A study on brucella inner lipopolysaccharide sections: Lipid a and coreUnderstanding chemical structure with genetics

Resumen

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.