Zhe Ma

Complete Genome Sequence of Streptococcus equi subsp. zooepidemicus Strain ATCC 35246

Streptococcus equi subsp. zooepidemicus is an opportunistic pathogen. It has caused a very large economic loss in the swine industry of China and has become a threat to human health. We announce the complete genome sequence of S. equi subsp. zooepidemicus strain ATCC 35246, which provides opportunities to understand its pathogenesis mechanism and genetic basis.

Insight into the specific virulence related genes and toxin-antitoxin virulent pathogenicity islands in swine streptococcosis pathogen Streptococcus equi ssp. zooepidemicus strain ATCC35246

BackgroundStreptococcus 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.ResultsGenome 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.ConclusionGenome 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.

A novel vaccine against Porcine circovirus type 2 (PCV2) and Streptococcus equi ssp. zooepidemicus (SEZ) co-infection.

To develop a vaccine against Porcine circovirus type 2 (PCV2) and Streptococcus equi ssp. zooepidemicus (SEZ) co-infection, the genes of porcine IL-18, capsid protein (Cap) of PCV2 and M-like protein (SzP) of SEZ were inserted into the swinepox virus (SPV) genome by homologous recombination. The recombinant swinepox virus rSPV-ICS was verified by PCR and indirect immunofluorescence assays. To evaluate the immunogenicity of rSPV-ICS, 28 PCV2 and SEZ seronegative Bama minipigs were immunized with rSPV-ICS (n=8), commercial PCV2 vaccine and SEZ vaccine (n=8) or wild type SPV (n=8). The results showed that SzP-specific antibody and PCV2 neutralizing antibody of the rSPV-ICS immunized group increased significantly compared to the wild type SPV treated group after vaccination and increased continuously over time. The levels of IL-4 and IFN-γ in the rSPV-ICS immunized group were significantly higher than the other three groups, respectively. After been co-challenged with PCV2 and SEZ, 87.5% piglets in rSPV-ICS immunized group were survived. Significant reductions in gross lung lesion score, histopathological lung lesion score, and lymph node lesion score were noticed in the rSPV-ICS immunized group compared with the wtSPV treated group. The results suggested that the recombinant rSPV-ICS provided piglets with significant protection against PCV2-SEZ co-infection; thus, it offers proof-of-principle for the development of a vaccine for the prevention of these swine diseases.

The identification of six novel proteins with fibronectin or collagen type I binding activity from Streptococcus suis serotype 2

Streptococcus suis, a major swine pathogen, is an emerging zoonotic agent that causes meningitis and septic shock. Bacterial cell wall and secreted proteins are often involved in interactions with extracellular matrix proteins (ECMs), which play important roles in the initial steps of pathogenesis. In this study, 2D SDS-PAGE, western blotting-based binding affinity measurements, and microtiter plate binding assays were used to identify cell wall and secreted proteins from S. suis that interact with fibronectin and collagen type I. We identified six proteins from S. suis, including three proteins (translation elongation factor G, oligopeptide-binding protein OppA precursor, and phosphoglycerate mutase) that show both fibronectin and collagen type I binding activity. To the best of our knowledge, these three newly identified proteins had no previously reported fibronectin or collagen type I binding activity. Overall, the aim in this study was to identify proteins with ECM binding activity from S. suis and it represents the first report of six new proteins from S. suis that interact with fibronectin or collagen type I.

Identification of a novel collagen type І-binding protein from Streptococcus suis serotype 2.

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.

Identification and characterization of a Streptococcus equi ssp . zooepidemicus immunogenic GroEL protein involved in biofilm formation

Streptococcus equi ssp. zooepidemicus (S. equi spp. zooepidemicus) is an opportunistic pathogen that causes major economic losses in the swine industry in China and is also a threat for human health. Biofilm formation by this bacterium has been previously reported. In this study, we used an immunoproteomic approach to search for immunogenic proteins expressed by biofilm-grown S. equi spp. zooepidemicus. Seventeen immunoreactive proteins were found, of which nine common immunoreactive proteins were identified in planktonic and biofilm-grown bacteria. The immunogenicity and protective efficacy of the S. equi spp. zooepidemicus immunoreactive GroEL chaperone protein was further investigated in mice. The protein was expressed in vivo and elicited high antibody titers following S. equi spp. zooepidemicus infections of mice. An animal challenge experiment with S. equi spp. zooepidemicus showed that 75% of mice immunized with the GroEL protein were protected. Using in vitro biofilm inhibition assays, evidence was obtained that the chaperonin GroEL may represent a promising target for the prevention and treatment of persistent S. equi spp. zooepidemicus biofilm infections. In summary, our results suggest that the recombinant GroEL protein, which is involved in biofilm formation, may efficiently stimulate an immune response, which protects against S. equi spp. zooepidemicus infections. It may therefore be a candidate of interest to be included in vaccines against S. equi spp. zooepidemicus infections.

Construction and immunogenicity of a recombinant swinepox virus expressing a multi-epitope peptide for porcine reproductive and respiratory syndrome virus

To characterize neutralizing mimotopes, phages were selected from a 12-mer phage display library using three anti-porcine reproductive and respiratory syndrome virus (PRRSV) neutralizing monoclonal antibodies: (1) A1; (2) A2; and (3) A7. Of these, A2 and A7 recognize the mimotope, P2, which contains the SRHDHIH motif, which has conserved consensus sequences from amino acid positions 156 to 161 in the N-terminal ectodomain of GP3. The artificial multi-epitope gene, mp2, was designed by combining three repeats of the mimotope P2. The resulting sequence was inserted into the swinepox virus (SPV) genome to construct a recombinant swinepox virus (rSPV-mp2). The rSPV-mp2 was able to stably express the multi-epitope peptide, mP2, in vitro. The rSPV-mp2 immunized pigs exhibited a significantly shorter fever duration compared with the wtSPV treated group (P < 0.05). There was an enhanced humoral and cellular immune response, decreased number of PRRSV genomic copies, and a significant reduction in the gross lung pathology (P < 0.05) was observed following PRRSV infection in rSPV-mp2-immunized animals. The results suggest that the recombinant rSPV-mp2 provided pigs with significant protection against PRRSV infection.

Differential Protein Analysis of IPEC-J2 Cells Infected with Porcine Epidemic Diarrhea Virus Pandemic and Classical Strains Elucidates the Pathogenesis of Infection

Porcine epidemic diarrhea (PED) re-emerged in China in late 2010 and has now become widespread. Accumulated evidence indicates that this large-scale outbreak of diarrhea was caused by variants of the highly virulent porcine epidemic diarrhea virus (PEDV). A pandemic PEDV YC2014 strain (YC2014) was isolated from clinical samples. An iTRAQ-based comparative quantitative proteomic study of IPEC-J2 cells infected with YC2014 and a classical CV777 strain (CV777) was performed to determine the differences between pandemic and classical PEDV strain infection. Totals of 353 and 299 differentially expressed proteins were identified upon YC2014 and CV777 infection, respectively. The canonical pathways and functional networks involved in both PEDV infections were analyzed. The results indicated that the PEDV suppressed protein synthesis of IPEC-J2 cells through down-regulation of the PI3K-AKT/mTOR signaling pathways. Infection with YC2014 could activate the JAK-STAT signaling pathway and the NF-κB pathway more intensively than CV777. YC2014 could activate NF-κB pathway more intensively than CV777. On the basis of differentially expressed proteins, we propose that PEDV might disrupt apoptosis and may elicit stronger inflammatory cascades as well. This study might contribute to an understanding of the pathogenesis of PEDV infection and aid in the development of effective preventive and control vaccines.

Streptococcus suis Serotype 2 Biofilms Inhibit the Formation of Neutrophil Extracellular Traps

Invasive infections caused by Streptococcus suis serotype 2 (SS2) has emerged as a clinical problem in recent years. Neutrophil extracellular traps (NETs) are an important mechanism for the trapping and killing of pathogens that are resistant to phagocytosis. Biofilm formation can protect bacteria from being killed by phagocytes. Until now, there have only been a few studies that focused on the interactions between bacterial biofilms and NETs. SS2 in both a biofilm state and a planktonic cell state were incubated with phagocytes and NETs, and bacterial survival was assessed. DNase I and cytochalasin B were used to degrade NET DNA or suppress phagocytosis, respectively. Extracellular DNA was stained with impermeable fluorescent dye to quantify NET formation. Biofilm formation increased up to 6-fold in the presence of neutrophils, and biofilms were identified in murine tissue. Both planktonic and biofilm cells induced neutrophils chemotaxis to the infection site, with neutrophils increasing by 85.1 and 73.8%, respectively. The bacteria in biofilms were not phagocytized. The bactericidal efficacy of NETs on the biofilms and planktonic cells were equal; however, the biofilm extracellular matrix can inhibit NET release. Although biofilms inhibit NETs release, NETs appear to be an important mechanism to eliminate SS2 biofilms. This knowledge advances the understanding of biofilms and may aid in the development of treatments for persistent infections with a biofilm component.

A novel suicide shuttle plasmid for Streptococcus suis serotype 2 and Streptococcus equi ssp. zooepidemicus gene mutation.

The mariner-based Himar1 system has been utilized for creating mutant libraries of many Gram-positive bacteria. Streptococcus suis serotype 2 (SS2) and Streptococcus equi ssp. zooepidemicus (SEZ) are primary pathogens of swine that threaten the swine industry in China. To provide a forward-genetics technology for finding virulent phenotype-related genes in these two pathogens, we constructed a novel temperature-sensitive suicide shuttle plasmid, pMar4s, which contains the Himar1 system transposon, TnYLB-1, and the Himar1 C9 transposase from pMarA and the repTAs temperature-sensitive fragment from pSET4s. The kanamycin (Kan) resistance gene was in the TnYLB-1 transposon. Temperature sensitivity and Kan resistance allowed the selection of mutant strains and construction of the mutant library. The SS2 and SEZ mutant libraries were successfully constructed using the pMar4s plasmid. Inverse-Polymerase Chain Reaction (Inverse-PCR) results revealed large variability in transposon insertion sites and that the library could be used for phenotype alteration screening. The thiamine biosynthesis gene apbE was screened for its influence on SS2 anti-phagocytosis; likewise, the sagF gene was identified to be a hemolytic activity-related gene in SEZ. pMar4s was suitable for mutant library construction, providing more information regarding SS2 and SEZ virulence factors and illustrating the pathogenesis of swine streptococcosis.