Immunochemical characterisation of Brucella LPS and its relation to virulence

Sammanfattning: Members of the genus Brucella are facultative intracellular pathogens which infect and produce disease in a wide variety of mammals. Although some factors have been implicated in the virulence of Brucella, the virulence mechanisms of this intracellular pathogen are not known. Brucella lipopolysaccharide (LPS), is both chemically and biologically different as compared to common enteropathogens. The peptidoglycan of Brucella is strongly associated with the outer membrane (OM). The OM proteins of Brucella are strongly associated to the LPS molecule. Brucella LPS is not stabilised by divalent cations, and it contains less amount of Kdo and phosphate. This makes the molecule less negatively charged, and less susceptible to the action of cationic bactericidal peptides. Other lipids that may contribute to the resistance of microbicidal substances are the ornithine-containing lipids, the presence of phosphatidylcholine in the OM of Brucella, the long chain fatty acids in the lipid A, and the native hapten (NH) polysaccharide, located in the outer leaflet of the OM. Immunochemical characterisation of the B. abortus LPS using monoclonal antibodies revealed nine epitopes in the LPS of B. abortus which were located: two in the O-chain; two in the core, three in the lipid A, two in the lipid associated OMP. Using these antibodies we could show that in addition to the heterogeneity in the O-polysaccharide of Brucella, the LPS of the genus is also heterogeneous in the core and lipid A. In contrast to other Gam-negative pathogens, Brucella do not show traditional aggressive virulence mechanisms such as exotoxins, anti-phagocytic capsules, fimbria, flagella or antigenic variation, even though it is highly pathogenic for preferred and accidental host. This virulence is must likely related to the cell envelope. Using well characterised preparations of Brucella LPS and lipid A, we have demonstrated a low activation of oxidative metabolism and Iysozyme release of human neutrophils, as compared to Salmonella LPS and lipid A preparations. These might be the factors contributing to survival and eventual reproduction of Brucella organisms within phagocytic cells. Brucella cells are resistant to polycationic peptides like defensins and polymyxin B, which destabilise the OM and usually have a lethal effect on enterobacterial strains. We have constructed a live model using rough (R)-Brucella-heterologous LPS chimeras as a tool to study the role of the LPS in the protection to cationic peptides and EDTA. This model showed that the resistance of R-Brucella/heterologous LPS chimeras was dependent on the origin of the LPS partitioned in the R-Brucella OM. The permeability of hydrophobic compounds into the OM of the hybrid cells showed similar pattern. Comparing the bactericidal effect and morphological alterations caused by cationic peptides on the natural rough B. ovis and mutant rough B. abortus with smooth B. abortus showed that rough Brucella variants have lower resistance compared to the smooth counterpart. This result supports the hypothesis that the presence of O polysaccharide and NH on the bacterial surface are factors that contribute to the increased OM resistance to the bactericidal action of cationic peptides. On the basis of these and accumulated experiences, it is proposed that the Brucella OM LPS layer is an efficient barrier for withstanding the digestive activities of phagocytes, and therefore this molecule should be considered as a virulence factor.