Gil Ramírez, Yolanda

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https://academica-e.unavarra.es/handle/2454/49144

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Gil Ramírez

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Yolanda

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Instituto de Agrobiotecnología (IdAB)

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810152

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2011 - 20183
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    PublicationOpen Access
    Identification of lptA, lpxE, and lpxO, three genes involved in the remodeling of Brucella cell envelope
    (Frontiers Media, 2018) Conde Álvarez, Raquel; Palacios Chaves, Leyre; Gil Ramírez, Yolanda; Salvador Bescós, Miriam; Bárcena-Varela, Marina; Aragón Aranda, Beatriz; Martínez Gómez, Estrella; Zúñiga Ripa, Amaia; Miguel López, María Jesús de; Bartholomew, Toby Leigh; Hanniffy, Sean; Grilló Dolset, María Jesús; Vences Guzmán, Miguel Ángel; Bengoechea Alonso, José Antonio; Arce Gorvel, Vilma; Gorvel, Jean-Pierre; Moriyón Uría, Ignacio; Iriarte, Maite; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
    The brucellae are facultative intracellular bacteria that cause a worldwide extended zoonosis. One of the pathogenicity mechanisms of these bacteria is their ability to avoid rapid recognition by innate immunity because of a reduction of the pathogen-associated molecular pattern (PAMP) of the lipopolysaccharide (LPS), free-lipids, and other envelope molecules. We investigated the Brucella homologs of lptA, lpxE, and lpxO, three genes that in some pathogens encode enzymes that mask the LPS PAMP by upsetting the core-lipid A charge/hydrophobic balance. Brucella lptA, which encodes a putative ethanolamine transferase, carries a frame-shift in B. abortus but not in other Brucella spp. and phylogenetic neighbors like the opportunistic pathogen Ochrobactrum anthropi. Consistent with the genomic evidence, a B. melitensis lptA mutant lacked lipid A-linked ethanolamine and displayed increased sensitivity to polymyxin B (a surrogate of innate immunity bactericidal peptides), while B. abortus carrying B. melitensis lptA displayed increased resistance. Brucella lpxE encodes a putative phosphatase acting on lipid A or on a free-lipid that is highly conserved in all brucellae and O. anthropi. Although we found no evidence of lipid A dephosphorylation, a B. abortus lpxE mutant showed increased polymyxin B sensitivity, suggesting the existence of a hitherto unidentified free-lipid involved in bactericidal peptide resistance. Gene lpxO putatively encoding an acyl hydroxylase carries a frame-shift in all brucellae except B. microti and is intact in O. anthropi. Free-lipid analysis revealed that lpxO corresponded to olsC, the gene coding for the ornithine lipid (OL) acyl hydroxylase active in O. anthropi and B. microti, while B. abortus carrying the olsC of O. anthropi and B. microti synthesized hydroxylated OLs. Interestingly, mutants in lptA, lpxE, or olsC were not attenuated in dendritic cells or mice. This lack of an obvious effect on virulence together with the presence of the intact homolog genes in O. anthropi and B. microti but not in other brucellae suggests that LptA, LpxE, or OL β-hydroxylase do not significantly alter the PAMP properties of Brucella LPS and free-lipids and are therefore not positively selected during the adaptation to intracellular life.
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    PublicationOpen Access
    Brucella abortus ornithine lipids are dispensable outer membrane components devoid of a marked pathogen-associated molecular pattern
    (Public Library of Science, 2011) Palacios Chaves, Leyre; Conde Álvarez, Raquel; Gil Ramírez, Yolanda; Zúñiga Ripa, Amaia; Barquero-Calvo, Elías; Chacón Díaz, Carlos; Chaves Olarte, Esteban; Arce Gorvel, Vilma; Gorvel, Jean-Pierre; Moreno, Edgardo; Miguel, María Jesús de; Grilló Dolset, María Jesús; Moriyón Uría, Ignacio; Iriarte, Maite; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
    The brucellae are alpha-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-alpha 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 alpha-Proteobacteria animal pathogens to reduce and eventually eliminate the envelope components susceptible of recognition by innate immunity.
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    PublicationOpen Access
    Mutants in the lipopolysaccharide of Brucella ovis are attenuated and protect against B. ovis infection in mice
    (BioMed Central, 2014) Soler Lloréns, Pedro; Gil Ramírez, Yolanda; Zabalza Baranguá, Ana; Iriarte, Maite; Conde Álvarez, Raquel; Zúñiga Ripa, Amaia; San Román Aberasturi, Beatriz; Zygmunt, Michel; Vizcaíno, Nieves; Cloeckaert, Axel; Grilló Dolset, María Jesús; Moriyón Uría, Ignacio; López Goñi, Ignacio; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
    Brucella spp. are Gram-negative bacteria that behave as facultative intracellular parasites of a variety of mammals. This genus includes smooth (S) and rough (R) species that carry S and R lipopolysaccharides (LPS), respectively. S-LPS is a virulence factor, and mutants affected in the S-LPS O-polysaccharide (R mutants), core oligosaccharide or both show attenuation. However, B. ovis is naturally R and is virulent in sheep. We studied the role of B. ovis LPS in virulence by mutating the orthologues of wadA, wadB and wadC, three genes known to encode LPS core glycosyltransferases in S brucellae. When mapped with antibodies to outer membrane proteins (Omps) and R-LPS, wadB and wadC mutants displayed defects in LPS structure and outer membrane topology but inactivation of wadA had little or no effect. Consistent with these observations, the wadB and wadC but not the wadA mutants were attenuated in mice. When tested as vaccines, the wadB and wadC mutants protected mice against B. ovis challenge. The results demonstrate that the LPS core is a structure essential for survival in vivo not only of S brucellae but also of a naturally R Brucella pathogenic species, and they confirm our previous hypothesis that the Brucella LPS core is a target for vaccine development. Since vaccine B. melitensis Rev 1 is S and thus interferes in serological testing for S brucellae, wadB mutant represents a candidate vaccine to be evaluated against B. ovis infection of sheep suitable for areas free of B. melitensis.