Conde-Alvarez, R. (Raquel)
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- 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.
- Disruption of pyruvate phosphate dikinase in Brucella ovis PA CO2-dependent and independent strains generates attenuation in the mouse model(2020) Moriyon, I. (Ignacio); Zuñiga-Ripa, A. (Amaia); Vizcaíno, N. (Nieves); Iriarte-Cilveti, M. (Maite); Pérez-Etayo, L. (Lara); Conde-Alvarez, R. (Raquel)Brucella ovis is a non-zoonotic rough Brucella that causes genital lesions, abortions and increased perinatal mortality in sheep and is responsible for important economic losses worldwide. Research on virulence factors of B. ovis is necessary for deciphering the mechanisms that enable this facultative intracellular pathogen to establish persistent infections and for developing a species-specific vaccine, a need in areas where the cross-protecting ovine smooth B. melitensis Rev1 vaccine is banned. Although several B. ovis virulence factors have been identified, there is little information on its metabolic abilities and their role in virulence. Here, we report that deletion of pyruvate phosphate dikinase (PpdK, catalyzing the bidirectional conversion pyruvate ⇌ phosphoenolpyruvate) in B. ovis PA (virulent and CO2-dependent) impaired growth in vitro. In cell infection experiments, although showing an initial survival higher than that of the parental strain, this ppdK mutant was unable to multiply. Moreover, when inoculated at high doses in mice, it displayed an initial spleen colonization higher than that of the parental strain followed by a marked comparative decrease, an unusual pattern of attenuation in mice. A homologous mutant was also obtained in a B. ovis PA CO2-independent construct previously proposed for developing B. ovis vaccines to solve the problem that CO2-dependence represents for large scale production. This CO2-independent ppdK mutant reproduced the growth defect in vitro and the multiplication/clearance pattern in mouse spleens, and is thus an interesting vaccine candidate for the immunoprophylaxis of B. ovis ovine brucellosis.
- 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
- Phylogenomic insights into brucellaceae: The Pseudochrobactrum algeriensis case(2024) Coscollá, M. (Mireia); Loperena-Barber, M. (Maite); Salvador-Bescós, M. (Miriam); Moriyon, I. (Ignacio); Bengoechea, J.A. (José A.); Pellegrini, J.M. (Joaquín Miguel); Renau-Mínguez, C. (Chantal); Zuñiga-Ripa, A. (Amaia); Ruiz-Rodríguez, P. (Paula); Gorvel, J.P. (Jean Pierre); Elizalde-Bielsa, A. (Aitor); Iriarte-Cilveti, M. (Maite); Lancaster, R. (Rebecca); Conde-Alvarez, R. (Raquel)The genus Pseudochrobactrum encompasses free-living bacteria phylogenetically close to Ochrobactrum opportunistic pathogens and to Brucella, facultative intracellular parasites causing brucellosis, a worldwide-extended and grave zoonosis. Recently, Pseudochrobactrum strains were isolated from Brucella natural hosts on Brucella selective media, potentially causing diagnostic confusions. Strikingly, P. algeriensis was isolated from cattle lymph nodes, organs that are inimical to bacteria. Here, we analyse P. algeriensis potential virulence factors in comparison with Ochrobactrum and Brucella. Consistent with genomic analyses, Western-Blot analyses confirmed that P. algeriensis lacks the ability to synthesize the N-formylperosamine O-polysaccharide characteristic of the lipopolysaccharide (LPS) of smooth Brucella core species. However, unlike other Pseudochrobactrum but similar to some early diverging brucellae, P. algeriensis carries genes potentially synthetizing a rhamnose-based O-polysaccharide LPS. Lipid A analysis by MALDI-TOF demonstrated that P. algeriensis LPS bears a lipid A with a reduced pathogen-associated molecular pattern, a trait shared with Ochrobactrum and Brucella that is essential to generate a highly stable outer membrane and to delay immune activation. Also, although not able to multiply intracellularly in macrophages, the analysis of P. algeriensis cell lipid envelope revealed the presence of large amounts of cationic aminolipids, which may account for the extremely high resistance of P. algeriensis to bactericidal peptides and could favor colonization of mucosae and transient survival in Brucella hosts. However, two traits critical in Brucella pathogenicity are either significantly different (T4SS [VirB]) or absent (erythritol catabolic pathway) in P. algeriensis. This work shows that, while diverging in other characteristics, lipidic envelope features relevant in Brucella pathogenicity are conserved in Brucellaceae. The constant presence of these features strongly suggests that reinforcement of the envelope integrity as an adaptive advantage in soil was maintained in Brucella because of the similarity of some environmental challenges, such as the action of cationic peptide antibiotics and host defense peptides. This information adds knowledge about the evolution of Brucellaceae, and also underlines the taxonomical differences of the three genera compared.
- Modulation of Haemophilus influenzae interaction with hydrophobic molecules by the VacJ/MlaA lipoprotein impacts strongly on its interplay with the airways(Nature, 2018) Garmendia, J. (Junkal); Bengoechea, J.A. (José A.); Fernández-Calvet, A. (Ariadna); Ortiz-de-Solorzano, C. (Carlos); Euba, B. (Begoña); Ramos-Vivas, J. (Jose); Moleres, J. (Javier); Caballero, L. (Lucía); Martí, S. (Sara); Almagro, G. (Goizeder); Yuste, J.E. (José Enrique); Leigh-Bartholomew, T. (Toby); Morales-Urteaga, X. (Xabier); Rodríguez-Arce, I. (Irene); Conde-Alvarez, R. (Raquel)Airway infection by nontypeable Haemophilus influenzae (NTHi) associates to chronic obstructive pulmonary disease (COPD) exacerbation and asthma neutrophilic airway inflammation. Lipids are key inflammatory mediators in these disease conditions and consequently, NTHi may encounter free fatty acids during airway persistence. However, molecular information on the interplay NTHi-free fatty acids is limited, and we lack evidence on the importance of such interaction to infection. Maintenance of the outer membrane lipid asymmetry may play an essential role in NTHi barrier function and interaction with hydrophobic molecules. VacJ/MlaA-MlaBCDEF prevents phospholipid accumulation at the bacterial surface, being the only system involved in maintaining membrane asymmetry identified in NTHi. We assessed the relationship among the NTHi VacJ/MlaA outer membrane lipoprotein, bacterial and exogenous fatty acids, and respiratory infection. The vacJ/mlaA gene inactivation increased NTHi fatty acid and phospholipid global content and fatty acyl specific species, which in turn increased bacterial susceptibility to hydrophobic antimicrobials, decreased NTHi epithelial infection, and increased clearance during pulmonary infection in mice with both normal lung function and emphysema, maybe related to their shared lung fatty acid profiles. Altogether, we provide evidence for VacJ/MlaA as a key bacterial factor modulating NTHi survival at the human airway upon exposure to hydrophobic molecules.
- 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.
- 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.
- Disruption of pyruvate phosphate dikinase in Brucella ovis PA CO2-dependent and independent strains generates attenuation in the mouse model(2020) Moriyon, I. (Ignacio); Zuñiga-Ripa, A. (Amaia); Vizcaíno, N. (Nieves); Iriarte-Cilveti, M. (Maite); Pérez-Etayo, L. (Lara); Conde-Alvarez, R. (Raquel)Brucella ovis is a non-zoonotic rough Brucella that causes genital lesions, abortions and increased perinatal mortal‑ ity in sheep and is responsible for important economic losses worldwide. Research on virulence factors of B. ovis is necessary for deciphering the mechanisms that enable this facultative intracellular pathogen to establish persistent infections and for developing a species-specifc vaccine, a need in areas where the cross-protecting ovine smooth B. melitensis Rev1 vaccine is banned. Although several B. ovis virulence factors have been identifed, there is little information on its metabolic abilities and their role in virulence. Here, we report that deletion of pyruvate phosphate dikinase (PpdK, catalyzing the bidirectional conversion pyruvate ⇌ phosphoenolpyruvate) in B. ovis PA (virulent and CO2-dependent) impaired growth in vitro. In cell infection experiments, although showing an initial survival higher than that of the parental strain, this ppdK mutant was unable to multiply. Moreover, when inoculated at high doses in mice, it displayed an initial spleen colonization higher than that of the parental strain followed by a marked comparative decrease, an unusual pattern of attenuation in mice. A homologous mutant was also obtained in a B. ovis PA CO2-independent construct previously proposed for developing B. ovis vaccines to solve the problem that CO2-dependence represents for large scale production. This CO2-independent ppdK mutant reproduced the growth defect in vitro and the multiplication/clearance pattern in mouse spleens, and is thus an interesting vaccine candidate for the immunoprophylaxis of B. ovis ovine brucellosis.
- A novel gluconeogenic route enables efficient use of erythritol in zoonotic Brucella(2024) Van-Schaftingen, E. (Emile); Moriyon, I. (Ignacio); Veiga-da-Cunha, M. (Maria); Zuñiga-Ripa, A. (Amaia); Lázaro-Antón, L. (Leticia); Elizalde-Bielsa, A. (Aitor); Iriarte-Cilveti, M. (Maite); Letesson, J.J. (Jean Jacques); Conde-Alvarez, R. (Raquel); Chevalier, N. (Nathalie)Brucellosis is a worldwide extended zoonosis caused by pathogens of the genus Brucella. While most B. abortus, B. melitensis, and B. suis biovars grow slowly in complex media, they multiply intensely in livestock genitals and placenta indicating high metabolic capacities. Mutant analyses in vitro and in infection models emphasize that erythritol (abundant in placenta and genitals) is a preferred substrate of brucellae, and suggest hexoses, pentoses, and gluconeogenic substrates use in host cells. While Brucella sugar and erythritol catabolic pathways are known, growth on 3–4 carbon substrates persists in Fbp- and GlpX-deleted mutants, the canonical gluconeogenic fructose 1,6-bisphosphate (F1,6bP) bisphosphatases. Exploiting the prototrophic and fast-growing properties of B. suis biovar 5, we show that gluconeogenesis requires fructose-bisphosphate aldolase (Fba); the existence of a novel broad substrate bisphosphatase (Bbp) active on sedoheptulose 1,7-bisphosphate (S1,7bP), F1,6bP, and other phosphorylated substrates; that Brucella Fbp unexpectedly acts on S1,7bP and F1,6bP; and that, while active in B. abortus and B. melitensis, GlpX is disabled in B. suis biovar 5. Thus, two Fba-dependent reactions (dihydroxyacetone-phosphate + glyceraldehyde 3-phosphate ⇌ F1,6bP; and dihydroxyacetone-phosphate + erythrose 4-phosphate ⇌ S1,7bP) can, respectively, yield fructose 6-phosphate and sedoheptulose 7-phosphate for classical gluconeogenesis and the Pentose Phosphate Shunt (PPS), the latter reaction opening a new gluconeogenic route. Since erythritol generates the PPS-intermediate erythrose 4-phosphate, and the Fba/Fbp-Bbp route predicts sedoheptulose 7-phosphate generation from erythrose 4-phosphate, we re-examined the erythritol connections with PPS. Growth on erythritol required transaldolase or the Fba/Fbp-Bbp pathway, strongly suggesting that Fba/Fbp-Bbp works as a PPS entry for both erythritol and gluconeogenic substrates in Brucella. We propose that, by increasing erythritol channeling into PPS through these peculiar routes, brucellae proliferate in livestock genitals and placenta in the high numbers that cause abortion and infertility, and make brucellosis highly contagious. These findings could be the basis for developing attenuated brucellosis vaccines safer in pregnant animals.
- 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.