Inhibition of TLR4 signaling by Brucella TIR-containing protein TcpB-derived decoy peptides.

Brucella spp. avoid host immune recognition and thus, weaken the immune response to infection. The Toll/interleukin-1 receptor (TIR) domain-containing protein (TcpB/Btp1) of Brucella spp. is thought to be involved in blocking host innate immune responses by binding to adaptors downstream of Toll-like receptors. In this study, based on the observation that TcpB binds to the host target proteins, MAL, through the TIR domain, we examined decoy peptides from TcpB TIR domains and found that TB-8 and TB-9 substantially inhibit lipopolysaccharide (LPS)-induced signaling in vitro and in vivo. Both these peptides share a common loop, the DD loop, indicating a novel structural region mediating TIR interactions. The inhibition of LPS signaling by TB-8 and TB-9 shows no preference to MyD88-dependent cytokines, such as TNF-α and IL-1ß or TRIF-dependent cytokines including IFN-ß and IL-6. Furthermore, these two peptides rescue the virulence of Brucella ΔtcpB mutants at the cellular level, indicating key roles of the DD loop in Brucella pathogenesis. In conclusion, identification of inhibitors from the bacterial TIR domains is helpful not only for illustrating interacting mechanisms between TIR domains and bacterial pathogenesis, but also for developing novel signaling inhibitors and therapeutics for human inflammatory diseases.

Development of a rapid recombinase polymerase amplification assay for detection of Brucella in blood samples.

A rapid and sensitive recombinase polymerase amplification (RPA) assay, Bruce-RPA, was developed for detection of Brucella. The assay could detect as few as 3 copies of Brucella per reaction within 20 min. Bruce-RPA represents a candidate point-of-care diagnosis assay for human brucellosis.

Changes of predominant species/biovars and sequence types of Brucella isolates, Inner Mongolia, China.

BACKGROUND: Human brucellosis incidence in China was divided into 3 stages, high incidence (1950-1960s), decline (1970-1980s) and re-emergence (1990-2000s). Human brucellosis has been reported in all the 32 provinces, of which Inner Mongolia has the highest prevalence, accounting for over 40% of the cases in China. To investigate the etiology alteration of human brucellosis in Inner Mongolia, the species, biovars and genotypes of 60 Brucella isolates from this province were analyzed. METHODS: Species and biovars of the Brucella strains isolated from outbreaks were determined based on classical identification procedures. Strains were genotyped by multi locus sequence typing (MLST). Sequences of 9 housekeeping genes were obtained and sequence types were defined. The distribution of species, biovars and sequence types (STs) among the three incidence stages were analyzed and compared. RESULTS: The three stages of high incidence, decline and re-emergence were predominated by B. melitensis biovar 2 and 3, B. abortus biovar 3, and B. melitensis biovar 1, respectively, implying changes in the predominant biovars. Genotyping by MLST revealed a total of 14 STs. Nine STs (from ST28 to ST36), accounting for 64.3% of all the STs, were newly defined and different from those observed in other countries. Different STs were distributed among the three stages. ST8 was the most common ST in 1950-1960s and 1990-2000s, while ST2 was the most common in 1970-1980s. CONCLUSIONS: The prevalence of biovars and sequence types of Brucella strains from Inner Mongolia has changed over time in the three stages. Compared with those from other countries, new sequence types of Brucella strains exist in China.

Complete genome sequence of Brucella canis BCB018, a strain isolated from a human patient.

Brucella canis is considered a rare cause of human brucellosis because of difficulties in presumptive diagnosis and underestimation of the incidence. Here, we report the draft genome sequence of a Brucella canis isolate, BCB018, isolated from a human patient, providing precious resources for comparative genomics analysis of Brucella field strains.

Complete genome sequence of Brucella suis field strain BCB025 of sequence type ST22.

Brucella is a genus of relatively conservative pathogenic bacteria. Brucella suis is the most diversified Brucella species. Strains of B. suis belong to different sequence types. Here, we report the genome sequence of B. suis strain BCB025, one isolate of the sequence type 22 epidemic in China.

Genome sequences of three live attenuated vaccine strains of Brucella species and implications for pathogenesis and differential diagnosis.

Live attenuated vaccines play essential roles in the prevention of brucellosis. Here, we report the draft genome sequences of three vaccine strains, Brucella melitensis M5-10, B. suis S2-30, and B. abortus 104M. Primary genome sequence analysis identified mutations, deletions, and insertions which have implications for attenuation and signatures for differential diagnosis.

Complete genome sequence of Brucella abortus 134, a biovar 1 strain isolated from human.

Brucella abortus is one of the common pathogens causing brucellosis in China. Here, we report the genome sequence of B. abortus strain 134, a strain isolated from a human patient and belonging to biovar 1, the most highly represented biovar among B. abortus strains in China.

Complete genome sequence of Brucella abortus strain BCB034, a strain of biovar 2 isolated from human.

Brucella abortus is divided into eight biovars, of which biovars 1 to 3 are the most frequently represented biovars in strains isolated from humans. Here, we report the genome sequence of B. abortus strain BCB034, a strain isolated from a human patient and that belongs to biovar 2.

Genome sequence of an OXA23-producing, carbapenem-resistant Acinetobacter baumannii strain of sequence type ST75.

The increase of Acinetobacter baumannii resistance to carbapenems is of great concern. OXA23 is one of the most prevalent carbapenemases of A. baumannii that causes outbreaks. Here, we announce the genome sequence of an OXA23-producing A. baumannii strain assigned ST75, a newly emerged sequence type harboring carbapenemase.

Altered Transcriptome of the B. melitensis Vaccine Candidate 16MΔvjbR, Implications for Development of Genetically Marked Live Vaccine.

The VjbR protein induced antibody responses in both human and animal brucellosis, and the vjbR mutant 16MΔvjbR is an ideal vaccine candidate because of the feasibility of using the VjbR as diagnostic antigen. To further characterize this vaccine candidate and provide information for vaccine development, in the present study, a whole genome DNA microarray of 16M were used to compare the transcriptome of the vjbR mutant to that of the wild type strains. A total of 126 genes were greatly differentially expressed in the vjbR mutant. A great proportion of virB and flagellar genes were differentially expressed in the vjbR mutant, implying that the vjbR regulate expression of virulence genes by sensing intracellular environments. Interestingly, the virB genes are regulated by the vjbR in independent manners as shown by their different fold changes and transcription abundances. A number of genes involved in translation, stress response, amino acid transport and metabolism, cell wall/membrane biogenesis, energy production and conversion, translation were differentially expressed. The vjbR mutant showed increased sensitivity to stresses of nutrition limitation, oxidative stress and acidification, and decreased survival in macrophage and mice, being consistent with its transcription profiles. These results indicated that the quorum sensing regulator vjbR could sense intracellular environments and response to them by regulate expression of virulence genes and other intracellular survival related genes, and therefore contribute to Brucella survival in host cells. This also provided direct evidence for the rational vaccine design by using antigenic global regulator for future development of genetically marked vaccine for brucellosis.

[Construction of BP26 tagged vaccine strain and development of discriminating PCR for Brucella].

OBJECTIVE: The wide application of live attenuated vaccine strains is limited because of drawbacks of residual virulence, similar antigenenicity to virulent strain and the difficulty to differentiate vaccination and natural infection. In this study, we modified the vaccine strain to prevent the drawbacks. METHODS: By using homologous recombination, we replaced the BP26 gene with the kanamycin gene in a live attenuated vaccine strain M5. The new tagged vaccine strain, M5DeltaBP26, was generated. The wild type strain and M5DeltaBP26 were used to infect macrophage and mice to compare their intracellular survival capability. According to the conservative sequence of dnaK and the deleted region of BP26, primers were designed to develop a duplex PCR for discriminating the wild type strain and M5DeltaBP26. RESULTS: A new tagged strain, M5DeltaBP26, was successfully constructed. The tagged strain could survive in both macrophage and mice, indicating the feasibility as live attenuated vaccine strain. Results from mice infection showed that, at 2 weeks p.i., 10(2.9) CFU of Brucella were isolated from M5 infected mice, whereas only 10(1.1) CFU of Brucella were isolated from M5DeltaBP26 infected mice (P < 0.01). At 3 weeks p. i., 10(2.2) CFU of Brucella whereas no M5DeltaBP26 were isolated. These results indicated that infection capability of M5DeltaBP26 was decreased. Based on the sequence differences between M5DeltaBP26 and M5, a new discriminating duplex PCR was developed. With the duplex PCR, only one product was amplified from M5DeltaBP26, by which it can be differentiated from wild type and virulent strains. CONCLUSION: The construction of tagged strain and the development of discriminating PCR provide a new candidate for further vaccine development.

Large-scale identification of small noncoding RNA with strand-specific deep sequencing and characterization of a novel virulence-related sRNA in Brucella melitensis.

Brucella is the causative agent of brucellosis, a worldwide epidemic zoonosis. Small noncoding RNAs (sRNAs) are important modulators of gene expression and involved in pathogenesis and stress adaptation of Brucella. In this study, using a strand-specific RNA deep-sequencing approach, we identified a global set of sRNAs expressed by B. melitensis 16M. In total, 1321 sRNAs were identified, ranging from 100 to 600 nucleotides. These sRNAs differ in their expression levels and strand and chromosomal distributions. The role of BSR0441, one of these sRNAs, in the virulence of B. melitensis 16M was further characterized. BSR0441 was highly induced during the infection of macrophages and mice. The deletion mutant of BSR0441 showed significantly reduced spleen colonization in the middle and late phases of infection. The expression of the BSR0441 target mRNA genes was also altered in the BSR0441 mutant strain during macrophage and mice infection, which is consistent with its reduced intracellular survival capacity. In summary, Brucella encodes a large number of sRNAs, which may be involved in the stress adaptation and virulence of Brucella. Further investigation of these regulators will extend our understanding of the Brucella pathogenesis mechanism and the interactions between Brucella and its hosts.

Immunization with individual proteins of the Lrp/AsnC family induces protection against Brucella melitensis 16M challenges in mice.

Brucellosis is one of the most common zoonoses worldwide. Subunit vaccines are promising for the prevention of human brucellosis. In our previous protective antigen screening studies, we identified a new protective antigen, BMEI0357, which belongs to the Lrp/asnC protein family, a conserved transcriptional regulator in bacteria that is absent in eukaryotes. In the present study, the Brucella genome annotation was screened and a total of six proteins were identified as members of the Lrp/AsnC family. Lrp/AsnC proteins have two domains that are conserved among the family members. However, sequence similarities between these proteins ranged from 9 to 50%, indicating high sequence heterogeneity. To test whether proteins of this family have similar characteristics, all six proteins were cloned and expressed in Escherichia coli. The recombinant proteins were purified and their protective efficacy was evaluated in BALB/c mice challenged with Brucella melitensis 16M. The results show that all six Lrp/AsnC proteins could induce a protective immune response against Brucella melitensis 16M. Antibodies against the Lrp/AsnC proteins were detected in the immunized mice. However, levels of antibodies against these proteins were relatively variable in human brucellosis sera. Taken together, our results show that these six proteins of the Lrp/AsnC family in Brucella could induce protective immune responses in mice.

Identification of a Novel Small Non-Coding RNA Modulating the Intracellular Survival of Brucella melitensis.

Bacterial small non-coding RNAs (sRNAs) are gene expression modulators respond to environmental changes, stressful conditions, and pathogenesis. In this study, by using a combined bioinformatic and experimental approach, eight novel sRNA genes were identified in intracellular pathogen Brucella melitensis. BSR0602, one sRNA that was highly induced in stationary phase, was further examined and found to modulate the intracellular survival of B. melitensis. BSR0602 was present at very high levels in vitro under stresses similar to those encountered during infection in host macrophages. Furthermore, BSR0602 was found to be highly expressed in the spleens of infected mice, suggesting its potential role in the control of pathogenesis. BSR0602 targets the mRNAs coding for gntR, a global transcriptional regulator, which is required for B. melitensis virulence. Overexpression of BSR0602 results in distinct reduction in the gntR mRNA level. B. melitensis with high level of BSR0602 is defective in bacteria intracellular survival in macrophages and defective in growth in the spleens of infected mice. Therefore, BSR0602 may directly inhibit the expression of gntR, which then impairs Brucellae intracellular survival and contributes to Brucella infection. Our findings suggest that BSR0602 is responsible for bacterial adaptation to stress conditions and thus modulate B. melitensis intracellular survival.

Sensitive and rapid detection of blaNDM-1 in clinical samples by isothermal cross-priming amplification.

A highly sensitive, specific diagnostic assay for detection of bla(NDM-1) based on cross priming amplification (CPA) was developed. The sensitivity ranged from 2.5 to 25 copies per reaction for different clinical samples. The highly and sensitive detection of bla(NDM-1) in clinical samples highlighted the potential clinical applications of CPA.

Draft Genome Sequence of Brucella abortus BCB027, a Strain Isolated from a Domestic Deer.

Many Brucella species are isolated from nonpreferred hosts, and these bacteria may show genetic differences from isolates from the preferred hosts. Here, we report the draft genome sequence of Brucella abortus BCB027, a novel strain isolated from a domestic deer.

Impact of Hfq on global gene expression and intracellular survival in Brucella melitensis.

Brucella melitensis is a facultative intracellular bacterium that replicates within macrophages. The ability of brucellae to survive and multiply in the hostile environment of host macrophages is essential to its virulence. The RNA-binding protein Hfq is a global regulator that is involved in stress resistance and pathogenicity. Here we demonstrate that Hfq is essential for stress adaptation and intracellular survival in B. melitensis. A B. melitensis hfq deletion mutant exhibits reduced survival under environmental stresses and is attenuated in cultured macrophages and mice. Microarray-based transcriptome analyses revealed that 359 genes involved in numerous cellular processes were dysregulated in the hfq mutant. From these same samples the proteins were also prepared for proteomic analysis to directly identify Hfq-regulated proteins. Fifty-five proteins with significantly affected expression were identified in the hfq mutant. Our results demonstrate that Hfq regulates many genes and/or proteins involved in metabolism, virulence, and stress responses, including those potentially involved in the adaptation of Brucella to the oxidative, acid, heat stress, and antibacterial peptides encountered within the host. The dysregulation of such genes and/or proteins could contribute to the attenuated hfq mutant phenotype. These findings highlight the involvement of Hfq as a key regulator of Brucella gene expression and facilitate our understanding of the role of Hfq in environmental stress adaptation and intracellular survival of B. melitensis.