O-chain expression in the rough Brucella melitensis strain B115: induction of O-polysaccharide-specific monoclonal antibodies and intracellular localization demonstrated by immunoelectron microscopy.

Spleen cells from mice infected with the rough Brucella melitensis strain B115 were fused with NSO myeloma cells. Hybridoma supernatants were screened in ELISA with cell walls (CW), sonicated cell extracts (CE) and rough lipopolysaccharide (R-LPS) of B. melitensis strain B115 and whole B. melitensis B115 cells. Surprisingly, 22 monoclonal antibodies (mAbs) reacting in ELISA with both CW and CE but not with R-LPS and bacterial cells were shown by immunoblot analysis and ELISA to react with smooth lipopolysaccharide (S-LPS). These mAbs also reacted in ELISA with O polysaccharides (OPS) from the smooth Brucella abortus strain 99 and the smooth B. melitensis strain 16M and thus recognize epitopes present on the O-chain. Proteinase K LPS preparations from B. melitensis B115 analysed by immunoblotting with one mAb (12G12) recognizing S-LPS of both A and M specificity displayed the typical S-LPS high-molecular-mass ladder pattern but no S-LPS was detected in the phenol/water/chloroform/light petroleum LPS preparation of the same strain. mAb 12G12, specific for S-LPS, and a mAb (A68/03F03/D05) specific for R-LPS were used to localize the O-chain and R-LPS expressed in B. melitensis strain B115 by immunoelectron microscopy. Immunogold labelling was observed at the surface of B. melitensis B115 cells with the anti-R-LPS mAb but not with the anti-S-LPS mAb. In ultrathin sections, immunogold labelling with the S-LPS specific mAb was observed in the cytoplasm and in the periphery of the cytoplasm, probably at the cytoplasmic membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of Brucella abortus and Brucella melitensis native haptens as outer membrane O-type polysaccharides independent from the smooth lipopolysaccharide.

Brucella native haptens (NHs) extracted with hot water from smooth (S)-type B. abortus and B. melitensis were purified to high levels of serological activity and compared with the polysaccharide obtained by acid hydrolysis (PS) of the S lipopolysaccharide (S-LPS). By 13C nuclear magnetic resonance analysis, NHs showed the spectrum of a homopolymer of alpha-1,2- or alpha-1,2- plus alpha-1,3-linked 4-formamido-4,6-dideoxy-D-mannose (N-formylperosamine) previously reported for the LPS O chain. However, while PS contained up to 0.6% 3-deoxy-D-manno-2-octulosonate, this LPS-core marker was absent from NH. High performance liquid chromatography and thin-layer chromatography showed heterogeneity in NH purified from whole cells but not in PS. By immunoprecipitation, polysaccharides indistinguishable from NH were demonstrated in extracts obtained with phenol-water, saline at 60 degrees C, and ether-water treatments, and none of these treatments caused S-LPS hydrolysis detectable with antibodies to the O chain and lipid A. Two lines of evidence showed that NH was in the cell surface. First, NH became biotinylated when B. abortus live cells were labelled with biotin-hydrazide, and the examination of cell fractions and electron microscopy sections with streptavidin-peroxidase and streptavidin-coloidal gold, respectively, showed that labelling was extrinsic. Moreover, whereas only traces of NH were found in cytosols, the amount of NH was enriched in cell envelopes and in the outer membrane blebs spontaneously released by brucellae during growth. Interactions between NH and S-LPS were observed in crude cell extracts, and such interactions could be reconstituted by using purified NH and LPS. The results demonstrate that NH is not a hydrolytic product of S-LPS and suggest a model in which LPS-independent O-type polysaccharides (NH) are intertwined with the O chain in the outer membrane of S-type brucellae.     

Interactions of lactoferricin-derived peptides with LPS and antimicrobial activity

Synthetic peptides derived from human and bovine lactoferricin, as well as tritrpticin sequences, were assayed for antimicrobial activity against wild-type Escherichia coli and LPS mutant strains. Antimicrobial activity was only obtained with peptides derived from the bovine lactoferricin sequence and peptides corresponding to chimeras of human and bovine sequences. None of the peptides corresponding to different regions of native human lactoferricin showed any antimicrobial activity. The results underline the importance of the content of tryptophan and arginine residues, and the relative location of these residues for antimicrobial activity. Results obtained for the same assays performed with LPS mutants suggest that lipid A is not the main binding site for lactoferricin which interacts first with the negative charges present in the inner core. Computer modelling of the most active peptides led to a model in which positively charged residues of the cationic peptide interact with negative charges carried by the LPS to disorganise the structure of the outer membrane and facilitate the approach of tryptophan residues to the lipid A in order to promote hydrophobic interactions.     

Lysosomal accumulation and recycling of lipopolysaccharide to the cell surface of murine macrophages, an in vitro and in vivo study.

In this study, we detailed in a time-dependent manner the trafficking, the recycling, and the structural fate of Brucella abortus LPS in murine peritoneal macrophages by immunofluorescence, ELISA, and biochemical analyses. The intracellular pathway of B. abortus LPS, a nonclassical endotoxin, was investigated both in vivo after LPS injection in the peritoneal cavity of mice and in vitro after LPS incubation with macrophages. We also followed LPS trafficking after infection of macrophages with B. abortus strain 19. After binding to the cell surface and internalization, Brucella LPS is routed from early endosomes to lysosomes with unusual slow kinetics. It accumulates there for at least 24 h. Later, LPS leaves lysosomes and reaches the macrophage cell surface. This recycling pathway is also observed for LPS released by Brucella S19 following in vitro infection. Indeed, by 72 h postinfection, bacteria are degraded by macrophages and LPS is located inside lysosomes dispersed at the cell periphery. From 72 h onward, LPS is gradually detected at the plasma membrane. In each case, the LPS present at the cell surface is found in large clusters with the O-chain facing the extracellular medium. Both the antigenicity and heterogenicity of the O-chain moiety are preserved during the intracellular trafficking. We demonstrate that LPS is not cleared by macrophages either in vitro or in vivo after 3 mo, exposing its immunogenic moiety toward the extracellular medium.     

Structure and properties of the outer membranes of Brucella abortus and Brucella melitensis.

The brucellae are Gram-negative bacteria characteristically able to multiply facultatively within phagocytic cells and which cause a zoonosis of world-wide importance. This article reviews the structure and topology of the main components (lipopolysaccharide, native hapten polysaccharide, free lipids and proteins) of the outer membranes of Brucella abortus and B. melitensis, as well as some distinctive properties (permeability and interactions with cationic peptides) of these membranes. On these data, an outer membrane model is proposed in which, as compared to other Gram-negatives, there is a stronger hydrophobic anchorage for the lipopolysaccharide, free lipids, porin proteins and lipoproteins, and a reduced surface density of anionic groups, which could be partially or totally neutralized by ornithine lipids. This model accounts for the permeability of Brucella to hydrophobic permeants and for its resistance to the bactericidal oxygen-independent systems of phagocytes.     

Cationic antimicrobial peptides activate a two-component regulatory system, PmrA-PmrB, that regulates resistance to polymyxin B and cationic antimicrobial peptides in Pseudomonas aeruginosa

The two-component regulatory system PhoP-PhoQ of Pseudomonas aeruginosa regulates resistance to cationic antimicrobial peptides, polymyxin B and aminoglycosides in response to low Mg2+ conditions. We have identified a second two-component regulatory system, PmrA-PmrB, that regulates resistance to polymyxin B and cationic antimicrobial peptides. This system responds to limiting Mg2+, and is affected by a phoQ, but not a phoP mutation. Inactivation of the pmrB sensor kinase and pmrA response regulator greatly decreased the expression of the operon encoding pmrA-pmrB while expression of the response regulator pmrA in trans resulted in increased activation suggesting that the pmrA-pmrB operon is autoregulated. Interposon mutants in pmrB, pmrA, or in an intergenic region upstream of pmrA-pmrB exhibited two to 16-fold increased susceptibility to polymyxin B and cationic antimicrobial peptides. The pmrA-pmrB operon was also found to be activated by a number of cationic peptides including polymyxins B and E, cattle indolicidin and synthetic variants as well as LL-37, a component of human innate immunity, whereas peptides with the lowest minimum inhibitory concentrations tended to be the weakest inducers. Additionally, we showed that the putative LPS modification operon, PA3552-PA3559, was also induced by cationic peptides, but its expression was only partially dependent on the PmrA-PmrB system. The discovery that the PmrA-PmrB two-component system regulates resistance to cationic peptides and that both it and the putative LPS modification system are induced by cationic antimicrobial peptides has major implications for the development of these antibiotics as a therapy for P. aeruginosa infections.     

Characterization of Brucella abortus lipopolysaccharide macrodomains as mega rafts

The lipopolysaccharides (LPS) of intracellular Proteobacteria such as Brucella, Chlamydia, Legionella and Rickettsia, have properties distinct from enterobacterial LPSs. These properties include deficient LPS induction of host cell activation, low endotoxicity and resistance to macrophage degradation. Together these constitute key virulence mechanisms for intracellular survival and replication. We previously demonstrated that B. abortus LPS captured by macrophages was recycled back to the plasma membrane where it was found associated with macrodomains. Furthermore, this LPS interferes with the MHC class II (MHC-II) presentation of peptides to specific T cell hybridomas. Here, we characterized the Brucella LPS macrodomains by microscopy and biochemistry approaches. We show for the first time that LPS macrodomains act as detergent resistant membranes (DRMs), segregating several lipid-raft components, LPS-binding proteins and MHC-II molecules. Brucella LPS macrodomains remain intact for several months in macrophages and are resistant to the disruptive effects of methyl beta-cyclodextrin. Fluorescent anisotropy measurements show that B. abortus LPS is responsible for the formation of rigid surface membrane complexes. In addition, relocalization of MHC-II molecules is observed in these structures. The effects of B. abortus LPS on membrane properties could be responsible for pathogenic effects such as the inhibition of MHC-II-dependent antigen presentation.     

Genomic island 2 is an unstable genetic element contributing to Brucella lipopolysaccharide spontaneous smooth-to-rough dissociation

Brucella is a Gram-negative bacterium that causes a worldwide-distributed zoonosis. The genus includes smooth (S) and rough (R) species that differ in the presence or absence, respectively, of the O-polysaccharide of lipopolysaccharide. In S brucellae, the O-polysaccharide is a critical diagnostic antigen and a virulence determinant. However, S brucellae spontaneously dissociate into R forms, a problem in antigen and S vaccine production. Spontaneous R mutants of Brucella abortus, Brucella melitensis, and Brucella suis carried the chromosomal scar corresponding to genomic island 2 (GI-2) excision, an event causing the loss of the wboA and wboB O-polysaccharide genes, and the predicted excised circular intermediate was identified in B. abortus, B. melitensis, and B. suis cultures. Moreover, disruption of a putative phage integrase gene in B. abortus GI-2 caused a reduction in O-polysaccharide loss rates under conditions promoting S-R dissociation. However, spontaneous R mutants not carrying the GI-2 scar were also detected. These results demonstrate that the phage integrase-related GI-2 excision is a cause of S-R brucella dissociation and that other undescribed mechanisms must also be involved. In the R Brucella species, previous works have shown that Brucella ovis but not Brucella canis lacks GI-2, and a chromosomal scar identical to those in R mutants was observed. These results suggest that the phage integrase-promoted GI-2 excision played a role in B. ovis speciation and are consistent with other evidence, suggesting that this species and B. canis have emerged as two independent lineages.     

Evidence for covalent bonding of native hapten-protein complexes to smooth lipopolysaccharide of Brucella abortus

Abstract Smooth lipopolysaccharide (S-LPS) of Brucella abortus was dissociated from noncovalently attached components by differential centrifugation and exhaustive treatment with guanidinium thiocyanate. Mild alkaline hydrolysis then released ester-linked fatty acids as well as native hapten (NH)-protein complexes. These complexes, comprising 15 to 20% by weight of the S-LPS, failed to dissociate in guanidinium thiocyanate or in boiling SDS, suggesting covalent attachment. Analyses for 2-keto-3-deoxyoctonate demonstrated that the released carbohydrate was NH, rather than degraded O -polysaccharide or intact S-LPS monomers. This provides strong evidence that NH-protein complexes are covalently linked to S-LPS, most likely through ester bonds.     

Anti‐inflammatory and anti‐endotoxin properties of peptides derived from the carboxy‐terminal region of a defensin from the tick Ornithodoros savignyi

Antimicrobial peptides are small cationic peptides that possess a large spectrum of bioactivities, including antimicrobial, anti‐inflammatory and antioxidant activities. Several antimicrobial peptides are known to inhibit lipopolysaccharide (LPS)‐induced inflammation in vitro and to protect animals from sepsis. In this study, the cellular anti‐inflammatory and anti‐endotoxin activities of Os and Os‐C, peptides derived from the carboxy‐terminal of a tick defensin, were investigated. Both Os and Os‐C were found to bind LPS in vitro, albeit to a lesser extent than polymyxin B and melittin, known endotoxin‐binding peptides. Binding to LPS was found to reduce the bactericidal activity of Os and Os‐C against Escherichia coli confirming the affinity of both peptides for LPS. At a concentration of 25 µM, the nitric oxide (NO) scavenging activity of Os was higher than glutathione, a known NO scavenger. In contrast, Os‐C showed no scavenging activity. Os and Os‐C inhibited LPS/IFN‐γ induced NO and TNF‐α production in RAW 264.7 cells in a concentration‐dependent manner, with no cellular toxicity even at a concentration of 100 µM. Although inhibition of NO and TNF‐α secretion was more pronounced for melittin and polymyxin B, significant cytotoxicity was observed at concentrations of 1.56 µM and 25 µM for melittin and polymyxin B, respectively. In addition, Os, Os‐C and glutathione protected RAW 264.7 cells from oxidative damage at concentrations as low as 25 µM. This study identified that besides previously reported antibacterial activity of Os and Os‐C, both peptides have in addition anti‐inflammatory and anti‐endotoxin properties. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Expression of Babesia bovis rhoptry-associated protein 1 (RAP1) in Brucella abortus S19

Brucella abortus strain 19 (live vaccine) induces a strong humoral and cellular immune response and therefore, it is an attractive vector for the delivery of heterologous antigens. The objective of the present study was to express the rhoptry-associated protein (RAP1) of Babesia bovis in B. abortus S19, as a model for heterologous expression of immunostimulatory antigens from veterinary pathogens. A plasmid for the expression of recombinant proteins fused to the aminoterminal of the outer membrane lipoprotein OMP19 was created, pursuing the objective of increasing the immunogenicity of the recombinant antigen being expressed by its association to a lipid moiety. Recombinant strains of B. abortus S19 expressing RAP1 as a fusion protein either with the first amino acids of beta-galactosidase (S19pBB-RAP1) or B. abortus OMP19 (S19pBB19-RAP1) were generated. Plasmid stability and the immunogenicity of the heterologous proteins were analyzed. Mice immunized with S19pBB-RAP1 or S19pBB19-RAP1 developed specific humoral immune response to RAP1, IgG2a being the predominant antibody isotype. Furthermore, a specific cellular immune response to recombinant RAP1 was elicited in vitro by lymphocytes from mice immunized with both strains. Therefore, we concluded that B. abortus S19 expressing RAP1 is immunostimulatory and may provide the basis for combined heterologous vaccines for babesiosis and brucellosis.     

Highly sensitive enzyme-linked assay based on monoclonal antibodies for detection of Brucella antigens

Mice monoclonal antibodies against lypopolysaccharides (LPS) of Brucella abortus has been obtained and characterized. The antibodies detected LPS of B. abortus, B. melitensis and B. suis with high sensivity and specificity and did not react with LPS of Yersinia enterocolitica O:3, Y. enterocolitica O:9, Salmonella typhimurium, and Francisella tularensis. It has been shown that interaction of monoclonal antibodies and LPS of Brucella species can be critically dependent from buffer system. Obtained monoclonal antibodies allowed to develop highly sensitive assay which was able to detect antigens of Brucella species in concentrations 0.05 - 0.1 ng/ml. The assay can be used for detection and identification of Brucella species.     

Studies on the antigenic S-type lipopolysaccharides of Brucella abortus strains 7 and Mustapha

Antigenic phenol phase S-type lipopolysaccharides (LPS) isolated from Brucella abortus (B. abortus) strains 7 and Mustapha were observed to have 13C n.m.r. spectra which were almost identical to the one reported for the Brucella abortus 1119-3. The glycosyl content of the lipid A obtained from the LPS of strain 7 was found to be 2-acetamido-2-deoxyglucose only while strain Mustapha was found to contain both 2-acetamido-2-deoxyglucose and 2-acetamido-2-deoxygalactose. The fatty acid present in the lipid A of both strains was mainly n-hexadecanoic acid. Octadecanoic acid, 3-hydroxytetradecanoic acid as well as small quantities of 3-hydroxydodecanoic acid were also identified. This contrasts with the earlier reports of the absence of 3-OH-14:0 in the LPS of Brucella abortus.     

Brucella spp noncanonical LPS: structure, biosynthesis, and interaction with host immune system

Brucella spp. are facultative intracellular pathogens that have the ability to survive and multiply in professional and non-professional phagocytes, and cause abortion in domestic animals and undulant fever in humans. Several species are recognized within the genus Brucella and this classification is mainly based on the difference in pathogenicity and in host preference. Brucella strains may occur as either smooth or rough, expressing smooth LPS (S-LPS) or rough LPS (R-LPS) as major surface antigen. This bacterium possesses an unconventional non-endotoxic lipopolysaccharide that confers resistance to anti-microbial attacks and modulates the host immune response. The strains that are pathogenic for humans (B. abortus, B. suis, B. melitensis) carry a smooth LPS involved in the virulence of these bacteria. The LPS O-chain protects the bacteria from cellular cationic peptides, oxygen metabolites and complement-mediated lysis and it is a key molecule for Brucella survival and replication in the host. Here, we review i) Brucella LPS structure; ii) Brucella genome, iii) genes involved in LPS biosynthesis; iv) the interaction between LPS and innate immunity.     

Antibodies to N-terminal peptides of gonococcal porin are bactericidal when gonococcal lipopolysaccharide is not sialylated

Six synthetic 25-mer peptides corresponding to certain presumed surface-exposed regions of gonococcal porin protein I (PI) were made from strains FA19 (PIA) and MS11 (PIB). Four peptides were immunogenic in rabbits. Affinity-purified antisera against both PIA and PIB N-terminal peptides were bactericidal for homologous gonococci and many heterologous PI serovars. However, sialylation of gonococcal lipopolysaccharide (LPS) by growth of gonococci in the presence of cytidine monophosphate-neuraminic acid (CMP-NANA) abrogated the bactericidal activity of these antisera. Binding of anti-PI monoclonal antibodies to whole gonococci was reduced two- to fourfold by sialylation of LPS, suggesting that sialylation may inhibit bactericidal activity by masking porin epitopes. However, binding of anti-PII (Opa) monoclonal antibodies was not inhibited, yet complement-mediated killing was inhibited by sialylated LPS. Binding of complement components C3 and C9 was inhibited in the presence of either anti-PI or anti-PII monoclonals when gonococci were grown in the presence of CMP-NANA. Thus sialylation inhibited both anti-PI antibody binding and complement deposition, with a resultant decrease in bactericidal activity.     

METABOLIC CHARACTERIZATION OF THE GENUS BRUCELLA IV. 3: Correlation of Oxidative Metabolic Patterns and Susceptibility to Brucella Bacteriophage, Type abortus, Strain.

Meyer, Margaret E. (University of California, Davis). Metabolic characterization of the genus Brucella. IV. Correlation of oxidative metabolic patterns and susceptibility to Brucella bacteriophage, type abortus, strain 3. J. Bacteriol. 82:950-953. 1961.-A total of 212 strains of brucellae that had been identified as Brucella melitensis, B. abortus, B. suis, or B. neotomae by their oxidative metabolism were tested for their susceptibility to Brucella bacteriophage, type abortus, strain 3. It was demonstrated that only those organisms that displayed the oxidative metabolic pattern that is singular for B. abortus were susceptible to this strain of phage, irrespective of their identity by the conventional methods usually employed for differentiating members of this genus. Strains of organisms that display the features of B. melitensis by the conventional determinative methods, but display the metabolic characteristics of B. abortus, are susceptible to lysis by this phage. These organisms are in fact B. abortus. Strains of organisms that display the features of B. melitensis by the classical methods, and display the metabolic pattern of B. melitensis, are not lysed by this phage. These organisms are B. melitensis. The conclusions then were drawn that B. abortus is the only species that can serve as host for this strain of phage, that oxidative metabolic patterns accurately identify the species in this genus, and that by the conventional methods of differentiation, many strains of B. abortus are misidentified as B. melitensis.     

4-Amino-4-deoxy-l-arabinose in LPS of enterobacterial R-mutants and its possible role for their polymyxin reactivity

Abstract The content of 4-amino-4-deoxy- l -arabinopyranose ( l -Arap4N) and the phosphate substitution pattern of the LPS of various strains from Salmonella minnesota, Yersinia enterocolitica and Proteus mirabilis was determined by GC/MS, HPLC and ³¹P-NMR. These data allowed us to examine the possible role of these components for the polymyxin B-binding capacity of LPS and for the minimal inhibiting concentration (MIC) and the minimal bactericidal concentration (MBC) of polymyxins B and E towards the respective R-mutants. Contrary to other investigated Re-, Rd- and Rc-mutants of S. minnesota , strain R595 (Re-mutant) showed about a 90% substitution of the ester-linked phosphate-group with l -Arap4N, whereas the l -Arap4N content of the other S. minnesota strains amounted to 17–25%. Neither the binding capacity of LPS to polymyxin B, determined by a bioassay, nor the MIC- and MBC-values of the R-mutants were significantly affected by this alteration. Similar results were obtained after using the temperature-dependent changes in the l -Ara p4N-content and phosphate substitution pattern of Y. enterocolitica 75R . In order to explore the relevant polymyxin B binding site, lipid A samples with or without substitution of their ester-linked phosphate group were prepared and subjected to the polymyxin-binding assay. The results obtained so far indicated that the inner core bound l -Arap4N, detected in all resistant strains investigated, may play a decisive role in the decreased binding of polymyxin B, responsible for the bacterial resistance towards polymyxin(s).     

Defensin Susceptibility and Colonization in the Mouse Model of AJ100, a Polymyxin B-Resistant, Brucella abortus RB51 Isolate

Intracellular pathogens selected for increased susceptibility to polycations are commonly attenuated, yet the effect of decreased susceptibility to polycations on pathogenicity has not been researched. The polymyxin-resistant mutant Brucella abortus AJ100 was characterized by comparing its susceptibility to the polycationic antibiotic polymyxin B, defensins, and lactoferricin, and its colonization and clearance in the mouse model to the parent strain RB51. MIC (minimum inhibitory concentration) values determined by Etest for AJ100 and RB51 were 1.5 and 0.25 μg/ml, respectively. Though AJ100 is less susceptible to polymyxin B than RB51, it was more susceptible than its parent strain to the cationic defensins melittin, magainin 2, and cecropin P1. In the mouse model, initial colonization of the spleen was lower for AJ100 than RB51, and the rate of clearance from the spleen was faster for AJ100 than RB51. However, initial colonization and clearance rates of AJ100 from the liver were indistinguishable from those of RB51. This study suggests that the susceptibility profile of Brucella to polycationic defensins rather than polymyxin B may be indicative of differential survival in the spleen and liver in the mouse and is indicative of spleen and liver residential macrophages’ differing ability to inactivate Brucella.     

Purification and characterization of smooth and rough lipopolysaccharides from Brucella abortus.

In an attempt to obtain pure and well characterized smooth lipopolysaccharide (S-LPS) and rough lipopolysaccharide (R-LPS), smooth and rough strains of Brucella abortus were extracted by two different modifications of the phenol-water method. S-LPS was obtained in the phenol phase, and R-LPS was obtained in the aqueous phase. Further purification was accomplished by treatment with enzymes, detergents, NaI as a chaotropic agent to separate non-covalently bound contaminants, and by gel filtration. The degree of purity of the molecules was determined by chemical and immunological analysis and by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Lipid identification by gas-liquid chromatography showed seven major fatty acids. Palmitic acid accounts for about 50%, stearic acid accounts for about 10%, and hydroxylated fatty acids account for less than 5% of total fatty acids. 2-Keto-3-deoxyoctonate but not heptose was detected in the sugar analysis. Protein was found to be firmly bound to S-LPS but not to R-LPS.