Newcastle disease virus-like particles containing the Brucella BCSP31 protein induce dendritic cell activation and protect mice against virulent Brucella challenge.

Brucellosis is a widespread zoonosis that poses a substantial threat to human and animal public health due to the absence of a sufficiently safe and efficient vaccine. Virus-like particles (VLPs) have been developed as novel vaccine candidates and suitable carrier platforms for the delivery of exogenous proteins. Herein, we constructed chimeric virus-like particles (cVLPs) assembled by a Newcastle disease virus (NDV) M protein and glycosylphosphatidylinositol-anchored Brucella BCSP31 protein (GPI-BCSP31). cVLPs-GPI-BCSP31 were highly efficient in murine dendritic cell (DC) activation, both in vitro and in vivo. Moreover, they elicited strong specific humoural immune responses detected through ELISA assay with inactivated Brucella and recombinant BCSP31 protein and by elevated cellular immune responses indicated by intracellular IFN-γ and IL-4 levels in CD3+CD4+ T and CD3+CD8+ T cells. Importantly, cVLPs-GPI-BCSP31 conferred protection against virulent Brucella melitensis strain 16 M challenge, comparable to the efficacy of Brucella vaccine strain M5. In summary, this study provides a new strategy for the development of a safe and effective vaccine candidate against virulent Brucella and further extends the application of NDV VLP-based vaccine platforms.

Yeast Surface Display of Escherichia coli Enterotoxin and Its Effects of Intestinal Microflora and Mucosal Immunity.

Enterotoxigenic Escherichia coli (ETEC) is a significant cause of infectious diarrhea in animals. In this study, yeast surface display technology was employed to investigate the effects of ETEC enterotoxin fusion protein on the intestinal flora and mucosal immunity of rats. ETEC estA, estB, and eltAB (heat-labile and heat-stable toxins) were expressed on the surface of yeast. Rats were divided into normal saline, yeast and display yeast groups. Fecal and jejunal content samples were collected on the 7th, 14th, and 21st days. Rats were then fed ETEC for 3 days before again collecting these samples. Levels of SIgA, IL-2, IL-4, IFN-γ, and microbial population density and diversity were documented by ELISA, T-RFLP and real-time PCR. The results demonstrated that estA, estB, and eltAB fusion proteins were expressed on the surface of yeast. Following ETEC challenge, levels of SIgA, IL-2, IL-4, IFN-γ, and, the numbers and variety of intestinal microbes were significantly increased in rats receiving display yeast and yeast. These factors were significantly decreased in rats given normal saline and yeast. Our results indicate that display yeast and yeast can increase the number and diversity of intestinal microbes in rats and improve intestinal immune function. After ETEC challenge, the display yeast can better maintain the balance of intestinal bacteria and mucosal immunity.

A safe and molecular-tagged Brucella canis ghosts confers protection against virulent challenge in mice.

Canine brucellosis, caused by Brucella canis, is a persistent infectious reproductive disease in dogs. The absence of effective treatment to the intracellular pathogen and the irreversible consequence of infection makes the need of a specific vaccine urgent. Bacterial ghosts (BGs) are the empty envelopes of bacteria with no genome content inside, which emerge as a proper vaccine candidate due to its intact outer antigen. It is generally derived from a genetically engineered strain, through the expression of Bacteriophage phiX174 lysis E gene upon induction. In this study, we combined the homologous recombination (HR) and bacterial ghost technologies, generating a genetically stable B. canis ghost strain which bears no drug resistance gene. When the ghost strain grows to OD600 of 0.6, 100% inactivation can be achieved under 42°C in 60h. The resultant BGs showed guaranteed safety and comparable immunogenicity to a live vaccine. The bacterial B0419 protein was depleted during HR process, which is subsequently proved to work as a molecular tag in distinguishing natural infection and BGs immunization through ELISA. Additionally, the BGs also conferred protection against B. canis RM6/66 and B. melitensis 16M. Therefore, the application of current BGs as a vaccine candidate and the corresponding serological diagnostic approach may provide better B. canis prevention strategy.

A novel virulence-associated protein, vapE, in Streptococcus suis serotype 2.

Streptococcus suis serotype 2 (SS2) is an important pathogen that affects pigs. However, neither its virulence nor its pathogenesis of infection has yet to be fully elucidated. The present study identifies a novel virulence­associated protein E gene (vapE) of SS2. To investigate the importance of vapE in SS2 infection, a vapE knock­out mutant based on SS2 wild­type strain ZY458 was designated 458ΔvapE. 458ΔvapE was generated through homologous recombination, using a combined plasmid with a vapE knock­out fragment and a pSET4s suicide vector. Additionally, the 458ΔvapE strain was transformed by a pAT18 shuttle plasmid containing the vapE gene. A functionally complemented strain for the vapE gene [termed 458ΔvapE (pvapE)] was constructed. Animal experiments demonstrated that mice infected with ZY458 and 458ΔvapE (pvapE) exhibited severe clinical symptoms, including depression, apathy, fever, anorexia, emaciation, swollen eyes and neural disorders, and died within two days of infection. All mice infected with ZY458, and 85% of mice infected with 458ΔvapE (pvapE), died within 2 days of infection. In contrast, mice inoculated with 458ΔvapE exhibited only mild clinical symptoms in the first 2 days following infection, and recovered within a week. A bacterial colonization assay demonstrated the ability of the 458ΔvapE mutant SS2 strain to colonize the heart, liver, spleen, lung and kidney of infected mice. PCR analysis of the vapE gene revealed that functional vapE was detected in virulent strains, but not in avirulent and carrier strains of S. suis SS2. These findings indicate that vapE is important for the pathogenesis of SS2.

Rapid method of luxS and pfs gene inactivation in enterotoxigenic Escherichia coli and the effect on biofilm formation.

Rapid and efficient inactivation of a target gene in Escherichia coli chromosomes is required to investigate metabolic engineering. In the present study, a multiple gene inactivation approach was demonstrated in four strains of enterotoxigenic E. coli (ETEC), which are the predominant pathogenic bacteria causing piglet diarrhea, mediated by λ Red and Xer recombination. The chromosomal genes, luxS and pfs were inactivated using the multiple gene inactivation approach in the wild­type strains of E. coli, K88, K99, 987P and F41. This indicated that dif sites may be reused to inactivate multiple chromosomal genes when no antibiotic­resistant selectable markers remain. Following inactivation of luxS and pfs, the ability of ETEC to produce the quorum sensing signal, and induce auto­inducer 2 activity and biofilm formation were significantly reduced. Furthermore, the multiple gene inactivation approach also exhibits a high recombination efficiency and follows a simple process.

Catabolite control protein A has an important role in the metabolic regulation of Streptococcus suis type 2 according to iTRAQ-based quantitative proteomic analysis.

The catabolite control protein A (ccpA) regulates the carbon metabolism in Streptococcus suis type 2 and has pleiotropic regulatory functions in bacterial virulence and transcription. The present study systematically investigated ccpA activity in Streptococcus suis type 2 using isobaric tag for relative and absolute quantification (iTRAQ) liquid chromatography­tandem mass spectrometry­based proteomics. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses demonstrated that ccpA is an important protein for the regulation of metabolism, virulence and immune pathways in Streptococcus suis type 2. The present study therefore expanded the current understanding of the effects of ccpA on virulence, metabolic regulation and transcription in Streptococcus suis type 2 and other important pathogens.

Investigation into the role of catabolite control protein A in the metabolic regulation of Streptococcus suis serotype 2 using gene expression profile analysis.

Catabolite control protein A (CcpA) serves a key function in the catabolism of Streptococcus suis serotype 2 (S. suis 2) by affecting the biological function and metabolic regulatory mechanisms of this bacterium. The aim of the present study was to identify variations in CcpA expression in S. suis 2 using gene expression profile analysis. Using sequencing and functional analysis, CcpA was demonstrated to play a regulatory role in the expression and regulation of virulence genes, carbon metabolism and immunoregulation in S. suis 2. Gene Ontology and Kyto Encyclopedia of Genes and Genomes analyses indicated that CcpA in S. suis 2 is involved in the regulation of multiple metabolic processes. Furthermore, combined analysis of the transcriptome and metabolite data suggested that metabolites varied due to the modulation of gene expression levels under the influence of CcpA regulation. In addition, metabolic network analysis indicated that CcpA impacted carbon metabolism to a certain extent. Therefore, the present study has provided a more comprehensive analysis of the role of CcpA in the metabolic regulation of S. suis 2, which may facilitate future investigation into this mechanism. Furthermore, the results of the present study provide a foundation for further research into the regulatory function of CcpA and associated metabolic pathways in S. suis 2.

Catabolite control protein A is an important regulator of metabolism in Streptococcus suis type 2.

Streptococcus suis (S. suis) type 2 is an extremely important Gram-positive bacterial pathogen that can cause human or swine endocarditis, meningitis, bronchopneumonia, arthritis and sepsis. Catabolite control protein A (CcpA) is a major transcriptional regulator in S. suis type 2 that functions in catabolite control, specifically during growth on glucose or galactose. The regulation of central metabolism can affect the virulence of bacteria. In the present study, a metabolomics approach was used along with principal components analysis (PCA) and partial least-squares-discriminant analysis (PLS-DA) models and 37 metabolites were found that differed substantially between native S. suis and a mutant lacking CcpA. These results showed that CcpA is an important protein in S. suis type 2 for studying bacterial protein function.

Effects of partial deletion of the wzm and wzt genes on lipopolysaccharide synthesis and virulence of Brucella abortus S19.

Brucellosis is a worldwide human and animal infectious disease, and the effective methods of its control are immunisation of animals by vaccination and elimination. Brucella abortus S19 is one of the popular vaccines with virulence in the control of cattle Brucellosis. In the present study, allelic exchange plasmids of wzm and wzt genes and partial knockout mutants of wzm and wzt were constructed to evaluate the resulting difference in virulence of B. abortus S19. PCR analysis revealed that the target genes were knocked out. The mutants were rough mutants and they could be differentiated from natural infection by the Rose Bengal plate and standard agglutination tests. The molecular weights of lipopolysaccharides of the Δwzm and Δwzt mutants were clustered between 25 and 40 kDa, and 30 and 35 kDa separately, and were markedly different from those in B. abortus S19. The virulence of B. abortus Δwzm and Δwzt was decreased compared with that of B. abortus S19 in mice. All these results identified that there were several differences between the wzm and wzt genes on lipopolysaccharide synthesis and on the virulence of B. abortus.

Immunogenic response induced by wzm and wzt gene deletion mutants from Brucella abortus S19.

Brucellosis is an infectious disease affecting humans and animals worldwide. Effective methods of control include inducing immunity in animals by vaccination and elimination. Brucella abortus S19 is one of the popular vaccines for control of cattle brucellosis, as it has low virulence. In this paper, allelic exchange plasmids of wzm and wzt genes were constructed and partially knocked out to evaluate the effects on the induction of immunity to Brucella abortus S19 mutants. Cytokine secretion in vitro, INF-γ induction in vivo and antibody dynamics were evaluated. These data suggested that the immunity-eliciting ability of the wzm and wzt gene deletion mutants was similar, although reduced compared with the S19 strain. The results demonstrated that the wzt gene may be more important in the regulation of the induction of immunity than the wzm gene.

Immunoproteomic analysis of Brucella melitensis and identification of a new immunogenic candidate protein for the development of brucellosis subunit vaccine.

In order to screen immunogenic candidate antigens for the development of a brucellosis subunit vaccine, an immunoproteomic assay was used to identify immunogenic proteins from Brucella melitensis 16 M soluble proteins. In this study, a total of 56 immunodominant proteins were identified from the two-dimensional electrophoresis immunoblot profiles by liquid chromatography tandem mass spectrometry (LC-MS/MS). Two proteins of interest, riboflavin synthase alpha chain (RS-α) and Loraine synthase (LS-2), which are both involved in riboflavin synthesis, were detected by two-dimensional immunoblots using antisera obtained from Brucella-infected human and goats. LS-2, however, is an already well-known vaccine candidate. Therefore, we focussed our studies on the novel vaccine candidate RS-α. B. melitensis RS-α and LS-2 were then expressed in Escherichia coli as fusion proteins with His tag. The humoral and cellular immune responses to the recombinant (r)RS-α was characterized. In response to in vitro stimulation by rRS-α, splenocytes from mice vaccinated with rRS-α were able to produce γ-interferon (IFN-γ) and interleukin (IL)-2 but not interleukin (IL)-4 and interleukin (IL)-10. Furthermore, rRS-α or rLS-2-vaccinated mice were partially protected against B. melitensis infection. Our results suggested that we have developed a high-throughout, accurate, rapid and highly efficient method for the identification of candidate antigens by a combination of immunoproteomics with immunisation and bacterial challenge and rRs-α could be a useful candidate for the development of subunit vaccines against B. melitensis.

Comparative proteomic analyses of Streptococcus suis serotype 2 cell wall-associated proteins.

Streptococcus suis serotype 2 (SS2) is a zoonotic pathogen that is distributed throughout the world. Virulence factors and/or markers of the virulent serotype 2 strains have not been fully identified. In this study a simple, rapid, and non-destructive method was used to extract cell wall-associated proteins from SS2 strains. Two virulent strains were compared with one avirulent strain by 2-dimensional electrophoresis (2DE). When the results of the 2DE analyses were combined with the results of mass spectrometry analyses, a total of 40 unique proteins were identified, including 26 antigens (2DE immunoblotting was used as a preliminary study). In addition to a known virulence factor, muramidase-released protein, two new proteins, catabolite control protein A and leucyl aminopeptidase, and nine potential virulence factors were also identified. The formers may be a potential virulence regulator or drug target, and the latter contains plasminogen-binding proteins and molecular chaperones. Our results complemented previous immunoproteomics studies of SS2 strains.

Immunization of mice with recombinant S-adenosyl-L-homocysteine hydrolase protein confers protection against Brucella melitensis infection.

Due to drawbacks of live attenuated vaccines, much attention has been focused on screening Brucella-protective antigens as subunit vaccine candidates. Here, an immunoproteomic assay was used to identify the immunogenic soluble proteins of Brucella melitensis 16M. In the present study, 27 unique immunogenic proteins were identified from the two-dimensional electrophoresis immunoblot profiles by liquid chromatography tandem MS (LC-MS/MS). From this set, the gene encoding one immunodominant protein of interest, S-adenosyl-l-homocysteine hydrolase (AdoHcyase), was expressed in Escherichia coli. The recombinant AdoHcyase induced a strong antibody response in BALB/c mice, and the polyclonal antibody could recognize a band of approximately 52 kDa in the immunoblots of soluble protein extracts from five Brucella strains. rAdoHcyase significantly stimulated the production of interferon-γ and interleukin-2, and induced a high level of protection against B. melitensis 16M challenge at 4 weeks postchallenge. Our results indicated that rAdoHcyase could be a useful candidate for the development of subunit vaccines against B. melitensis.

Preparation and application of microarrays for the detection of antibiotic resistance genes in samples isolated from Changchun, China.

The emergence of antibiotic-resistant bacteria, especially tetracycline- and beta-lactam-resistant bacteria, poses a great threat to human health. The purpose of this study was to develop and apply a suitable gene microarray for the detection of antibiotic resistance genes. We isolated 463 strains of bacteria from a hospital, a veterinary station, an animal nursery, and living environment of Changchun, China. After screening, it was found that 93.9% of these bacteria were resistant to tetracycline, 74.9% to ampicillin, 55.6% to deoxycycline, and 41.7% to ciprofloxacin. For amplification of antibiotic genes, we designed 28 pairs of primers. In addition, 28 hybridization probes for these genes were developed. The DNA microarray analysis was performed at 42 degrees C for 5 h. We were successful in detecting 12 resistance genes by microarray analysis. After detection, we also evaluated the sensitivity of the microarray analysis. The LDL (Lowest Detection Level) of the microarray was 1 x 10(6) copies/ml of template DNA. It is believed that such microarray-based determination of tetracycline and beta-lactam resistance genes can have a potential application in clinical studies in the future.