BRUCELOSIS:TESTS DIAGNOSTICOS Y VACUNAS DIVA FRENTE A BRUCELLA OVIS Y BRUCELLA SUIS

AGL2014-58795-C4-1-R

Nombre agencia financiadora Ministerio de Economía y Competitividad
Acrónimo agencia financiadora MINECO
Programa Programa Estatal de I+D+I Orientada a los Retos de la Sociedad
Subprograma Todos los retos
Convocatoria Retos Investigación: Proyectos de I+D+I (2014)
Año convocatoria 2014
Unidad de gestión Dirección General de Investigación Científica y Técnica
Centro beneficiario UNIVERSIDAD DE NAVARRA
Centro realización FACULTAD DE MEDICINA
Identificador persistente http://dx.doi.org/10.13039/501100003329

Publicaciones

Found(s) 13 result(s)
Found(s) 2 page(s)

Disruption of pyruvate phosphate dikinase in Brucella ovis PA CO2-dependent and independent strains generates attenuation in the mouse model

Dadun. Depósito Académico Digital de la Universidad de Navarra
  • Vizcaíno, N. (Nieves)
  • Pérez-Etayo, L. (Lara)
  • Conde-Alvarez, R. (Raquel)
  • Iriarte, M. (Maite)
  • Moriyon, I. (Ignacio)
  • Zuñiga-Ripa, A. (Amaia)
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.




Immunomodulatory properties of Brucella melitensis lipopolysaccharide determinants on mouse dendritic cells in vitro and in vivo

Dadun. Depósito Académico Digital de la Universidad de Navarra
  • Zhao, Y. (Yun)
  • Hanniffy, S. (Sean)
  • Arce-Gorvel, V. (Vilma)
  • Conde-Alvarez, R. (Raquel)
  • Oh, S. (SangKon)
  • Moriyon, I. (Ignacio)
  • Mémet, S. (Sylvie)
  • Gorvel, J.P. (Jean Pierre)
The lipopolysaccharide (LPS) is a major virulence factor of Brucella, a facultative intracellular pathogenic Gram-negative bacterium. Brucella LPS exhibits a low toxicity and its atypical structure was postulated to delay the host immune response, favouring the establishment of chronic disease. Here we carried out an in-depth in vitro and in vivo characterisation of the immunomodulatory effects of Brucella LPS on different dendritic cell (DC) subpopulations. By using LPSs from bacteria that share some of Brucella LPS
structural features, we demonstrated that the core component of B. melitensis wild-type (Bm-wt) LPS accounts for the low activation potential of Brucella LPS in mouse GM-CSF-derived (GM-) DCs. Contrary to the accepted dogma considering Brucella LPS a poor TLR4 agonist and DC activator, Bm-wt LPS selectively induced expression of surface activation markers and cytokine secretion from Flt3-Ligandderived (FL-) DCs in a TLR4-dependent manner. It also primed in vitro T cell proliferation by FL-DCs. In contrast, modified LPS with a defective core purified from Brucella carrying a mutated wadC gene (BmwadC), efficiently potentiated mouse and human DC activation and T cell proliferation in vitro. In vivo,
Bm-wt LPS promoted scant activation of splenic DC subsets and limited recruitment of monocyte- DC like cells in the spleen, conversely to Bm-wadC LPS. Bm-wadC live bacteria drove high cytokine secretion levels in sera of infected mice. Altogether, these results illustrate the immunomodulatory properties of Brucella LPS and the enhanced DC activation ability of the wadC mutation with potential for vaccine development targeting Brucella core LPS structure.




Development of attenuated live vaccine candidates against swine brucellosis in a non‑zoonotic B. suis biovar 2 background

Dadun. Depósito Académico Digital de la Universidad de Navarra
  • Aragón-Aranda, B. (Beatriz)
  • de-Miguel, M.J. (María Jesús)
  • Lázaro-Antón, L. (Leticia)
  • Salvador-Bescós, M. (Miriam)
  • Zuñiga-Ripa, A. (Amaia)
  • Moriyon, I. (Ignacio)
  • Iriarte, M. (Maite)
  • Muñoz, P. (Pilar)
  • 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.




The fast-growing Brucella suis Biovar 5 depends on phosphoenolpyruvate carboxykinase and pyruvate phosphate dikinase but not on Fbp and GlpX fructose-1, 6-bisphosphatases or isocitrate lyase for full virulence in laboratory models

Zaguán. Repositorio Digital de la Universidad de Zaragoza
  • Zúñiga-Ripa, A.
  • Barbier, T.
  • Lázaro-Antón, L.
  • de Miguel, M.J.
  • Conde-álvarez, R.
  • Muñoz, P.M.
  • Letesson, J.J.
  • Iriarte, M.
  • Moriyón, I.
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.




Identification of lptA, lpxE, and lpxO, three genes involved in the remodeling of Brucella cell envelope

Zaguán. Repositorio Digital de la Universidad de Zaragoza
  • Conde-álvarez, R.
  • Palacios-Chaves, L.
  • Gil-Ramírez, Y.
  • Salvador-Bescós, M.
  • Bárcena-Varela, M.
  • Aragón-Aranda, B.
  • Martínez-Gómez, E.
  • Zúñiga-Ripa, A.
  • de Miguel, M.J.
  • Bartholomew, T.L.
  • Hanniffy, S.
  • Grilló, M.-J.
  • Vences-Guzmán, M.Á.
  • Bengoechea, J.A.
  • Arce-Gorvel, V.
  • Gorvel, J.-P.
  • Moriyón, I.
  • Iriarte, M.
The brucellae are facultative intracellular bacteria that cause a worldwide extended zoonosis. One of the pathogenicity mechanisms of these bacteria is their ability to avoid rapid recognition by innate immunity because of a reduction of the pathogen-associated molecular pattern (PAMP) of the lipopolysaccharide (LPS), free-lipids, and other envelope molecules. We investigated the Brucella homologs of lptA, lpxE, and lpxO, three genes that in some pathogens encode enzymes that mask the LPS PAMP by upsetting the core-lipid A charge/hydrophobic balance. Brucella lptA, which encodes a putative ethanolamine transferase, carries a frame-shift in B. abortus but not in other Brucella spp. and phylogenetic neighbors like the opportunistic pathogen Ochrobactrum anthropi. Consistent with the genomic evidence, a B. melitensis lptA mutant lacked lipid A-linked ethanolamine and displayed increased sensitivity to polymyxin B (a surrogate of innate immunity bactericidal peptides), while B. abortus carrying B. melitensis lptA displayed increased resistance. Brucella lpxE encodes a putative phosphatase acting on lipid A or on a free-lipid that is highly conserved in all brucellae and O. anthropi. Although we found no evidence of lipid A dephosphorylation, a B. abortus lpxE mutant showed increased polymyxin B sensitivity, suggesting the existence of a hitherto unidentified free-lipid involved in bactericidal peptide resistance. Gene lpxO putatively encoding an acyl hydroxylase carries a frame-shift in all brucellae except B. microti and is intact in O. anthropi. Free-lipid analysis revealed that lpxO corresponded to olsC, the gene coding for the ornithine lipid (OL) acyl hydroxylase active in O. anthropi and B. microti, while B. abortus carrying the olsC of O. anthropi and B. microti synthesized hydroxylated OLs. Interestingly, mutants in lptA, lpxE, or olsC were not attenuated in dendritic cells or mice. This lack of an obvious effect on virulence together with the presence of the intact homolog genes in O. anthropi and B. microti but not in other brucellae suggests that LptA, LpxE, or OL ß-hydroxylase do not significantly alter the PAMP properties of Brucella LPS and free-lipids and are therefore not positively selected during the adaptation to intracellular life.




The CO2-dependence of Brucella ovis and Brucella abortus biovars is caused by defective carbonic anhydrases

Zaguán. Repositorio Digital de la Universidad de Zaragoza
  • Perez-Etayo, L.
  • de Miguel, M.J.
  • Conde-Alvarez, R.
  • Muñoz, P.M.
  • Khames, M.
  • Iriarte, M.
  • Moriyon, I.
  • Zuniga-Ripa, A.
Brucella bacteria cause brucellosis, a major zoonosis whose control requires efficient diagnosis and vaccines. Identification of classical Brucella spp. has traditionally relied on phenotypic characterization, including surface antigens and 5-10% CO2 necessity for growth (CO2-dependence), a trait of Brucella ovis and most Brucella abortus biovars 1-4 strains. Although molecular tests are replacing phenotypic methods, CO2-dependence remains of interest as it conditions isolation and propagation and reflects Brucella metabolism, an area of active research. Here, we investigated the connection of CO2-dependence and carbonic anhydrases (CA), the enzymes catalyzing the hydration of CO2 to the bicarbonate used by anaplerotic and biosynthetic carboxylases. Based on the previous demonstration that B. suis carries two functional CAs (CAI and CAII), we analyzed the CA sequences of CO2-dependent and -independent brucellae and spontaneous mutants. The comparisons strongly suggested that CAII is not functional in CO2-dependent B. abortus and B. ovis, and that a modified CAII sequence explains the CO2-independent phenotype of spontaneous mutants. Then, by mutagenesis and heterologous plasmid complementation and chromosomal insertion we proved that CAI alone is enough to support CO2-independent growth of B. suis in rich media but not of B. abortus in rich media or B. suis in minimal media. Finally, we also found that insertion of a heterologous active CAII into B. ovis reverted the CO2-dependence but did not alter its virulence in the mouse model. These results allow a better understanding of central aspects of Brucella metabolism and, in the case of B. ovis, provide tools for large-scale production of diagnostic antigens and vaccines.




WadD, a New Brucella Lipopolysaccharide Core Glycosyltransferase Identified by Genomic Search and Phenotypic Characterization

Zaguán. Repositorio Digital de la Universidad de Zaragoza
  • Salvador-Bescos, M.
  • Gil-Ramirez, Y.
  • Zuniga-Ripa, A.
  • Martinez-Gomez, E.
  • de Miguel, M.J.
  • Muñoz, P.M.
  • Cloeckaert, A.
  • Zygmunt, M.S.
  • Moriyon, I.
  • Iriarte, M.
  • Conde-Alvarez, R.
Brucellosis, an infectious disease caused by Brucella, is one of the most extended bacterial zoonosis in the world and an important cause of economic losses and human suffering. The lipopolysaccharide (LPS) of Brucella plays a major role in virulence as it impairs normal recognition by the innate immune system and delays the immune response. The LPS core is a branched structure involved in resistance to complement and polycationic peptides, and mutants in glycosyltransferases required for the synthesis of the lateral branch not linked to the O-polysaccharide (O-PS) are attenuated and have been proposed as vaccine candidates. For this reason, the complete understanding of the genes involved in the synthesis of this LPS section is of particular interest. The chemical structure of the Brucella LPS core suggests that, in addition to the already identified WadB and WadC glycosyltransferases, others could be implicated in the synthesis of this lateral branch. To clarify this point, we identified and constructed mutants in 11 ORFs encoding putative glycosyltransferases in B. abortus. Four of these ORFs, regulated by the virulence regulator MucR (involved in LPS synthesis) or the ByrR/ByrS system (implicated in the synthesis of surface components), were not required for the synthesis of a complete LPS neither for virulence or interaction with polycationic peptides and/or complement. Among the other seven ORFs, six seemed not to be required for the synthesis of the core LPS since the corresponding mutants kept the O-PS and reacted as the wild type with polyclonal sera. Interestingly, mutant in ORF BAB1_0953 (renamed wadD) lost reactivity against antibodies that recognize the core section while kept the O-PS. This suggests that WadD is a new glycosyltransferase adding one or more sugars to the core lateral branch. WadD mutants were more sensitive than the parental strain to components of the innate immune system and played a role in chronic stages of infection. These results corroborate and extend previous work indicating that the Brucella LPS core is a branched structure that constitutes a steric impairment preventing the elements of the innate immune system to fight against Brucella.




Comparative performance of lateral flow immunochromatography, iELISA and rose Bengal tests for the diagnosis of cattle, sheep, goat and swine brucellosis

Zaguán. Repositorio Digital de la Universidad de Zaragoza
  • Gusi, Amahyel M.
  • Bertu, Wilson J.
  • Miguel, M. Jesús de
  • Dieste-Pérez, Lucía
  • Smits, Henk L.
  • Ocholi, Reuben A.
  • Blasco, José M.
  • Moriyón, Ignacio
  • Muñoz, Pilar M.
Background Brucellosis is a world-wide extended zoonosis that causes a grave problem in developing economies. Animal vaccination and diagnosis are essential to control brucellosis, and the need for accurate but also simple and low-cost tests that can be implemented in low-infra-structure laboratories has been emphasized. Methodology We evaluated bovine, sheep, goat and swine lateral flow immunochromatography assay kits (LFA), the Rose Bengal test (RBT) and a well-validated protein G indirect ELISA (iELISA) using sera of Brucella culture-positive and unvaccinated brucellosis free livestock. Sera from cattle vaccinated with S19 and RB51 brucellosis vaccines were also tested. Finally, we compared RBT and LFA using sera of white Fulani cattle of unknown bacteriological status from a brucellosis endemic area of Nigeria. Results and conclusions Although differences were not statistically significant, RBT showed the highest values for diagnostic sensitivity/specificity in cattle (LFA, 96.6/98.8; RBT, 98.9/100; and iELISA, 96.6/100) and the iELISA yielded highest values in sheep (LFA, 94.0/100; RBT, 92.0/100; iELISA, 100/100), goats (LFA, 95.7/96.2; RBT, 97.8/100; iELISA, 100/100) and pigs (LFA, 92.3/100; RBT, 92.3/100; iELISA, 100/100). Vaccine S19 administered subcutaneously interfered in all tests but conjunctival application minimized the problem. Although designed not to interfere in serodiagnosis, vaccine RB51 interfered in LFA and iELISA but not in the RBT. We found closely similar apparent prevalence results when testing the Nigerian Fulani cattle by RBT and LFA. Although both RBT and LFA (showing similar diagnostic perfor-mance) are suitable for small laboratories in resource-limited areas, RBT has the advantage that a single reagent is useful in all animal species. Considering these advantages, its low cost and that it is also useful for human brucellosis diagnosis, RBT might be a good choice for resource-limited laboratories.




Rev1 wbdR tagged vaccines against Brucella ovis

Zaguán. Repositorio Digital de la Universidad de Zaragoza
  • Aragón-Aranda, Beatriz
  • De Miguel, María Jesús
  • Martínez-Gómez, Estrella
  • Zúñiga-Ripa, Amaia
  • Salvador-Bescós, Miriam
  • Moriyón, Ignacio
  • Iriarte, Maite
  • Muñoz, Pilar M.
  • Conde-Álvarez, 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.




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

Zaguán. Repositorio Digital de la Universidad de Zaragoza
  • Lazaro-Anton, L.
  • de Miguel, M.J.
  • Barbier, T.
  • Conde-Alvarez, R.
  • Munoz, P.M.
  • Letesson, J.J.
  • Iriarte, M.
  • Moriyon, I.
  • Zuniga-Ripa, A.
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.