[show abstract][hide abstract] ABSTRACT: BACKGROUND: Streptococcus equi ssp. zooepidemicus (S. zooepidemicus) is an important pathogen causing swine streptococcosis in China. Pathogenicity islands (PAIs) of S. zooepidemicus have been transferred among bacteria through horizontal gene transfer (HGT) and play important roles in the adaptation and increased virulence of S. zooepidemicus. The present study used comparative genomics to examine the different pathogenicities of S. zooepidemicus. RESULTS: Genome of S. zooepidemicus ATCC35246 (Sz35246) comprises 2,167,264-bp of a single circular chromosome, with a GC content of 41.65%. Comparative genome analysis of Sz35246, S. zooepidemicus MGCS10565 (Sz10565), Streptococcus equi. ssp. equi. 4047 (Se4047) and S. zooepidemicus H70 (Sz70) identified 320 Sz35246-specific genes, clustered into three toxin-antitoxin (TA) systems PAIs and one restriction modification system (RM system) PAI. These four acquired PAIs encode proteins that may contribute to the overall pathogenic capacity and fitness of this bacterium to adapt to different hosts. Analysis of the in vivo and in vitro transcriptomes of this bacterium revealed differentially expressed PAI genes and non-PAI genes, suggesting that Sz35246 possess mechanisms for infecting animals and adapting to a wide range of host environments. Analysis of the genome identified potential Sz35246 virulence genes. Genes of the Fim III operon were presumed to be involved in breaking the host-restriction of Sz35246. CONCLUSION: Genome wide comparisons of Sz35246 with three other strains and transcriptome analysis revealed novel genes related to bacterial virulence and breaking the host-restriction. Four specific PAIs, which were judged to have been transferred into Sz35246 genome through HGT, were identified for the first time. Further analysis of the TA and RM systems in the PAIs will improve our understanding of the pathogenicity of this bacterium and could lead to the development of diagnostics and vaccines.
[show abstract][hide abstract] ABSTRACT: Autotransporter (AT) proteins constitute a large family of extracellular proteins that contribute to bacterial virulence. A novel AT adhesin gene aatB was identified in avian pathogenic E. coli (APEC) DE205B via genomic analyses. The open reading frame of aatB was 1017 bp, coding a putative 36.3 kDa protein, which contained structural motifs characteristic for AT proteins: a signal peptide, a passenger domain, and a translocator domain. The predicted three-dimensional structure of AatB consisted of two distinct domains, the C-terminal β-barrel translocator domain and an N-terminal passenger domain. The prevalence analyses of aatB in APEC indicated that aatB was detected in 26.4% (72/273) of APEC strains, which was strongly associated with phylogenetic groups D and B2. Quantitative real-time reverse transcription-PCR analyses revealed that AatB expression was increased during infection in vitro and in vivo. Moreover, AatB could elicit antibodies in infected ducks, suggesting AatB was involved in APEC pathogenicity. Thus, mutant and complement strains of aatB gene in APEC DE205B were constructed. Inactivation of aatB resulted in the reduced capacity to adhere to DF-1 cells, defective virulence in vivo, and decreased colonization capacity in lung during the systemic infection compared with wild-type strain. Furthermore, these capacities were restored in the complementation strains. These results indicated that AatB makes a significant contribution to APEC virulence through bacterial adherence to host tissues in vivo and in vitro. In addition, biofilm formation assays for strain AAEC189 expressing AatB indicated that AatB mediates biofilm formation.
Infection and immunity 04/2013; · 4.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: Streptococcus suis, a major pathogen of pigs, is an emerging zoonotic agent that causes meningitis and septic shock. cbp40 is a putative virulent gene that has been identified using suppression subtractive hybridization performed on the virulent S. suis serotype 2 strain HA9801 and the avirulent S. suis serotype 2 strain T15. Based on predicted protein features showing a shared conserved domain with the collagen-binding protein Cna of Staphylococcus aureus, Cbp40 is likely to function as a direct mediator of collagen adhesion. Here, the cbp40 gene was cloned and the recombinant protein purified. Western blotting using swine convalescent sera confirmed its role as an immunogenic protein. Collagen binding activity could be detected by western affinity blot and ELISA. Conversely, deletion of the cbp40 gene reduced bacterial adhesion to HEp-2 cells, capacity for biofilm formation, and virulence in a zebrafish infection model. The response of the bEnd.3 cell line to infection with the S. suis serotype 2 strain ZY05719 and the cbp40-knockout strain was evaluated using gene expression arrays. The differentially expressed genes were involved in inflammatory and immune responses, leukocyte adhesion and heterophilic cell adhesion. Collectively, these data suggest that Cbp40 plays an important role as an extracellular matrix adhesion protein that interacts with host cells during infection.
The Veterinary Journal 03/2013; · 2.42 Impact Factor
[show abstract][hide abstract] ABSTRACT: Streptococcus equi ssp. zooepidemicus (SEZ) is a Gram-positive bacterium responsible for respiratory tract infection, septicemia, meningitis, endocarditi and arthritis in swine and humans. However, the expression and regulation of SEZ genes during an infection in vivo is poorly understood. In this study, we focused on the identification of SEZ genes preferentially expressed in vivo during infection in pigs. This study identified 45 SEZ genes that were up-regulated in infected porcine lung tissues using the selective capture of transcribed sequences (SCOTS) technique and comparative dot blot analysis, followed by quantitative RT-PCR validation. The identified genes were characterized into 6 functional categories: metabolism, cell wall-associated, stress response, transporters, regulators and unknown functions. Our study successfully identified multiple genes, which can deepen our understanding about SEZ pathogenesis and infer probable virulence factors. It will promote the development of novel vaccines and therapies about this pathogen for further study.
[show abstract][hide abstract] ABSTRACT: One hundred and two Streptococcus agalactiae (group B streptococcus [GBS]) isolates were collected from dairy cattle with subclinical mastitis in Eastern China during 2011. Clonal groups were established by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE), respectively. Capsular polysaccharides (CPS), pilus and alpha-like-protein (Alp) family genes were also characterized by molecular techniques. MLST analysis revealed that these isolates were limited to three clonal groups and were clustered in six different lineages, i.e. ST (sequence type) 103, ST568, ST67, ST301, ST313 and ST570, of which ST568 and ST570 were new genotypes. PFGE analysis revealed this isolates were clustered in 27 PFGE types, of which, types 7, 8, 14, 15, 16, 18, 23 and 25 were the eight major types, comprising close to 70% (71/102) of all the isolates. The most prevalent sequence types were ST103 (58% isolates) and ST568 (31% isolates), comprising capsular genotype Ia isolates without any of the detected Alp genes, suggesting the appearance of novel genomic backgrounds of prevalent strains of bovine S. agalactiae. All the strains possessed the pilus island 2b (PI-2b) gene and the prevalent capsular genotypes were types Ia (89% isolates) and II (11% isolates), the conserved pilus type providing suitable data for the development of vaccines against mastitis caused by S. agalactiae.
PLoS ONE 01/2013; 8(7):e67755. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: It has been shown that ORF5a protein in EAV is important but not essential for virus infectivity. In this study, we found that RNA changes in the overlapping region (1-104 nucleotide, nt) between ORF5 and ORF5a introduced by codon-optimized GP5 was lethal for virus viability, suggesting that the nt changes or amino acid (aa) mutations in the GP5 or ORF5a protein did not permit the production of infectious virus. Furthermore, inactivation of ORF5a expression in the context of type 1 (pSHE) and type 2 (pAJXM and pAPRRS) full-length PRRSV cDNA clones was lethal for the production of infectious virus, while viable PRRSV could be recovered by expressing ORF5a protein in trans, suggesting that ORF5a protein was essential for virus viability. Finally, ORF5a protein could be putatively extended to 63 aas by inactivation of the downstream stop codon candidates, thereby demonstrating that the C-terminus of ORF5a may be variable.
[show abstract][hide abstract] ABSTRACT: Torque teno sus virus 1 (TTSuV1) is a novel virus that has been found widely distributed in the swine population in recent years. Analysis of codon usage can reveal much about the molecular evolution of TTSuV1. In this study, synonymous codon usage patterns and the key determinants in the coding region of 29 available complete TTSuV1 genome sequences were examined. By calculating the nucleotide content and relative synonymous codon usage (RSCU) of TTSuV1 coding sequences, we found that the preferentially used codons were mostly those ending with A or C nucleotides; less-used codons were mostly codons ending with U or G nucleotides, and these were mainly affected by composition constraints. Although there was a variation in codon usage bias among different TTSuV1 genomes, the codon usage bias and GC content in the TTSuV1 coding region was lower, which was mainly determined by the base composition in the third codon position and the effective number of codons (ENC) value. Moreover, the results of correspondence analysis (COA) indicated that the codon usage patterns of TTSuV1 isolated from different countries varied greatly and had significant differences. In addition, Spearman's rank correlation analysis and an ENC plot revealed that apart from mutation pressure, which was critical in determining the codon usage pattern, other factors were involved in shaping the evolution of codon usage bias in TTSuV1, such as natural selection. Those results suggested that synonymous codon usage patterns of TTSuV1 genomes were the result of interaction between mutation pressure and natural selection. The information from this study not only provides important insights into the synonymous codon usage pattern of TTSuV1, but also helps to identify the main factors affecting codon usage by this virus.
Archives of Virology 09/2012; · 2.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: Riemerella anatipestifer (RA) is one of the most important bacterial pathogens of ducks and other avian species worldwide. Twenty-one serotypes of RA have been identified, with RA serotype two (RA2) being reported as one of the most predominant serotypes underlying infections in China. Current approaches to the control of RA are hindered by the absence of effective vaccines, particularly those that exhibit cross-protection between different serotypes. In this study, a combination of two-dimensional electrophoresis, Western blot analysis and mass spectrometry were used to identify the antigenic proteins of RA2. A total of 16 immunoreactive proteins, representing 12 distinct proteins, were identified. These included OmpA, a known immunogenic protein of RA, as well as novel immunogens. PCR analysis also indicated that genes corresponding to each of the 12 distinct proteins were conserved among different RA serotypes. Eleven genes encoding these proteins were cloned and expressed in Escherichia coli BL21 (DE3). Eight of the 11 expressed proteins were able to react with hyperimmune rabbit serum against RAf153. One of these, recombinant elongation factor G, responded to RA2 sera but not RA1, whereas recombinant OmpA responded to both RA1 and RA2 sera. These data form a basis for the development of vaccine for both homologous and heterogeneous RA serotypes in addition to the production of target antigens for the development of diagnostic antibodies with the potential to distinguish between RA serotypes.
[show abstract][hide abstract] ABSTRACT: To evaluate the effect of natural infection with TTSuV1 on the antibody response to vaccination with PRRS vaccine and clinical signs when co-infected with virulent PRRSV, 15 4-week-old TTSuV1-positive piglets and 20 TTSuV1-negative piglets were selected by PCR from two pig farms in Jiangsu province. TTSuV1-negative pigs were divided into four groups, and TTSuV1-positive pigs were divided into three groups. Experimental pigs were vaccinated with a PRRSV modified live virus (MLV) at 6 weeks of age and subsequently challenged with a virulent strain of PRRSV at 10 weeks of age. A TTSuV1-negative control group and an unvaccinated PRRS MLV control group were tested at the same time. The levels of antibody/cytokine and protective efficiency against PRRS MLV vaccine were evaluated. TTSuV1-infected/PRRSV-vaccinated pigs had lower levels of PRRSV antibody, as well as IFN-γ, IL-10 and T lymphocyte proliferation, than the TTSuV1-uninfected/PRRSV-vaccinated group (P < 0.05, except IL-10) after vaccination at only one time point. TTSuV1-infected/PRRS MLV-vaccinated/PRRSV-challenged pigs had more severe clinical signs (P > 0.05), more macroscopic lung lesions (P < 0.05) and lower levels of PRRSV antibody (P < 0.05 at 7 to 14 days post-PRRSV-challenge) than TTSuV1-uninfected/PRRSV-vaccinated/PRRSV-challenged pigs. These data indicate that TTSuV1 natural infection has an adverse effect on the development of host immune responses, suppresses immunization by the PRRS MLV vaccine, and exacerbates PRRS to a certain extent in pigs.
Archives of Virology 02/2012; 157(5):927-33. · 2.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: Streptococcus suis (SS) is a zoonotic pathogen that causes severe disease symptoms in pigs and humans. Biofilms of SS bind to extracellular matrix proteins in both endothelial and epithelial cells and cause persistent infections. In this study, the differences in the protein expression profiles of SS grown either as planktonic cells or biofilms were identified using comparative proteomic analysis. The results revealed the existence of 13 proteins of varying amounts, among which six were upregulated and seven were downregulated in the Streptococcus biofilm compared with the planktonic controls. The convalescent serum from mini-pig, challenged with SS, was applied in a Western blot assay to visualize all proteins from the biofilm that were grown in vitro and separated by two-dimensional gel electrophoresis. A total of 10 immunoreactive protein spots corresponding to nine unique proteins were identified by MALDI-TOF/TOF-MS. Of these nine proteins, five (Manganese-dependent superoxide dismutase, UDP-N-acetylglucosamine 1-carboxyvinyltransferase, ornithine carbamoyltransferase, phosphoglycerate kinase, Hypothetical protein SSU05_0403) had no previously reported immunogenic properties in SS to our knowledge. The remaining four immunogenic proteins (glyceraldehyde-3-phosphate dehydrogenase, hemolysin, pyruvate dehydrogenase and DnaK) were identified under both planktonic and biofilm growth conditions. In conclusion, the protein expression pattern of SS, grown as biofilm, was different from the SS grown as planktonic cells. These five immunogenic proteins that were specific to SS biofilm cells may potentially be targeted as vaccine candidates to protect against SS biofilm infections. The four proteins common to both biofilm and planktonic cells can be targeted as vaccine candidates to protect against both biofilm and acute infections.
PLoS ONE 01/2012; 7(4):e33371. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Streptococcus equi ssp. zooepidemicus (S. zooepidemicus, S.z) is one of the common pathogens that can cause septicemia, meningitis, and mammitis in domesticated species. M-like protein (SzP) is an important virulence factor of S. zooepidemicus and contributes to bacterial infection and antiphagocytosis. The interaction between SzP of S. zooepidemicus and porcine thioredoxin (TRX) was identified by the yeast two-hybrid and further confirmed by co-immunoprecipitation. SzP interacted with both reduced and the oxidized forms of TRX without inhibiting TRX activity. Membrane anchored SzP was able to recruit TRX to the surface, which would facilitate the antiphagocytosis of the bacteria. Further experiments revealed that TRX regulated the alternative complement pathway by inhibiting C3 convertase activity and associating with factor H (FH). TRX alone inhibited C3 cleavage and C3a production, and the inhibitory effect was additive when FH was also present. TRX inhibited C3 deposition on the bacterial surface when it was recruited by SzP. These new findings indicated that S. zooepidemicus used SzP to recruit TRX and regulated the alternative complement pathways to evade the host immune phagocytosis.
PLoS ONE 01/2012; 7(2):e32099. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Streptococcus equi subsp. zooepidemicus (S. zooepidemicus), which belongs to Lancefield group C streptococci, is an important pathogen of domesticated species, causing septicemia, meningitis and mammitis. M-like protein (SzP) is an important virulence factor of S. zooepidemicus and contributes to bacterial infection and antiphagocytosis. To increase our knowledge of the mechanism of SzP in infection, we profiled the response of porcine pulmonary alveolar macrophage (PAM) to infection with S. zooepidemicus ATCC35246 wild strain (WD) and SzP-knockout strain (KO) using the Roche NimbleGen Porcine Genome Expression Array. We found SzP contributed to differential expression of 446 genes, with upregulation of 134 genes and downregulation of 312 genes. Gene Ontology category and KEGG pathway were analyzed for relationships among differentially expressed genes. These genes were represented in a variety of functional categories, including genes involved in immune response, regulation of chemokine production, signal transduction and regulation of apoptosis. The reliability of the data obtained from the microarray was verified by performing quantitative real-time PCR on 12 representative genes. The data will contribute to understanding of SzP mediated mechanisms of S. zooepidemicus pathogenesis.
PLoS ONE 01/2012; 7(5):e36452. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Streptococcus suis type 2 (SS2) is an important swine pathogen and zoonosis agent. A/J mice are significantly more susceptible than C57BL/6 (B6) mice to SS2 infection, but the genetic basis is largely unknown. Here, alterations in gene expression in SS2 (strain HA9801)-infected mice were identified using Illumina mouse BeadChips. Microarray analysis revealed 3,692 genes differentially expressed in peritoneal macrophages between A/J and B6 mice due to SS2 infection. Between SS2-infected A/J and control A/J mice, 2646 genes were differentially expressed (1469 upregulated; 1177 downregulated). Between SS2-infected B6 and control B6 mice, 1449 genes were differentially expressed (778 upregulated; 671 downregulated). These genes were analyzed for significant Gene Ontology (GO) categories and signaling pathways using the Kyoto Encylopedia of Genes and Genomes (KEGG) database to generate a signaling network. Upregulated genes in A/J and B6 mice were related to response to bacteria, immune response, positive regulation of B cell receptor signaling pathway, type I interferon biosynthesis, defense and inflammatory responses. Additionally, upregulated genes in SS2-infected B6 mice were involved in antigen processing and presentation of exogenous peptides, peptide antigen stabilization, lymphocyte differentiation regulation, positive regulation of monocyte differentiation, antigen receptor-mediated signaling pathway and positive regulation of phagocytosis. Downregulated genes in SS2-infected B6 mice played roles in glycolysis, carbohydrate metabolic process, amino acid metabolism, behavior and muscle regulation. Microarray results were verified by quantitative real-time PCR (qRT-PCR) of 14 representative deregulated genes. Four genes differentially expressed between SS2-infected A/J and B6 mice, toll-like receptor 2 (Tlr2), tumor necrosis factor (Tnf), matrix metalloproteinase 9 (Mmp9) and pentraxin 3 (Ptx3), were previously implicated in the response to S. suis infection. This study identified candidate genes that may influence susceptibility or resistance to SS2 infection in A/J and B6 mice, providing further validation of these models and contributing to understanding of S. suis pathogenic mechanisms.
PLoS ONE 01/2012; 7(2):e32150. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: To investigate the prevalence and genetic characterization of Pigeon circovirus (PiCV) circulating in Chinese flocks, the genomic DNA of 144 samples collected from pigeons in 6 different geographic regions of eastern China between 2009 and 2010 were amplified using previously published PiCV primers. The PiCV sequence was detected in 83 of 104 unhealthy pigeons (79.8%) and 25 of 40 healthy pigeons (62.5%). The overall positive rate was 75% for all samples. An inverse primer polymerase chain reaction (IP-PCR) assay was performed to amplify the full-length sequence from a random sample of each region, and 6 specific DNA fragments were gel-purified and sequenced. The 6 full-length sequences were designated as SHWH-AB4 (2,031 bp), NJPK-21 (2,035 bp), HBLF-E2 (2,031 bp), JSJN (2,039 bp), SDDZ (2,037 bp), and AHBZ (2,035 bp) after BLAST analyses. The phylogenic tree and amino acid comparison indicated that all the strains examined were derived from a common strain, but had undergone genetic mutations through time. Pairwise comparisons revealed 93.4%-100% amino acid identity for the putative replication-associated proteins and 67.5%-100% for the putative capsid proteins.
Journal of veterinary diagnostic investigation: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 07/2011; 23(4):665-72. · 1.18 Impact Factor
[show abstract][hide abstract] ABSTRACT: The aim of this work is to construct a safe and effective drug candidate against Streptococcus suis infection. A panel of chitosan-based polymer conjugates with branched galabiose (Galalpha1-4Gal) side chains was synthesized as inhibitors of S. suis adhesion. The synthesis was achieved by using an aldehyde-functionalized galabiose derivative to graft it onto chitosan amino groups. Structural compositions of the conjugates were verified by 1H NMR spectroscopy and CHN elemental analyses. Potent inhibitory activities of the conjugates against S. suis adhesion to human erythrocytes were determined at low nanomolar concentration by HAI assay. An SPR study revealed a high affinity binding (Kd=39.6 nM) of the conjugate with BSI-B4 lectin. By using biocompatible chitosan as the scaffold for presenting S. suis -specific galabiose units, as well as the concise route tailored for the conjugate syntheses, the present study provides a practical way for explorations of new anti- S. suis therapies.
[show abstract][hide abstract] ABSTRACT: The trag is a novel infection-related factor identified using in vivo-induced antigen technology (IVIAT) from SS2 expression libraries with swine convalescent sera. PCR-based studies of various S. suis strains collected from different sources revealed that the trag gene was present in all the virulent SS2 strains, but absent in the avirulent T15 strain. PCR and Southern hybridization analyses of a trag-knockout strain created through homologous recombination confirmed the coding sequence of trag replaced by spc (R) cassette in the ∆trag mutant. Zebrafish was used to identify the role of trag in SS2 virulence. The reduction of virulence in the trag mutant compared to the wild-type in animal model systems, laid the foundation for further studies.
Current Microbiology 04/2010; 61(6):494-9. · 1.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: The M-like protein, also known as SzP, is expressed on the surface of Streptococcus equi subsp. zooepidemicus (S. zooepidemicus). Previous studies demonstrated that SzP is similar to M protein of group A Streptococcus in the structure and characteristics of antiphagocytosis. The M protein is an adhesin that can bind to the host cells, however it is not known whether the SzP of S. zooepidemicus also functions as an adhesin. We conducted an investigation to determine SzP as an adhesin, and one SzP epitope was identified to be responsible for mediating binding to HEp-2 cells.
The gene encoding SzP was expressed in E. coli, and the purified recombinant SzP (rSzP) was recognized by rabbit anti-S. zooepidemicus antibodies using immunoblot. Furthermore, the adherence of S. zooepidemicus to HEp-2 cells was inhibited by anti-rSzP antibodies in a dose-dependent manner. We employed a random 12-peptide phage display library for screening of immunodominant mimics of the SzP, which were recognized by an anti-SzP specific monoclonal antibody (mAb 2C8). Initial positive phage clones were identified by ELISA, followed by assays to determine the adherence-inhibiting ability of the peptide.
Ten out of fourteen selected positive clones showed high reactivity that effectively inhibited the binding of mAb 2C8 to rSzP. The motif XSLSRX was highly conserved among six of the ten clones.
Collectively, our findings suggest that the motif XSLSRX may represent an immunodominant mimic epitope of the SzP of S. zooepidemicus strain ATCC 35246, and that the same epitope may be used to mediate SzP binding to HEp-2 cells.
[show abstract][hide abstract] ABSTRACT: A proteomic approach combining two-dimensional electrophoresis, Western blot and matrix-assisted laser desorption tandem time-of-flight mass spectrometry has been used to map the extracellular proteins of Streptococcus equi ssp. zooepidemicus (S. zooepidemicus) strain ATCC 35246. These bioinformatic technologies facilitated the identification of novel S. zooepidemicus vaccine candidate antigens and therapeutic agents. Despite the limitations posed by the unavailability of complete genome and proteome data for S. zooepidemicus, seven of 15 chosen immunogenic spots were successfully identified as streptococcal proteins (AE1 and AE4 c. 10) from homologous Streptococcus species. Among these, AE6 and AE7 were identified as S. zooepidemicus UDP-N-acetyl-glucosamine pyrophosphorylase and UDP-glucose pyrophosphorylase proteins. In addition, AE4 was determined to be glyceraldehyde-3-phosphate dehydrogenase from Enterococcus faecalis. Following SIGNALIP 3.0 (http://www.cbs.dtu.dk/servicess/SignalIP) prediction, data suggested that AE5, AE7 and AE9 contained signal peptides. BLAST (http://www.sanger.ac.uk) results found that nucleotide sequences of all identified proteins shared high homology (> or = 65%) with S. zooepidemicus. The majority of proteins identified in our study remain formally unreported in S. zooepidemicus. However, these proteins serve a vital role in the immune system and reproduction of host species. Therefore, we further evaluated the proteins as vaccine candidates in this study.