and -independent processes. Although the involvement of the ISBm1 copies flanking wbkA suggested a transpositional event, the predicted transpositional joint could not be detected. This absence of detectable transposition was consistent with the presence of polymorphism in the inverted repeats of one of the ISBm1 copies. The spontaneous excision of wbkA represents a novel dissociation mechanism of smooth brucellae that adds to the previously described excision of genomic island-2. This ISBm1-mediated wbkA excision and the different %GC levels of the excised fragment and of other wbk genes suggest that the Brucella wbk locus is the result of at least two horizontal acquisition events.
- Comunicar la ciencia: desafíos y oportunidades para los investigadores(2023-11) Lopez-Goñi, I. (Ignacio); Armentia, J. (Javier); Sevilla, J. (Joaquín); Grau-Gumbau, P. (Paloma)Con motivo de la Semana Internacional del Acceso abierto de 2023, Paloma Grau, Vicerrectora de Investigación y Sostenibilidad de la Universidad de Navarra, conversará con los investigadores y divulgadores Ignacio López-Goñi, Javier Armentia y Joaquín Sevilla acerca de los retos de la divulgación científica en el nuevo panorama de la Ciencia abierta.
- Brucella abortus transits through the autophagic pathway and replicates in the endoplasmic reticulum of nonprofessional phagocytes(American Society for Microbiology, 1998) Lopez-Goñi, I. (Ignacio); Goot, G. (Gisou) van der; Gorvel, J.P. (Jean Pierre); Parton, R.G. (Robert G.); Moreno, E. (Edgardo); Sola-Landa, A. (Alberto); Pizarro-Cerda, J. (Javier); Meresse, S. (Stéphane)Brucella abortus is an intracellular pathogen that replicates within a membrane-bounded compartment. In this study, we have examined the intracellular pathway of the virulent B. abortus strain 2308 (S2308) and the attenuated strain 19 (S19) in HeLa cells. At 10 min after inoculation, both bacterial strains are transiently detected in phagosomes characterized by the presence of early endosomal markers such as the early endosomal antigen 1. At approximately 1 h postinoculation, bacteria are located within a compartment positive for the lysosome-associated membrane proteins (LAMPs) and the endoplasmic reticulum (ER) marker sec61beta but negative for the mannose 6-phosphate receptors and cathepsin D. Interestingly, this compartment is also positive for the autophagosomal marker monodansylcadaverin, suggesting that S2308 and S19 are located in autophagic vacuoles. At 24 h after inoculation, attenuated S19 is degraded in lysosomes, while virulent S2308 multiplies within a LAMP- and cathepsin D-negative but sec61beta- and protein disulfide isomerase-positive compartment. Furthermore, treatment of infected cells with the pore-forming toxin aerolysin from Aeromonas hydrophila causes vacuolation of the bacterial replication compartment. These results are compatible with the hypothesis that pathogenic B. abortus exploits the autophagic machinery of HeLa cells to establish an intracellular niche favorable for its replication within the ER.
- Structure and properties of the outer membranes of Brucella abortus and Brucella melitensis(Springer, 1998) Lopez-Goñi, I. (Ignacio); Moriyon, I. (Ignacio)The brucellae are Gram-negative bacteria characteristically able to multiply facultatively within phagocytic cells and which cause a zoonosis of world-wide importance. This article reviews the structure and topology of the main components (lipopolysaccharide, native hapten polysaccharide, free lipids and proteins) of the outer membranes of Brucella abortus and B. melitensis, as well as some distinctive properties (permeability and interactions with cationic peptides) of these membranes. On these data, an outer membrane model is proposed in which, as compared to other Gram-negatives, there is a stronger hydrophobic anchorage for the lipopolysaccharide, free lipids, porin proteins and lipoproteins, and a reduced surface density of anionic groups, which could be partially or totally neutralized by ornithine lipids. This model accounts for the permeability of Brucella to hydrophobic permeants and for its resistance to the bactericidal oxygen-independent systems of phagocytes.
- Specific detection of Brucella DNA by PCR(American Society for Microbiology, 1995) Lopez-Goñi, I. (Ignacio); Gamazo, C. (Carlos); Romero, C. (Conchi); Pardo, M. (Marisa)A PCR assay with primers derived from the 16S rRNA sequence of Brucella abortus was developed. Nine different combinations between six primers were tested. One pair of primers, which amplified a 905-bp fragment, was selected. As little as 80 fg of Brucella DNA was detected by this method. DNAs from all of the representative strains of the species and biovars of Brucella and from 23 different Brucella isolates were analyzed and yielded exclusively the 905-bp fragment. No amplification was detected with DNAs from 10 strains phylogenetically related to Brucella spp., 5 gram-negative bacteria showing serological cross-reactions with Brucella spp., and 36 different clinical isolates of non-Brucella species. Only Ochrobactrum anthropi biotype D yielded a PCR product of 905 bp, suggesting a closer relationship between Brucella spp. and O. anthropi biotype D. The specificity and high sensitivity of the PCR assay may provide a valuable tool for the diagnosis of brucellosis.
- Characterization of Brucella abortus O-polysaccharide and core lipopolysaccharide mutants and demonstration that a complete core is required for rough vaccines to be efficient against Brucella abortus and Brucella ovis in the mouse model(American Society for Microbiology, 2003) Lopez-Goñi, I. (Ignacio); Cloeckaert, A. (Axel); Miguel, M.J. (María Jesús) de; Moriyon, I. (Ignacio); González-Fernández, D. (David); Marin, C.M. (C. M.); Blasco, J.M. (J. M.); Monreal, D. (Daniel); Grillo, M.J. (María Jesús)Brucella abortus rough lipopolysaccharide (LPS) mutants were obtained by transposon insertion into two wbk genes (wbkA [putative glycosyltransferase; formerly rfbU] and per [perosamine synthetase]), into manB (pmm [phosphomannomutase; formerly rfbK]), and into an unassigned gene. Consistent with gene-predicted roles, electrophoretic analysis, 2-keto-3-manno-D-octulosonate measurements, and immunoblots with monoclonal antibodies to O-polysaccharide, outer and inner core epitopes showed no O-polysaccharide expression and no LPS core defects in the wbk mutants. The rough LPS of manB mutant lacked the outer core epitope and the gene was designated manB(core) to distinguish it from the wbk manB(O-Ag). The fourth gene (provisionally designated wa**) coded for a putative glycosyltransferase involved in inner core synthesis, but the mutant kept the outer core epitope. Differences in phage and polymyxin sensitivity, exposure or expression of outer membrane protein, core and lipid A epitopes, and lipid A acylation demonstrated that small changes in LPS core caused significant differences in B. abortus outer membrane topology. In mice, the mutants showed different degrees of attenuation and induced antibodies to rough LPS and outer membrane proteins. Core-defective mutants and strain RB51 were ineffective vaccines against B. abortus in mice. The mutants per and wbkA induced protection but less than the standard smooth vaccine S19, and controls suggested that anti O-polysaccharide antibodies accounted largely for the difference. Whereas no core-defective mutant was effective against B. ovis, S19, RB51, and the wbkA and per mutants afforded similar levels of protection. These results suggest that rough Brucella vaccines should carry a complete core for maximal effectiveness.
- Generation of the Brucella melitensis ORFeome version 1.1.(Cold Spring Habor Laboratory Press, 2004) Lopez-Goñi, I. (Ignacio); Bolle, X. (Xavier) de; Bertin, N. (Nicolas); Moriyon, I. (Ignacio); Lamesch, P. (Philippe); Delroisse, J.M. (Jean Marc); Whatmore, A.M. (Adrian M.); Sangari, F.J. (Félix Javier); Hill, D.E. (David E.); Sequerra, R. (Reynaldo); MacMillan, A.P. (Alastair P.); Vidal, M. (Marc); Hao, T. (Tong); Garcia-Lobo, J.M. (Juan María); Lambert, C. (Christophe); Hallez, R. (Régis); Rual, J. J. (Jean François); Cutler, S.J. (Sally J.); Letesson, J.J. (Jean Jacques); Dupuy, D. (Denis); Vandenhaute, J. (Jean); Doucettte-Stamm, L. (Lynn); Bozak, S. (Stephanie); Dricot, A. (Amélie)The bacteria of the Brucella genus are responsible for a worldwide zoonosis called brucellosis. They belong to the alpha-proteobacteria group, as many other bacteria that live in close association with a eukaryotic host. Importantly, the Brucellae are mainly intracellular pathogens, and the molecular mechanisms of their virulence are still poorly understood. Using the complete genome sequence of Brucella melitensis, we generated a database of protein-coding open reading frames (ORFs) and constructed an ORFeome library of 3091 Gateway Entry clones, each containing a defined ORF. This first version of the Brucella ORFeome (v1.1) provides the coding sequences in a user-friendly format amenable to high-throughput functional genomic and proteomic experiments, as the ORFs are conveniently transferable from the Entry clones to various Expression vectors by recombinational cloning. The cloning of the Brucella ORFeome v1.1 should help to provide a better understanding of the molecular mechanisms of virulence, including the identification of bacterial protein-protein interactions, but also interactions between bacterial effectors and their host's targets.
- Encefalopatías espongiformes transmisibles: un reto científico para el nuevo siglo(Servicio de Publicaciones de la Universidad de Navarra, 2001) Lopez-Goñi, I. (Ignacio)