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.     

BmaC, a novel autotransporter of Brucella suis, is involved in bacterial adhesion to host cells

Brucella is an intracellular pathogen responsible of a zoonotic disease called brucellosis. Brucella survives and proliferates within several types of phagocytic and non-phagocytic cells. Like in other pathogens, adhesion of brucellae to host surfaces was proposed to be an important step in the infection process. Indeed, Brucella has the capacity to bind to culture human cells and key components of the extracellular matrix, such as fibronectin. However, little is known about the molecular bases of Brucella adherence. In an attempt to identify bacterial genes encoding adhesins, a phage display library of Brucella suis was panned against fibronectin. Three fibronectin-binding proteins of B. suis were identified using this approach. One of the candidates, designated BmaC was a very large protein of 340 kDa that is predicted to belong to the type I (monomeric) autotransporter family. Microscopy studies showed that BmaC is located at one pole on the bacterial surface. The phage displaying the fibronectin-binding peptide of BmaC inhibited the attachment of brucellae to both, HeLa cells and immobilized fibronectin in vitro. In addition, a bmaC deletion mutant was impaired in the ability of B. suis to attach to immobilized fibronectin and to the surface of HeLa and A549 cells and was out-competed by the wild-type strain in co-infection experiments. Finally, anti-fibronectin or anti-BmaC antibodies significantly inhibited the binding of wild-type bacteria to HeLa cells. Our results highlight the role of a novel monomeric autotransporter protein in the adhesion of B. suis to the extracellular matrix and non-phagocytic cells via fibronectin binding.     

Type III secretion homologs are present in Brucella melitensis,B. ovis,and B. suis biovars 1, 2, and 3

Protein sequences from characterized type III secretion (TTS) systems were used as probes in silico to identify several TTS gene homologs in the genome sequence of Brucella suis biovar 1 strain 1330. Four of the genes, named flhB, fliP, fliR, and fliF on the basis of greatest homologies to known flagellar apparatus proteins, were targeted in PCR and hybridization assays to determine their distribution among other Brucella nomen species and biovars. The results indicated that flhB, fliP, fliR and fliF are present in Brucella melitensis, Brucella ovis, and Brucella suis biovars 1, 2 and 3. Similar homologos have been reported previously in Brucella abortus. Using RT-PCR assays, we were unable to detect any expression of these genes. It is not yet known whether the genes are the cryptic remnants of a flagellar system or are actively involved in a process contributing to pathogenicity or previously undetected motility, but they are distributed widely in Brucella and merit further study to determine their role.     

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。     

细菌学方法和HOOF-Prints技术在绵羊种布鲁氏菌019株鉴定中的比较研究

应用细菌学常规方法和分子生物学检测方法对绵羊种布鲁氏菌非典型株019进行分类研究。利用高变8聚核苷酸DNA指纹技术(HOOF-Prints)对绵羊种布鲁氏菌019株可变数目重复片段(VNTR)的8个位点进行PCR扩增和序列测定,将测定结果与GenBank数据库比较分析,应用DNAMAN进行同源性分析,并构建系统进化树。结果表明,绵羊种布鲁氏菌019株和绵羊种布鲁氏菌参考株63/290的亲缘关系高于绵羊种布鲁氏菌019株与其他参考株的亲缘关系,该结论与细菌学常规鉴定结果一致。应用HOOF-Prints技术可以对绵羊种布鲁氏菌非典型株019进行鉴定,该技术有望弥补传统分类方法的不足。     

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.     

Cloning, expression, and purification of Brucella suis outer membrane proteins

Brucella, an aerobic, nonsporeforming, nonmotile Gram-negative coccobacillus, is a NIH/CDC category B bioterror threat agent that causes incapacitating human illness. Medical defense against the bioterror threat posed by Brucella would be strengthened by development of a human vaccine and improved diagnostic tests. Central to advancement of these goals is discovery of bacterial constituents that are immunogenic or antigenic for humans. Outer membrane proteins (OMPs) are particularly attractive for this purpose. In this study, we cloned, expressed, and purified seven predicted OMPs of Brucella suis. The recombinant proteins were fused with 6-His and V5 epitope tags at their C termini to facilitate detection and purification. The B. suis surface genes were PCR synthesized based on their ORF sequences and directly cloned into an entry vector. The recombinant entry constructs were propagated in TOP 10 cells, recombined into a destination vector, pET-DEST42, then transformed into Escherichia coli BL21 cells for IPTG-induced protein expression. The expressed recombinant proteins were confirmed with Western blot analysis using anti-6-His antibody conjugated with alkaline phosphatase. These B. suis OMPs were captured and purified using a HisGrab plate. The purified recombinant proteins were examined for their binding activity with antiserum. Serum derived from a rabbit immunized intramuscularly with dialyzed cell lysate of Brucella rough mutant WRR51. The OMPs were screened using the rabbit antiserum and purified IgG. The results suggested that recombinant B. suis OMPs were successfully cloned, expressed and purified. Some of the expressed OMPs showed high binding activity with immunized rabbit antiserum.     

Central role of MyD88-dependent dendritic cell maturation and proinflammatory cytokine production to control Brucella abortus infection

Brucella abortus is a facultative intracellular bacterium that infects humans and domestic animals. The enhanced susceptibility to virulent B. abortus observed in MyD88 knockout (KO) mice led us to investigate the mechanisms involved in MyD88-dependent immune responses. First, we defined the role of MyD88 in dendritic cell (DC) maturation. In vitro as well as in vivo, B. abortus-exposed MyD88 KO DCs displayed a significant impairment on maturation as observed by expression of CD40, CD86, and MHC class II on CD11c+ cells. In addition, IL-12 and TNF-alpha production was totally abrogated in MyD88 KO DCs and macrophages. Furthermore, B. abortus-induced IL-12 production was found to be dependent on TLR2 in DC, but independent on TLR2 and TLR4 in macrophages. Additionally, we investigated the role of exogenous IL-12 and TNF-alpha administration on MyD88 KO control of B. abortus infection. Importantly, IL-12, but not TNF-alpha, was able to partially rescue host susceptibility in MyD88 KO-infected animals. Furthermore, we demonstrated the role played by TLR9 during virulent B. abortus infection. TLR9 KO-infected mice showed 1 log Brucella CFU higher than wild-type mice. Macrophages and DC from TLR9 KO mice showed reduced IL-12 and unaltered TNF-alpha production when these cells were stimulated with Brucella. Together, these results suggest that susceptibility of MyD88 KO mice to B. abortus is due to impaired DC maturation and lack of IL-12 synthesis. Additionally, DC activation during Brucella infection plays an important regulatory role by stimulating and programming T cells to produce IFN-gamma.     

Immunization of mice with recombinant protein CobB or AsnC confers protection against Brucella abortus infection

Due to drawbacks of live attenuated vaccines, much more attention has been focused on screening of Brucella protective antigens as subunit vaccine candidates. Brucella is a facultative intracellular bacterium and cell mediated immunity plays essential roles for protection against Brucella infection. Identification of Brucella antigens that present T-cell epitopes to the host could enable development of such vaccines. In this study, 45 proven or putative pathogenesis-associated factors of Brucella were selected according to currently available data. After expressed and purified, 35 proteins were qualified for analysis of their abilities to stimulate T-cell responses in vitro. Then, an in vitro gamma interferon (IFN-γ) assay was used to identify potential T-cell antigens from B. abortus. In total, 7 individual proteins that stimulated strong IFN-γ responses in splenocytes from mice immunized with B. abortus live vaccine S19 were identified. The protective efficiencies of these 7 recombinant proteins were further evaluated. Mice given BAB1_1316 (CobB) or BAB1_1688 (AsnC) plus adjuvant could provide protection against virulent B. abortus infection, similarly with the known protective antigen Cu-Zn SOD and the license vaccine S19. In addition, CobB and AsnC could induce strong antibodies responses in BALB/c mice. Altogether, the present study showed that CobB or AsnC protein could be useful antigen candidates for the development of subunit vaccines against brucellosis with adequate immunogenicity and protection efficacy.     

Lamins A and C but not lamin B1 regulate nuclear mechanics

Mutations in the nuclear envelope proteins lamins A and C cause a broad variety of human diseases, including Emery-Dreifuss muscular dystrophy, dilated cardiomyopathy, and Hutchinson-Gilford progeria syndrome. Cells lacking lamins A and C have reduced nuclear stiffness and increased nuclear fragility, leading to increased cell death under mechanical strain and suggesting a potential mechanism for disease. Here, we investigated the contribution of major lamin subtypes (lamins A, C, and B1) to nuclear mechanics by analyzing nuclear shape, nuclear dynamics over time, nuclear deformations under strain, and cell viability under prolonged mechanical stimulation in cells lacking both lamins A and C, cells lacking only lamin A (i.e. "lamin C-only" cells), cells lacking wild-type lamin B1, and wild-type cells. Lamin A/C-deficient cells exhibited increased numbers of misshapen nuclei and had severely reduced nuclear stiffness and decreased cell viability under strain. Lamin C-only cells had slightly abnormal nuclear shape and mildly reduced nuclear stiffness but no decrease in cell viability under strain. Interestingly, lamin B1-deficient cells exhibited normal nuclear mechanics despite having a significantly increased frequency of nuclear blebs. Our study indicates that lamins A and C are important contributors to the mechanical stiffness of nuclei, whereas lamin B1 contributes to nuclear integrity but not stiffness.     

An essential tyrosine phosphatase homolog regulates cell separation, outer membrane integrity, and morphology in Caulobacter crescentus

Although reversible phosphorylation on tyrosine residues regulates the activity of many eukaryotic proteins, there are few examples of this type of regulation in bacteria. We have identified the first essential tyrosine phosphatase homolog in a bacterium, Caulobacter crescentus CtpA. ctpA mutants with altered active-site residues are nonviable, and depletion of CtpA yields chains of cells with blebbed outer membranes, linked by unresolved peptidoglycan. CtpA overexpression reduces cell curvature in a manner similar to deleting the intermediate filament protein crescentin, but it does not disrupt crescentin localization or membrane attachment. Although it has no obvious signal sequence or transmembrane-spanning domains, CtpA associates with the Caulobacter inner membrane. Immunolocalization experiments suggest that CtpA accumulates at the division site during the last quarter of the cell cycle. We propose that CtpA dephosphorylates one or more proteins involved in peptidoglycan biosynthesis or remodeling, which in turn affect cell separation, cell envelope integrity, and vibrioid morphology.     

The role of the vacB gene in the pathogenesis of Brucella abortus

Brucella species are important zoonotic pathogens affecting a wide variety of mammals. Therefore, the identification of new Brucella virulence factors is of great interest in understanding bacterial pathogenesis and immune evasion. In this study, we have identified Brucella abortus vacB gene that presents 2343 nucleotides and 781 amino acids and it shows 39% identity with Shigella flexneri vacB gene that encodes an exoribonuclease RNase R involved in bacterial virulence. Further, we have inactivated Brucella vacB by gene replacement strategy generating a deletion mutant strain. In order to test the role of Brucella vacB in pathogenesis, BALB/c and interferon regulatory factor-1 (IRF-1) knockout (KO) mice received Brucella vacB mutant, the virulent parental strain 2308 or the vaccine strain RB51 and the bacterial CFU numbers in spleens and mous survival were monitored. Our results demonstrated that the B. abortus DeltavacB mutant and the wild type strain 2308 showed similar CFU numbers in BALB/c mice. Additionally, IRF-1 KO mice that received either the vacB mutant or S2308 strain died in 12-14 days postinfection; in contrast, all animals that received the RB51 vaccine strain survived for 30 days postinoculation. In summary, this study reports that the vacB gene in B. abortus has no impact on bacterial pathogenesis.     

Denitrification genes regulate Brucella virulence in mice

Brucella is the causative agent of the zoonotic disease brucellosis, which is endemic in many parts of the world. Genome sequencing of B. suis and B. melitensis revealed that both are complete denitrifiers. To learn more about the role of denitrification in these animal pathogens, a study of the role of denitrification in the closely related B. neotomae was undertaken. In contrast to B. suis and B. melitensis, it was found that B. neotomae is a partial denitrifier that can reduce nitrate to nitrite but no further. Examination of the B. neotomae genome showed that a deletion in the denitrification gene cluster resulted in complete loss of nirV and the partial deletion of nirK and nnrA. Even though the nor operon is intact, a norC-lacZ promoter fusion was not expressed in B. neotomae. However, the norC-lacZ fusion was expressed in the related denitrifier Agrobacterium tumefaciens, suggesting that the lack of expression in B. neotomae is due to inactivation of NnrA. A narK-lacZ promoter fusion was found to exhibit nitrate-dependent expression consistent with the partial denitrifier phenotype. Complementation of the deleted region in B. neotomae by using nirK, nirV, and nnrA from B. melitensis restored the ability of B. neotomae to reduce nitrite. There was a significant difference in the death of IRF-1-/- mice when infected with B. neotomae containing nirK, nirV, and nnrA and those infected with wild-type B. neotomae. The wild-type strain killed all the infected mice, whereas most of the mice infected with B. neotomae containing nirK, nirV, and nnrA survived.     

Brucella abortus synthesizes phosphatidylcholine from choline provided by the host

The Brucella cell envelope is characterized by the presence of phosphatidylcholine (PC), a common phospholipid in eukaryotes that is rare in prokaryotes. Studies on the composition of Brucella abortus 2308 phospholipids revealed that the synthesis of PC depends on the presence of choline in the culture medium, suggesting that the methylation biosynthetic pathway is not functional. Phospholipid composition of pmtA and pcs mutants indicated that in Brucella, PC synthesis occurs exclusively via the phosphatidylcholine synthase pathway. Transformation of Escherichia coli with an expression vector containing the B. abortus pcs homologue was sufficient for PC synthesis upon induction with IPTG (isopropyl-beta-d-thiogalactopyranoside), while no PC formation was detected when bacteria were transformed with a vector containing pmtA. These findings imply that Brucella depends on choline provided by the host cell to form PC. We could not detect any obvious associated phenotype in the PC-deficient strain under vegetative or intracellular growth conditions in macrophages. However, the pcs mutant strain displays a reproducible virulence defect in mice, which suggests that PC is necessary to sustain a chronic infection process.     

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.     

Comparative proteome analysis of Brucella abortus 2308 and its virB type IV secretion system mutant reveals new T4SS-related candidate proteins

Brucella abortus is an alpha-2 proteobacteria with a type IV secretion system (T4SS) known as virB, which is necessary to gain virulence by building up a replicative vacuole associated with the endoplasmic reticulum of the host cell. A virB T4SS mutant of the B. abortus 2308 strain and its wild-type strain were grown in acid medium in order to obtain and analyze their proteomes, looking for putative proteins that may serve as T4SS substrates and those that may be subjected to T4SS regulation. A total of 47 overexpressed and 22 underexpressed proteins from the virB T4SS mutant strain were selected and sequenced. Some of the 69 analyzed proteins have not been described before either as over or under-expressed in relation to a virB T4SS mutation, whereas some of them have been already described by other groups as potentially important secretory proteins in other Brucella species. An important number of the proteins identified are outer membrane and periplasmic space protein, which makes them become particularly important new T4SS-related candidate proteins.     

Whole genome sequences of four Brucella strains

Brucella melitensis and Brucella suis are intracellular pathogens of livestock and humans. Here we report four genome sequences, those of the virulent strain B. melitensis M28-12 and vaccine strains B. melitensis M5 and M111 and B. suis S2, which show different virulences and pathogenicities, which will help to design a more effective brucellosis vaccine.     

Characterization of Brucella suis clpB and clpAB mutants and participation of the genes in stress responses

Pathogens often encounter stressful conditions inside their hosts. In the attempt to characterize the stress response in Brucella suis, a gene highly homologous to Escherichia coli clpB was isolated from Brucella suis, and the deduced amino acid sequence showed features typical of the ClpB ATPase family of stress response proteins. Under high-temperature stress conditions, ClpB of B. suis was induced, and an isogenic B. suis clpB mutant showed increased sensitivity to high temperature, but also to ethanol stress and acid pH. The effects were reversible by complementation. Simultaneous inactivation of clpA and clpB resulted in a mutant that was sensitive to oxidative stress. In B. suis expressing gfp, ClpA but not ClpB participated in degradation of the green fluorescent protein at 42 degrees C. We concluded that ClpB was responsible for tolerance to several stresses and that the lethality caused by harsh environmental conditions may have similar molecular origins.