GrpE and ComD contribute to the adherence, biofilm formation, and pathogenicity of Streptococcus suis.

Streptococcus suis is a major bacterial pathogen of swine and an emerging zoonotic agent that has to date resulted in substantial economic losses to the swine industry worldwide, and can cause persistent infection by forming biofilms. GrpE and histidine protein kinase ComD are important proteins implicated in the pathogenicity of S. suis, although whether they play roles in adhesion and biofilm formation has yet to be sufficiently clarified. In this study, we constructed grpE and comD deletion strains of S. suis by homologous recombination, and examined their cell adhesion and biofilm formation capacities compared with those of the wild-type strain. The pathogenicity of the grpE and comD deletion strains was evaluated using a mouse infection model, which revealed that compared with the wild-type, these deletion strains induced milder symptoms and lower bacteremia, as well as comparatively minor organ (brain, spleen, liver, and lung) lesions, in the infected mice. Moreover, the deletion of grpE and comD significantly reduced the pro-inflammatory cytokine (IL-6, IL-1ß, and TNF-α) induction capacity of S. suis. Collectively, the findings of this study indicate that the GrpE and ComD proteins of Streptococcus suis play key roles in the adherence to PK-15 cells and the formation of biofilms, thereby contributing to the virulence of this pathogen.

Relationship between L-lactate dehydrogenase and multidrug resistance in Staphylococcus xylosus.

Staphylococcus xylosus is a gram-positive bacterium that has attracted much attention due to its increasing clinical appearance, frequently associated with serious multidrug resistance cases. L-lactate dehydrogenase (LDH) has been related to drug resistance in several bacterial species. However, the mechanism of multidrug resistance in S. xylosus remains unclear as well as the involvement of LDH in such resistance. To explore the relationship between multidrug resistance and LDH in S. xylosus, we used tylosin-resistant S. xylosus as the parent strain to construct ldh knockout and complemented strains. Then, we tested their resistance to macrolides, lincosamides, tetracyclines, and aminoglycosides. In addition, the enzyme activity, metabolite content, and transcriptional level of key genes involved in the TCA cycle and thioredoxin system were determined to clarify the mechanism of resistance. We observed that the resistance to multiple antibiotics increased significantly after ldh knockout, especially that to lincomycin, whereas antibiotic sensitivity was partially restored in the complemented strain. The levels of pyruvate, nicotinamide adenine dinucleotide, and reactive oxygen species decreased significantly upon ldh knockout, and the activity of isocitrate dehydrogenase and malate dehydrogenase decreased. These results indicate that the lack of LDH promotes multidrug resistance in S. xylosus by inhibiting the TCA cycle and regulating the thioredoxin system.

Comparative proteomic analysis reveals drug resistance of Staphylococcus xylosus ATCC700404 under tylosin stress.

BACKGROUND: As a kind of opportunist pathogen, Staphylococcus xylosus (S. xylosus) can cause mastitis. Antibiotics are widely used for treating infected animals and tylosin is a member of such group. Thus, the continuous use of antibiotics in dairy livestock enterprise will go a long way in increasing tylosin resistance. However, the mechanism of tylosin-resistant S. xylosus is not clear. Here, isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomics methods was used to find resistance-related proteins. RESULTS: We compared the differential expression of S. xylosus in response to tylosin stress by iTRAQ. A total of 155 proteins (59 up-regulated, 96 down-regulated) with the fold-change of >1.2 or <0.8 (p value ≤0.05) were observed between the S. xylosus treated with 1/2 MIC (0.25 µg/mL) tylosin and the untreated S. xylosus. Bioinformatic analysis revealed that these proteins play important roles in stress-response and transcription. Then, in order to verify the relationship between the above changed proteins and mechanism of tylosin-resistant S. xylosus, we induced the tylosin-resistant S. xylosus, and performed quantitative PCR analysis to verify the changes in the transcription proteins and the stress-response proteins in tylosin-resistant S. xylosus at the mRNA level. The data displayed that ribosomal protein L23 (rplw), thioredoxin(trxA) and Aldehyde dehydrogenase A(aldA-1) are up-regulated in the tylosin-resistant S. xylosus, compared with the tylosin-sensitive strains. CONCLUSION: Our findings demonstrate the important of stress-response and transcription in the tylosin resistance of S. xylosus and provide an insight into the prevention of this resistance, which would aid in finding new medicines .

New Characterization of Multi-Drug Resistance of Streptococcus suis and Biofilm Formation from Swine in Heilongjiang Province of China.

The aim of this study was to investigate the antimicrobial resistance profiles and genotypes of Streptococcus suis in Heilongjiang Province, China. A total of 29 S. suis were isolated from 332 samples collected from 6 pig farms. The results showed that serotypes 2, 4 and 9 were prevalent, and all the clinical isolates were resistant to at least two antibacterial drugs. The most resisted drugs were macrolides, and the least resisted drugs were fluoroquinolones. Resistant genes ermB and aph (3')-IIIa were highly distributed among the isolates, with the detection rates of 79.31% and 75.86%. The formation of biofilm could be observed in all the isolated S. suis, among which D-1, LL-1 and LL-3 strains formed stronger biofilm structure than other strains. The results indicate that S. suis in Heilongjiang Province presents a multi-drug resistance to commonly used antimicrobial drugs, which was caused by the same target gene, the dissemination of drug resistance genes, and bacterial biofilm.

Molecular Mechanism of Staphylococcus xylosus Resistance Against Tylosin and Florfenicol.

Purpose: Drug resistance presents an ever-increasing global public health threat that involves all major microbial pathogens and antimicrobial drugs. Strains that are resistant to multiple drugs pose severe clinical problems and cost lives. However, systematic studies on cross-resistance of Staphylococcus xylosus have been missing. Methods: Here, we investigated various mutations in the sequence of ribosomal proteins involved in cross-resistance. To understand this effect on a molecular basis and to further elucidate the role of cross-resistance, we computationally constructed the 3D model of the large ribosomal subunit from S. xylosus as well as its complexes with both tylosin and florfenicol. Meanwhile, all-atom molecular dynamics simulations was used. In addition, the regulation of protein networks also played an essential role in the development of cross-resistance in S. xylosus. Results: We discovered that the minimum inhibitory concentration against both tylosin and florfenicol of the mutant strain containing the insertion L22 97KRTSAIN98 changed dramatically. Further, we found that unique structural changes in the ß-hairpin of L22 played a central role in this variant in the development of antibiotic resistance in S. xylosus. The regulation of protein networks also played an essential role in the development of cross-resistance in S. xylosus. Conclusion: Our work provides insightful views into the mechanism of S. xylosus resistance that could be useful for the development of the next generation of antibiotics.

Tylosin Inhibits Streptococcus suis Biofilm Formation by Interacting With the O-acetylserine (thiol)-lyase B CysM.

Streptococcus suis (S. suis) can decrease its virulence or modify local conditions through biofilm formation, which promotes infection persistence in vivo. Biofilm formation is an important cause of chronic drug-resistant S. suis infection. The aim of this study was to evaluate whether tylosin effectively inhibits S. suis biofilm formation by interacting with O-acetylserine (thiol)-lyase B (CysM), a key enzymatic regulator of cysteine synthesis. Biofilm formation of the mutant (ΔcysM) strain was significantly lower compared to the wild-type ATCC 700794 strain. Tylosin inhibited cysM gene expression, decreased extracellular matrix contents, and reduced cysteine, homocysteine, and S-adenosylmethionine levels, indicating its potential value as an effective inhibitor of S. suis biofilm formation. Furthermore, using biolayer interferometry technology and fourier-transform infrared spectroscopy, we found that tylosin and CysM could be combined directly. Overall, these results provide evidence that tylosin inhibits S. suis biofilm formation by interacting with CysM.

In vitro activity and In vivo efficacy of Isoliquiritigenin against Staphylococcus xylosus ATCC 700404 by IGPD target.

Staphylococcus xylosus (S. xylosus) is a type of coagulase-negative Staphylococcus, which was previously considered as non-pathogenic. However, recent studies have linked it with cases of mastitis in cows. Isoliquiritigenin (ISL) is a bioactive compound with pharmacological functions including antibacterial activity. In this study, we evaluated the effect of ISL on S. xylosus in vitro and in vivo. The MIC of ISL against S. xylosus was 80 µg/mL. It was observed that sub-MICs of ISL (1/2MIC, 1/4MIC, 1/8MIC) significantly inhibited the formation of S. xylosus biofilm in vitro. Previous studies have observed that inhibiting imidazole glycerol phosphate dehydratase (IGPD) concomitantly inhibited biofilm formation in S. xylosus. So, we designed experiments to target the formation of IGPD or inhibits its activities in S. xylosus ATCC 700404. The results indicated that the activity of IGPD and its histidine content decreased significantly under 1/2 MIC (40 µg/mL) ISL, and the expression of IGPD gene (hisB) and IGPD protein was significantly down-regulated. Furthermore, Bio-layer interferometry experiments showed that ISL directly interacted with IGPD protein (with strong affinity; KD = 234 µM). In addition, molecular docking was used to predict the binding mode of ISL and IGPD. In vivo tests revealed that, ISL significantly reduced TNF-α and IL-6 levels, mitigated the destruction of the mammary glands and reversed the production of inflammatory cells in mice. The results of the study suggest that, ISL may inhibit S. xylosus growth by acting on IGPD, which can be used as a target protein to treat infections caused by S. xylosus.

Analysis of multidrug resistance in Streptococcus suis ATCC 700794 under tylosin stress.

Streptococcus suis is an important zoonotic pathogen. The massive use of tylosin and other antibiotics in swine production has led to the emergence of resistant phenotypes of S. suis. However, there are no adequate measures available to address the problem of bacterial resistance. This study involved the use of 1/4 MIC (0.125 µg/mL) of tylosin to investigate resistance-related proteins by S. suis ATCC 700794. Our results showed that 171 proteins were differentially expressed in S. suis tested with 1/4 MIC (0.125 µg/mL) of tylosin using iTRAQ-based quantitative proteomic methods. TCS, heat shock protein and elongation factors were differentially expressed at 1/4 MIC (0.125 µg/mL) of tylosin compared to non treated, control cells. Using quantitative RT-PCR analysis, we verified the relationship between the differentially expressed proteins in S. suis with different MIC values. The data showed that expression profile for elongation factor G (fusA), elongation factor Ts (tsf), elongation factor Tu (tuf), putative histidine kinase of the competence regulon, ComD (comD), putative competence-damage inducible protein (cinA) and protein GrpE (grpE), observed in tylosin-resistant S. suis, correlated with that of S. suis ATCC 700794 at 1/4 MIC (0.125 µg/mL). The MIC of tylosin-resistant showed high-level resistance in terramycin, chlortetracycline, ofloxacin and enrofloxacin. Our findings demonstrated the importance of elongation factors, TCS and heat shock protein during development of tylosin resistance in S. suis. Thus, our study will provide insight into new drug targets and help reduce bacterial multidrug resistance through development of corresponding inhibitors.

FGF21 exerts comparable pharmacological efficacy with Adalimumab in ameliorating collagen-induced rheumatoid arthritis by regulating systematic inflammatory response.

Previous studies have reported that Fibroblast growth factor 21 (FGF21) can regulate inflammation and may play an important role in inflammatory and immune-mediated diseases, such as autoimmune diseases. Adalimumab is one of the clinically effective anti-rheumatoid arthritis (RA) drugs. The aim of this study was to compare the therapeutic efficacy of FGF21 and Adalimumab on collagen-induced arthritis (CIA) model mice. Mice with CIA were subcutaneously treated with FGF21 or Adalimumab at dose of 1mgkg-1d-1, respectively. Our results showed that FGF21 significantly alleviated the severity of arthritis by reducing cellular immune responses and exerted the similar anti-inflammatory effects with Adalimumab in decreasing the mRNA and protein expression levels of IL-2, IL-6 and IL-17. However, the expression levels of IL-1ß, RANKL and IL-10 in the mice treated with FGF21 were decreased 2.2-fold, 2.5-fold and increased 4.3-fold compared with Adalimumab, respectively. However, the levels of TNF-α in the mice treated with Adalimumab were lower than those in the mice treated with FGF21. Western blotting results demonstrated that FGF21 displayed equivalent effects with Adalimumab by inhibiting NF-κB/IκBα signaling pathway. However, FGF21 could also regulate systematic inflammatory response and the mechanism maybe related to other signal pathway. In summary, FGF21 exerts comparable pharmacological efficacy with Adalimumab by regulating systematic inflammatory response, providing that FGF21 may be a promising therapeutic agent for RA patients.

The pharmacological efficacy of the anti-IL17 scFv and sTNFR1 bispecific fusion protein in inflammation mouse stimulated by LPS.

Acute lung injury (ALI) is still a leading cause of morbidity and mortality in critically ill patients. Recently, our study found that a bispecific fusion protein treatment can ameliorate the lung injury induced by LPS. However, the molecular mechanisms which bispecific fusion protein ameliorates acute lung injury remain unclear. In this study, we found that the bispecific fusion protein treatment inhibited the nuclear transcription of NF-κB in confocal laser scanning fluorescence microscopy, the bispecific fusion protein exert protective effects in the cell model of ALI induced by lipopolysaccharide (LPS) via inhibiting the nuclear factor κB (NF-κB) signaling pathway and mediate inflammation. Moreover, the treatment of the bispecific fusion protein show its efficacy in animal models stimulated by LPS, the results of real-time PCR and ELISA demonstrate that bispecific fusion protein treatment effectively inhibited the over-expression of inflammatory cytokines(tumor necrosis factor α, interleukin 1ß and interleukin 17). In addition, LPS-challenged mice exhibited significant lung injury characterized by the deterioration of histopathology, which was meliorated by bispecific fusion protein treatment. Collectively, these results demonstrate that bispecific fusion protein treatment ameliorates LPS-induced ALI through reducing inflammatory cytokines and lung inflammation, which may be associated with the decreased the nuclear transcription of NF-κB. The bispecific fusion protein may be useful as a novel therapy to treat ALI.

Correction: Identification of Optimal Insertion Site in Recombinant Newcastle Disease Virus (rNDV) Vector Expressing Foreign Gene to Enhance Its Anti-Tumor Effect.

[This corrects the article DOI: 10.1371/journal.pone.0164723.].

A recombinant avian antibody against VP2 of infectious bursal disease virus protects chicken from viral infection.

A stable cell-line was established that expressed the recombinant avian antibody (rAb) against the infectious bursal disease virus (IBDV). rAb exhibited neutralization activity to IBDV-B87 strain in DF1 cells. The minimum rAb concentration required for inhibition of the cytopathic effect (CPE) was 1.563µg/mL. To test the efficacy of rAb, a 168-h cohabitation challenge experiment was performed to transmit the disease from the chickens challenged with vvIBDV (HLJ0504 strain) to three test groups of chickens, i.e. (1) chickens treated with rAb, (2) chickens treated with yolk antibody, and (3) non-treatment chickens. The survival rates of chickens treated with rAb, yolk antibody and without treatment were 73%, 67% and 20%, respectively. Another batch of chickens was challenged with IBDV (BC6/85 strain) and then injected with rAb (1.0mg/kg) 6, 24 and 36h post-challenge. Non-treatment chickens had 100% morbidity, whereas those administered with rAb exhibited only 20% morbidity. Morbidity was evaluated using clinical indicators and bursal histopathological section. This study provides a new approach to treating IBDV and the rAb represents a promising candidate for this IBDV therapy.

Identification of Optimal Insertion Site in Recombinant Newcastle Disease Virus (rNDV) Vector Expressing Foreign Gene to Enhance Its Anti-Tumor Effect.

Recombinant Newcastle disease virus (rNDV) is tumor selective and intrinsically oncolytic, which has been developed as a vector to express exogenous genes to enhance its oncolytic efficacy. Our previous studies found that insertion sites of foreign gene in rNDV vector affected its expression and anti-tumor activities. However, the optimal insertion site for foreign genes remains unknown. In this study, we inserted the enhanced green fluorescence protein (EGFP) and IL2 genes into four different intergenic regions of the rNDV using reverse genetics technology. Recombinants rNDV-EGFPs and rNDV-IL2s were successfully rescued, which displayed the similar growth kinetics with parental virus. Both EGFP mRNA and protein levels were most abundant in HepG2 cells, when EGFP gene was inserted between the NP/P site of the rNDV. Similarly, the IL-2 expressed by HepG2 cells infected with rNDV-IL2 was highest, when IL2 was inserted into NP/P site. To test whether these rNDVs that express higher foreign genes could induce stronger anti-tumor response, we treated the H22-oxter-tumor-bearing C57BL/6J mice with rNDV-IL2s and then examined the oncolytic efficacy. The results showed that rNDV-IL2-NP/P had the strongest inhibition of murine hepatoma carcinoma tumors. The splenocytes isolated from the mice treated with rNDV-IL2-NP/P reached the highest degrees of CD4+ T and CD8+ T cells. In addition, animals' survival rate in rNDV-IL2-NP/P-treated group was higher than that of other groups. Taken together, these results demonstrate that NP and P gene junction in rNDV is the optimal insertion site for foreign genes expression to enhance rNDV's anti-tumor effects.

Newcastle disease virus chimeras expressing the Hemagglutinin- Neuraminidase protein of mesogenic strain exhibits an enhanced anti-hepatoma efficacy.

Newcastle disease virus (NDV) is an intrinsically tumor-specific virus, many researchers have reported that lentogenic NDV is a safe and effective agent for human cancer therapy. It had been demonstrated that the amino acid sequence of the fusion protein cleavage site is a major factor in the pathogenicity and anti-tumor efficacy of rNDV. However, the role of Hemagglutinin-Neuraminidase (HN) gene that contributes to virulence and anti-tumor efficacy remains undefined. To assess the role of HN gene in virus pathogenicity and anti-tumor efficacy, a reverse genetic system was developed using the lentogenic NDV Clone30 strain to provide backbone for gene exchange. Chimeric virus (rClone30-Anh(HN)) created by exchange of the HN gene of lentogenic strain Clone30 with HN gene of mesogenic strain produce no significant changes in virus pathogenicity as assessed by conducting the mean death time (MDT) and intracerebral pathogenicity index (ICPI) assays. In vitro, infection with chimeras could induce the formation of syncytium relative significantly in HepG2 cells. Furthermore, chimeras was shown to induce the cell apoptosis via MTT and Annexin V-PI assays, reduce mitochondrial membrane potential and increase the mRNA transcription level of caspase 3. In vivo, ICR mice carrying tumor of hepatoma H22 cells were treated via intratumoral injection of chimeric virus. The treatment of chimera shows an obvious suppression in tumor volume. These results suggest that it could be an ideal approach to enhance the antitumor ability of Newcastle disease virus and highlighted the potential therapeutic application of rClone30-Anh(HN) as a viral vector to deliver foreign genes for treatment of cancers.