Letesson, J.J. (Jean Jacques)

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    Brucellosis and one health: inherited and future challenges
    (2023) Moriyon, I. (Ignacio); De-Massis, F. (Frabizio); Letesson, J.J. (Jean Jacques); Blasco, J.M. (J. M.); Moreno, E. (Edgardo)
    One Health is the collaborative efforts of multiple disciplines to attain optimal health for people, animals and the environment, a concept that historically owes much to the study of brucellosis, including recent political and ethical considerations. Brucellosis One Health actors include Public Health and Veterinary Services, microbiologists, medical and veterinary practitioners and breeders. Brucellosis awareness, and the correct use of diagnostic, epidemiological and prophylactic tools is essential. In brucellosis, One Health implementation faces inherited and new challenges, some aggravated by global warming and the intensification of breeding to meet growing food demands. In endemic scenarios, disease awareness, stakeholder sensitization/engagement and the need to build breeder trust are unresolved issues, all made difficult by the protean characteristics of this zoonosis. Extended infrastructural weaknesses, often accentuated by geography and climate, are critically important. Capacity-building faces misconceptions derived from an uncritical adoption of control/eradication strategies applied in countries with suitable means, and requires additional reference laboratories in endemic areas. Challenges for One Health implementation include the lack of research in species other than cattle and small ruminants, the need for a safer small ruminant vaccine, the need to fill in the infrastructure gap, the need for realistic capacity-building, the creation of reference laboratories in critical areas, and the stepwise implementation of measures not directly transposed from the so-called developed countries.
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    Generation of the Brucella melitensis ORFeome version 1.1.
    (Cold Spring Habor Laboratory Press, 2004) Lopez-Goñi, I. (Ignacio); Bolle, X. (Xavier) de; Bertin, N. (Nicolas); Moriyon, I. (Ignacio); Lamesch, P. (Philippe); Delroisse, J.M. (Jean Marc); Whatmore, A.M. (Adrian M.); Sangari, F.J. (Félix Javier); Hill, D.E. (David E.); Sequerra, R. (Reynaldo); MacMillan, A.P. (Alastair P.); Vidal, M. (Marc); Hao, T. (Tong); Garcia-Lobo, J.M. (Juan María); Lambert, C. (Christophe); Hallez, R. (Régis); Rual, J. J. (Jean François); Cutler, S.J. (Sally J.); Letesson, J.J. (Jean Jacques); Dupuy, D. (Denis); Vandenhaute, J. (Jean); Doucettte-Stamm, L. (Lynn); Bozak, S. (Stephanie); Dricot, A. (Amélie)
    The bacteria of the Brucella genus are responsible for a worldwide zoonosis called brucellosis. They belong to the alpha-proteobacteria group, as many other bacteria that live in close association with a eukaryotic host. Importantly, the Brucellae are mainly intracellular pathogens, and the molecular mechanisms of their virulence are still poorly understood. Using the complete genome sequence of Brucella melitensis, we generated a database of protein-coding open reading frames (ORFs) and constructed an ORFeome library of 3091 Gateway Entry clones, each containing a defined ORF. This first version of the Brucella ORFeome (v1.1) provides the coding sequences in a user-friendly format amenable to high-throughput functional genomic and proteomic experiments, as the ORFs are conveniently transferable from the Entry clones to various Expression vectors by recombinational cloning. The cloning of the Brucella ORFeome v1.1 should help to provide a better understanding of the molecular mechanisms of virulence, including the identification of bacterial protein-protein interactions, but also interactions between bacterial effectors and their host's targets.
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    DNA polymorphism analysis of Brucella lipopolysaccharide genes reveals marked differences in O-polysaccharide biosynthetic genes between smooth and rough Brucella species and novel species-specific markers
    (BioMed Central, 2009) Cloeckaert, A. (Axel); Moriyon, I. (Ignacio); Zygmunt, M. (Michel); Letesson, J.J. (Jean Jacques); Blasco, J.M. (J. M.)
    Background: The lipopolysaccharide is a major antigen and virulence factor of Brucella, an important bacterial pathogen. In smooth brucellae, lipopolysaccharide is made of lipid A-core oligosaccharide and N-formylperosamine O-polysaccharide. B. ovis and B. canis (rough species) lack the O-polysaccharide. Results: The polymorphism of O-polysaccharide genes wbkE, manA(O-Ag), manB(O-Ag), manC(O-Ag), wbkF and wbkD) and wbo (wboA and wboB), and core genes manB(core) and wa** was analyzed. Although most genes were highly conserved, species- and biovar-specific restriction patterns were found. There were no significant differences in putative N-formylperosamyl transferase genes, suggesting that Brucella A and M serotypes are not related to specific genes. In B. pinnipedialis and B. ceti (both smooth), manB(O-Ag) carried an IS711, confirming its dispensability for perosamine synthesis. Significant differences between smooth and rough species were found in wbkF and wbkD, two adjacent genes putatively related to bactoprenol priming for O-polysaccharide polymerization. B. ovis wbkF carried a frame-shift and B. canis had a long deletion partially encompassing both genes. In smooth brucellae, this region contains two direct repeats suggesting the deletion mechanism. Conclusion: The results define species and biovar markers, confirm the dispensability of manB(O-Ag) for O-polysaccharide synthesis and contribute to explain the lipopolysaccharide structure of rough and smooth Brucella species.
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    The fast-growing Brucella suis Biovar 5 depends on phosphoenolpyruvatecCarboxykinase and pyruvate phosphate dikinase but not on Fbp and GlpX fructose-1,6-bisphosphatases or isocitrate lyase for full virulence in laboratory models
    (NCBI, 2018) Miguel, M.J. (María Jesús) de; Moriyon, I. (Ignacio); Zuñiga-Ripa, A. (Amaia); Muñoz, P. (Pilar); Lázaro-Antón, L. (Leticia); Iriarte-Cilveti, M. (Maite); Letesson, J.J. (Jean Jacques); Raquel; Barbier, T. (Thibault)
    Bacteria of the genus Brucella infect a range of vertebrates causing a worldwide extended zoonosis. The best-characterized brucellae infect domestic livestock, behaving as stealthy facultative intracellular parasites. This stealthiness depends on envelope molecules with reduced pathogen-associated molecular patterns, as revealed by the low lethality and ability to persist in mice of these bacteria. Infected cells are often engorged with brucellae without signs of distress, suggesting that stealthiness could also reflect an adaptation of the parasite metabolism to use local nutrients without harming the cell. To investigate this, we compared key metabolic abilities of Brucella abortus 2308 Wisconsin (2308W), a cattle biovar 1 virulent strain, and B. suis 513, the reference strain of the ancestral biovar 5 found in wild rodents. B. suis 513 used a larger number of C substrates and showed faster growth rates in vitro, two features similar to those of B. microti, a species phylogenomically close to B. suis biovar 5 that infects voles. However, whereas B. microti shows enhanced lethality and reduced persistence in mice, B. suis 513 was similar to B. abortus 2308W in this regard. Mutant analyses showed that B. suis 513 and B. abortus 2308W were similar in that both depend on phosphoenolpyruvate synthesis for virulence but not on the classical gluconeogenic fructose-1,6-bisphosphatases Fbp-GlpX or on isocitrate lyase (AceA). However, B. suis 513 used pyruvate phosphate dikinase (PpdK) and phosphoenolpyruvate carboxykinase (PckA) for phosphoenolpyruvate synthesis in vitro while B. abortus 2308W used only PpdK. Moreover, whereas PpdK dysfunction causes attenuation of B. abortus 2308W in mice, in B. suis, 513 attenuation occurred only in the double PckA-PpdK mutant. Also contrary to what occurs in B. abortus 2308, a B. suis 513 malic enzyme (Mae) mutant was not attenuated, and this independence of Mae and the role of PpdK was confirmed by the lack of attenuation of a double Mae-PckA mutant. Altogether, these results decouple fast growth rates from enhanced mouse lethality in the brucellae and suggest that an Fbp-GlpX-independent gluconeogenic mechanism is ancestral in this group and show differences in central C metabolic steps that may reflect a progressive adaptation to intracellular growth.
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    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.
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    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.
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    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.
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    Brucellosis vaccines: assessment of Brucella melitensis lipopolysaccharide rough mutants defective in core and O-polysaccharide synthesis and export
    (Public Library of Science, 2008) Lopez-Goñi, I. (Ignacio); Ali, T. (Tara); Miguel, M.J. (María Jesús) de; Moriyon, I. (Ignacio); Widmalm, G. (Göran); González-Fernández, D. (David); Zygmunt, M. (Michel); Arce-Gorvel, V. (Vilma); Marin, C.M. (C. M.); Muñoz, P. (Pilar); Gorvel, J.P. (Jean Pierre); Delrue, R.M. (Rose May); Iriarte-Cilveti, M. (Maite); Letesson, J.J. (Jean Jacques); Blasco, J.M. (J. M.); Weintraub, A. (Andrej); Raquel; Grillo, M.J. (María Jesús)
    The brucellae are facultative intracellular bacteria that cause brucellosis, one of the major neglected zoonoses. In endemic areas, vaccination is the only effective way to control this disease. Brucella melitensis Rev 1 is a vaccine effective against the brucellosis of sheep and goat caused by B. melitensis, the commonest source of human infection. However, Rev 1 carries a smooth lipopolysaccharide with an O-polysaccharide that elicits antibodies interfering in serodiagnosis, a major problem in eradication campaigns. Because of this, rough Brucella mutants lacking the O-polysaccharide have been proposed as vaccines. METHODOLOGY/PRINCIPAL FINDINGS: To examine the possibilities of rough vaccines, we screened B. melitensis for lipopolysaccharide genes and obtained mutants representing all main rough phenotypes with regard to core oligosaccharide and O-polysaccharide synthesis and export. Using the mouse model, mutants were classified into four attenuation patterns according to their multiplication and persistence in spleens at different doses. In macrophages, mutants belonging to three of these attenuation patterns reached the Brucella characteristic intracellular niche and multiplied intracellularly, suggesting that they could be suitable vaccine candidates. Virulence patterns, intracellular behavior and lipopolysaccharide defects roughly correlated with the degree of protection afforded by the mutants upon intraperitoneal vaccination of mice. However, when vaccination was applied by the subcutaneous route, only two mutants matched the protection obtained with Rev 1 albeit at doses one thousand fold higher than this reference vaccine. These mutants, which were blocked in O-polysaccharide export and accumulated internal O-polysaccharides, stimulated weak anti-smooth lipopolysaccharide antibodies. CONCLUSIONS/SIGNIFICANCE: The results demonstrate that no rough mutant is equal to Rev 1 in laboratory models and question the notion that rough vaccines are suitable for the control of brucellosis in endemic areas.
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    Glucose Oxidation to Pyruvate Is Not Essential for Brucella suis Biovar 5 Virulence in the Mouse Model
    (2021) Moriyon, I. (Ignacio); Zuñiga-Ripa, A. (Amaia); de-Miguel, M.J. (María Jesús); Muñoz, P. (Pilar); Lázaro-Antón, L. (Leticia); Iriarte-Cilveti, M. (Maite); Letesson, J.J. (Jean Jacques); Conde-Alvarez, R. (Raquel); Barbier, T. (Thibault)
    Brucella species cause brucellosis, a worldwide extended zoonosis. The brucellae are related to free-living and plant-associated α2-Proteobacteria and, since they multiply within host cells, their metabolism probably reflects this adaptation. To investigate this, we used the rodent-associated Brucella suis biovar 5, which in contrast to the ruminant-associated Brucella abortus and Brucella melitensis and other B. suis biovars, is fast-growing and conserves the ancestral Entner-Doudoroff pathway (EDP) present in the plant-associated relatives. We constructed mutants in Edd (glucose-6-phosphate dehydratase; first EDP step), PpdK (pyruvate phosphate dikinase; phosphoenolpyruvate ⇌ pyruvate), and Pyk (pyruvate kinase; phosphoenolpyruvate → pyruvate). In a chemically defined medium with glucose as the only C source, the Edd mutant showed reduced growth rates and the triple Edd-PpdK-Pyk mutant did not grow. Moreover, the triple mutant was also unable to grow on ribose or xylose. Therefore, B. suis biovar 5 sugar catabolism proceeds through both the Pentose Phosphate shunt and EDP, and EDP absence and exclusive use of the shunt could explain at least in part the comparatively reduced growth rates of B. melitensis and B. abortus. The triple Edd-PpdK-Pyk mutant was not attenuated in mice. Thus, although an anabolic use is likely, this suggests that hexose/pentose catabolism to pyruvate is not essential for B. suis biovar 5 multiplication within host cells, a hypothesis consistent with the lack of classical glycolysis in all Brucella species and of EDP in B. melitensis and B. abortus. These results and those of previous works suggest that within cells, the brucellae use mostly 3 and 4 C substrates fed into anaplerotic pathways and only a limited supply of 5 and 6 C sugars, thus favoring the EDP loss observed in some species.