Cloning, nucleotide sequence, and expression of the Brucella melitensis bp26 gene coding for a protein immunogenic in infected sheep

Abstract We have previously identified a Brucella melitensis 28 kDa cytosoluble protein (CP28) which was highly immunogenic in infected sheep and which in addition made possible the serological differentiation between infected and B. melitensis Rev.l vaccinated sheep. Monoclonal antibodies against CP28 were used to screen a B. melitensis 16M genomic library and to clone the corresponding gene. DNA sequencing of the gene encoding CP28 of B. melitensis 16M revealed that it was nearly identical to that of the recently published bp26 gene of Brucella abortus vaccine strain S19 coding for a periplasmic protein. The differences between the B. melitensis 16M gene and that of B. abortus S19 consisted of single nucleotide substitutions, one or two codon deletions, one codon addition, and most importantly a 21-bp deletion. The corresponding region of B. abortus S19 contains two 10-bp direct repeats which could have been involved in the genesis of the deletion. Expression of the B. melitensis 16M bp26 gene in Escherichia coli studied by the use of the monoclonal antibodies showed the same characteristics as reported for the B. abortus S19 bp26 gene, i.e. the presence of a higher molecular mass preprotein and a lower molecular mass band which probably corresponds to the mature protein exported to the periplasm. Immunoblotting performed with sera from either naturally infected or B. melitensis H38 experimentally infected sheep confirmed the importance of the B. melitensis CP28/BP26 protein as diagnostic antigen.     

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.     

Parallel gene loss and acquisition among strains of different Brucella species and biovars

The genus Brucella is divided into six species; of these, B. melitensis and B. abortus are pathogenic to humans, and B. ovis and B. neotomae are nonpathogenic to humans. The definition of gene loss and acquisition is essential for understanding Brucella's ecology, evolutionary history, and host relationships. A DNA microarray containing unique genes of B. melitensis Type strain 16MT and B. abortus 9-941 was constructed and used to determine the gene contents of the representative strains of Brucella. Phylogenetic relationships were inferred from sequences of housekeeping genes. Gene loss and acquisition of different Brucella species were inferred. A total of 214 genes were found to be differentially distributed, and 173 of them were clustered into 15 genomic islands (GIs). Evidence of horizontal gene transfer was observed for 10 GIs. Phylogenetic analysis indicated that the 19 strains formed five clades, and some of the GIs had been lost or acquired independently among the different lineages. The derivation of Brucella lineages is concomitant with the parallel loss or acquisition of GIs, indicating a complex interaction between various Brucella species and hosts.     

Adaptor long-range PCR procedure for clone-specific characterization and chromosomal localization

An efficient adaptor long-range PCR (ALR-PCR) procedure was developed to detect genomic rearrangements in high-plasticity genomic regions between closely related strains of bacteria. The method was precisely optimized using a combination of high-speed experimental steps for the chromosomal localization and elucidation of deletions, inversions, duplications, or inserted sequences within a clone-specific flanking region. The advantages of this strategy are: (i) ready-to-use polymerase mixtures and Master mix (ready-to-use reaction mixtures with polymerase MasterAmp and buffer 2x Premix 4); (ii) a 5-min ligation procedure; (iii) rapid purification of DNA digests; (iv) optimized DNA template concentration protocol to avoid nonspecific amplification and high backgrounds; (v) long-range PCR protocol to obtain at least 9.6 kb single PCR products; (vi) two-step PCR cycling with the same annealing and extension temperature at 68 degrees C; (vii) simple design of the adaptors according to the preferred restriction endonuclease enzyme; and (viii) simple technology and equipment required. The application of this method for a tester-specific suppressive subtractive hybridization (SSH) clone of Brucella melitensis 16M revealed an 837-bp deletion and a 7255-bp DNA transfer from one chromosomal location to another for Brucella abortus 2308 used as a driver.     

Comparative whole-genome hybridization reveals genomic islands in Brucella species

Brucella species are responsible for brucellosis, a worldwide zoonotic disease causing abortion in domestic animals and Malta fever in humans. Based on host preference, the genus is divided into six species. Brucella abortus, B. melitensis, and B. suis are pathogenic to humans, whereas B. ovis and B. neotomae are nonpathogenic to humans and B. canis human infections are rare. Limited genome diversity exists among Brucella species. Comparison of Brucella species whole genomes is, therefore, likely to identify factors responsible for differences in host preference and virulence restriction. To facilitate such studies, we used the complete genome sequence of B. melitensis 16M, the species highly pathogenic to humans, to construct a genomic microarray. Hybridization of labeled genomic DNA from Brucella species to this microarray revealed a total of 217 open reading frames (ORFs) altered in five Brucella species analyzed. These ORFs are often found in clusters (islands) in the 16M genome. Examination of the genomic context of these islands suggests that many are horizontally acquired. Deletions of genetic content identified in Brucella species are conserved in multiple strains of the same species, and genomic islands missing in a given species are often restricted to that particular species. These findings suggest that, whereas the loss or gain of genetic material may be related to the host range and virulence restriction of certain Brucella species for humans, independent mechanisms involving gene inactivation or altered expression of virulence determinants may also contribute to these differences.     

检测OMP28抗体不能有效诊断羊布鲁氏菌病

【目的】探索布鲁氏菌外膜蛋白OMP28作为羊布鲁氏菌病特异性检测方法的可行性。【方法】体外表达和纯化OMP28蛋白,建立并优化以OMP28重组蛋白为包被抗原的布鲁氏菌病ELISA诊断方法。以3个不同种属的4株布鲁氏菌(羊种布鲁氏菌16M和M28,猪种布鲁氏菌S1330,牛种布鲁氏菌2308)分别感染山羊和绵羊至42周,期间每隔2周收集血清,分别用布鲁氏菌LPS包被的ELISA和OMP28 ELISA方法对不同阶段的分离血清进行检测,比较2种不同ELISA对4株布鲁氏菌感染的山羊和绵羊的检测敏感性。【结果】4株布鲁氏菌感染的山羊和绵羊均产生高水平针对LPS的抗体,但是仅有B. melitensis 16M和B. melitensis M28感染的绵羊与B. melitensis 16M和B. abortus 2308感染的山羊可产生针对OMP28的抗体。【结论】基于OMP28的间接ELISA具有细菌属特异性和宿主动物品种特异性,通过检测OMP28抗体不能有效诊断羊布鲁氏菌病。     

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.     

Antigenic, immunologic and genetic characterization of rough strains B. abortus RB51, B. melitensis B115 and B. melitensis B18

The lipopolysaccharide (LPS) is considered the major virulent factor in Brucella spp. Several genes have been identified involved in the synthesis of the three LPS components: lipid A, core and O-PS. Usually, Brucella strains devoid of O-PS (rough mutants) are less virulent than the wild type and do not induce undesirable interfering antibodies. Such of them proved to be protective against brucellosis in mice. Because of these favorable features, rough strains have been considered potential brucellosis vaccines. In this study, we evaluated the antigenic, immunologic and genetic characteristics of rough strains B. abortus RB51, B. melitensis B115 and B. melitensis B18. RB51 derived from B. abortus 2308 virulent strain and B115 is a natural rough strain in which the O-PS is present in the cytoplasm. B18 is a rough rifampin-resistan mutant isolated in our laboratory. The surface antigenicity of RB51, B115 and B18 was evaluated by testing their ability to bind antibodies induced by rough or smooth Brucella strains. The antibody response induced by each strain was evaluated in rabbits. Twenty-one genes, involved in the LPS-synthesis, were sequenced and compared with the B. melitensis 16M strain. The results indicated that RB51, B115 and B18 have differences in antigenicity, immunologic and genetic properties. Particularly, in B115 a nonsense mutation was detected in wzm gene, which could explain the intracellular localization of O-PS in this strain. Complementation studies to evaluate the precise role of each mutation in affecting Brucella morphology and its virulence, could provide useful information for the assessment of new, attenuated vaccines for brucellosis.     

DNA polymorphism in the omp25/omp31 family of Brucella spp.: identification of a 1.7-kb inversion in Brucella cetaceae and of a 15.1-kb genomic island, absent from Brucella ovis, related to the synthesis of smooth lipopolysaccharide

Five genes homologous to the well-known omp25 and omp31 genes, that code for two major Brucella spp. outer membrane proteins (OMPs), have been detected in the genome of Brucella melitensis 16M and Brucella suis 1330. In this work we have determined the nucleotide sequence of these five genes, named omp31b, omp25b, omp25c, omp25d and omp22, in the six classical Brucella species reference strains and in representative strains of the recently proposed species Brucella cetaceae and Brucella pinnipediae that classify the Brucella strains isolated in the last years from marine mammals. Although these genes are quite conserved in the genus Brucella, several important differences have been found between species (i) omp31b contains a premature stop codon in B. canis and B. ovis truncating the encoded protein; (ii) the 5' end of omp31b is deleted in the three biovars of B. melitensis which probably prevents synthesis of Omp31b in this species; (iii) only B. melitensis, B. suis and B. neotomae would be able to synthesize the Omp25b protein with the characteristics shared by the Omp25/Omp31 group of proteins (characteristic signal sequence and C-terminal phenylalanine); (iv) a DNA inversion of 1747 bp including omp25b was detected in B. cetaceae strains; (v) a DNA deletion of about 15 kb was detected in all the six B. ovis strains tested. This deletion in B. ovis includes, among other genes, omp25b and wboA, a gene that has been shown to be required for the synthesis of the O-polysaccharide chain of the Brucella spp. smooth lipopolysaccharide. Several features of the DNA region absent from B. ovis suggest that this DNA fragment is a genomic island acquired by the Brucella ancestor by horizontal transfer and later deleted from B. ovis. The DNA polymorphism we have found in this work within the genus Brucella might be involved in the differences in pathogenicity and host preference displayed by the Brucella species.     

Restriction site polymorphisms in the genes encoding new members of group 3 outer membrane protein family of Brucella spp

Thirty-seven Brucella reference and field strains representing all the species and their biovars were analysed by PCR-RFLP to determine the degree of variation in the genes encoding the new members of group 3 outer membrane protein (Omp) family. Analysis of the omp22 and omp25c/omp25d genes indicated that the restriction patterns were identical for all species and biovars with all restriction enzymes tested, except for Brucella ovis that showed a short 30 bp deletion close to omp22 gene, and for B. abortus biovar 6 and B. ovis that lacked a DdeI site and a HinfI site, respectively, in the omp25c/omp25d genes. Analysis of PCR products of the omp31b gene digested with 20 restriction enzymes revealed that this gene has a greater level of DNA polymorphism than the other genes encoding the new members of group 3 Omp family. A deletion of 232bp was detected in fourteen B melitensis strains from different hosts and from different geographic origins, confirming that this feature is indeed a hallmark of B. melitensis. PCR-RFLP analysis of omp31b with DdeI allowed us to identify species-specific markers for B. abortus, B. melitensis, and B. ovis. Finally, by PCR analysis, Southern blot hybridization and DNA sequencing we showed that a large deletion of 15 kb, comprising the entire omp25b gene and 21 more genes, is present in all B. ovis strains, thus confirming previous observations from other authors.     

Identification of the A and M antigens of Brucella as the O-polysaccharides of smooth lipopolysaccharides

Rabbit antisera, cross-absorbed serotype-specific for the Brucella A and M antigens, precipitated respectively the smooth lipopolysaccharides from B. abortus 1119-3 and B. melitensis 16M. The antigenic A and M activity of these lipopolysaccharides was shown to reside within the O-chain region of the smooth lipopolysaccharides by inhibition experiments. Homologous O-polysaccharides showed the highest inhibitory activity in ELISA, although both the A and M antigens were active as heterologous inhibitors, showing that the antigenic determinants of the classical A and M antigens are therefore within the respective O-polysaccharide structures. Their cross-serological activity may be explained in terms of the distinct but related chemical structures of these polysaccharides.     

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.     

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.     

羊布鲁氏菌非编码小RNA分子BSR-2的预测与鉴定

利用RT-PCR和RACE等方法对生物信息学预测的羊布鲁氏菌非编码小RNA(smallnon-codingRNA,sRNA)BSR-2进行了实验鉴定,并通过分析BSR-2在胞内生存缺陷株中的转录情况以及预测BSR-2的靶标基因对BSR-2的功能进行初步探讨.RT-PCR结果表明,BSR-2在羊布鲁氏菌的总RNA中存在转录本,而且在不同的应激条件下转录水平不同.RACE结果表明,BSR-2长224nt,位于Ⅱ号染色体的BMEII0742和BMEII0743之间的基因间区.进一步的实验结果表明,BSR-2可能与布鲁氏菌的胞内生存能力相关.     

Early Appearance of Colonies of Brucella Spp. on Solid Media containing Erythritol

S ummary . The incorporation of erythritol (1 μ M /ml) in Morris medium caused the earlier appearance of colonies of virulent strains of Brucella abortus and B. melitensis. On Albimi agar erythritol accelerated the growth of B. melitensis but not that of B. abortus. There was no acceleration of the growth of a virulent strain of B. suis on either medium.     

Use of western immunoblot analysis for testing moose serum for Brucella suis biovar 4 specific antibodies.

To determine if 12 moose (Alces alces) from northern Alaska with agglutinating antibodies specific for Brucella spp. had been exposed to either B. suis biovar 4 or B. abortus biovar 1, western immnnoblot serologic analysis was performed. Differential serologic responses to strain specific A and M antigenic variances of the lipopolysaccharide O-polysaccharide sugar allowed strain identification. Prior to examination, test sera were absorbed with killed whole cells from either B. abortus biovar 1, containing predominately A antigen (A+ M-); B. melitensis biovar 1, containing essentially M antigen (A- M+); or B. suis biovar 4, containing both antigenic tyes (A+ M+). The resulting sera were then examined by western immunoblot for recognition of either B. abortus biovar 1, B. melitensis biovar 1, or B. suis biovar 4 cell lysates. The results of this study indicate that these moose were exposed to B. suis biovar 4, a known pathogen of caribou (Rangifer tarandus) from arctic Alaska.     

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.     

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.     

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)     

Surface Antigens of Smooth Brucellae

Surface antigens of smooth brucellae were extracted by ether-water, phenol-water, trichloroacetic acid, and saline and examined by immunoelectrophoresis and gel diffusion with antisera from infected and immunized rabbits. Ether-water extracts of Brucella melitensis contained a lipopolysaccharide protein component, which was specific for the surface of smooth brucellae and was correlated with the M agglutinogen of Wilson and Miles, a polysaccharide protein component devoid of lipid which was not restricted to the surface of smooth brucellae and was not correlated with the smooth agglutinogen (component 1), and several protein components which were associated with internal antigens of rough and smooth brucellae. Immunoelectrophoretic analysis of ether-water extracts of B. abortus revealed only two components, a lipopolysaccharide protein component, which was correlated with the A agglutinogen, and component 1. Component 1 from B. melitensis and B. abortus showed identity in gel diffusion tests, whereas component M from B. melitensis and component A from B. abortus showed partial identity with unabsorbed antisera and no cross-reactions with monospecific sera. Attempts to prepare monospecific sera directly by immunization of rabbits with cell walls or ether-water extracts were unsuccessful. Absorption of antisera with heavy fraction of ether-water extracts did not always result in monospecific sera. It was concluded (as has been described before) that the A and M antigens are present on a single antigenic complex, in different proportions depending upon the species and biotype, and that this component is a lipopolysaccharide protein complex of high molecular weight that diffuses poorly through agar gel. Components 1, A, and M were also demonstrated in trichloroacetic acid and phenol-water extracts. With all extracts, B. melitensis antigen showed greater diffusibility in agar than B. abortus antigens. After mild acid hydrolysis, B. abortus ether-water extract was able to diffuse more readily.