Glucose Oxidation to Pyruvate Is Not Essential for Brucella suis Biovar 5 Virulence in the Mouse Model

Brucella species cause brucellosis, a worldwide extended zoonosis. The brucellae are related to free-living and plant-associated alpha 2-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

A Brucella melitensis H38¿wbkF rough mutant protects against Brucella ovis in rams

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

Evolution towards pathogenicity: Comparative analysis between Brucella and a recently isolated Pseudochrobactrum

Póster presentado Online en el XXVIII Congreso Nacional de Microbiología 28 de junio al 2 de julio, 2021 .- https://hal.inrae.fr/hal-03337244/documentLa brucelosis es una de las zoonosis bacterianas más ampliamente distribuidas en el mundo, contribuyendo de forma importante a la pobreza de muchos países en desarrollo. Ninguna de las vacunas actuales proporciona una protección completa, son virulentas para humanos e interfieren en el diagnóstico serológico. Por ello, para mejorar las vacunas actuales, es imprescindible seguirestudiando los mecanismos de virulencia de Brucella, el agente etiológico de la brucelosis. Ésta pertenece a las α-2 Proteobacteria, grupo que incluye desde bacterias ambientales hasta bacterias que han evolucionado a patógenos animales difícilmente detectados por el sistema inmune, como es el caso de Brucella. Para estudiar la evolución que se ha dado hacia la patogenicidad en esta familia y poder determinar mejor así los factores de virulencia de Brucella, es interesante comparar bacterias filogenéticamente próximas que son biológicamente diferentes. Por primera vez, se han aislado bacterias del género Pseudochrobactrum en un huésped natural de Brucella.Pseudochrobactrum es un género filogenéticamente cercano a Brucella que, hasta la fecha, sólo incluye bacterias ambientales. En este trabajo se han caracterizado estos nuevos aislamientos y teniendo en cuenta sus propiedades fisiológicas, su perfil de lípidos polares y ácidos grasos, y el análisis filogenético, se ha propuesto una nueva especie con el nombre de Pseudochrobactrum bovis. Los estudios comparativos con Brucella han revelado diferencias en la composición de la envoltura celular, el contenido genético y virulencia. Por ello, P. bovis puede representar un nuevo modelo para estudiar la evolución de Brucella hacia la virulenciaN

Mutants in the lipopolysaccharide of Brucella ovis are attenuated and protect against B. ovis infection in mice

Incluye 1 fichero de datosBrucella spp. are Gram-negative bacteria that behave as facultative intracellular parasites of a variety of mammals. This genus includes smooth (S) and rough (R) species that carry S and R lipopolysaccharides (LPS), respectively. S-LPS is a virulence factor, and mutants affected in the S-LPS O-polysaccharide (R mutants), core oligosaccharide or both show attenuation. However, B. ovis is naturally R and is virulent in sheep. We studied the role of B. ovis LPS in virulence by mutating the orthologues of wadA, wadB and wadC, three genes known to encode LPS core glycosyltransferases in S brucellae. When mapped with antibodies to outer membrane proteins (Omps) and R-LPS, wadB and wadC mutants displayed defects in LPS structure and outer membrane topology but inactivation of wadA had little or no effect. Consistent with these observations, the wadB and wadC but not the wadA mutants were attenuated in mice. When tested as vaccines, the wadB and wadC mutants protected mice against B. ovis challenge. The results demonstrate that the LPS core is a structure essential for survival in vivo not only of S brucellae but also of a naturally R Brucella pathogenic species, and they confirm our previous hypothesis that the Brucella LPS core is a target for vaccine development. Since vaccine B. melitensis Rev 1 is S and thus interferes in serological testing for S brucellae, wadB mutant represents a candidate vaccine to be evaluated against B. ovis infection of sheep suitable for areas free of B. melitensis.This work was funded by Ministerio de Economía y Competitividad of Spain (reference project AGL2011-30453-C04) and Fundación para la Investigación Médica Aplicada (FIMA). Financial support to PS-L from Ministerio de Economía y Competitividad of Spain (reference BES-2009-015656), AZ-B from Universidad Pública de Navarra, and BSR from CSIC and European Social Fund (Programa JAE-Doc) are also gratefully acknowledged

A Brucella melitensis H38ΔwbkF rough mutant protects against Brucella ovis in rams

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.Publishe