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

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

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 BvrR/BvrS 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 Brucell

Development and evaluation of the Galleria mellonella (greater wax moth) infection model to study Brucella host-pathogen interaction

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

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

A study of Brucella inner lipopolysaccharide sections: lipid A and core. Understanding chemical structure with genetics

Brucellosis is a zoonotic disease caused by Brucella. Its lipopolysaccharide (LPS) is a modified Pathogen-Associated Molecular Pattern (PAMP) that plays a major role in virulence since impairs normal recognition by the innate immune system, and delays the Th1-mediated immune response, allowing the bacteria to reach a safe replicative niche. The core and lipid A LPS sections play a crucial role in this strategy. In contrast to most gram-negative bacteria, Brucella lipid A presents a diaminoglucose disaccharide backbone, but its biosynthetic pathway remains unknown. We have identified in its genome the orthologues of gnnA and gnnB, responsible for the synthesis of diaminoglucose in other bacteria. Following a classical protocol, proven to be successful for many years, we were unable to construct mutants in any of these genes and we concluded that they are probably essential for outer membrane stability and Brucella viability. This was confirmed in parallel by a group of collaborators using a Tn-seq that enables straightforward saturating transposon mutagenesis of Brucella and the identification of genes that strongly contribute to the bacterium fitness. Although we were able to purify the proteins encoded by both genes, the assays to prove their enzymatic activity were not conclusive. The core region of Brucella LPS also contributes to its ability to escape from innate immune system and is crucial for virulence. Mutants in glycosyltransferases involved in the synthesis of the core lateral branch not linked to the O-polysaccharide (O-PS) are attenuated, induce a significantly stronger immune response, and are good vaccine candidates against brucellosis. The chemical structure of the Brucella LPS core, recently elucidated, suggests that, in addition to the already identified WadB and WadC, other glycosyltransferases could be implicated in its synthesis. To clarify the genetics of core synthesis is thus crucial for the development of Brucella vaccines. In this work, we analysed B. abortus genome, a species that presents smooth LPS, to find new genes encoding putative glycosyltransferases involved in LPS synthesis. We constructed mutants in a total of 12 identified genes and analysed their LPS structure. Among them, 11 were not implicated in the synthesis of a complete LPS. Moreover, a mucR mutant in B. abortus also presented a LPS similar to that of the parental strain. This is in accordance with the fact that the mucR-reguladed hypothetical glycosyltransferases were neither essential for the synthesis of a complete LPS, nor for interaction with elements of innate immunity or virulence in Brucella. Interestingly, mutant in BAB1_0953 (renamed wadD) lost reactivity against the antibodies that recognize the core section, but kept the O-PS. This suggests that WadD is a new glycosyltransferase adding one or more sugars to the core ramification of Brucella LPS that is not linked to the O-PS. wadD mutants were more sensitive than the parental strain to components of the innate immune system and in vivo studies suggest that WadD plays a role in chronic stages of infection. Since mutants in genes involved in the synthesis of the core lateral branch are attenuated, induce a stronger immune response and protect against brucellosis, modification of these lateral branch has been proposed as a new strategy for the development of brucellosis vaccines. In this work, we have applied this strategy to modify a B. ovis genetically engineered strain able to grow in atmospheric conditions for the development of a B. ovis specific vaccine. We have also clarified an open question and demonstrated that Open Reading Frame (ORF) BMEI0999 // BAB1_0998, situated immediately upstream the O-PS genes wboA and wboB is not required for the synthesis of a smooth LPS in B. melitensis or B. abortus