Aragón-Aranda, B. (Beatriz)
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- Development and evaluation of the Galleria mellonella (greater wax moth) infection model to study Brucella host-pathogen interaction(2023) Loperena-Barber, M. (Maite); Salvador-Bescós, M. (Miriam); Moriyon, I. (Ignacio); Zuñiga-Ripa, A. (Amaia); Aragón-Aranda, B. (Beatriz); Elizalde-Bielsa, A. (Aitor); Conde-Alvarez, R. (Raquel)Brucellosis is a zoonotic disease caused by Gram-negative bacteria of the genus Brucella. These pathogens cause long-lasting infections, a process in which Brucella modifications in the lipopolysaccharide (LPS) and envelope lipids reduce pathogen-associated molecular pattern (PAMP) recognition, thus hampering innate immunity activation. In vivo models are essential to investigate bacterial virulence, mice being the most used model. However, ethical and practical considerations impede their use in high-throughput screening studies. Although lacking the complexity of the mammalian immune system, insects share key-aspects of innate immunity with mammals, and Galleria mellonella has been used increasingly as a model. G. mellonella larvae have been shown useful in virulence analyses, including Gram-negative pathogens like Klebsiella pneumoniae and Legionella pneumophila. To assess its potential to study Brucella virulence, we first evaluated larva survival upon infection with representative Brucella species (i.e.B. abortus 2308W, B. microti CCM4915 and B. suis biovar 2) and mutants in the VirB type-IV secretion system (T4SS) or in the LPS-O-polysaccharide (O-PS). As compared to K.pneumoniae, the Brucella spp. tested induced a delayed and less severe mortality profile consistent with an escape of innate immunity detection. Brucella replication within larvae was affected by the lack of O-PS, which is reminiscent of their attenuation in natural hosts. On the contrary, replication was not affected by T4SS dysfunction and the mutant induced only slightly less mortality (not statistically significant) than its parental strain. We also evaluated G. mellonella to efficiently recognise Brucella and their LPS by quantification of the pro-phenoloxidase system and melanisation activation, using Pseudomonas LPS as a positive control. Among the brucellae, only B. microti LPS triggered an early-melanisation response consistent with the slightly increased endotoxicity of this species in mice. Therefore, G. mellonella represents a tool to screen for potential Brucella factors modulating innate immunity, but its usefulness to investigate other mechanisms relevant in Brucella intracellular life is limited.
- Rev1 wbdR tagged vaccines against Brucella ovis(Springer Science and Business Media LLC, 2019) Miguel, M.J. (María Jesús) de; Salvador-Bescós, M. (Miriam); Moriyon, I. (Ignacio); Zuñiga-Ripa, A. (Amaia); Aragón-Aranda, B. (Beatriz); Muñoz, P. (Pilar); Martinez-Gomez, E. (Estrella); RaquelSheep brucellosis is a worldwide extended disease caused by B. melitensis and B. ovis, two species respectively carrying smooth or rough lipopolysaccharide. Vaccine B. melitensis Rev1 is used against B. melitensis and B. ovis but induces an anti-smooth-lipopolysaccharide response interfering with B. melitensis serodiagnosis, which precludes its use against B. ovis where B. melitensis is absent. In mice, Rev1 deleted in wbkC (Brucella lipopolysaccharide formyltransferase) and carrying wbdR (E. coli acetyl-transferase) triggered antibodies that could be diferentiated from those evoked by wild-type strains, was comparatively attenuated and protected against B. ovis, suggesting its potential as a B. ovis vaccine.
- Corrigendum: genetic and phenotypic characterization of the etiological agent of canine orchiepididymitis smooth brucella sp. BCCN84.3(Frontiers Media SA, 2019) Suárez-Esquivel, M. (Marcela); Cloeckaert, A. (Axel); Chacon-Diaz, C. (Carlos); Moriyon, I. (Ignacio); Gonnet, M. (Mathieu); Víquez-Ruiz, E. (Eunice); Campos, E. (Elena); Zygmunt, M. (Michel); Thomson, N.R. (Nicholas R.); Aragón-Aranda, B. (Beatriz); Muñoz, P. (Pilar); Blasco, J.M. (J. M.); Baker, K.S. (Kate S.); Moreno, E. (Edgardo); Ruíz-Villalobos, N. (Nazareth); Guzman-Verri, C. (Caterina); RaquelIn the original article, there was an error. In the Funding statement is written that MZ was granted with a fellowship from SEP, Universidad de Costa Rica. The correct Initials are MS-E.
- Correction: Rev1 wbdR tagged vaccines against Brucella ovis(Springer Nature, 2020) Salvador-Bescós, M. (Miriam); Moriyon, I. (Ignacio); Zuñiga-Ripa, A. (Amaia); Aragón-Aranda, B. (Beatriz); de-Miguel, M.J. (María Jesús); Muñoz, P. (Pilar); Iriarte-Cilveti, M. (Maite); Martinez-Gomez, E. (Estrella); Conde-Alvarez, R. (Raquel)Correction to: Vet Res (2019) 50:95
- 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.
- Genetic and Phenotypic Characterization of the Etiological Agent of Canine Orchiepididymitis Smooth Brucella sp. BCCN84.3(Frontiers Media SA, 2019) Suárez-Esquivel, M. (Marcela); Cloeckaert, A. (Axel); Chacon-Diaz, C. (Carlos); Moriyon, I. (Ignacio); Gonnet, M. (Mathieu); Víquez-Ruiz, E. (Eunice); Campos, E. (Elena); Zygmunt, M. (Michel); Thomson, N.R. (Nicholas R.); Aragón-Aranda, B. (Beatriz); Muñoz, P. (Pilar); Blasco, J.M. (J. M.); Baker, K.S. (Kate S.); Moreno, E. (Edgardo); Ruíz-Villalobos, N. (Nazareth); Guzman-Verri, C. (Caterina); RaquelMembers of the genus Brucella cluster in two phylogenetic groups: classical and non-classical species. The former group is composed of Brucella species that cause disease in mammals, including humans. A Brucella species, labeled as Brucella sp. BCCN84.3, was isolated from the testes of a Saint Bernard dog suffering orchiepididymitis, in Costa Rica. Following standard microbiological methods, the bacterium was first defined as “Brucella melitensis biovar 2.” Further molecular typing, identified the strain as an atypical “Brucella suis.” Distinctive Brucella sp. BCCN84.3 markers, absent in other Brucella species and strains, were revealed by fatty acid methyl ester analysis, high resolution melting PCR and omp25 and omp2a/omp2b gene diversity. Analysis of multiple loci variable number of tandem repeats and whole genome sequencing demonstrated that this isolate was different from the currently described Brucella species. The smooth Brucella sp. BCCN84.3 clusters together with the classical Brucella clade and displays all the genes required for virulence. Brucella sp. BCCN84.3 is a species nova taxonomical entity displaying pathogenicity; therefore, relevant for differential diagnoses in the context of brucellosis. Considering the debate on the Brucella species concept, there is a need to describe the extant taxonomical entities of these pathogens in order to understand the dispersion and evolution.
- A Brucella melitensis H38ΔwbkF rough mutant protects against Brucella ovis in rams(2022) Salvador-Bescós, M. (Miriam); Moriyon, I. (Ignacio); Zuñiga-Ripa, A. (Amaia); Aragón-Aranda, B. (Beatriz); Vizcaíno, N. (Nieves); de-Miguel, M.J. (María Jesús); Muñoz, P. (Pilar); Andrés-Barranco, S. (Sara); Barberan, M. (Montserrat); Iriarte-Cilveti, M. (Maite); Blasco, J.M. (J. M.); Martinez-Gomez, E. (Estrella); Conde-Alvarez, R. (Raquel)Brucella melitensis and Brucella ovis are gram-negative pathogens of sheep that cause severe economic losses and, although B. ovis is non-zoonotic, B. melitensis is the main cause of human brucellosis. B. melitensis carries a smooth (S) lipopolysaccharide (LPS) with an N-formyl-perosamine O-polysaccharide (O-PS) that is absent in the rough LPS of B. ovis. Their control and eradication require vaccination, but B. melitensis Rev 1, the only vaccine available, triggers anti-O-PS antibodies that interfere in the S-brucellae serodiagnosis. Since eradication and serological surveillance of the zoonotic species are priorities, Rev 1 is banned once B. melitensis is eradicated or where it never existed, hampering B. ovis control and eradication. To develop a B. ovis specific vaccine, we investigated three Brucella live vaccine candidates lacking N-formyl-perosamine O-PS: Bov::CAΔwadB (CO2-independent B. ovis with truncated LPS core oligosaccharide); Rev1::wbdRΔwbkC (carrying N-acetylated O-PS); and H38ΔwbkF (B. melitensis rough mutant with intact LPS core). After confirming their attenuation and protection against B. ovis in mice, were tested in rams for efficacy. H38ΔwbkF yielded similar protection to Rev 1 against B. ovis but Bov::CAΔwadB and Rev1::wbdRΔwbkC conferred no or poor protection, respectively. All H38ΔwbkF vaccinated rams developed a protracted antibody response in ELISA and immunoprecipitation B. ovis diagnostic tests. In contrast, all remained negative in Rose Bengal and complement fixation tests used routinely for B. melitensis diagnosis, though some became positive in S-LPS ELISA owing to LPS core epitope reactivity. Thus, H38ΔwbkF is an interesting candidate for the immunoprophylaxis of B. ovis in B. melitensis-free areas.
- Development of attenuated live vaccine candidates against swine brucellosis in a non‑zoonotic B. suis biovar 2 background(2020) Salvador-Bescós, M. (Miriam); Moriyon, I. (Ignacio); 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); Conde-Alvarez, R. (Raquel)Brucella is a genus of gram-negative bacteria that cause brucellosis. B. abortus and B. melitensis infect domestic rumi‑ nants while B. suis (biovars 1–3) infect swine, and all these bacteria but B. suis biovar 2 are zoonotic. Live attenuated B. abortus S19 and B. melitensis Rev1 are efective vaccines in domestic ruminants, though both can infect humans. How‑ ever, there is no swine brucellosis vaccine. Here, we investigated the potential use as vaccines of B. suis biovar 2 rough (R) lipopolysaccharide (LPS) mutants totally lacking O-chain (Bs2ΔwbkF) or only producing internal O-chain precursors (Bs2Δwzm) and mutants with a smooth (S) LPS defective in the core lateral branch (Bs2ΔwadB and Bs2ΔwadD). We also investigated mutants in the pyruvate phosphate dikinase (Bs2ΔppdK) and phosphoenolpyruvate carboxykinase (Bs2ΔpckA) genes encoding enzymes bridging phosphoenolpyruvate and the tricarboxylic acid cycle. When tested in the OIE mouse model at the recommended R or S vaccine doses (108 and 105 CFU, respectively), CFU/spleen of all LPS mutants were reduced with respect to the wild type and decreased faster for the R than for the S mutants. At those doses, protection against B. suis was similar for Bs2ΔwbkF, Bs2Δwzm, Bs2ΔwadB and the Rev1 control (105 CFU). As described before for B. abortus, B. suis biovar 2 carried a disabled pckA so that a double mutant Bs2ΔppdKΔpckA had the same metabolic phenotype as Bs2ΔppdK and ppdK mutation was enough to generate attenuation. At 105 CFU, Bs2ΔppdK also conferred the same protection as Rev1. As compared to other B. suis vaccine candidates described before, the mutants described here simultaneously carry irreversible deletions easy to identify as vaccine markers, lack antibiotic-resistance markers and were obtained in a non-zoonotic background. Since R vaccines should not elicit antibodies to the S-LPS and wzm mutants carry immunogenic O-chain precursors and did not improve Bs2ΔwbkF, the latter seems a better R vaccine candidate than Bs2Δwzm. However, taking into account that all R vaccines interfere in ELISA and other widely used assays, whether Bs2ΔwbkF is advantageous over Bs2ΔwadB or Bs2ΔppdK requires experi‑ ments in the natural host.
- Rev1 wbdR tagged vaccines against Brucella ovis(2019) Salvador-Bescós, M. (Miriam); Moriyon, I. (Ignacio); Zuñiga-Ripa, A. (Amaia); Aragón-Aranda, B. (Beatriz); de-Miguel, M.J. (María Jesús); Muñoz, P. (Pilar); Iriarte-Cilveti, M. (Maite); Martinez-Gomez, E. (Estrella); Conde-Alvarez, R. (Raquel)Sheep brucellosis is a worldwide extended disease caused by B. melitensis and B. ovis, two species respectively carrying smooth or rough lipopolysaccharide. Vaccine B. melitensis Rev1 is used against B. melitensis and B. ovis but induces an anti-smooth-lipopolysaccharide response interfering with B. melitensis serodiagnosis, which precludes its use against B. ovis where B. melitensis is absent. In mice, Rev1 deleted in wbkC (Brucella lipopolysaccharide formyl-transferase) and carrying wbdR (E. coli acetyl-transferase) triggered antibodies that could be differentiated from those evoked by wild-type strains, was comparatively attenuated and protected against B. ovis, suggesting its potential as a B. ovis vaccine.