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.     

Signal sequences are not uniformly hydrophobic

A sample of 34 hydrophobia core-segments from known signal sequences have been analysed with regard to the mean hydrophobicity at each position in the segment. The results show that these core-segments are not uniformly hydrophobic; instead, the more hydrophobic residues are concentrated around the midpoints of the segments. A similar analysis carried out on a sample of 12 membrane-spanning segments from transmembrane proteins indicates that the hydrophobic residues are much more evenly distributed along the segments in this case.     

High-speed railways are not barriers to Pyronia tithonus butterfly movements

Linear infrastructures such as railways and roads can be barriers to the movements of individuals and, hence, may have strong impacts on populations. We tested the barrier effect of a high-speed railway for Pyronia tithonus, a butterfly species showing homing behaviour when displaced. We captured, marked and displaced 152?individuals in two different locations. One-third of the butterflies were released at a capture plot, one-third on the other side of the railway (in a similar habitat) and one-third on the same side but 100?m away from the capture plot. We obtained recapture rates of 40 and 23?% per location. Many (31?%) butterflies crossed the railway, showing homing behaviour. Thus, contrary to wide, busy roads, high-speed railways do not seem to be barriers for these butterflies. We suggest that in an intensive agrarian landscape, railway verges can play a substitution habitat role for grassland butterflies.     

Polymorphism in Brucella spp. due to highly repeated DNA.

The species of Brucella are very closely related, but Brucella ovis does not express detectable amounts of a protein, designated BCSP31, that is common to the other species. We studied the lack of expression of BCSP31 by Southern analysis. DNAs from the B. ovis culture collection strains and field isolates were probed with a 1.3-kb HindIII fragment encoding BCSP31 of Brucella abortus. The probe hybridized to a 1.6-kb HindIII fragment of all B. ovis strains tested, showing that the gene is present in B. ovis but occurs on a larger restriction fragment. DNA linkage studies and restriction mapping of the cloned polymorphic region of B. ovis showed that the polymorphism was due to a DNA insertion of approximately 0.9 kb at a site downstream of the BCSP31-coding region. When the 1.6-kb polymorphic B. ovis fragment was used to probe a HindIII Southern blot of cellular DNA of strains of B. ovis and of B. abortus, at least 24 fragments of B. ovis and 6 fragments of B. abortus hybridized to the inserted DNA. Specimens of B. ovis collected over a 30-year period on two continents had similar hybridization patterns. The large difference between B. ovis and B. abortus in the number of copies of the repeated DNA is interesting in the context of the closeness of the Brucella species.     

Isolation of potentially novel Brucella spp. from frogs

Bacterial isolates from frogs were phenotypically identified as Ochrobactrum anthropi, but 16S rRNA sequencing showed up to 100% identity with Brucella inopinata. Further analysis of recA, omp2a, omp2b, bcsp31, and IS711 and multilocus sequence analysis (MLSA) verified a close relationship with Brucella, suggesting the isolates may actually represent novel members of this growing genus of zoonotic pathogens.     

Antibodies to Brucella spp. among blue wildebeest and African buffalo in Kenya

A serologic survey of blue wildebeest (Connochaetes taurinus Burchell) and African buffalo (Syncerus caffer Sparrman) in the Masai Mara area was conducted. Antibodies to Brucella spp. were found in 18% of the blue wildebeest and 30% of the African buffalo examined. There were titers in all age groups and in both sexes. Hygromata were seen in both species. The increase in numbers of blue wildebeest and African buffalo which share grazing and watering areas with cattle of the Masai people, makes the presence of infections by Brucella spp. in wildlife an important consideration in any program for control of brucellosis.     

Antibodies to Brucella spp. in Pacific bottlenose dolphins from the Solomon Islands

Brucella spp. have been recently isolated from a variety of marine mammals. Serum samples from 58 Pacific bottlenose dolphins (Tursipa aduncus) from the Solomon Islands were tested for antibodies to Brucella spp. by the tube agglutination test (TAT), enzymed-linked immunosorbent assay (ELISA), and immunoblotting. Anti-Brucella spp. antibodies were detected by TAT and ELISA in 31 and 40 of 58 samples, respectively. These results suggest that Pacific bottlenose dolphins from the Solomon Islands are infected with Brucella spp. or a Brucella-like organism.     

Identification of Brucella spp. by using the polymerase chain reaction.

The application of two synthetic oligonucleotides as probes and as primers in the polymerase chain reaction is presented for a specific, sensitive, and quick identification of Brucella spp. The specific oligonucleotide sequences were chosen on the basis of a 16S rRNA sequence alignment between Brucella abortus and Agrobacterium tumefaciens.     

Alkanes are not innocuous vehicles for hydrophobic reagents in membrane studies

Alkanes (C6-C16) are often used as vehicles for hydrophobic reagents, e.g. long-chain ceramides, in cell biology studies. It is generally understood that they are inert solvents, particularly when added in small volumes. However, simple calculations show that, under standard experimental conditions in cell studies, alkane: phospholipid molar ratios in excess of 1000:1 may be found. Even at much smaller ratios (close to 1:1) our studies with liposomes show that alkanes induce vesicle aggregation. Differential scanning calorimetry shows marked changes in both the gel-fluid and the lamellar-hexagonal transitions. Alkanes inhibit bacterial sphingomyelinase when acting on large unilamellar vesicles, and activate bacterial phospholipase C under the same conditions. Thus, the use of alkanes in cell studies requires strict control experiments to avoid artefactual results.     

Distribution of small hydrophobic molecules in membranes. I. Artificial lipid membranes]

A hydrophobic uncharged fluorescent probe of 4-dimethylaminochalcone (DMC) interacted with synthetic phospholipid membranes. Comparison of absorption spectra and fluorescence of DMC in the membranes and organic solvents shows that in the membranes the DMC molecules are located not in the hydrocarbon layer but in the polar regions near the surface. The probe is distributed regularly along the surface forming no dimers and clusters. Polar groups which surround the probe in the membrane are less mobile than the molecules of organic solvents at the same temperature. The evaluation shows that the relaxation time of polar groups in the probe environment is longer than 0.15-10(-9) sec. The DMC molecules may be located in different sites of the membrane surface, which seem to differ from one another in the mobility of polar groups.     

Brucella abortus ornithine lipids are dispensable outer membrane components devoid of a marked pathogen-associated molecular pattern

The brucellae are α-Proteobacteria facultative intracellular parasites that cause an important zoonosis. These bacteria escape early detection by innate immunity, an ability associated to the absence of marked pathogen-associated molecular patterns in the cell envelope lipopolysaccharide, lipoproteins and flagellin. We show here that, in contrast to the outer membrane ornithine lipids (OL) of other Gram negative bacteria, Brucella abortus OL lack a marked pathogen-associated molecular pattern activity. We identified two OL genes (olsB and olsA) and by generating the corresponding mutants found that olsB deficient B. abortus did not synthesize OL or their lyso-OL precursors. Liposomes constructed with B. abortus OL did not trigger IL-6 or TNF-α release by macrophages whereas those constructed with Bordetella pertussis OL and the olsB mutant lipids as carriers were highly active. The OL deficiency in the olsB mutant did not promote proinflammatory responses or generated attenuation in mice. In addition, OL deficiency did not increase sensitivity to polymyxins, normal serum or complement consumption, or alter the permeability to antibiotics and dyes. Taken together, these observations indicate that OL have become dispensable in the extant brucellae and are consistent within the trend observed in α-Proteobacteria animal pathogens to reduce and eventually eliminate the envelope components susceptible of recognition by innate immunity.     

The interaction of hydrophobic ions with lipid bilayer membranes

Summary Electrical relaxation studies have been made on lecithin bilayer membranes of varying chain length and degree of unsaturation, in the presence of dipicrylamine. Results obtained are generally consistent with a model for the transport of hydrophobic ions previously proposed by Ketterer, Neumcke, and Läuger (J. Membrane Biol. 5:225, 1971). This model visualizes as three distinct steps the interfacial adsorption, translocation, and desorption of ions. Measurements at high electric field yield directly the density of ions adsorbed to the membrane-solution interface. Variation of temperature has permitted determination of activation enthalpies for the translocation step which are consistent with the assumption of an electrostatic barrier in the hydrocarbon core of the membrane. The change of enthalpy upon adsorption of ions is, however, found to be negligible, the process being driven instead by an increase of entropy. It is suggested that this increase may be due to the destruction, upon adsorption, of a highly ordered water structure which surrounds the hydrophobic ion in the aqueous phase. Finally, it is shown that a decrease of transient membrane conductance observed at high concentration of hydrophobic ions, previously interpreted in terms of interfacial saturation, must instead be attributed to a more complex effect equivalent to a reduction of membrane fluidity.Research performed while on sabbatical leave April-September, 1974.     

Bison PRNP genotyping and potential association with Brucella spp. seroprevalence

The implication that host cellular prion protein (PrP(C)) may function as a cell surface receptor and/or portal protein for Brucella abortus in mice prompted an evaluation of nucleotide and amino acid variation within exon 3 of the prion protein gene (PRNP) for six US bison populations. A non-synonymous single nucleotide polymorphism (T50C), resulting in the predicted amino acid replacement M17T (Met --> Thr), was identified in each population. To date, no variation (T50; Met) has been detected at the corresponding exon 3 nucleotide and/or amino acid position for domestic cattle. Notably, 80% (20 of 25) of the Yellowstone National Park bison possessing the C/C genotype were Brucella spp. seropositive, representing a significant (P = 0.021) association between seropositivity and the C/C genotypic class. Moreover, significant differences in the distribution of PRNP exon 3 alleles and genotypes were detected between Yellowstone National Park bison and three bison populations that were either founded from seronegative stock or previously subjected to test-and-slaughter management to eradicate brucellosis. Unlike domestic cattle, no indel polymorphisms were detected within the corresponding regions of the putative bison PRNP promoter, intron 1, octapeptide repeat region or 3'-untranslated region for any population examined. This study provides the first evidence of a potential association between nucleotide variation within PRNP exon 3 and the presence of Brucella spp. antibodies in bison, implicating PrP(C) in the natural resistance of bison to brucellosis infection.     

Identification of a unique gene cluster of Brucella spp. that mediates adhesion to host cells

Brucella, the causative agent of brucellosis, a major zoonotic disease affecting a broad range of mammals, is a gram-negative bacterium whose virulence is dependent on the capacity to attach and invade different cells of the host. The bacterium is able to infect through a diverse repertoire of epitheliums: skin, airways or gastric. Although much has been studied on the mechanisms Brucella uses to establish an intracellular replication niche, almost none is known on how the bacterium adheres and invades host cells. We report here the identification of a pathogenicity island that harbors a gene homologous to proteins with bacterial immunoglobulin-like domains present in other pathogens that play a role in attachment and invasion. Deletion of the entire island results in a mutant with a reduced attachment capacity measured by intracellular replication and adhesion assays. Intraperitoneal and oral experimental infection of mice strongly suggests that this island plays a role during the oral infection probably mediating attachment and trespassing of the gastric epithelium to establish a systemic infection.     

Attachment to membranes of exogenous immunoglobulin conjugated to a hydrophobic anchor

A novel method has been developed for the attachment of exogenous protein to liposomal and other membranes. A new phospholipid-containing alkylating reagent, N-(N^α-idoacetyl, N^?-dansyl lysyl)-phosphatidylethanolamine, was synthesized for conjugation to sulfhydryl groups of proteins. Model experiments were carried out with a Bence-Jones dimer which was reduced to generate one sulfhydryl group per monomer. After alkylation with the reagent the modified protein spontaneously attached to preformed liposomes and red cell ghosts. The attachment and accessibility of the protein were demonstrated by the association of the protein with the liposomal fraction in gel filtration and the agglutination of treated vesicles and red cell ghosts with antisera to human λ chain and to the dansyl group.     

Use of hydrophobic membranes to supply hydrogen to sulphate reducing bioreactors

This paper reports on the application of hydrophobic membranes to supply the gaseous substrates hydrogen/carbon dioxide (H₂/CO₂)to a sulphate reducing bioreactor. For this, two flat 0.016m² sheets of flouroplast microporous (0.45m) membranes were inserted in a 3.6 dm016m³ bioreactor for the supply of H H₂/CO₂ gas as small gas bubbles. Thebioreactor was operated at 30 °C and pH 7.0 and was also equipped with an external ultra filtration module for biomass retention. At a sulphate loading rate (SLR) of 1.32 g SO₄^2- dm^-3 day^-1 and a hydraulic retention time (HRT) of 61 h, a sulphate reduction rate (SRR) of 0.90 g SO₄^2- dm^-3 day^-1 was achieved. When the influent sulphate concentration was reduced from 3.36 to 0.75 g SO₄^2- dm^-3 by lowering the HRT to 10.3 h (SLR of 1.75 g SO₄^2- dm^-3 day ^-1), the SRR dropped to 0.22 g SO₄^2- dm^-3 day ^-1. The lower sulphate reduction efficiency was most probably caused by a too short biomass-substrate contact time or by irreversible sulphide inhibition. Mass transfer limitation of H₂ and improper mixing of the reactor liquid were shown not to contribute to the lowsulphate reduction efficiency.     

Purified outer membranes of Serpulina hyodysenteriae contain cholesterol.

We have isolated outer and inner membranes of Serpulina hyodysenteriae by using discontinuous sucrose density gradients. The outer and inner membrane fractions contained less than 1 and 2%, respectively, of the total NADH oxidase activity (soluble marker) in the cell lysate. Various membrane markers including lipooligosaccharide (LOS), the 16-kDa outer membrane lipoprotein (SmpA), and the C subunit of the F1F0 ATPase indicated that the lowest-density membrane fraction contained outer membranes while the high-density membrane fraction contained inner membranes and that both are essentially free of contamination by the periplasmic flagella, a major contaminant of membranes isolated by other techniques. The outer membrane fractions (rho = 1.10 g/cm3) contained 0.25 mg of protein/mg (dry weight), while the inner membrane samples (rho = 1.16 g/cm3) contained significantly more protein (0.55 mg of protein/mg [dry weight]). Lipid analysis revealed that the purified outer membranes contained cholesterol as a major component of the membrane lipids. Treatment of intact S. hyodysenteriae with different concentrations of digitonin, a steroid glycoside that interacts with cholesterol, indicated that the outer membrane could be selectively removed at concentrations as low as 0.125%.