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.     

Differences in chromosome number and genome rearrangements in the genus Brucella

We have studied the genomic structure and constructed the Spe I, Pac I and I- Ceu I restriction maps of the four biovars of the pathogenic bacterium Brucella suis . B . suis biovar 1 has two chromosomes of 2.1 Mb and 1.15 Mb, similar to those of the other Brucella species: B . melitensis , B . abortus , B . ovis and B . neotomae . Two chromosomes were also observed in the genome of B . suis biovars 2 and 4, but with sizes of 1.85 Mb and 1.35 Mb, whereas only one chromosome with a size of 3.1 Mb was found in B . suis biovar 3. We show that the differences in chromosome size and number can be explained by rearrangements at chromosomal regions containing the three rrn genes. The location and orientation of these genes confirmed that these rearrangements are due to homologous recombination at the rrn loci. This observation allows us to propose a scheme for the evolution of the genus Brucella in which the two chromosome-containing strains can emerge from an hypothetical ancestor with a single chromosome, which is probably similar to that of B . suis biovar 3. As the genus Brucella is certainly monospecific, this is the first time that differences in chromosome number have been observed in strains of the same bacterial species.     

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.     

Identification of species of Brucella using Fourier transform infrared spectroscopy

Fourier transform infrared spectroscopy (FTIR) is a technique that has been used over the years in chemical analysis for the identification of substances and is one that may be applied to the characterisation of microorganisms. The marked tendency of Brucella towards variation in the smooth rough phase, together with the laboriousness and risk involved in the methods used in their identification, make their classification difficult. We studied the type strains of the different species and biovars of Brucella and 11 isolates of human origin of Brucella melitensis, six corresponding to biovar 1, one to biovar 2 and five to biovar 3. The results of linear discriminant analysis performed using the data provide an above 95% likelihood of correct classification, over half of which are in fact above 99% for the vast majority of Brucella strains. Only one case of B. melitensis biovar 1 has been incorrectly classified. The rest of the microorganisms studied (Staphylococcus aureus, Strteptococcus pyogenes, Enterococcus faecalis, Corynebacterium pseudodiphtheriticum, Clostridium perfringens, Escherichia coli, Acinetobacter calcoaceticus and Pseudomonas aeruginosa) have been classified correctly in all cases to a likelihood of over 80%. In the graphic representation of the analysis, a grouping of these can be seen in clusters, which include the different species. One of these comprises B. melitensis, another Brucella abortus, and another wider one is made up of Brucella suis. The Brucella canis, Brucella ovis and Brucella neotomae strains appear separate from the previously described groups.     

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.     

Polynucleotide Homologies of Brucella Deoxyribonucleic Acids

Deoxyribonucleic acids (DNA's) extracted from organisms presently placed in the genus Brucella (B. abortus, B. melitensis, B. neotomae, and B. suis) possessed very similar polynucleotide sequences. Unlabeled, single-stranded DNA fragments from B. abortus, B. melitensis, B. neotomae, and B. suis were equally effective in competing with the interaction of corresponding radiolabeled, single-stranded DNA fragments with their homologous DNA-agars. Unlabeled fragments of B. ovis, however, did not compete as effectively as the homologous, unlabeled DNA's, and this organism, therefore, had a detectably different polynucleotide composition. The mole percentages of guanine plus cytosine in Brucella DNA's (56 to 58%) were also similar. DNA's from Francisella tularensis, Escherichia coli, and the slow loris did not compete.     

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.     

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.     

Phospholipids of bacteria of Brucella genus

The phospholipid composition of 6 Brucella species (B. melitensis, B. abortus, B. suis, B. ovis. B. canis, B. neotomae) and Australian mouse-derived strains of Brucella N 4, 11, 12 were studied. Comparison of phospholipid composition of Brucella cells with that of serologically related microorganisms revealed that all Brucella biotypes contain phosphatidyl-(N-methyl)ethanolamine and phosphatidylcholine while Y. enterocolitica, Sh. disenteriae, E. coli cells do not contain these two substances. It is concluded that the specific phospholipid pattern of Brucella biotypes may be useful in typing of new Brucella strains.     

上海地区散发布氏杆菌感染的细菌学及分子鉴定

目的 本研究对我院的1例散发布氏杆菌病患者进行细菌学及分子生物学的分析,并在国内首次尝试了用数目可变串联重复单元(VNTR)分子指纹分析法对其进行了基因分型并和国际流行株进行了分子流行病学比较分析。方法 对临床疑似布氏杆菌病病例作血液细菌培养与生化鉴定,进一步作布氏杆菌特异性基因片段的序列分析鉴定以及利用布氏杆菌基因组中的8个位点构建VNTR指纹图谱,参照国际布氏杆菌VNTR数据库,构建布氏杆菌基因系统树。结果 用细菌学方法确定散发疑似布氏杆菌病病例体内分离到的为布氏杆菌,通过基因序列分析进一步得到证实,但不能鉴定到生物种和生物型。对分离株作VNTR指纹分析提示该散发布氏杆菌病为猪2型布氏杆菌感染所致。结论 通过传统细菌培养方法与布氏杆菌VNTR指纹分析可用于我国布氏杆菌病分子流行病学的系统调查。     

Experimental infection of white-tailed deer with rangiferine brucellosis

Experimental infections of white-tailed deer (Odocoileus virginianus) with Brucella suis biovar 4 were evaluated over a period of 6 wk. Five adult male hand-raised white-tailed deer were inoculated with 1 x 10(7) colony forming units of B. suis biovar 4 in the conjunctiva and serologically evaluated over 6 wk by the card test (CARD), rivanol test (RIV), serum agglutination test (SAT), complement fixation test (CFT), particle concentration fluorescence immunoassay (PCFIA), and competitive enzyme linked immunosorbent assay (cELISA), as routinely used for bovine samples. Six weeks postinoculation the animals were euthanized and cultured for B. suis biovar 4. One deer became serologically positive 4 wk postinoculation on CFT, CARD, PCFIA, and cELISA. At 6 wk postinoculation, CFT was positive in four infected deer, CARD was positive in three deer; RIV, SAT, and PCFIA was positive in two deer; and cELISA was positive in one deer. Only the CFT was 100% sensitive. At necropsy B. suis biovar 4 was isolated from four of five deer, and representative colonies were biologically similar to the challenge organism.     

Homologies of Deoxyribonucleic Acids from Brucella ovis, Canine Abortion Organisms, and other Brucella Species

The bacterium that causes canine abortion has polynucleotide sequences similar, in deoxyribonucleic acid (DNA)-DNA homology studies, to those of Brucella suis and, by inference from previous data, those of B. abortus and B. melitensis as well as B. neotomae. Therefore, the organism causing canine abortion appears to be a member of the genus Brucella. DNA preparations from Serratia marcescens, Alcaligenes faecalis, and Bordetella bronchiseptica, 58, 62, and 66 mole% guanine plus cytosine, respectively, do not have detectable polynucleotide sequence homologies with B. suis DNA which is 56 mole% guanine plus cytosine. B. ovis DNA lacks some of the polynucleotide sequences present in B. suis DNA and appears to be a deletion mutant. However, a large proportion of B. ovis polynucleotides are similar to those of other Brucella species, which supports the inclusion of B. ovis in the genus.     

Activity of native vs. synthetic promoters in Brucella

Brucellosis caused by Brucella species is reportedly the most common zoonotic infection worldwide. The bacterial pathogen is also classified by the Centers for Disease Control and Prevention as a category (B) pathogen that has the potential for development as a bioweapon. Although eight genomes of Brucella have been sequenced, little information is available regarding the regulation of gene expression and promoter activity in Brucella spp. We therefore constructed a set of broad-host-range vectors expressing the lacZ reporter gene from various promoters. Four groups of promoters (Brucella native, antibiotic resistant, bacteriophage and synthetic promoters) were tested in vivo and in vitro in Brucella suis. The highest level of heterologous gene expression was achieved with synthetic hybrid trc promoter carrying the adenine-rich upstream element. Furthermore, this demonstrates the usefulness of synthetic promoters for enhanced level of gene expression in Brucella spp.     

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.     

Experimental Brucella suis biovar 4 infection in a moose

A moose (Alces alces gigas) was inoculated with Brucella suis biovar 4 to better understand the effects of brucellosis in this species. Serum antibody titers increased rapidly and peaked within 21 to 56 days. Fever, leukocytosis, recumbency, anorexia and depression were observed starting 42 days post inoculation. Brucella suis biovar 4 was isolated from blood, lymph nodes, liver and spleen.     

Brucella suis infections in collared peccaries in Venezuela

A bacteriologic and serologic study was conducted on two ranches in the states of Apure and Guarico, Venezuela for brucellosis in collared peccaries (Tayassu tajacu). One hundred thirty-nine peccaries were necropsied and tissues were cultured. Forty-three isolations of Brucella suis biovar 1, were made from lymph nodes and spleens of 25 males and 18 females. Antibody to Brucella sp. was detected in sera from 122 animals by the rapid plate agglutination, standard tube agglutination, 2-mer-captoethanol, rivanol, complement fixation and card tests. Young animals had infection and reactor rates nearly as high as the older animals indicating most were infected at a relative early age. Results suggest that this species may transmit brucellosis when living with domestic animals. This is the first report of B. suis biovar 1 from collared peccaries in Venezuela.     

Brucella outer membrane protein Omp31 is a haemin-binding protein

The expression of haemin-binding proteins (HBPs) in the outer membrane is one of the strategies used by Gram-negative bacteria to obtain iron from the host. No HBP has been described in Brucella spp. We investigated whether Omp31, an outer membrane protein from Brucella with homology to HBPs from Bartonella quintana, is an HBP. Soluble recombinant Omp31 bound specifically to haemin-agarose, while an unrelated Brucella protein (SurA) did not. A similar experiment showed that native Omp31 found in the Brucella suis membrane fraction also binds to haemin-agarose. Recombinant Omp31 was electrophoresed by SDS-PAGE, transferred to nitrocellulose, and incubated with a haemin solution. Haemin bound to Omp31 and to albumin (positive control) but not to SurA. IPTG-induced recombinant Escherichia coli cells expressing Omp31 on their membrane bound significantly more haemin than uninduced cells or controls carrying a similar plasmid without the omp31 gene, showing that Omp31 also binds haemin in a bacterial membrane environment. Viable Brucella ovis cells bound haemin in solution, and this binding was markedly inhibited by preincubation of cells with antibodies to Omp31 and to an exposed prominent loop of the protein, thus showing that Omp31 functions as an HBP in brucellae. To test whether the expression of Omp31 is iron-regulated, B. suis was grown in trypticase-soy broth (TSB) and in iron-depleted TSB. The expression of Omp31, as assessed by Western blot, was significantly higher in bacteria grown under iron limitation. Overall, these results show that Omp31 from B. suis, B. melitensis and B. ovis is an HBP, whose expression seems to be induced by iron limitation.