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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
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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; · 2.24 Impact Factor
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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.
Microbial Pathogenesis 02/2013; · 1.94 Impact Factor
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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; · 2.11 Impact Factor
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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. · 2.11 Impact Factor
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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. · 4.09 Impact Factor
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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. · 4.09 Impact Factor
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PLoS ONE 01/2012; 7(5). · 4.09 Impact Factor
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PLoS ONE 01/2012; 7(5). · 4.09 Impact Factor
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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. · 1.21 Impact Factor
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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.
Biomacromolecules 07/2010; 11(7):1701-4. · 5.48 Impact Factor
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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. · 1.82 Impact Factor
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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.
BMC Microbiology 11/2008; 8:170. · 3.04 Impact Factor
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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.
FEMS Microbiology Letters 06/2008; 286(1):103-9. · 2.04 Impact Factor
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ABSTRACT: A PCR assay was developed to study the distributional characteristics of phage integrase gene in Streptococcus suis serotype 2 (SS2). A 323bp distinct DNA target can be amplified in 25 strains of virulent SS2, while can not be amplified in avirulent strain T15, 5 strains of other serotypes (SS1, SS7, SS9) and strains of group C Streptococcus strains from pigs, which suggested that the phage integrase gene may be related to the pathogenicity of SS2 and can be consider as a detection factor of the virulent gene of SS2. The sequencing and restriction endonuclease analysis of the PCR products were also done. Comparisons between the sequences of phage integrase gene with that of SS2 strain, showed a high homology with SS2 China strains 98HAH33, 05ZYH33 and North American strain 89-1591. Complete cell lysis was observed with SS2 virulent strains but not with avirulent strain T15 after the induction by mitomycin C. Electron microscopy analysis of the lysate from SS2 virulent strains HA9801 and ZY05719 revealed the presence of phage particles. The induced phage, named SS2-HA and SS2-ZY, both have a small isometric nucleocapsid approximately 50 nm in diameter and have no tail and is therefore a member of the Tectiviridae family. The phage integrase gene sequence of phage SS2-HA and SS2-ZY shared high homologue identities with virulent SS2 strains, which suggested that the phage integrase gene of SS2 has high specify. The temperate phage and phage integrase gene can only detected from SS2 virulent strains but not from avirulent strain, and the detection of phage integrase gene was related to the virulence-associate factors of SS2, such as the muramidase-released protein gene (mrp), which suggested that the temperate phage of SS2 may be related to the pathogenicity of SS2.
ACTA MICROBIOLOGICA SINICA 05/2008; 48(4):508-13.
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ABSTRACT: A mastitis model in rats, induced by Escherichia coli infection, was established and the protective effect of Cytosine-phosphate-Guanosine (CpG)-DNA was determined. An E. coli suspension containing either 2 x 10(3) colony forming units (CFU)mL(-1)(EL group), 2 x 10(5)CFU mL(-1) (EH group), or (as controls) 100 microL phosphate buffer saline (CON group), was inoculated into the mammary glands 72 h after parturition. The rats were euthanased 24 h post-infection. The histopathological changes in mammary tissue in the EL group were mild, whereas the structural changes in the EH group were severe and polymorphonuclear leukocytes (PMNs) had accumulated in the mammary alveoli. Interleukin (IL)-6 and tumour necrosis factor (TNF)-alpha and N-acetyl-beta-d-glucosaminidase (NAGase) were significantly increased in the mammary tissue from the EH group but not significantly changed in the EL group. On the basis of these findings, the potential protective effect of CpG-DNA on mammary glands was tested using a 2 x 10(5)CFU mL(-1) suspension. An intramuscular injection of either CpG-DNA (200 microg) or PBS (100 microL) was given immediately after parturition. At 72 h post-partum, 2 x 10(5)CFU mL(-1)E. coli (100 microL) were inoculated into the mammary glands of all rats. At pre-infection (0 h), and 8, 16, 24, 48 and 72 h after inoculation six rats were euthanased. CpG-DNA induced more rapid migration of PMNs from the blood to mammary tissue at the initial stage of infection, stimulated the secretion of IL-6 and TNF-alpha at different time points, reduced viable E. coli in mammary tissues and decreased the activity of NAGase. CpG-DNA also promoted the expression of its specific receptor TLR-9 mRNA in mammary tissue. The study showed that CpG-DNA protected against E. coli mastitis in this rat model.
The Veterinary Journal 04/2008; 175(3):369-78. · 2.24 Impact Factor
