Genome-wide association study identifies the virulence-associated marker in Streptococcus suis serotype 2.

Streptococcus suis (S. suis) has been reported to be a highly invasive pathogen in swine, which causes severe infections like meningitis, arthritis and septicemia, and also a zoonotic agent for humans. Although many putative virulence factors (VFs) have been identified, the exact and wildly accepted virulence associated marker and pathogenesis mechanism of S. suis are still unclear. To establish connection of the genotypes with virulence phenotypes, we performed an "internal standard" method based on the zebrafish model to assess the virulence phenotypes of S. suis and did the genome-wide association study (GWAS) based on the genomes of 68 S. suis isolates. Through GWAS, a total number of 172 genes were identified. Among these genes, 143 of them distribute in virulent isolates. Further VFs interaction network analysis based on protein-protein interaction database found that 71 genes identified in this study could interact with known VFs and some of them even played an important role as the bridge between known VFs or formed important hub. In addition, 12 genes were found conserved in virulent isolates and 3 genes were conserved in avirulent isolates, 8 genes of the virulent conserved genes were belonging to a srtBCD pili cluster. Considering that sbp2', a member of the srtBCD pili cluster has been reported as a virulence-associated factor, we predict that sbp2' could be a fitness virulence-associated marker of virulent isolates. Taken together, our findings contribute to the insights in S. suis pathogenesis, enhance the knowledge of the genomic evolution of S. suis and provide several novel virulence-associated candidates.

Pan-genome analysis of Streptococcus suis serotype 2 revealed genomic diversity among strains of different virulence.

Streptococcus suis (SS) is an emerging zoonotic pathogen that causes severe infections in swine and humans. Among the 33 known serotypes, serotype 2 is most frequently associated with infections in pigs and humans. To better understand the virulence characterization of S. suis serotype 2 (SS2) and discriminate the difference between virulent and avirulent strains in SS2, characterization of the genomic features of strains with different virulence is required. The result showed that Streptococcus suis have an open pan-genome. The pan-genome shared by the 19 S. suis serotype 2 strains was composed of 1,239 core genes and 2,436 accessory genes. COG analysis indicated that core genes are involved in the basic physiological function, but accessory genes related to tachytely evolution. Comparative analysis between core genomes of virulent strains and 9 avirulent strains suggested that srtBCD pilus cluster was a significant discrepancy between virulent and avirulent strains. Analysis between high virulent and group B low virulent strains showed 53 and 58 genes specific to each other. Moreover, genomes of avirulent strains tend to be larger than virulent strains; avirulent strains tend to possess more prophages sequences than virulent strains. Our findings could be contributed to a better understanding of the genomics of S. suis serotype 2.

Corrigendum: Identification of Novel Laminin- and Fibronectin-Binding Proteins by Far-Western Blot: Capturing the Adhesins of Streptococcus suis Type 2.

[This corrects the article DOI: 10.3389/fcimb.2015.00082.].

Therapeutic efficacy of acupuncture combined with neuromuscular joint facilitation in treatment of hemiplegic shoulder pain.

BACKGROUND: Stroke is a type of cerebrovascular disease with high prevalence, mortality, and onset of disability. As a neurodevelopmental therapy, neuromuscular joint facilitation (NJF) is widely used in the treatment of orthopedic and neurological disorders in the clinical practice. It is mainly used for central nervous system diseases or orthopedic diseases, movement disorders, and pain rehabilitation. According to related studies, NJF can also be used as a rehabilitation treatment in patients with hemiplegic shoulder pain (HSP). AIM: To investigate the clinical efficacy of acupuncture combined with NJF in patients with HSP. METHODS: Forty patients with HSP were randomly divided into a treatment group and a control group. The treatment group was treated with acupuncture combined with NJF and the control group was treated with acupuncture alone. All patients were assessed by using the visual analogue scale (VAS), Fugl-Meyer assessment (FMA), Barthel index (BI), and passive range of motion (PROM) before and after the training. All the clinical data were analyzed using SPSS 20.0 statistical software. RESULTS: There was no statistical difference in the general characteristics between the two groups. In the terms of duration of treatment, age, and pre-treatment indicators, the two groups were comparable (P > 0.05). After the treatment, VAS, PROM, BI, and FMA scores were significantly improved in the two groups of patients (P < 0.05). The VAS, PROM and FMA scores were significantly higher in the treatment group than in the control group (P < 0.05). However, there was no significant difference in BI scores between the two groups (P > 0.05). CONCLUSION: Both acupuncture alone and acupuncture combined with NJF in the treatment of HSP are effective, and can improve the clinical symptoms of patients. Acupuncture combined with NJF can improve the upper limb motor function, relieve pain, and increase joint mobility in patients with HSP. The combination therapy is better than acupuncture alone. However, there is no significant difference in improving the score of patients' self-care ability.

Infection and adaption-based proteomic changes of Streptococcus suis serotype 2 in a pig model.

Streptococcus suis (S. suis) is an emerging zoonotic agent that is responsible for significant economic losses to the porcine industry worldwide. However, most research regarding the pathogenic mechanisms has used in vitro cultures of S. suis, which may not provide an accurate representation of the in vivo biological activities. In this study, 188 differential abundance S. suis proteins were identified in in vivo samples obtained from the blood of the infected pigs. These were compared with in vitro samples by a Tandem Mass Tags (TMT) experiment. Thus, a virulence associated network was established using the enriched differential abundance proteins (obtained via bioinformatics analysis in this study) and the previously reported putative virulence factors associated with in vivo infection. One of the most important up-regulated hubs in this network, adhE (an acetaldehyde-CoA/alcohol dehydrogenase) was found. Furthermore, knocking out adhE in S. suis serotype 2 strain ZY05719 decreased virulence. Cell culture experiments and far-western blot analysis showed that adhE is involved in adhesion to Caco-2 cells; Hsp60 could be one of the receptors for this protein. SIGNIFICANCE: This study is a systematical research to identify in vivo regulated virulence associated proteins of S. suis in pigs. It constructs a network consisting of in vivo infection related factors for the first time to get to know the coordinated actions of a multitude of factors that lead to host pathogenicity and filter the most important hubs. The individual factors that contribute to infection is also identified. A novel differential protein adhE which is one of the most important hubs of this network and is up-regulated in abundance in vivo is found to moonlight as an important adhesion by binding Hsp60 and finally contributes to virulence.

The non-conserved region of MRP is involved in the virulence of Streptococcus suis serotype 2.

Muramidase-released protein (MRP) of Streptococcus suis serotype 2 (SS2) is an important epidemic virulence marker with an unclear role in bacterial infection. To investigate the biologic functions of MRP, 3 mutants named Δmrp, Δmrp domain 1 (Δmrp-d1), and Δmrp domain 2 (Δmrp-d2) were constructed to assess the phenotypic changes between the parental strain and the mutant strains. The results indicated that MRP domain 1 (MRP-D1, the non-conserved region of MRP from a virulent strain, a.a. 242-596) played a critical role in adherence of SS2 to host cells, compared with MRP domain 1* (MRP-D1*, the non-conserved region of MRP from a low virulent strain, a.a. 239-598) or MRP domain 2 (MRP-D2, the conserved region of MRP, a.a. 848-1222). We found that MRP-D1 but not MRP-D2, could bind specifically to fibronectin (FN), factor H (FH), fibrinogen (FG), and immunoglobulin G (IgG). Additionally, we confirmed that mrp-d1 mutation significantly inhibited bacteremia and brain invasion in a mouse infection model. The mrp-d1 mutation also attenuated the intracellular survival of SS2 in RAW246.7 macrophages, shortened the growth ability in pig blood and decreased the virulence of SS2 in BALB/c mice. Furthermore, antiserum against MRP-D1 was found to dramatically impede SS2 survival in pig blood. Finally, immunization with recombinant MRP-D1 efficiently enhanced murine viability after SS2 challenge, indicating its potential use in vaccination strategies. Collectively, these results indicated that MRP-D1 is involved in SS2 virulence and eloquently demonstrate the function of MRP in pathogenesis of infection.

Factor H specifically capture novel Factor H-binding proteins of Streptococcus suis and contribute to the virulence of the bacteria.

Factor H (FH), a regulatory protein of the complement system, can bind specifically to factor H-binding proteins (FHBPs) of Streptococcus suis serotype 2 (SS2), which contribute to evasion of host innate immune defenses. In the present study, we aimed to identify novel FHBPs and characterize the biological functions of FH in SS2 pathogenesis. Here, a method that combined proteomics and Far-western blotting was developed to identify the surface FHBPs of SS2. With this method, fourteen potential novel FHBPs were identified among SS2 surface proteins. We selected eight newly identified proteins and further confirmed their binding activity to FH. The binding of SS2 to immobilized FH decreased dramatically after pre-incubation with anti-FHBPs polyclonal antibodies. We showed for the first time that SS2 also interact specifically with mouse FH. Furthermore, we found that FH play an important role in adherence and invasion of SS2 to HEp-2 cells. Additionally, using a mouse model of intraperitoneal challenge, we confirmed that SS2 pre-incubated with FH enhanced bacteremia and brain invasion, compared with SS2 not pretreated with FH. Taken together, this study provides a useful method to characterize the host-bacteria interactions. These results first indicated that binding of FH to the cell surface improved the adherence and invasion of SS2 to HEp-2 cells, promoting SS2 to resist killing and leading to enhance virulence.

SBP2 plays an important role in the virulence changes of different artificial mutants of Streptococcus suis.

Streptococcus suis (SS) is an important bacterial zoonotic pathogen, which can cause infections in pigs and humans. However, the pathogenesis of this bacterium remains unclear, even though some putative virulence factors (VFs) have been reported. Comparative proteomics could be used to identify markers that can distinguish bacterial strains with different virulence; however, the application of this method is restricted by the genome diversities existing in different strains. In this study, two mutants, WT ΔpepT and WT ΔrfeA, which were generated from the same wild-type (WT) strain, ZY05719, and showed opposite virulence tendencies, were constructed. Combining two proteomics assays, two-dimensional difference gel electrophoresis (2D-DIGE) and label-free proteomics, we identified 38 differentially abundant proteins in the mutants compared with their parent, including five known VFs of S. suis and 33 novel elements. One of the novel proteins, a putative pilus protein, named SBP2, was considered as the most promising VF, because SBP2 was not only linked with the known VFs in the virulence interaction network and was proposed to be located on the cell surface, but also showed enriched distribution among highly virulent strains of SS. SBP2 could also bind fibronectin and laminin, two important extracellular matrix proteins of the host, to facilitate the process of adhesion. Thus, spb2 was identified as encoding a promising virulence-associated candidate associated with the pathogenesis of SS, and a comprehensive virulence interaction network of SS was established for the first time.

Identification of Novel Laminin- and Fibronectin-binding Proteins by Far-Western Blot: Capturing the Adhesins of Streptococcus suis Type 2.

Bacterial cell wall (CW) and extracellular (EC) proteins are often involved in interactions with extracellular matrix (ECM) proteins such as laminin (LN) and fibronectin (FN), which play important roles in adhesion and invasion. In this study, an efficient method combining proteomic analysis and Far-Western blotting assays was developed to screen directly for bacterial surface proteins with LN- and FN-binding capacity. With this approach, fifteen potential LN-binding proteins and five potential FN-binding proteins were identified from Streptococcus suis serotype 2 (SS2) CW and EC proteins. Nine newly identified proteins, including oligopeptide-binding protein OppA precursor (OppA), elongation factor Tu (EF-Tu), enolase, lactate dehydrogenase (LDH), fructose-bisphosphate aldolase (FBA), 3-ketoacyl-ACP reductase (KAR), Gly ceraldehyde-3-phosphate dehydrogenase (GAPDH), Inosine 5'-monophosphate dehydrogenase (IMPDH), and amino acid ABC transporter permease (ABC) were cloned, expressed, purified and further confirmed by Far-Western blotting and ELISA. Five proteins (OppA, EF-Tu, enolase, LDH, and FBA) exhibited specifically binding activity to both human LN and human FN. Furthermore, seven important recombinant proteins were selected and identified to have the ability to bind Hep-2 cells by the indirect immunofluorescent assay. In addition, four recombinant proteins, and their corresponding polyclonal antibodies, were observed to decrease SS2 adhesion to Hep-2 cells, which indicates that these proteins contribute to the adherence of SS2 to host cell surface. Collectively, these results show that the approach described here represents a useful tool for investigating the host-pathogen interactions.