5'-Nucleotidase is dispensable for the growth of Salmonella Typhimurium but inhibits the bactericidal activity of macrophage extracellular traps.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a zoonotic pathogen that causes severe gastroenteritis. The 5'-nucleotidases of pathogens can dephosphorylate adenosine phosphates, boost adenosine levels and suppress the pro-inflammatory immune response. In our previous study, an extracellular nuclease, 5'-nucleotidase, was identified in the extracellular proteins of S. Typhimurium. However, the nuclease activity and the function of the 5'-nucleotidase of S. Typhimurium have not been explored. In the present study, deletion of the 5'-nucleotidase gene is dispensable for S. Typhimurium growth, even under environmental stress. Fluorescence microscopy revealed that the 5'-nucleotidase mutant induced more macrophage extracellular traps (METs) than the wild type did. Furthermore, recombinant 5'-nucleotidase protein (r5Nuc) could degrade λDNA, and the nuclease activity of r5Nuc was optimum at 37 °C and pH 6.0-7.0. The Mg2+ enhanced the nuclease activity of r5Nuc, whereas Zn2+ inhibited it. Meanwhile, deletion of the 5'-nucleotidase gene increased the bactericidal activity of METs, and r5Nuc could degrade METs and inhibit the bactericidal activity of METs. In conclusion, S. Typhimurium growth was independent of 5'-nucleotidase, but the nuclease activity of 5'-nucleotidase assisted S. Typhimurium to evade macrophage-mediated extracellular killing through degrading METs.

Identification and Characterization of the Nuclease Activity of the Extracellular Proteins from Salmonella enterica Serovar Typhimurium.

Pathogens have evolved an array of strategies to establish a productive infection. The extracellular proteins secreted by pathogens are one of unique mechanisms to evade the host innate immune response. Many secretory proteins transported by the bacterial secretion systems have been widely investigated in Salmonella. Certain extracellular nucleases are essential for bacterial pathogenesis. However, there is no current data available for the enzymatic properties of the proteins secreted by Salmonella. Therefore, in the present study we have identified and characterized the nuclease activity of the extracellular proteins from Salmonella enterica serovar Typhimurium. It was demonstrated that the extracellular proteins from S. Typhimurium exhibited the deoxyribonucleases activity against λDNA by agarose gel electrophoresis and agar plate diffusion method. The activity was observed at 16 °C, 37 °C and 42 °C, and found to be highest at 42 °C and inhibited at temperatures over 60 °C. The nuclease activity was stable under alkaline conditions (pH 7-10) and the optimum pH was 9.0. The nuclease activity was promoted at high ionic strength of Ba2+, Ca2+, Mg2+, and Ni2+. Nuclease zymography analysis revealed that there were four activity bands in the extracellular proteins; followed by LC-ESI/MS/MS analysis seven proteins were identified. As demonstrated by nuclease zymography, the recombinant 5'-nucleotidase protein expressed in the prokaryotic expression system displayed the DNase activity. To our knowledge, the present findings represent the first direct and unambiguous demonstration of the nuclease activity of the extracellular proteins from S. Typhimurium, and it provides an important fundamental for further investigation of the role of the extracellular proteins in pathogenicity and immune evasion.

Effect of gga-miR-155 on cell proliferation, apoptosis and invasion of Marek's disease virus (MDV) transformed cell line MSB1 by targeting RORA.

BACKGROUND: Marek's disease (MD) is caused by the oncogenic Marek's disease virus (MDV), and is a highly contagious avian infection with a complex underlying pathology that involves lymphoproliferative neoplasm formation. MicroRNAs (miRNAs) act as oncogenes or tumor suppressors in most cancers. The gga-miR-155 is downregulated in the MDV-infected chicken tissues or lymphocyte lines, although its exact role in tumorigenesis remains unclear. The aim of this study was to analyze the effects of gga-miR-155 on the proliferation, apoptosis and invasiveness of an MDV-transformed lymphocyte line MSB1 and elucidate the underlying mechanisms. RESULTS: The expression level of gga-miR-155 was manipulated in MSB1 cells using specific mimics and inhibitors. While overexpression of gga-miR-155 increased proliferation, decreased the proportion of G1 phase cells relative to that in S and G2 phases, reduced apoptosis rates and increased invasiveness. However, its downregulation had the opposite effects. Furthermore, gga-miR-155 directly targeted the RORA gene and downregulated its expression in the MSB1 cells. CONCLUSION: The gga-miR-155 promotes the proliferation and invasiveness of the MDV-transformed lymphocyte line MSB1 and inhibits apoptosis by targeting the RORA gene.

Identification and characterization of a Streptococcus suis immunogenic ornithine carbamoytransferase involved in bacterial adherence.

BACKGROUND: Streptococcus suis (SS) is a major swine pathogen and a serious zoonotic pathogen causing septicemia and meningitis in piglets and humans. Using an immunoproteomic approach, we previously brought evidence that ornithine carbamoytransferase (OCT) may represent a vaccine candidate to protect against S. suis biofilm-related and acute infections. METHOD: In this study, the gene encoding OCT was cloned into the expression vector pET-28a and the recombinant protein was expressed in Escherichia coli BL21. The immunogenicity and protective efficacy of the SS OCT was further investigated in a mouse model. RESULTS: The protein was found to be expressed in vivo and elicited high antibody titers following SS infections in mice. An animal challenge experiment with SS showed that 62.5% of mice immunized with the OCT protein were protected. Using an in vitro competitive adherence inhibition assay of adherence, evidence was obtained that OCT could significantly reduce the number of SS cells adhered to porcine kidney PK-15 cells. The bacterial levels recovered in mice of the OCT immunized group were significantly decreased in some organs, compared with the control group. CONCLUSION: In summary, our results suggest that the recombinant SS OCT protein, which is involved in bacterial adherence, may efficiently stimulate an immune response conferring protection against SS infections. It may therefore be considered as a potential vaccine candidate, although further studies are necessary to evaluate their use in swine.

Recombinant-attenuated Salmonella Pullorum strain expressing the hemagglutinin-neuraminidase protein of Newcastle disease virus (NDV) protects chickens against NDV and Salmonella Pullorum challenge.

Newcastle disease virus (NDV) and Salmonella Pullorum have significant damaging effects on the poultry industry, but no previous vaccine can protect poultry effectively. In this study, a recombinant-attenuated S. Pullorum strain secreting the NDV hemagglutinin-neuraminidase (HN) protein, C79-13ΔcrpΔasd (pYA-HN), was constructed by using the suicide plasmid pREasd-mediated bacteria homologous recombination method to form a new bivalent vaccine candidate against Newcastle disease (ND) and S. Pullorum disease (PD). The effect of this vaccine candidate was compared with those of the NDV LaSota and C79-13ΔcrpΔasd (pYA) strains. The serum hemagglutination inhibition antibody titers, serum immunoglobulin G (IgG) antibodies, secretory IgA, and stimulation index in lymphocyte proliferation were increased significantly more (p < 0.01) in chickens inoculated with C79-13ΔcrpΔasd (pYA-HN) than with C79-13ΔcrpΔasd (pYA) but were not significantly increased compared with the chickens immunized with the LaSota live vaccine (p > 0.05). Moreover, the novel strain provides 60% and 80% protective efficacy against the NDV virulent strain F48E9 and the S. Pullorum virulent strain C79-13. In summary, in this study, a recombinant-attenuated S. Pullorum strain secreting NDV HN protein was constructed. The generation of the S. Pullorum C79-13ΔcrpΔasd (pYA-HN) strain provides a foundation for the development of an effective living-vector double vaccine against ND and PD.

[Nuclease activity of the recombinant plancitoxin-1-like proteins with mutations in the active site from Trichinella spiralis].

Although there are 125 predicted DNase Ⅱ-like family genes in the Trichinella spiralis genome, plancitoxin-1-like (Ts-Pt) contains the HKD motif, a typical conserved region of DNase Ⅱ, in N- and C-terminal. It is generally believed that histidine is the active site in DNase Ⅱ. To study the nuclease activity of recombinant Ts-Pt with mutations in the active site from T. spiralis, different fragments of the mutated Ts-Pt genes were cloned using overlap PCR technique and inserted into the expressing vector pET-28a(+), and transformed into Escherichia coli Rosseta (DE3). The fusion proteins were purified by Ni-NTA affinity chromatography and SDS-PAGE. Nuclease activity of the recombinant proteins was detected by agarose gel electrophoresis and nuclease-zymography. The recombinant plasmids harboring the mutated Ts-Pt genes were constructed and expressed as inclusive body in a prokaryotic expression system. After renaturation in vitro, the recombinant proteins had no nuclease activity according to agarose gel electrophoresis. However, the expressed proteins as inclusive body displayed the ability to degrade DNA after renaturation in gel. And the nuclease activity was not affected after subjected to mutation of active site in N- and C-termini of Ts-Pt. These results provide the basis to study the relationship between DNase Ⅱ-like protein family and infection of T. spiralis.

[Construction of host-vector balanced lethal system of Salmonella typhimurium SL1344Δcya mutant and immune protection test of chickling].

OBJECTIVE: To develop a host-vector balanced lethal system of Salmonella typhimurium adenylate cyclase mutant, and detect its biological characteristics. METHODS: We constructed SL1344ΔcyaΔasd mutant strain by recombinant suicide plasmid (pREAasd), and screened by two-step method, transformed pYA3493 plasmid containing the asd gene without resistance electric into the lack of SL1344 AcyaΔasd, then the recombinant strains SL1344 ΔcyaΔasd (pYA3493) was constructed successfully. RESULTS: The biochemical characteristics and growth rate of the mutant were different from that of the wild strain SL1344, but almost the same as that of the parent strain SL1344Δcya. The mutant strain could neither ferment maltose, lactose, and sorbitol, nor decompose H2S, galactose and rat lee sugar, but still retained the ability to use glucose. The one-day chicken lethal test showed that SL1344ΔcyaΔasd (pYA3493) mutant was at least 104 times lower than SL1344 strain. The protection rate induced by the SL1344ΔcyaΔasd (pYA3493) mutant was 62. 5%. CONCLUSION: The SL1344ΔcyaΔasd (pYA3493) mutant was successfully constructed, and had good immune protection, it laid a foundation for developing potential oral vaccines.

[Expression and adjuvant effects of the fusion peptide TBP5].

Thymopentin (TP5) and bursopentin (BP5) are both immunopotentiators. To explore whether the TP5-BP5 fusion peptide (TBP5) has adjuvant activity or not, we cloned the TBP5 gene and confirmed that the TBP5 gene in a recombinant prokaryotic expression plasmid was successfully expressed in Escherichia coli BL21. TBP5 significantly promoted the proliferation of thymic and splenic lymphocytes of mice. The potential adjuvant activity of the TBP5 was examined in mice by coinjecting TBP5 and H9N2 avian influenza virus (AIV) inactivated vaccine. HI antibody titers, HA antibodies and cytokines levels (IL-4 and IFN-γ) were determined. We found that TBP5 markedly elevated serum HI titers and HA antibody levels, induced the secretion of both IL-4 and IFN-γ cytokines. Furthermore, virus challenge experiments confirmed that TBP5 contributed to inhibition replication of the virus [H9N2 AIV (A/chicken/Jiangsu/NJ07/05)] from mouse lungs. Altogether, these findings suggest that TBP5 may be an effective adjuvant for avian vaccine and that this study provides a reference for further research on new vaccine adjuvants.

[Antitumor mechanism of Bursopentin (BP5)].

OBJECTIVE: Bursopentin (BP5) is a multi-functional bioactive peptide with functions of immunomodulatory, antioxidant and antitumor. However, the antitumor mechanism of BP5 is still unclear. METHODS: We constructed T7 phage cDNA library of DT40 cells, and the proteins interacted with BP5 were identified. Then, the expression profile of BP5-treated DT40 cells were analyzed using gene microarray, p53 Luciferase activity was detected. RESULTS: The results of the expression profiling revealed that BP5 regulated expression of 1078 genes, of which 537 were up-regulated and 541 were down-regulated. Differentially expressed genes involved in various pathways were identified, of which 25 pathways were associated with immune responses and tumorigenic processes, including the p53 signaling. Furththmore, BP5 significantly enhanced p53 luciferase activity and stimulated expression of p53 protein in HCT116 cells. CONCLUSION: These results suggest that BP5 exerted antitumor activity through p53 signaling and that this study provides novel insights on the antitumor mechanism of BP5.

A eukaryotic expression plasmid carrying chicken interleukin-18 enhances the response to newcastle disease virus vaccine.

Interleukin-18 (IL-18) is an important cytokine involved in innate and acquired immunity. In this study, we cloned the full-length chicken IL-18 (ChIL-18) gene from specific-pathogen-free (SPF) chicken embryo spleen cells and provided evidence that the ChIL-18 gene in a recombinant plasmid was successfully expressed in chicken DT40 cells. ChIL-18 significantly enhanced gamma interferon (IFN-γ) mRNA expression in chicken splenocytes, which increased IFN-γ-induced nitric oxide (NO) synthesis by macrophages. The potential genetic adjuvant activity of the ChIL-18 plasmid was examined in chickens by coinjecting ChIL-18 plasmid and inactivated Newcastle disease virus (NDV) vaccine. ChIL-18 markedly elevated serum hemagglutination inhibition (HI) titers and anti-hemagglutinin-neuraminidase (anti-HN)-specific antibody levels, induced the secretion of both Th1- (IFN-γ) and Th2- (interleukin-4) type cytokines, promoted the proliferation of T and B lymphocytes, and increased the populations of CD3(+) T cells and their subsets, CD3(+) CD4(+) and CD3(+) CD8(+) T cells. Furthermore, a virus challenge revealed that ChIL-18 contributed to protection against Newcastle disease virus challenge. Taken together, our data indicate that the coadministration of ChIL-18 plasmid and NDV vaccine induces a strong immune response at both the humoral and cellular levels and that ChIL-18 is a novel immunoadjuvant suitable for NDV vaccination.

Bursin-like peptide (BLP) enhances H9N2 influenza vaccine induced humoral and cell mediated immune responses.

Vaccination with H9N2 avian influenza whole-inactivated virus (WIV) has been shown to be ineffective at eliciting sufficient humoral and cellular immunity against H9N2 avian influenza virus. This study assessed the effects of a synthetic Bursin-like epitope peptide (BLP) as adjuvant for H9N2 WIV in mice. Titers HI and avian influenza virus neutralizing antibodies, subtypes of HA antibodies, T helper (Th) cytokine levels, cytotoxic T-lymphocyte activities and changes in spleen T-cell subsets and natural killer cells were determined. We found that BLP induced a balance between IgG1 and IgG2a secretion levels. WIV antigen alone induced mainly Th1 cytokines secretion, whereas BLP showed increased secretion of Th1 and Th2 cytokines, including interleukin (IL)-2, interferon-γ (IFN-γ) and IL-4, but not IL-10, and may be resembles a Th0 like response. BLP significantly promoted growth and expansion of natural killer cells and of CD4(+) and CD8(+) T-cell subsets in the spleen. Meanwhile, BLP induced a better cytotoxic T-lymphocyte response to H9N2 virus. Furthermore, virus challenge experiments confirmed that BLP contributed to inhibition replication of the virus from mouse lungs. Taken together, these findings suggest that BLP may be an effective adjuvant for H9N2 avian influenza vaccine.

Synonymous codon usage of the VP2 gene of a very virulent infectious bursal disease virus isolate serial passaged in chicken embryos.

Three very virulent infectious bursal disease virus (vvIBDV) strains were isolated from a single farm and shown to be phylogenetically related to the vvIBDV isolate UK661. In this study, a comparative analysis of the synonymous codon usage in the hypervariable region of theVP2 (vVP2) gene of the vvIBDV strains was done on viruses serially passaged in chicken embryos. Sequencing demonstrated that codons change during the serial passage in the vVP2 gene of the viruses. Nine codon mutations resulted in amino acids changes. The amino acid changes were I256V, I296L 6in isolate XA1989, A222P, I242V, Q253H, I256V in isolate XA1998, and Q253H, I256V, I296L in isolate XA2004. Three of the nine amino acid changes occurred at residue 256. The codons of the amino acids A232, N233, I234, T269, T283 and H338 changed to the synonymous codons in XA1989 after the 16th passage, in XA1998 after the 24th passage and in XA2004 22nd passage viruses. These mutations change the key amino acid residues Q253H and I256V in the domains which are essential for its virulence, and the synonymous codons were observed compared to classical virulent IBDV. The results indicated that the codon changes during the serial passage comprised of synonymous codon usage in the vVP2 gene of IBDV, and this synonymous codon bias was correlated with pathotypes. The extent of synonymous codon usage bias in the IBDV-vVP2 gene maybe influence the gene expression level and secondary structure of protein as well as hydrophobicity, therefore the results provide useful perspectives for evolution and understanding of the pathogenesis of IBDV.

[Immunogenicity and protective efficacy of pertactin recombinants against Bordetella bronchiseptica challenge].

OBJECTIVE: In this study we showed the immunogenicity and protective efficacy of five pertactin recombinants against Bordetella bronchiseptica (Bb) challenge. METHODS AND RESULTS: The complete coding sequence (2040 bp) of the prn gene (PRN) and its fragments,5'-terminal 1173 bp fragment (PN),3'-terminal 867 bp fragment (PC), two copies of region I (654 bp; PR I) in PN, and 2 copies of region II (678 bp; PR II) in PC, were separately cloned into the prokaryotic expression vector pGEX-KG, and expressed in the Eschierichia coli BL21 (DE3) using induction by isopropyl-beta-D-thiogalactopyranoside. The recombinant proteins were named GST-PRN, GST-PN, GST-PC, GST-2PR I and GST-2PR II. All five recombinant proteins showed immunological reactivity in the Western-blot analysis. Mice, immunized subcutaneously with two doses of the purified proteins mixed with an equal volume of Freund's adjuvant,produced robust PRN-specific IgG antibody levels. When challenged, 6 of 9 mice in GST-2PR I group and all 9 mice in the other groups survived intranasal challenge with three times the 50% lethal dose (LD50) of virulent Bb HH0809. After challenge with 10 LD50 7/9,3/9,6/9,1/10 and 6/10 of the mice survived. Furthermore, complete protection against intraperitoneal (i.p.) challenge with 10 LD50 of HH0809 was observed in mice that were injected i.p. with 0.5 ml rabbit anti-GST-PRN, GST-PN,GST-PC or GST-2PR II serum. Only 1 of 10 mice survived in the group of mice that received anti-GST-2PR I, and no survivors were noted in the group of mice that received PRN-absorbed rabbit antiserum (0/5). CONCLUSION: In this study,we showed that all of five pertactin recombinants had differential immunogenicity and protective efficacy against Bb challenge. Mice immunized with GST-PC had better survival against fatal Bb challenge than did those immunized with GST-PN. In addition, GST-2PR II and GST-2PR I provided the similar results These data may have implications for the development of safe and efficacious subunit vaccines for the prevention of bordetellosis on the basis of these five pertactin recombinants.

[Expression and analysis of recombinant chicken IL-18 in Pichia pastoris.].

AIM: Expression and analysis of recombinant chicken IL-18 in Pichia pastoris. METHODS: Chicken IL-18 mature peptide gene was amplified from the recombinant plasmid pMD18-T-ChIL-18 by PCR, and was subcloned into Pichia pastoris expression vector pPICZalphaA to construct the recombinant plasmid pPICZalphaA-ChIL-18. After identified by restriction enzymes digestion analysis, PCR and DNA sequencing, the recombinant plasmid was transformed into Pichia pastoris X-33.Then choosing the multi-copy recombinant strains to be induced for expression.Then the bioactivity of rchIL-18 was analysed by Western blot, ELISA and MTT after purified by Sephadex G-100 column. RESULTS: The chicken IL-18 with the immunogenicity was secreted by Pichia pastoris. It could induce T lymphocytes proliferation and secreting IFN-gamma in vitro. CONCLUSION: The chicken IL-18 with obvious biological activity is secreted by Pichia pastoris X-33.

Molecular characteristic of VP2 gene of infectious bursal disease viruses isolated from a farm in two decades.

Infectious bursal disease virus (IBDV) is a double-stranded RNA virus in the Birnaviridae family. Four pathotypes, attenuated, virulent, antigenic variant, and very virulent, have been identified. In this study, we examined the phylogenetic relationship of 25 field isolates that were collected from a single farm during 1989-2008. A sequence analysis of PCR amplified 714 bp VP2 region showed that all the samples were derived from very virulent infectious bursal disease virus (vvIBDV) and were more closely related to the vvIBDV isolate UK661. From 1999, the isolate XA1999 had amino acids I228 and T394. XA2000, XA2001, XA2002, and XA2003-09 had amino acids E279 and T394. From 2004 to 2008, the isolates had amino acids H320, I349, S375, and R381 while the UK661 virus had T228, D279, Q320, V349, P375, K381, and A394. Such mutations do not change key amino acid residues in the domains which are essential for its virulence. It suggests that a virulent IBDV strain could maintain its virulence for a long period in the same chicken farm and the strain is highly stable under normal environments.