Palacios-Chaves, L. (Leyre)
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- The identification of wadB, a new glycosyltransferase gene, confirms the branched structure and the role in virulence of the lipopolysaccharide core of Brucella abortus(2014) Moriyon, I. (Ignacio); Palacios-Chaves, L. (Leyre); Gil-Ramirez, Y. (Yolanda); Arce-Gorvel, V. (Vilma); Zuñiga-Ripa, A. (Amaia); Hanniffy, S. (Sean); Iriarte-Cilveti, M. (Maite); Raquel; Grillo, M.J. (María Jesús)
- The Phospholipid N-Methyltransferase and Phosphatidylcholine Synthase Pathways and the ChoXWV Choline Uptake System Involved in Phosphatidylcholine Synthesis Are Widely Conserved in Most, but Not All Brucella Species(2021) Salvador-Bescós, M. (Miriam); Moriyon, I. (Ignacio); Palacios-Chaves, L. (Leyre); Sholenkamp, C. (Christian); Zuñiga-Ripa, A. (Amaia); Aragón-Aranda, B. (Beatriz); de-Miguel, M.J. (María Jesús); Muñoz, P. (Pilar); Lázaro-Antón, L. (Leticia); Iriarte-Cilveti, M. (Maite); Vences-Guzmán, M.A. (Miguel Ángel); Conde-Alvarez, R. (Raquel)The brucellae are facultative intracellular bacteria with a cell envelope rich in phosphatidylcholine (PC). PC is abundant in eukaryotes but rare in prokaryotes, and it has been proposed that Brucella uses PC to mimic eukaryotic-like features and avoid innate immune responses in the host. Two PC synthesis pathways are known in prokaryotes: the PmtA-catalyzed trimethylation of phosphatidylethanolamine and the direct linkage of choline to CDP-diacylglycerol catalyzed by the PC synthase Pcs. Previous studies have reported that B. abortus and B. melitensis possess non-functional PmtAs and that PC is synthesized exclusively via Pcs in these strains. A putative choline transporter ChoXWV has also been linked to PC synthesis in B. abortus. Here, we report that Pcs and Pmt pathways are active in B. suis biovar 2 and that a bioinformatics analysis of Brucella genomes suggests that PmtA is only inactivated in B. abortus and B. melitensis strains. We also show that ChoXWV is active in B. suis biovar 2 and conserved in all brucellae except B. canis and B. inopinata. Unexpectedly, the experimentally verified ChoXWV dysfunction in B. canis did not abrogate PC synthesis in a PmtA-deficient mutant, which suggests the presence of an unknown mechanism for obtaining choline for the Pcs pathway in Brucella. We also found that ChoXWV dysfunction did not cause attenuation in B. suis biovar 2. The results of these studies are discussed with respect to the proposed role of PC in Brucella virulence and how differential use of the Pmt and Pcs pathways may influence the interactions of these bacteria with their mammalian hosts.
- The identification of wadB, a new glycosyltransferase gene, confirms the branched structure and the role in virulence of the lipopolysaccharide core of Brucella abortus(2014) Moriyon, I. (Ignacio); Palacios-Chaves, L. (Leyre); Gil-Ramirez, Y. (Yolanda); Arce-Gorvel, V. (Vilma); Zuñiga-Ripa, A. (Amaia); Hanniffy, S. (Sean); Iriarte-Cilveti, M. (Maite); Raquel; Grillo, M.J. (María Jesús)Brucellosis is a worldwide extended zoonosis caused by Brucella spp. These gram-negative bacteria are not readily detected by innate immunity, a virulence-related property largely linked to their surface lipopolysaccharide (LPS). The role of the LPS lipid A and O-polysaccharide in virulence is well known. Moreover, mutation of the glycosyltransferase gene wadC of Brucella abortus, although not affecting O-polysaccharide assembly onto the lipid-A core section causes a core oligosaccharide defect that increases recognition by innate immunity. Here, we report on a second gene (wadB) encoding a LPS core glycosyltransferase not involved in the assembly of the O-polysaccharide-linked core section. As compared to wild-type B. abortus, a wadB mutant was sensitive to bactericidal peptides and non-immune serum, and was attenuated in mice and dendritic cells. These observations show that as WadC, WadB is also involved in the assembly of a branch of Brucella LPS core and support the concept that this LPS section is a virulence-related structure.
- Brucella abortus depends on pyruvate phosphate dikinase and malic enzyme but not on Fbp and GlpX fructose-1,6-bisphosphatases for full virulence in laboratory models(American Society for Microbiology, 2014) Moriyon, I. (Ignacio); Palacios-Chaves, L. (Leyre); Gil-Ramirez, Y. (Yolanda); Zuñiga-Ripa, A. (Amaia); Iriarte-Cilveti, M. (Maite); Letesson, J.J. (Jean Jacques); Martinez-Gomez, E. (Estrella); Raquel; Barbier, T. (Thibault); Grillo, M.J. (María Jesús)The brucellae are the etiological agents of brucellosis, a worldwide-distributed zoonosis. These bacteria are facultative intracellular parasites and thus are able to adjust their metabolism to the extra- and intracellular environments encountered during an infectious cycle. However, this aspect of Brucella biology is imperfectly understood, and the nutrients available in the intracellular niche are unknown. Here, we investigated the central pathways of C metabolism used by Brucella abortus by deleting the putative fructose-1,6-bisphosphatase (fbp and glpX), phosphoenolpyruvate carboxykinase (pckA), pyruvate phosphate dikinase (ppdK), and malic enzyme (mae) genes. In gluconeogenic but not in rich media, growth of ppdK and mae mutants was severely impaired and growth of the double fbp- glpX mutant was reduced. In macrophages, only the ppdK and mae mutants showed reduced multiplication, and studies with the ppdK mutant confirmed that it reached the replicative niche. Similarly, only the ppdK and mae mutants were attenuated in mice, the former being cleared by week 10 and the latter persisting longer than 12 weeks. We also investigated the glyoxylate cycle. Although aceA (isocitrate lyase) promoter activity was enhanced in rich medium, aceA disruption had no effect in vitro or on multiplication in macrophages or mouse spleens. The results suggest that B. abortus grows intracellularly using a limited supply of 6-C (and 5-C) sugars that is compensated by glutamate and possibly other amino acids entering the Krebs cycle without a critical role of the glyoxylate shunt.
- Brucella abortus ornithine lipids are dispensable outer membrane components devoid of a marked pathogen-associated molecular pattern(Public Library of Science, 2011) Chacon-Diaz, C. (Carlos); Miguel, M.J. (María Jesús) de; Moriyon, I. (Ignacio); Barquero-Calvo, E. (Elías); Chaves-Olarte, E. (Esteban); Palacios-Chaves, L. (Leyre); Gil-Ramirez, Y. (Yolanda); Arce-Gorvel, V. (Vilma); Zuñiga-Ripa, A. (Amaia); Gorvel, J.P. (Jean Pierre); Iriarte-Cilveti, M. (Maite); Moreno, E. (Edgardo); Raquel; Grillo, M.J. (María Jesús)The brucellae are α-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-α 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 α-Proteobacteria animal pathogens to reduce and eventually eliminate the envelope components susceptible of recognition by innate immunity.
- Identification and functional analysis of the cyclopropane fatty acid synthase of Brucella abortus(Society for General Microbiology, 2012) Moriyon, I. (Ignacio); Palacios-Chaves, L. (Leyre); Gil-Ramirez, Y. (Yolanda); Zuñiga-Ripa, A. (Amaia); Iriarte-Cilveti, M. (Maite); Gutierrez, A. (Ana); RaquelThe brucellae are facultative intracellular pathogens of mammals that are transmitted by contact with infected animals or contaminated materials. Several major lipidic components of the brucella cell envelope are imperfectly recognized by innate immunity, thus contributing to virulence. These components carry large proportions of acyl chains of lactobacillic acid, a long chain cyclopropane fatty acid (CFA). CFAs result from addition of a methylene group to unsaturated acyl chains and contribute to resistance to acidity, dryness and high osmolarity in many bacteria and to virulence in mycobacteria. We examined the role of lactobacillic acid in Brucella abortus virulence by creating a mutant in ORF BAB1_0476, the putative CFA synthase gene. The mutant did not incorporate [(14)C]methyl groups into lipids, lacked CFAs and synthesized the unsaturated precursors, proving that BAB1_0476 actually encodes a CFA synthase. BAB1_0476 promoter-luxAB fusion studies showed that CFA synthase expression was promoted by acid pH and high osmolarity. The mutant was not attenuated in macrophages or mice, strongly suggesting that CFAs are not essential for B. abortus intracellular life. However, when the mutant was tested under high osmolarity on agar and acid pH, two conditions likely to occur on contaminated materials and fomites, they showed reduced ability to grow or survive. Since CFA synthesis entails high ATP expenses and brucellae produce large proportions of lactobacillic acyl chains, we speculate that the CFA synthase has been conserved because it is useful for survival extracellularly, thus facilitating persistence in contaminated materials and transmission to new hosts.
- The lipopolysaccharide core of Brucella abortus acts as a shield against innate immunity recognition(Public Library of Science, 2012) Chacon-Diaz, C. (Carlos); Brandenburg, K. (Klaus); Moriyon, I. (Ignacio); Barquero-Calvo, E. (Elías); Bengoechea, J.A. (José A.); Chaves-Olarte, E. (Esteban); Palacios-Chaves, L. (Leyre); Mancek-Keber, M. (Mateja); Jerala, R. (Roman); Arce-Gorvel, V. (Vilma); Gorvel, J.P. (Jean Pierre); Iriarte-Cilveti, M. (Maite); Moreno, E. (Edgardo); Martirosyan, A. (Anna); Llobet, E. (Enrique); Raquel; Bargen, K. (Kristine) von; Grillo, M.J. (María Jesús)Innate immunity recognizes bacterial molecules bearing pathogen-associated molecular patterns to launch inflammatory responses leading to the activation of adaptive immunity. However, the lipopolysaccharide (LPS) of the gram-negative bacterium Brucella lacks a marked pathogen-associated molecular pattern, and it has been postulated that this delays the development of immunity, creating a gap that is critical for the bacterium to reach the intracellular replicative niche. We found that a B. abortus mutant in the wadC gene displayed a disrupted LPS core while keeping both the LPS O-polysaccharide and lipid A. In mice, the wadC mutant induced proinflammatory responses and was attenuated. In addition, it was sensitive to killing by non-immune serum and bactericidal peptides and did not multiply in dendritic cells being targeted to lysosomal compartments. In contrast to wild type B. abortus, the wadC mutant induced dendritic cell maturation and secretion of pro-inflammatory cytokines. All these properties were reproduced by the wadC mutant purified LPS in a TLR4-dependent manner. Moreover, the core-mutated LPS displayed an increased binding to MD-2, the TLR4 co-receptor leading to subsequent increase in intracellular signaling. Here we show that Brucella escapes recognition in early stages of infection by expressing a shield against recognition by innate immunity in its LPS core and identify a novel virulence mechanism in intracellular pathogenic gram-negative bacteria. These results also encourage for an improvement in the generation of novel bacterial vaccines.