lipid-a and Brucellosis

Brucellaceae are Gram-negative bacteria that cause brucellosis, one of the most distributed worldwide zoonosis, transmitted to humans by contact with either infected animals or their products. The lipopolysaccharide exposed on the cell surface has been intensively studied and is considered a major virulence factor of Brucella. In the last years, structural studies allowed the determination of new structures in the core oligosaccharide and the O-antigen of this lipopolysaccharide. In this work, we have reinvestigated the lipid A structure isolated from B. suis and B. abortus lipopolysaccharides. A detailed study by MALDI-TOF mass spectrometry in the positive and negative ion modes of the lipid A moieties purified from both species was performed. Interestingly, a new feature was detected: the presence of a pyrophosphorylethanolamine residue substituting the backbone. LID-MS/MS analysis of some of the detected ions allowed assurance that the Lipid A structure composed by the diGlcN3N disaccharide, mainly hexa-acylated and penta-acylated, bearing one phosphate and one pyrophosphorylethanolamine residue. Graphical abstract ᅟ.

Topics: Acylation; Brucella abortus; Brucella suis; Brucellosis; Disaccharides; Ethanolamines; Humans; Lipid A; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tandem Mass Spectrometry

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.

Topics: Animals; Bacterial Secretion Systems; Brucella abortus; Brucellosis; Dendritic Cells; Female; Immune Evasion; Immunity, Innate; Inflammation; Lipid A; Lipopolysaccharides; Macrophages; Membrane Proteins; Mice; Mice, Inbred BALB C

In a series of studies in SPF and conventional guinea pigs, various adjuvants (larifan, polyoxidonium-PO, natrium thiosulphate-NT, TNF-β and Ribi adjuvant system-RAS) were evaluated for their ability to enhance immune responses to the live brucellosis vaccine, Brucella abortus strain 82-PS (penicillin-sensitive). Combining adjuvants with S82-PS increased synthesis of antibodies against rough (R) and smooth (S) Brucella antigens. Dynamics and levels of antibodies differed dependent upon the adjuvant. Adjuvants enhanced cell-mediated responses to S82-PS, and phagocytosis by macrophages. Humoral and cellular immune responses stimulated by the adjuvants correlated with increased vaccine protection against experimental challenge. The highest protection was demonstrated by combining TNF-β or PO with S82-PS. Our data demonstrates the potential of adjuvants to improve immunogenic properties of live brucellosis vaccines.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Bacterial; Brucella abortus; Brucella Vaccine; Brucellosis; Cell Proliferation; Cell Wall Skeleton; Cord Factors; Guinea Pigs; Immunity, Cellular; Immunity, Humoral; Lipid A; Lymphotoxin-alpha; Macrophages, Peritoneal; Male; Organic Chemicals; Phagocytosis; Piperazines; Polymers

The two-component BvrS/BvrR system is essential for Brucella abortus virulence. It was shown previously that its dysfunction abrogates expression of some major outer membrane proteins and increases bactericidal peptide sensitivity. Here, we report that BvrS/BvrR mutants have increased surface hydrophobicity and susceptibility to killing by nonimmune serum. The bvrS and bvrR mutant lipopolysaccharides (LPSs) bound more polymyxin B, chimeras constructed with bvrS mutant cells and parental LPS showed augmented polymyxin B resistance, and, conversely, parental cells and bvrS mutant LPS chimeras were more sensitive and displayed polymyxin B-characteristic outer membrane lesions, implicating LPS as being responsible for the phenotype of the BvrS/BvrR mutants. No qualitative or quantitative changes were detected in other envelope and outer membrane components examined: periplasmic beta(1-2) glucans, native hapten polysaccharide, and phospholipids. The LPS of the mutants was similar to parental LPS in O-polysaccharide polymerization and fine structure but showed both increased underacylated lipid A species and higher acyl-chain fluidity that correlated with polymyxin B binding. These lipid A changes did not alter LPS cytokine induction, showing that in contrast to other gram-negative pathogens, recognition by innate immune receptors is not decreased by these changes in LPS structure. Transcription of Brucella genes required for incorporating long acyl chains into lipid A (acpXL and lpxXL) or implicated in lipid A acylation control (bacA) was not affected. We propose that in Brucella the outer membrane homeostasis depends on the functioning of BvrS/BvrR. Accordingly, disruption of BvrS/BvrR damages the outer membrane, thus contributing to the severe attenuation manifested by bvrS and bvrR mutants.

Topics: Acylation; Animals; Antimicrobial Cationic Peptides; Bacterial Proteins; beta-Glucans; Brucella abortus; Brucellosis; Cytokines; Gene Expression Regulation, Bacterial; Haptens; Hydrophobic and Hydrophilic Interactions; Immune Sera; Lipid A; Macrophages; Mice; Mice, Inbred C57BL; Mutation; Phenotype; Phospholipids; Protein Binding; Transcription, Genetic; Virulence

Initial host defense to bacterial infection is executed by innate immunity, and therefore the main goal of this study was to examine the contribution of Toll-like receptors (TLRs) during Brucella abortus infection. CHO reporter cell lines transfected with CD14 and TLRs showed that B. abortus triggers both TLR2 and TLR4. In contrast, lipopolysaccharide (LPS) and lipid A derived from Brucella rough (R) and smooth (S) strains activate CHO cells only through TLR4. Consistently, macrophages from C3H/HePas mice exposed to R and S strains and their LPS produced higher levels of tumor necrosis factor alpha (TNF-alpha) and interleukin-12 compared to C3H/HeJ, a TLR4 mutant mouse. The essential role of TLR4 for induction of proinflammatory cytokines was confirmed with diphosphoryl lipid A from Rhodobacter sphaeroides. Furthermore, to determine the contribution of TLR2 and TLR4 in bacterial clearance, numbers of Brucella were monitored in the spleen of C3H/HeJ, C3H/HePas, TLR2 knockout, and wild-type mice at 1, 3, and 6 weeks following B. abortus infection. Interestingly, murine brucellosis was markedly exacerbated at weeks 3 and 6 after infection in animals that lacked functional TLR4 (C3H/HeJ) compared to C3H/HePas that paralleled the reduced gamma interferon production by this mouse strain. Finally, by mass spectrometry analysis we found dramatic differences on the lipid A profiles of R and S strains. In fact, S lipid A was shown to be more active to trigger TLR4 than R lipid A in CHO cells and more effective in inducing dendritic cell maturation. In conclusion, these results indicate that TLR4 plays a role in resistance to B. abortus infection and that S lipid A has potent adjuvant activity.

Topics: Animals; Brucella abortus; Brucellosis; CHO Cells; Cricetinae; Dendritic Cells; Flow Cytometry; Immunity, Cellular; Lipid A; Lipopolysaccharides; Macrophages; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Receptors, Cell Surface; Signal Transduction; Spleen; Toll-Like Receptor 2; Toll-Like Receptor 4; Toll-Like Receptors

The study compared the immune and protective responses induced in BALB/c mice vaccinated with six salt-extractable periplasmic protein fractions (Brucella cell surface proteins [BCSP]) of Brucella abortus 19 and later challenge exposed with B. abortus 2308. BCSP70 was precipitated with ammonium sulfate at 70% saturation, and BCSP100 was precipitated with ammonium sulfate at 100% saturation by use of supernatant fluid of BCSP70 that had been precipitated with 70% ammonium sulfate. Four subfractions were separated from BCSP100 by anion-exchange high-performance liquid chromatography (HPLC). Monophosphoryl lipid A (MPL) from Salmonella typhimurium Re mutant strain was used as a potential immune response modifier in some vaccines. Reduced or increased numbers of CFU and increased spleen size in the principal groups of mice relative to that of the nonvaccinated control group were considered protectiveness or virulence (survival) criteria. Results indicated that vaccines prepared from BCSP70 and BCSP100 were moderately protective and immunogenic. The subfractions designated BCSP100-A through BCSP100-D purified by anion-exchange HPLC were not protective when MPL was not used as an immune response modifier. However, two subfractions were associated with significant (P < 0.05) increases in CFU per spleen and splenomegaly in vaccinated mice compared with those in nonvaccinated challenge-exposed mice. MPL enhanced protection or was neutral when used with BCSP70, BCSP100, BCSP100-C, and BCSP100-D. Serologic results of an enzyme-linked immunosorbent assay indicated that MPL modulated the immunoglobulin G responses induced by BCSP70, BCSP100, and subfraction BCSP100-B vaccines only. The overall results suggest that certain proteinaceous periplasmic fractions might serve as virulence or survival factors in B. abortus infections.

Topics: Animals; Antibodies, Bacterial; Bacterial Proteins; Bacterial Vaccines; Brucella abortus; Brucellosis; Female; Immunoglobulin G; Immunoglobulin M; Lipid A; Mice; Mice, Inbred BALB C; Organ Size; Spleen; Vaccination