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

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

The Lipopolysaccharide Core of Brucella abortus Acts as a Shield Against Innate Immunity Recognition

Innate immunity recognizes bacterial molecules bearing pathogen-associated molecular patterns to launch inflammatory responses leading to the activation of adaptive immunity. However, the lipopolysaccharide (LPS) of the gram-negative bacterium Brucella lacks a marked pathogen-associated molecular pattern, and it has been postulated that this delays the development of immunity, creating a gap that is critical for the bacterium to reach the intracellular replicative niche. We found that a B. abortus mutant in the wadC gene displayed a disrupted LPS core while keeping both the LPS O-polysaccharide and lipid A. In mice, the wadC mutant induced proinflammatory responses and was attenuated. In addition, it was sensitive to killing by non-immune serum and bactericidal peptides and did not multiply in dendritic cells being targeted to lysosomal compartments. In contrast to wild type B. abortus, the wadC mutant induced dendritic cell maturation and secretion of pro-inflammatory cytokines. All these properties were reproduced by the wadC mutant purified LPS in a TLR4-dependent manner. Moreover, the core-mutated LPS displayed an increased binding to MD-2, the TLR4 co-receptor leading to subsequent increase in intracellular signaling. Here we show that Brucella escapes recognition in early stages of infection by expressing a shield against recognition by innate immunity in its LPS core and identify a novel virulence mechanism in intracellular pathogenic gram-negative bacteria. These results also encourage for an improvement in the generation of novel bacterial vaccines

Effect of polymorphisms in the Slc11a1 coding region on resistance to brucellosis by macrophages in vitro and after challenge in two Bos breeds (Blanco Orejinegro and Zebu)

The resistance/susceptibility of selected cattle breeds to brucellosis was evaluated in an F1 population generated by crossing animals classified as resistant (R) and susceptible (S) (R x R, R x S, S x R, S x S) based on challenges in vitro and in vivo. The association between single nucleotide polymorphisms identified in the coding region of the Slc11a1 gene and resistance/susceptibility was estimated. The trait resistance or susceptibility to brucellosis, evaluated by a challenge in vitro, showed a high heritable component in terms of additive genetic variance (h2 = 0.54 ± 0.11). In addition, there was a significant association (p < 0.05) between the control of bacterial survival and two polymorphisms (a 3'UTR and SNP4 located in exon 10). The antibody response of animals classified as resistant to infection by Brucella abortus differed significantly (p < 0.05) from that of susceptible animals. However, there was no significant association between single nucleotide polymorphisms located in the Slc11a1 gene and the antibody response stimulated by a challenge in vivo

Brucellosis Vaccines: Assessment of Brucella melitensis Lipopolysaccharide Rough Mutants Defective in Core and O-Polysaccharide Synthesis and Export

Background: 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.This work was funded by the European Commission (Research Contract QLK2-CT-2002-00918) and the Ministerio de Ciencia y Tecnología of Spain (Proyecto AGL2004-01162/GAN)

Brucella abortus Uses a Stealthy Strategy to Avoid Activation of the Innate Immune System during the Onset of Infection

To unravel the strategy by which Brucella abortus establishes chronic infections, we explored its early interaction with innate immunity. Methodology/Principal Findings Brucella did not induce proinflammatory responses as demonstrated by the absence of leukocyte recruitment, humoral or cellular blood changes in mice. Brucella hampered neutrophil (PMN) function and PMN depletion did not influence the course of infection. Brucella barely induced proinflammatory cytokines and consumed complement, and was strongly resistant to bactericidal peptides, PMN extracts and serum. Brucella LPS (BrLPS), NH-polysaccharides, cyclic glucans, outer membrane fragments or disrupted bacterial cells displayed low biological activity in mice and cells. The lack of proinflammatory responses was not due to conspicuous inhibitory mechanisms mediated by the invading Brucella or its products. When activated 24 h post-infection macrophages did not kill Brucella, indicating that the replication niche was not fusiogenic with lysosomes. Brucella intracellular replication did not interrupt the cell cycle or caused cytotoxicity in WT, TLR4 and TLR2 knockout cells. TNF-α-induction was TLR4- and TLR2-dependent for live but not for killed B. abortus. However, intracellular replication in TLR4, TLR2 and TLR4/2 knockout cells was not altered and the infection course and anti-Brucella immunity development upon BrLPS injection was unaffected in TLR4 mutant mice. Conclusion/Significance We propose that Brucella has developed a stealth strategy through PAMPs reduction, modification and hiding, ensuring by this manner low stimulatory activity and toxicity for cells. This strategy allows Brucella to reach its replication niche before activation of antimicrobial mechanisms by adaptive immunity. This model is consistent with clinical profiles observed in humans and natural hosts at the onset of infection and could be valid for those intracellular pathogens phylogenetically related to Brucella that also cause long lasting infections

Brucellosis in Sub-Saharan Africa:Current challenges for management, diagnosis and control

Brucellosis is a highly contagious zoonosis caused by bacteria of the genus Brucella and affecting domestic and wild mammals. In this paper, the bacteriological and serological evidence of brucellosis in Sub-Saharan Africa (SSA) and its epidemiological characteristics are discussed. The tools available for the diagnosis and treatment of human brucellosis and for the diagnosis and control of animal brucellosis and their applicability in the context of SSA are presented and gaps identified. These gaps concern mostly the need for simpler and more affordable antimicrobial treatments against human brucellosis, the development of a B. melitensis vaccine that could circumvent the drawbacks of the currently available Rev 1 vaccine, and the investigation of serological diagnostic tests for camel brucellosis and wildlife. Strategies for the implementation of animal vaccination are also discussed.Publishe

Brucellosis as an Emerging Threat in Developing Economies:Lessons from Nigeria

Nigeria is the most populous country in Africa, has a large proportion of the world's poor livestock keepers, and is a hotspot for neglected zoonoses. A review of the 127 accessible publications on brucellosis in Nigeria reveals only scant and fragmented evidence on its spatial and temporal distribution in different epidemiological contexts. The few bacteriological studies conducted demonstrate the existence of Brucella abortus in cattle and sheep, but evidence for B. melitensis in small ruminants is dated and unclear. The bulk of the evidence consists of seroprevalence studies, but test standardization and validation are not always adequately described, and misinterpretations exist with regard to sensitivity and/or specificity and ability to identify the infecting Brucella species. Despite this, early studies suggest that although brucellosis was endemic in extensive nomadic systems, seroprevalence was low, and brucellosis was not perceived as a real burden; recent studies, however, may reflect a changing trend. Concerning human brucellosis, no studies have identified the Brucella species and most reports provide only serological evidence of contact with Brucella in the classical risk groups; some suggest brucellosis misdiagnoses as malaria or other febrile conditions. The investigation of a severe outbreak that occurred in the late 1970s describes the emergence of animal and human disease caused by the settling of previously nomadic populations during the Sahelian drought. There appears to be an increasing risk of re-emergence of brucellosis in sub-Saharan Africa, as a result of the co-existence of pastoralist movements and the increase of intensive management resulting from growing urbanization and food demand. Highly contagious zoonoses like brucellosis pose a threat with far-reaching social and political consequences

Taxonomía, estructura antigénica y características genéticas de Brucella melitensis y Brucella ovis

Las brucelas pertenecen a las -2 Proteobacteria, junto con patógenos y endosimbiontes peri o intracelulares de vegetales o animales. La mayoría de las brucelas poseen dos cromosomas, pero carecen de plásmidos y fagos lisogénicos, lo que, junto con su confinamiento ecológico, explica su uniformidad genética. Aunque controvertida, las especies clásicas (como B. melitensis y B. ovis) tienen validez científica y práctica. Los lípidos de la envoltura de Brucella difieren de los de muchos gram-negativos y se relacionan con la ácido-alcohol resistencia (Stamp). El periplasma contiene proteínas, mureína y glucanos cíclicos. En la membrana externa destacan el lipopolisacárido (LPS), las proteínas de los grupos 2 (porinas Omp2a y 2b) y 3 (Omp31, Omp3A [u Omp25] y Omp3B) y lipoproteínas (Omp19, 16 y 10). El LPS de B. melitensis lleva un polisacárido O con epitopos M (melitensis), A (abortus, sólo en el biotipo 3) y C (comunes a todas las brucelas lisas). Existe, además, un hapteno nativo de estructura semejante, pero no idéntica, al polisacárido O. Ambos polisacáridos muestran inmunorreactividad con los de algunos gram-negativos como Y. enterocolítica O: 9. El LPS de B. ovis no tiene polisacárido O y sí un oligosacárido con epitopos propios y otros compartidos con la parte interna del LPS de B. melitensis y el de algunas -2 Proteobacteria. En B. melitensis, el polisacárido O cubre la superficie; en la de B. ovis, están expuestos los epitopos proteicos. El epitopo C del polisacárido O es inmunodominante en la respuesta serológica frente a B. melitensis pero en B. ovis, tanto el del LPS como las proteínas de membrana externa son importantes. A nivel citoplasmático, no existen diferencias antigénicas significativas entre ambas especies. Las pruebas serológicas difieren en el antígeno reconocido según empleen suspensiones bacterianas (sólo en B. melitensis), o extractos y antígenos purificados (B. melitensis y B. ovis)

Taxonomía, estructura antigénica y características genéticas de Brucella melitensis y Brucella ovis

Las brucelas pertenecen a las -2 Proteobacteria, junto con patógenos y endosimbiontes peri o intracelulares de vegetales o animales. La mayoría de las brucelas poseen dos cromosomas, pero carecen de plásmidos y fagos lisogénicos, lo que, junto con su confinamiento ecológico, explica su uniformidad genética. Aunque controvertida, las especies clásicas (como B. melitensis y B. ovis) tienen validez científica y práctica. Los lípidos de la envoltura de Brucella difieren de los de muchos gram-negativos y se relacionan con la ácido-alcohol resistencia (Stamp). El periplasma contiene proteínas, mureína y glucanos cíclicos. En la membrana externa destacan el lipopolisacárido (LPS), las proteínas de los grupos 2 (porinas Omp2a y 2b) y 3 (Omp31, Omp3A [u Omp25] y Omp3B) y lipoproteínas (Omp19, 16 y 10). El LPS de B. melitensis lleva un polisacárido O con epitopos M (melitensis), A (abortus, sólo en el biotipo 3) y C (comunes a todas las brucelas lisas). Existe, además, un hapteno nativo de estructura semejante, pero no idéntica, al polisacárido O. Ambos polisacáridos muestran inmunorreactividad con los de algunos gram-negativos como Y. enterocolítica O: 9. El LPS de B. ovis no tiene polisacárido O y sí un oligosacárido con epitopos propios y otros compartidos con la parte interna del LPS de B. melitensis y el de algunas -2 Proteobacteria. En B. melitensis, el polisacárido O cubre la superficie; en la de B. ovis, están expuestos los epitopos proteicos. El epitopo C del polisacárido O es inmunodominante en la respuesta serológica frente a B. melitensis pero en B. ovis, tanto el del LPS como las proteínas de membrana externa son importantes. A nivel citoplasmático, no existen diferencias antigénicas significativas entre ambas especies. Las pruebas serológicas difieren en el antígeno reconocido según empleen suspensiones bacterianas (sólo en B. melitensis), o extractos y antígenos purificados (B. melitensis y B. ovis)