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.     

MLVA16 Typing of Portuguese Human and Animal Brucella melitensis and Brucella abortus Isolates

To investigate the epidemiological relationship of isolates from different Portuguese geographical regions and to assess the diversity among isolates, the MLVA16(Orsay) assay (panels 1, 2A and 2B) was performed with a collection of 126 Brucella melitensis (46 human and 80 animal isolates) and 157 B. abortus field isolates, seven vaccine strains and the representative reference strains of each species. The MLVA16(Orsay) showed a similar high discriminatory power (HGDI 0.972 and 0.902) for both species but panel 1 and 2A markers displayed higher diversity (HGDI 0.693) in B. abortus compared to B. melitensis isolates (HGDI 0.342). The B. melitensis population belong to the "Americas" (17%) and "East Mediterranean" (83%) groups. No isolate belonged to the "West Mediterranean" group. Eighty-five percent of the human isolates (39 in 46) fit in the "East-Mediterranean" group where a single lineage known as MLVA11 genotype 116 is responsible for the vast majority of Brucella infections in humans. B. abortus isolates formed a consistent group with bv1 and bv3 isolates in different clusters. Four MLVA11 genotypes were observed for the first time in isolates from S. Jorge and Terceira islands from Azores. From the collection of isolates analysed in this study we conclude that MLVA16(Orsay) provided a clear view of Brucella spp. population, confirming epidemiological linkage in outbreak investigations. In particular, it suggests recent and ongoing colonisation of Portugal with one B. melitensis lineage usually associated with East Mediterranean countries.     

Comparative proteome analysis of laboratory grown Brucella abortus 2308 and Brucella melitensis 16M

Brucella species are pathogenic agents that cause brucellosis, a debilitating zoonotic disease that affects a large variety of domesticated animals and humans. Brucella melitensis and Brucella abortus are considered major health threats because of their highly infectious nature and worldwide occurrence. The availability of the annotated genomes for these two species has allowed a comparative proteomics study of laboratory grown B. melitensis 16M and B. abortus 2308 by two-dimensional (2-D) gel electrophoresis and peptide mass fingerprinting. Computer-assisted analysis of the different 2-D gel images of strains 16M and 2308 revealed significant quantitative and qualitative differences in their protein expression patterns. Proteins involved in membrane transport, particularly the high affinity amino acids binding proteins, and those involved in Sec-dependent secretion systems related to type IV and type V secretion systems, were differentially expressed. Differential expression of these proteins may be responsible for conferring specific host preference in the two strains 2308 and 16M.     

Completion of the genome sequence of Brucella abortus and comparison to the highly similar genomes of Brucella melitensis and Brucella suis

Brucellosis is a worldwide disease of humans and livestock that is caused by a number of very closely related classical Brucella species in the alpha-2 subdivision of the Proteobacteria. We report the complete genome sequence of Brucella abortus field isolate 9-941 and compare it to those of Brucella suis 1330 and Brucella melitensis 16 M. The genomes of these Brucella species are strikingly similar, with nearly identical genetic content and gene organization. However, a number of insertion-deletion events and several polymorphic regions encoding putative outer membrane proteins were identified among the genomes. Several fragments previously identified as unique to either B. suis or B. melitensis were present in the B. abortus genome. Even though several fragments were shared between only B. abortus and B. suis, B. abortus shared more fragments and had fewer nucleotide polymorphisms with B. melitensis than B. suis. The complete genomic sequence of B. abortus provides an important resource for further investigations into determinants of the pathogenicity and virulence phenotypes of these bacteria.     

CtrA controls cell division and outer membrane composition of the pathogen Brucella abortus

Brucella abortus is a pathogen infecting cattle, able to survive, traffic, and proliferate inside host cells. It belongs to the Alphaproteobacteria, a phylogenetic group comprising bacteria with free living, symbiotic, and pathogenic lifestyles. An essential regulator of cell cycle progression named CtrA was described in the model bacterium Caulobacter crescentus. This regulator is conserved in many alphaproteobacteria, but the evolution of its regulon remains elusive. Here we identified promoters that are CtrA targets using ChIP‐seq and we found that CtrA binds to promoters of genes involved in cell cycle progression, in addition to numerous genes encoding outer membrane components involved in export of membrane proteins and synthesis of lipopolysaccharide. Analysis of a conditional B. abortus ctrA loss of function mutant confirmed that CtrA controls cell division. Impairment of cell division generates elongated and branched morphologies, that are also detectable inside HeLa cells. Surprisingly, abnormal bacteria are able to traffic to the endoplasmic reticulum, the usual replication niche of B. abortus in host cells. We also found that CtrA depletion affected outer membrane composition, in particular the abundance and spatial distribution of Omp25. Control of the B. abortus envelope composition by CtrA indicates the plasticity of the CtrA regulon along evolution.     

Cloning and sequencing of 28 kDa outer membrane protein gene of Brucella melitensis Rev. 1

Brucella melitensis is an organism of paramount zoonotic importance. The 28 kDa outer membrane protein (OMP) is one of the immunodominant antigens of B. melitensis. The gene encoding 28 kDa OMP (omp28) has been amplified from B. melitensis Rev. 1 strain. A PCR product of 753 bp, encoding complete omp28 gene of B. melitensis, was obtained. The gene was further cloned and sequenced. The nucleotide sequence of B. melitensis Rev. 1 strain showed substitution of 2 nucleotides from that of 16M strain.     

In vitro markers and biological activity in mice of seed lot strains and commercial Brucella melitensis Rev 1 and Brucella abortus B19 vaccines.

Four seed lots and fourteen batches of Brucella melitensis Rev 1 and B. abortus B19 living anti-Brucella commercial vaccines obtained from six Spanish laboratories were tested in vitro and in vivo in the reference mouse model for quality control. All the strains tested showed the characteristic morphology of their respective Rev 1 or B19 reference strains with the exception of three Rev 1 strains: seed lot SL2 and commercial vaccine R3, in which giant colonies were predominant, and commercial vaccine R5, in which 5% rough colonies were detected. Strains SL2 and R5 (but not the R3) had a deficient activity when tested in the mouse model. All strains but two (Rev 1 strain R1 and B19 strain B2) had standard resistance/ sensitivity patterns to streptomycin and penicillin G. Strains R1 and B2 had an increased resistance to penicillin when incubated in a 10% CO2 atmosphere and both strains showed an increased residual virulence in mice. As residual virulence and immunogenicity in mice were not always correlated one another nor with the in vitro tests, all tests should be performed to control properly the anti-Brucella live vaccines. A computerized statistical procedure to calculate the residual virulence of vaccines is proposed as an alternative to that used in the current method.     

2308、M28、S1330、16M四株布鲁氏菌灭活参数研究

【目的】比较不同灭活方法对布鲁氏菌灭活的效果,确定灭活参数,为制备布鲁氏菌灭活抗原提供参考。【方法】将4株布鲁氏菌参考强毒株2308(牛种)、M28(羊种)、S1330(猪种)以及16M(羊种)分别经大豆酶消化蛋白胨(TSA)培养基培养繁殖后,用生理盐水制成(4-8)×1010 CFU/m L的菌悬液,分成等份于80 oC灭活不同时间,另将同样浓度的菌悬液分别用不同浓度甲醛于37 oC灭活不同时间,通过灭活检验,确定灭活效果。取经甲醛和热灭活的16M抗原,分别以1×1010 CFU/只剂量皮下注射1.5-2.0 kg家兔2只,免疫6周内,每周采血用虎红平板凝集试验(RBT)和试管凝集试验(SAT)测定抗体效价。【结果】80 oC、5 min可灭活2308、S1330和16M三种菌株,80 oC、10 min可灭活全部4种菌株。0.2%甲醛灭活7 d,4种试验菌株均不能被彻底灭活;0.4%甲醛在12 h内只能灭活16M,72 h可灭活M28;0.4%甲醛灭活2308和S1330两次试验结果差异较大。0.6%甲醛可在72 h内灭活4种试验菌。不同方法灭活的16M抗原免疫家兔后,其血清抗体虎红平板凝集和试管凝集效价消长趋势基本一致,甲醛灭活的抗原免疫原性略高于热灭活抗原。【结论】80 oC热灭活和0.6%甲醛灭活均可用于对布鲁氏菌的灭活,且不影响布鲁氏菌的免疫原性。     

Antigenic Relationship of Brucella ovis and Brucella melitensis

Immune sera were prepared in rabbits by the injection of living and acetone-killed cells of Brucella ovis and smooth and rough B. melitensis. The use of whole-cell antigens in agglutination and agglutinin-absorption tests revealed little relationship between B. ovis and smooth B. melitensis, although there was extensive cross-agglutination between B. ovis and rough B. melitensis. The use of water-soluble antigens prepared from ultrasonically treated cells of the three strains revealed extensive cross-reactions in indirect hemagglutination, agar gel precipitation, and immunoelectrophoresis tests, as well as in allergic skin tests in rabbits. The most definitive results were obtained with the immunoelectrophoresis technique. B. ovis antigen produced at least 11 lines with its homologous serum. All were removed by absorption of the serum with rough B. melitensis antigen. All but three were removed by absorption with smooth B. melitensis antigen. Smooth B. melitensis antigen produced 11 lines with its homologous serum, and all but 3 were removed by absorption with B. ovis antigen. Rough B. melitensis produced nine lines with its homologous serum, and eight were removed by B. ovis antigen. The extensive cross-reactions between soluble antigens of B. ovis and B. melitensis are added evidence that B. ovis belongs in the genus Brucella.     

Erythritol catabolism by Brucella abortus.

Cell extracts of Brucella abortus (British 19) catabolized erythritol through a series of phosphorylated intermediates to dihydroxyacetonephosphate and CO-2. Cell extracts required adenosine 5'-triphosphate (ATP), nicotinamide adenine dinucleotide (NAD), Mg2+, inorganic orthophosphate, and reduced glutathione for activity. The first reaction in the pathway was the phosphorylation of mesoerythritol with an ATP-dependent kinase which formed d-erythritol 1-phosphate (d-erythro-tetritol 1-phosphate). d-Erythritol 1-phosphate was oxidized by an NAD-dependent dehydrogenase to d-erythrulose 1-phosphate (d-glycero-2-tetrulose 1-phosphate). B. abortus (US-19) was found to lack the succeeding enzyme in the pathway and was used to prepare substrate amounts of d-erythrulose 1-phosphate. d-Erythritol 1-phosphate dehydrogenase (d-erythro-tetritol 1-phosphage: NAD 2-oxidoreductase) is probably membrane bound. d-Erythrulose 1-phosphate was oxidized by an NAD-dependent dehydrogenase to 3-keto-l-erythrose 4-phosphate (l-glycero-3-tetrosulose 4-phosphate) which was further oxidized at C-1 by a membrane-bound dehydrogenase coupled to the electron transport system. Either oxygen or nitrate had to be present as a terminal electron acceptor for the oxidation of 3-keto-l-erythrose 4-phosphate to 3-keto-l-erythronate 4-phosphate (l-glycero-3-tetrulosonic acid 4-phosphate). The beta-keto acid was decarboxylated by a soluble decarboxylase to dihydroxyacetonephosphate and CO-2. Dihydroxyacetonephosphate was converted to pyruvic acid by the final enzymes of glycolysis. The apparent dependence on the electron transport system of erythritol catabolism appears to be unique in Brucella and may play an important role in coupling metabolism to active transport and generation of ATP.     

Detoxification and immunogenic properties of endotoxin-containing precipitate of Brucella abortus

Badakhsh, Fred F. (University of Georgia, Athens), and John W. Foster. Detoxification and immunogenic properties of endotoxin-containing precipitate of Brucella abortus. J. Bacteriol. 91:494-498. 1966.-Endotoxin-containing precipitates (ECP) were prepared from Brucella abortus strain 19A by aqueous ether extraction followed by ethyl alcohol precipitation. Lysozyme was the most effective of several enzymes tried for detoxification of endotoxin present in the precipitate. Trypsin was shown to reduce mouse lethal toxicity but not rabbit dermal toxicity. Immunological studies of ECP and enzyme-treated ECP demonstrated that lysozyme did not harm the immunogenic property of ECP, whereas heat, ribonuclease, lipase, and proteolytic enzymes had an adverse effect. Serological reactivity of ECP was increased after lysozyme treatment, whereas ribonuclease reduced serological activity.     

Persistent properties of Brucella abortus isolated from various sources

Persistence factors in 70 B. abortus strains isolated from different sources were studied. Depending on the source of the isolation of B. abortus, the spread of their antilysozyme, anticomplementary, antiintercidal activity was determined and intraspecific differences in the manifestation of these signs were established. The highest spread and manifestation of persistence factors was registered in B. abortus populations isolated from brucellosis patients and from cattle.     

Antitumor properties of chemically detoxified killed Brucella abortus organisms

Summary Killed Brucella abortus organisms of the vaccinal strain B19 were detoxified by incubation in NaOH. A 24-h incubation in 0.01 M NaOH increased the LD₅₀ of smooth (S) and rough (R) organisms 2–3 times in normal mice and 50–100 times in adrenalectomized mice. This NaOH treatment did not alter the antitumor activity of Brucella abortus as shown in EL4 lymphoma- and Lewis carcinoma-grafted mice. After incubation in NaOH, S bacteria injected IV retained their ability to provoke spleen hypertrophy and antibody synthesis, and S and R organisms injected into he footpad had comparable ability to induce granulomas. NaOH treatment tended to diminish the mitogenic activity of R bacteria for spleen cell cultures.     

Rapid multiplex PCR assay for the simultaneous detection of the Brucella Genus, B. abortus, B. melitensis, and B. suis

The routine identification and differentiation of Brucella species is a time-consuming and labor-intensive process, which frequently places personnel at risk of laboratoryacquired infection. Here, we describe the development of a rapid multiplex PCR assay for the confirmation of presumptive Brucella isolates. The assay was able to identify and differentiate major human pathogens, namely B. abortus, B. melitensis, and B. suis, in a single test of less than an hour and a half.     

The outer membranes of Brucella spp. are not barriers to hydrophobic permeants.

The patterns of susceptibility to hydrophobic and hydrophilic drugs and the uptake of the fluorescent probe N-phenyl-naphthylamine in Brucella spp., Haemophilus influenzae, Escherichia coli, and deep rough Salmonella minnesota mutants were compared. The results show that the outer membranes of smooth and naturally rough Brucella spp. do not represent barriers to hydrophobic permeants and that this absence of a barrier relates at least in part to the properties of Brucella lipopolysaccharide.     

Purification, refolding and characterization of the trimeric Omp2a outer membrane porin from Brucella melitensis

Brucella melitensis is a gram-negative bacteria known to cause brucellosis and to produce severe infections in humans. Whilst brucella's outer membrane proteins have been extensively studied due to their potential role as antigens or virulence factors, their function is still poorly understood at the structural level, as the 3D structure of Brucella β-barrel membrane proteins are still unknown. In this context, the B. melitensis trimeric Omp2a porin has been overexpressed and refolded in n-dodecyl-β-d-maltopyranoside. We here show that this refolding process is insensitive to urea but is temperature- and ionic strength-dependent. Reassembled species were characterized by fluorescence, size-exclusion chromatography and circular dichroism. A refolding mechanism is proposed, suggesting that Omp2a first refolds under a monomeric form and then self-associates into a trimeric state. This first complete in vitro refolding of a membrane protein from B. melitensis shall eventually lead to functional and 3D structure determination.