Barquero-Calvo, E. (Elías)
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- Brucella abortus S19 GFP-tagged vaccine allows the serological identification of vaccinated cattle(2021) Hernández-Mora, G. (Gabriela); Chacon-Diaz, C. (Carlos); Blasco, J.M. (José María); San-Roman, B. (Beatriz); Barquero-Calvo, E. (Elías); Chaves-Olarte, E. (Esteban); Salas-Alfaro, D. (Dariana); Iriarte-Cilveti, M. (Maite); Moreno, E. (Edgardo); Zabalza-Barangua, A. (Ana); Guzman-Verri, C. (Caterina); Grillo, M.J. (María Jesús)Bovine brucellosis induces abortion in cows, produces important economic losses, and causes a widely distributed zoonosis. Its eradication was achieved in several countries after sustained vaccination with the live attenuated Brucella abortus S19 vaccine, in combination with the slaughtering of serologically positive animals. S19 induces antibodies against the smooth lipopolysaccharide (S-LPS), making difficult the differentiation of infected from vaccinated bovines. We developed an S19 strain constitutively expressing the green fluorescent protein (S19-GFP) coded in chromosome II. The S19-GFP displays similar biological characteristics and immunogenic and protective efficacies in mice to the parental S19 strain. S19-GFP can be distinguished from S19 and B. abortus field strains by fluorescence and multiplex PCR. Twenty-five heifers were vaccinated withS19-GFP (5×109 CFU) by the subcutaneous or conjunctival routes and some boosted with GFP seven weeks thereafter. Immunized animals were followed up for over three years and tested for anti-S-LPS antibodies by both the Rose Bengal test and a competitive ELISA. Anti-GFP antibodies were detected by an indirect ELISA and Western blotting. In most cases, anti-S-LPS antibodies preceded for several weeks those against GFP. The anti-GFP antibody response was higher in the GFP boosted than in the non-boosted animals. In all cases, the anti-GFP antibodies persisted longer, or at least as long, as those against S-LPS. The drawbacks and potential advantages of using the S19-GFP vaccine for identifying vaccinated animals in infected environments are discussed.
- The differential interaction of Brucella and Ochrobactrum with innate immunity reveals traits related to the evolution of stealthy pathogens(Public Library of Science, 2009) Chacon-Diaz, C. (Carlos); Moriyon, I. (Ignacio); Barquero-Calvo, E. (Elías); Chaves-Olarte, E. (Esteban); Quesada-Lobo, L. (Lucía); Mancek-Keber, M. (Mateja); Jerala, R. (Roman); Gorvel, J.P. (Jean Pierre); Iriarte-Cilveti, M. (Maite); Moreno, E. (Edgardo); Martirosyan, A. (Anna); Guzman-Verri, C. (Caterina); RaquelBackground: During evolution, innate immunity has been tuned to recognize pathogen-associated molecular patterns. However, some alpha-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-kappaB 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 alpha-Proteobacteria like Bartonella, Rickettsia, Anaplasma, Ehrlichia and Wolbachia.
- BvrR/BvrS-controlled outer membrane proteins Omp3a and Omp3b are not essential for Brucella abortus virulence(American Society for Microbiology, 2007) Lopez-Goñi, I. (Ignacio); Miguel, M.J. (María Jesús) de; Moriyon, I. (Ignacio); Barquero-Calvo, E. (Elías); Chaves-Olarte, E. (Esteban); Moreno, E. (Edgardo); Guzman-Verri, C. (Caterina); Manterola, L. (Lorea); Grillo, M.J. (María Jesús)The Brucella abortus two-component regulatory system BvrR/BvrS controls the expression of outer membrane proteins (Omp) Omp3a (Omp25) and Omp3b (Omp22). Disruption of bvrS or bvrR generates avirulent mutants with altered cell permeability, higher sensitivity to microbicidal peptides, and complement. Consequently, the role of Omp3a and Omp3b in virulence was examined. Similar to bvrS or bvrR mutants, omp3a and omp3b mutants displayed increased attachment to cells, indicating surface alterations. However, they showed unaltered permeability; normal expression of Omp10, Omp16, Omp19, Omp2b, and Omp1; native hapten polysaccharide; and lipopolysaccharide and were resistant to complement and polymyxin B at ranges similar to those of the wild-type (WT) counterpart. Likewise, omp3a and omp3b mutants were able to replicate in murine macrophages and in HeLa cells, were resistant to the killing action of human neutrophils, and persisted in mice, like the WT strain. Murine macrophages infected with the omp3a mutant generated slightly higher levels of tumor necrosis factor alpha than the WT, whereas the bvrS mutant induced lower levels of this cytokine. Since the absence of Omp3a or Omp3b does not result in attenuation, it can be concluded that BvrR/BvrS influences additional Brucella properties involved in virulence. Our results are discussed in the light of previous works suggesting that disruption of omp3a generates attenuated Brucella strains, and we speculate on the role of group 3 Omps.
- Brucella abortus uses a stealthy strategy to avoid activation of the innate immune system during the onset of infection(Public Library of Science, 2007) Chacon-Diaz, C. (Carlos); Moriyon, I. (Ignacio); Barquero-Calvo, E. (Elías); Chaves-Olarte, E. (Esteban); Weiss, D.S. (David S.); Moreno, E. (Edgardo); Rucavado, A. (Alexandra); Guzman-Verri, C. (Caterina)To unravel the strategy by which Brucella abortus establishes chronic infections, we explored its early interaction with innate immunity. Methodology/Principal Findings Brucella did not induce proinflammatory responses as demonstrated by the absence of leukocyte recruitment, humoral or cellular blood changes in mice. Brucella hampered neutrophil (PMN) function and PMN depletion did not influence the course of infection. Brucella barely induced proinflammatory cytokines and consumed complement, and was strongly resistant to bactericidal peptides, PMN extracts and serum. Brucella LPS (BrLPS), NH-polysaccharides, cyclic glucans, outer membrane fragments or disrupted bacterial cells displayed low biological activity in mice and cells. The lack of proinflammatory responses was not due to conspicuous inhibitory mechanisms mediated by the invading Brucella or its products. When activated 24 h post-infection macrophages did not kill Brucella, indicating that the replication niche was not fusiogenic with lysosomes. Brucella intracellular replication did not interrupt the cell cycle or caused cytotoxicity in WT, TLR4 and TLR2 knockout cells. TNF-α-induction was TLR4- and TLR2-dependent for live but not for killed B. abortus. However, intracellular replication in TLR4, TLR2 and TLR4/2 knockout cells was not altered and the infection course and anti-Brucella immunity development upon BrLPS injection was unaffected in TLR4 mutant mice. Conclusion/Significance We propose that Brucella has developed a stealth strategy through PAMPs reduction, modification and hiding, ensuring by this manner low stimulatory activity and toxicity for cells. This strategy allows Brucella to reach its replication niche before activation of antimicrobial mechanisms by adaptive immunity. This model is consistent with clinical profiles observed in humans and natural hosts at the onset of infection and could be valid for those intracellular pathogens phylogenetically related to Brucella that also cause long lasting infections.
- If You're Not Confused, You're Not Paying Attention: Ochrobactrum Is Not Brucella(2023) Bertu, W.J. (Wilson J.); Güler, L. (Leyla); Caswell, C.C. (Clayton C.); Araj, G.F. (George F.); Suárez-Esquivel, M. (Marcela); Lopez-Goñi, I. (Ignacio); Al-Dahouk, S. (Sascha); Roop, M. (Martin); Pembroke, J.T. (J. Tony); Chacon-Diaz, C. (Carlos); Middlebrook, E.A. (Edgar A.); Blasco, J.M. (José María); Loperena-Barber, M. (Maite); Keriel, A. (Anne); Salvador-Bescós, M. (Miriam); Dadar, M. (Maryam); O'Callaghan, D. (David); Moriyon, I. (Ignacio); De-Massis, F. (Frabizio); Altamirano-Silva, P. (Pamela); Barquero-Calvo, E. (Elías); Chaves-Olarte, E. (Esteban); Neubauer, H. (Heinrich); Whatmore, A.M. (Adrian M.); Wareth, G. (Gamal); De-Lima-Santos, R. (Renato); Arenas-Gamboa, A. (Ángela); Welburn, S.C. (Susan C.); Godfroid, J. (Jacques); Diaz, R. (Ramón); Splitter, G. (Gary); Garin-Bastuji, B. (B.); Gusi, A.M. (Amahyel M.); Sangari, F.J. (Félix Javier); Melzer, F. (Falk); Comerci, D.J. (Diego J.); Salcedo, S.P. (Suzana P.); Arce-Gorvel, V. (Vilma); Zuñiga-Ripa, A. (Amaia); Vizcaíno, N. (Nieves); Ruiz-Villalonos, N. (Nazaret); Erdenlig-Gürbilek, S. (Sevil); Muñoz, P. (Pilar); Tsolis, R.M. (Renee M.); Mora-Cartin, R. (Ricardo); Gorvel, J.P. (Jean Pierre); Ryan, M.P. (Michael P.); Iriarte-Cilveti, M. (Maite); Seimenis, A. (Aristarchos); Tabbaa, D. (Darem); Khames, M. (Mammar); Cravero, S. (Silvio); Celli, J. (Jean); Moran-Gilad, J. (Jacob); Bosilkovski, M. (Mile); Letesson, J.J. (Jean Jacques); Cook, E. (Elizabeth); Oñate-Landa, A.(A.); Moreno, E. (Edgardo); Ariza, J. (J.); Pandey, P. (Piyush); Escobar, G.I. (Gabriela I.); McGiven, J. (John); Guzman-Verri, C. (Caterina); Trangoni, M.D. (Marcos David); Pappas, G. (Georgios); Köhler, S. (Stephan); Foster, J.T. (Jeffrey T.); De-Boelle, X. (Xavier); Hernández-Mora, G. (Gabriela); Conde-Alvarez, R. (Raquel); Cadmus, S. (Simeon); Battelli, G. (Giorgio); Ficht, T.A. (Thomas A.); Hai, J. (Jiang); Jacob, N.R. (Nestor R.); Ocholi, R.A. (Reuben A.); Fernandez-Lago, L. (Luis)Bacteria of the genus Brucella are facultative intracellular parasites that cause brucellosis, a severe animal and human disease. Recently, a group of taxonomists merged the brucellae with the primarily free-living, phylogenetically related Ochrobactrum spp. in the genus Brucella. This change, founded only on global genomic analysis and the fortuitous isolation of some opportunistic Ochrobactrum spp. from medically compromised patients, has been automatically included in culture collections and databases. We argue that clinical and environmental microbiologists should not accept this nomenclature, and we advise against its use because (i) it was presented without in-depth phylogenetic analyses and did not consider alternative taxonomic solutions; (ii) it was launched without the input of experts in brucellosis or Ochrobactrum; (iii) it applies a non-consensus genus concept that disregards taxonomically relevant differences in structure, physiology, population structure, core-pangenome assemblies, genome structure, genomic traits, clinical features, treatment, prevention, diagnosis, genus description rules, and, above all, pathogenicity; and (iv) placing these two bacterial groups in the same genus creates risks for veterinarians, medical doctors, clinical laboratories, health authorities, and legislators who deal with brucellosis, a disease that is particularly relevant in low- and middle-income countries. Based on all this information, we urge microbiologists, bacterial collections, genomic databases, journals, and public health boards to keep the Brucella and Ochrobactrum genera separate to avoid further bewilderment and harm.
- 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.
- 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.