Lung Resident Memory T Cells Activated by Oral Vaccination Afford Comprehensive Protection against Pneumonic Yersinia pestis Infection.

A growing body of evidence has shown that resident memory T (TRM) cells formed in tissue after mucosal infection or vaccination are crucial for counteracting reinfection by pathogens. However, whether lung TRM cells activated by oral immunization with Yptb1(pYA5199) play a protective role against pneumonic plague remains unclear. In this study, we demonstrated that lung CD4+ and CD8+ TRM cells significantly accumulated in the lungs of orally Yptb1(pYA5199)-vaccinated mice and dramatically expanded with elevated IL-17A, IFN-γ, and/or TNF-α production after pulmonary Yersinia pestis infection and afforded significant protection. Short-term or long-term treatment of immunized mice with FTY720 did not affect lung TRM cell formation and expansion or protection against pneumonic plague. Moreover, the intratracheal transfer of both lung CD4+ and CD8+ TRM cells conferred comprehensive protection against pneumonic plague in naive recipient mice. Lung TRM cell-mediated protection was dramatically abolished by the neutralization of both IFN-γ and IL-17A. Our findings reveal that lung TRM cells can be activated via oral Yptb1(pYA5199) vaccination, and that IL-17A and IFN-γ production play an essential role in adaptive immunity against pulmonary Y. pestis infection. This study highlights an important new target for developing an effective pneumonic plague vaccine.

Remodeling Yersinia pseudotuberculosis to generate a highly immunogenic outer membrane vesicle vaccine against pneumonic plague.

SignificanceYersinia pestis, the etiologic agent of plague, has been responsible for high mortality in several epidemics throughout human history. This plague bacillus has been used as a biological weapon during human history and is currently one of the deadliest biological threats. Currently, no licensed plague vaccines are available in the Western world. Since an array of immunogens are enclosed in outer membrane vesicles (OMVs), immune responses elicited by OMVs against a diverse range of antigens may reduce the likelihood of antigen circumvention. Therefore, self-adjuvanting OMVs from a remodeled Yersinia pseudotuberculosis strain as a type of plague vaccine could diversify prophylactic choices and solve current vaccine limitations.

Protection Induced by Oral Vaccination with a Recombinant Yersinia pseudotuberculosis Delivering Yersinia pestis LcrV and F1 Antigens in Mice and Rats against Pneumonic Plague.

A newly attenuated Yersinia pseudotuberculosis strain (designated Yptb1) with triple mutation Δasd ΔyopK ΔyopJ and chromosomal insertion of the Y. pestis caf1R-caf1M-caf1A-caf1 operon was constructed as a live vaccine platform. Yptb1 tailored with an Asd+ plasmid (pYA5199) (designated Yptb1[pYA5199]) simultaneously delivers Y. pestis LcrV and F1. The attenuated Yptb1(pYA5199) localized in the Peyer's patches, lung, spleen, and liver for a few weeks after oral immunization without causing any disease symptoms in immunized rodents. An oral prime-boost Yptb1(pYA5199) immunization stimulated potent antibody responses to LcrV, F1, and Y. pestis whole-cell lysate (YPL) in Swiss Webster mice and Brown Norway rats. The prime-boost Yptb1(pYA5199) immunization induced higher antigen-specific humoral and cellular immune responses in mice than a single immunization did, and it provided complete short-term and long-term protection against a high dose of intranasal Y. pestis challenge in mice. Moreover, the prime-boost immunization afforded substantial protection for Brown Norway rats against an aerosolized Y. pestis challenge. Our study highlights that Yptb1(pYA5199) has high potential as an oral vaccine candidate against pneumonic plague.

Dissecting psa Locus Regulation in Yersinia pestis.

The pH 6 antigen (PsaA) of Yersinia pestis is a virulence factor that is expressed in response to high temperature (37°C) and low pH (6.0). Previous studies have implicated the PsaE and PsaF regulators in the temperature- and pH-dependent regulation of psaA. Here, we show that PsaE levels are themselves controlled by pH and temperature, explaining the regulation of psaA. We identify hundreds of binding sites for PsaE across the Y. pestis genome, with the majority of binding sites located in intergenic regions bound by the nucleoid-associated protein H-NS. However, we detect direct regulation of only two transcripts by PsaE, likely due to displacement of H-NS from the corresponding promoter regions; our data suggest that most PsaE binding sites are nonregulatory or that they require additional environmental cues. We also identify the precise binding sites for PsaE that are required for temperature- and pH-dependent regulation of psaA and psaE. Thus, our data reveal the critical role that PsaE plays in the regulation of psaA and suggest that PsaE may have many additional regulatory targets. IMPORTANCE Y. pestis, the etiologic agent of plague, has been responsible for high mortality in several epidemics throughout human history. The plague bacillus has been used as a biological weapon during human history and is currently one of the most likely biological threats. PsaA and PsaE appear to play important roles during Y. pestis infection. Understanding their regulation by environmental cues would facilitate a solution to impede Y. pestis infection.

Recombinant Pseudomonas Bionanoparticles Induce Protection against Pneumonic Pseudomonas aeruginosa Infection.

To develop an effective Pseudomonas aeruginosa outer-membrane-vesicle (OMV) vaccine, we eliminated multiple virulence factors from a wild-type (WT) P. aeruginosa strain, PA103, to generate a recombinant strain, PA-m14. Strain PA-m14 was tailored with a pSMV83 plasmid carrying the pcrV-hitAT fusion gene to produce OMVs. The recombinant OMVs (termed OMV-PH) enclosed increased amounts of the PcrV-HitAT bivalent antigen (PH) and exhibited lower toxicity than did the OMVs from PA103. Intramuscular vaccination with OMV-PH from PA-m14(pSMV83) afforded 70% protection against intranasal challenge with 6.5 × 106 CFU (∼30 50% lethal doses [LD50]) of PA103, while immunization using OMVs without the PH antigen (termed OMV-NA) or the PH antigen alone failed to offer effective protection against the same challenge. Further immune analysis showed that OMV-PH immunization significantly stimulated potent antigen-specific humoral and T-cell (Th1/Th17) responses over those with PH or OMV-NA immunization in mice and that these more-potent responses can effectively hinder P. aeruginosa infection. Undiluted antisera from OMV-PH-immunized mice displayed significantly more opsonophagocytic killing of WT PA103 than antisera from PH antigen- or OMV-NA-immunized mice. Moreover, OMV-PH immunization afforded significant antibody-independent cross-protection to mice against PAO1 and the AMC-PA10 clinical isolate. Taking our findings together, the recombinant P. aeruginosa OMV delivering the bivalent PH antigen exhibits high immunogenicity and may be a promising next-generation vaccine candidate against P. aeruginosa infection.

Induction of Protective Antiplague Immune Responses by Self-Adjuvanting Bionanoparticles Derived from Engineered Yersinia pestis.

A Yersinia pestis mutant synthesizing an adjuvant form of lipid A (monophosphoryl lipid A, MPLA) displayed increased biogenesis of bacterial outer membrane vesicles (OMVs). To enhance the immunogenicity of the OMVs, we constructed an Asd-based balanced-lethal host-vector system that oversynthesized the LcrV antigen of Y. pestis, raised the amounts of LcrV enclosed in OMVs by the type II secretion system, and eliminated harmful factors like plasminogen activator (Pla) and murine toxin from the OMVs. Vaccination with OMVs containing MPLA and increased amounts of LcrV with diminished toxicity afforded complete protection in mice against subcutaneous challenge with 8 × 105 CFU (80,000 50% lethal dose [LD50]) and intranasal challenge with 5 × 103 CFU (50 LD50) of virulent Y. pestis This protection was significantly superior to that resulting from vaccination with LcrV/alhydrogel or rF1-V/alhydrogel. At week 4 postimmunization, the OMV-immunized mice showed more robust titers of antibodies against LcrV, Y. pestis whole-cell lysate (YPL), and F1 antigen and more balanced IgG1:IgG2a/IgG2b-derived Th1 and Th2 responses than LcrV-immunized mice. Moreover, potent adaptive and innate immune responses were stimulated in the OMV-immunized mice. Our findings demonstrate that self-adjuvanting Y. pestis OMVs provide a novel plague vaccine candidate and that the rational design of OMVs could serve as a robust approach for vaccine development.

Oil body bound oleosin-rhFGF9 fusion protein expressed in safflower (Carthamus tinctorius L.) stimulates hair growth and wound healing in mice.

BACKGROUND: Fibroblast growth factor 9 (FGF9) is a heparin-binding growth factor, secreted by both mesothelial and epithelial cells, which participates in hair follicle regeneration, wound healing, and bone development. A suitable source of recombinant human FGF9 (rhFGF9) is needed for research into potential clinical applications. We present that expression of oleosin-rhFGF9 fusion protein in safflower (Carthamus tinctorius L.) seeds stimulates hair growth and wound healing. RESULTS: The oleosin-rhFGF9 expressed in safflower seeds, in which it localizes to the surface of oil bodies. The expression of oleosin-rhFGF9 was confirmed by polyacrylamide gel electrophoresis and western blotting. According to BCA and Enzyme-linked immunosorbent assay (ELISA) assay, the results show that the expression level of oleosin-rhFGF9 was 0.14% of oil body protein. The oil body bound oleosin-rhFGF9 showed mitogenic activity towards NIH3T3 cells in a methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. The efficacy of oil body bound oleosin-rhFGF9 in promoting hair growth and wound healing was investigated in C57BL/6 mice. In a hair regeneration experiment, 50 µg/µl oil body bound oleosin-rhFGF9 was applied to the dorsal skin of mice in the resting phase of the hair growth cycle. After 15 days, thicker hair and increased number of new hairs were seen compared with controls. Furthermore, the number of new hairs was greater compared with rhFGF9-treated mice. The hair follicles of mice treated with oil body bound oleosin-rhFGF9 expressed ß-catenin more abundantly. In a wound healing experiment, dorsal skin wounds were topically treated with 50 µg/µl oil body bound oleosin-rhFGF9. Wound healing was quicker compared with mice treated with rhFGF9 and controls, especially in the earlier stages of healing. CONCLUSIONS: The oil body bound oleosin-rhFGF9 promotes both hair growth and wound healing. It appears to promote hair growth, at least in part, by up-regulating ß-catenin expression. The potential of oil body bound oleosin-rhFGF9 as an external drug can treat the alopecia and wounds or use in further clinical application.

Jilinibacillus soli gen. nov., sp. nov., a novel member of the family Bacillaceae.

A Gram-positive, aerobic, rod-shaped, motile, endospore-forming bacterium, designated strain A12(T), was isolated from a saline and alkali soil samples in Baicheng City, western of Jilin Province, China. Growth occurred in 15-45 °C (optimum, 30 °C) and at pH 7.0-11.5 (optimum, pH 9.0) and in the presence of 0-10 % (w/v) NaCl [optimum, 1-3 % (w/v) NaCl]. Meso-DAP was present in the peptidoglycan. The predominant menaquinone was MK-7. The major polar lipid profile was phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidyl inositol-methyl and phosphotidylinositol dimannosid. The major fatty acid (>10 % of total fatty acids) was anteiso-C15:0. DNA G + C content was 36.2 mol %. The level of 16S rRNA gene sequence similarity between strain A12(T) and other recognized species of the family was below 95.6 %. Phylogenetic analysis based on 16S rRNA gene sequence data indicated that the strain A12(T) fell with the family Bacillaceae and formed a distinct taxon. Based on physiological, chemotaxonomic and phylogenetic analyses, strain A12(T) was considered to represent a novel species of a new genus, for which the name Jilinibacillus soli gen. nov., sp. nov. was proposed. The type strain of Jilinibacillus soli was A12(T) (=GIMN1.014(T) = CCTCC M2011164(T) = KCTC 33417(T)).

Recombinant VirB5 protein as a potential serological marker for the diagnosis of bovine brucellosis.

The molecular tag vaccine against Brucella abortus and serological testing are the main methods of prevention of brucellosis used currently. They can discriminate vaccinated animals and humans from those naturally infected. In this study, we constructed a gene deletion mutant strain, B. abortus S19 virB5 with a molecular tag. Recombinant VirB5 was expressed and purified for evaluation as a diagnostic reagent for bovine brucellosis. In total, 400 sera samples were tested using a VirB5 antigen-based enzyme-linked immunosorbent assay (ELISA) and the results were compared with those of the standard tube agglutination test (SAT). This showed that the sensitivity was 88.2%, specificity was 97.8% and accuracy was 94.8%. Recombinant VirB5 could also be used to discriminate B. abortus-infected mice from mice infected with the B. abortus S19 virB5 mutant strain. It was concluded that recombinant VirB5 could be used as a potential antigen and serological marker for the diagnosis of bovine brucellosis.

Dietary Bacillus licheniformis improves the effect of Astragalus membranaceus extract on blood glucose by regulating antioxidation activity and intestinal microbiota in InR[E19]/TM2 Drosophila melanogaster.

BACKGROUND: The diabetes mellitus prevalence is rapidly increasing in most parts of the world and has become a vital health problem. Probiotic and herbal foods are valuable in the treatment of diabetes. METHODS AND PERFORMANCE: In this study, Bacillus licheniformis (BL) and Astragalus membranaceus extract (AE) were given with food to InR[E19]/TM2 Drosophila melanogaster, and the blood glucose, antioxidation activity and intestinal microbiota were investigated. The obtained results showed that BA (BL and AE combination) supplementation markedly decreased the blood glucose concentration compared with the standard diet control group, accompanied by significantly increased enzymatic activities of catalase (CAT), decreased MDA levels and prolonged lifespan of InR[E19]/TM2 D. melanogaster. The treatments with BL, AE and BA also ameliorated intestinal microbiota equilibrium by increasing the population of Lactobacillus and significantly decreasing the abundance of Wolbachia. In addition, clearly different evolutionary clusters were found among the control, BL, AE and BA-supplemented diets, and the beneficial microbiota, Lactobacillaceae and Acetobacter, were found to be significantly increased in male flies that were fed BA. These results indicated that dietary supplementation with AE combined with BL not only decreased blood glucose but also extended the lifespan, with CAT increasing, MDA decreasing, and intestinal microbiota improving in InR[E19]/TM2 D. melanogaster. CONCLUSION: The obtained results showed that dietary supplementation with BL and AE, under the synergistic effect of BL and AE, not only prolonged the lifespan of InR[E19]/TM2 D. melanogaster, increased body weight, and improved the body's antiaging enzyme activity but also effectively improved the types and quantities of beneficial bacteria in the intestinal flora of InR[E19]/TM2 D. melanogaster to improve the characteristics of diabetes symptoms. This study provides scientific evidence for a safe and effective dietary therapeutic method for diabetes mellitus.

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.

Outer Membrane Vesicles Displaying a Heterologous PcrV-HitA Fusion Antigen Promote Protection against Pulmonary Pseudomonas aeruginosa Infection.

Along with surging threats and antibiotic resistance of Pseudomonas aeruginosa in health care settings, it is imperative to develop effective vaccines against P. aeruginosa infection. In this study, we used an Asd (aspartate-semialdehyde dehydrogenase)-based balanced-lethal host-vector system of a recombinant Yersinia pseudotuberculosis mutant to produce self-adjuvanting outer membrane vesicles (OMVs). The OMVs were used as a carrier to deliver the heterologous PcrV-HitAT (PH) fusion antigen of P. aeruginosa for vaccine evaluation. Intramuscular vaccination with OMVs carrying the PH antigen (referred to rOMV-PH) afforded 73% protection against intranasal challenge with 5 × 106 (25 50% lethal doses) of the cytotoxic PA103 strain and complete protection against a noncytotoxic PAO1 strain. In contrast, vaccination with the PH-deficient OMVs or PH antigen alone failed to offer effective protection against the same challenge. Immune analysis showed that the rOMV-PH vaccination induced potent humoral and Th1/Th17 responses compared to the PH vaccination. The rOMV-PH vaccination rapidly cleared P. aeruginosa burdens with coordinated production of proinflammatory cytokines in mice. Moreover, antigen-specific CD4+ and CD8+ T cells and their producing cytokines (tumor necrosis factor alpha and interleukin-17A), rather than antibodies, were essential for protection against pneumonic P. aeruginosa infection. Our studies demonstrated that the recombinant Y. pseudotuberculosis OMVs delivering heterologous P. aeruginosa antigens could be a new promising vaccine candidate for preventing the spread of drug-resistant P. aeruginosa. IMPORTANCE Hospital- and community-acquired infections with Pseudomonas aeruginosa cause a high rate of morbidity and mortality in patients who have underlying medical conditions. The spread of multidrug-resistant P. aeruginosa strains is becoming a great challenge for treatment using antibiotics. Thus, a vaccine as one of the alternative strategies is urgently required to prevent P. aeruginosa infection.

Protection and Safety Evaluation of Live Constructions Derived from the Pgm- and pPCP1- Yersinia pestis Strain.

Based on a live attenuated Yersinia pestis KIM10(pCD1Ap) strain (Pgm-, pPCP1-), we attempted to engineer its lipid A species to achieve improvement of immunogenicity and safety. A mutant strain designated as YPS19(pCD1Ap), mainly synthesizing the hexa-acylated lipid A, and another mutant strain designated as YPS20(pCD1Ap), synthesizing 1-dephosphalated hexa-acylated lipid A (detoxified lipid A), presented relatively low virulence in comparison to KIM10(pCD1Ap) by intramuscular (i.m.) or subcutaneous (s.c.) administration. The i.m. administration with either the KIM10(pCD1Ap) or YPS19(pCD1Ap) strain afforded significant protection against bubonic and pneumonic plague compared to the s.c. administration, while administration with completely attenuated YPS20(pCD1Ap) strain failed to afford significant protection. Antibody analysis showed that i.m. administration induced balanced Th1 and Th2 responses but s.c. administration stimulated Th2-biased responses. Safety evaluation showed that YPS19(pCD1Ap) was relatively safer than its parent KIM10(pCD1Ap) in Hfe-/- mice manifesting iron overload in tissues, which also did not impair its protection. Therefore, the immune activity of hexa-acylated lipid A can be harnessed for rationally designing bacteria-derived vaccines.

Oral vaccination with live attenuated Yersinia pseudotuberculosis strains delivering a FliC180-LcrV fusion antigen confers protection against pulmonary Y. Pestis infection.

We incorporated the ΔPfur::TT araC PBADfur deletion-insertion mutation on top of a previous Yersinia pseudotuberculosis mutant (Δasd ΔyopJ ΔyopK) to construct a new mutant designated as Yptb5, which manifests the arabinose-dependent regulated delayed fur (encoding ferric uptake regulator) shut-off. The Yptb5 strain was used to deliver an adjuvanted fusion protein, FliC180-LcrV. Levels of FliC180-LcrV synthesis were same in Yptb5 either harboring pSMV4, a p15A ori plasmid or pSMV8, a pSC101 ori plasmid containing the fliC180-lcrV fusion gene driven by Ptrc promoter. Tissue burdens of both Yptb5(pSMV4) and Yptb5(pSMV8) in mice had similar patterns. Mice vaccinated orally with 5 × 108 CFU of either Yptb5(pSMV4) or Yptb5(pSMV8) strain were primed high antibody titers with a balanced Th1/Th2 response, also developed potent T-cell responses with significant productions of IFN-γ, IL-17A and TNF-α. Immunization with each mutant strain conferred complete protection against pulmonary challenge with 5.5 × 103 CFU (55 LD50) of Y. pestis, but partial protection (50% survival) against 100 LD50 of Y. pestis. Our results demonstrate that arabinose-dependent regulated delayed fur shut-off is an effective strategy to develop live attenuated bacterial vaccines while retaining strong immunogenicity.

Size-controllable preparation and antibacterial mechanism of thermo-responsive copolymer-stabilized silver nanoparticles with high antimicrobial activity.

The emergence of bacterial resistance has become one of the top global concern, and silver nanoparticles (AgNPs) provide alternative strategies for the development of new antimicrobial agent. Herein, three small sizes (1.5-4.0 nm) of well-dispersed AgNPs were successfully synthesized using a thermo-sensitive P(NIPAM-co-MQ) copolymer with coordination ability as a stabilizer. The copolymer stabilized silver nanoparticles (AgNPs@P) displayed good thermo-sensitive characteristics and solution stability at pH = 6.5-8.0. AgNPs@P had high-efficiency and long-term antimicrobial properties for Gram-positive bacteria (S. aureus) and Gram-negative bacteria (E. coli). In particular, AgNPs@P3 with ultrasmall size (1.59 nm) exhibited better antimicrobial activity against both normal bacteria and antibiotic-resistant bacteria with a very low MIC value of 4.05 µg/mL. Moreover, AgNPs@P also showed an interesting temperature-dependent antibacterial activity mainly owing to the effect of thermo-sensitive copolymer on AgNPs. It was found that the antibacterial activity of the AgNPs@P also was affected by the proportion of copolymer, sizes of AgNPs, and experimental temperature. The antibacterial mechanism of AgNPs@P involved a variety of ways including destroying cell membranes, internalization of AgNPs and generation of ROS. Our research provides a new perspective for the preparation of effective nanosilver antimicrobial agents.

Downregulated miR-21 mediates matrine-induced apoptosis via the PTEN/Akt signaling pathway in FTC-133 human follicular thyroid cancer cells.

Matrine is an alkaloid extracted from the leguminous plant Sophora flavescens. Matrine has clinical effects in the treatment of tumors, including those in lung cancer, nasopharyngeal cancer and liver cancer. However, the effect of matrine on follicular thyroid cancer has not been reported. The aim of the present study was to investigate the effect of matrine on follicular thyroid cancer and its potential mechanism. FTC-133 follicular thyroid cancer cells were treated with different concentrations of matrine, and an MTT assay showed that matrine inhibited the growth of FTC-133 cells in a dose- and time-dependent manner with an IC50 value of 154.8 µM. Cell apoptosis was analyzed by flow cytometry and the results showed that matrine effectively induced the apoptosis of FTC-133 cells. The expression level of microRNA (miR)-21 was analyzed by reverse transcription-quantitative PCR (RT-qPCR) analysis, and the mRNA and protein expression levels of PTEN, Akt and phosphorylated (p)-Akt were detected by RT-qPCR analysis and western blotting, respectively. The expression of miR-21 was significantly downregulated, PTEN was upregulated at the mRNA and protein expression levels, and p-Akt was downregulated in the FTC-133 cells. The effects of miR-21 mimics and miR-21 inhibitor on the expression of miR-21, PTEN and Akt in FTC-133 cells, and the effect of miR-21 mimics/matrine on the expression of PTEN were also investigated. The results of the present study suggested that matrine inhibited the growth and induced apoptosis of FTC-133 cells via the miR-21/PTEN/Akt signaling pathway.

Terpenoids and sterols from Saussurea cauloptera.

A total of 27 natural products were isolated from Saussurea cauloptera, including two new eudesmane-type sesquiterpenoids, the gerin derivatives 2 and 3, and one new ent-labdane diterpenoid, compound 9. The known compounds included six sesquiterpenoids, eleven triterpenoids, six sterols, and one lignan. Their structures were elucidated on the basis of extensive spectroscopic and mass-spectrometric analyses, as well as by X-ray crystallography in the case of gerin (1). The structurally related compounds 1-4 were found to exhibit strong inhibitory activities against human gastric carcinoma (SGC-7901) cells.

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.

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.