Structure-guided discovery of novel dUTPase inhibitors with anti-Nocardia activity by computational design.

The zoonosis caused by Nocardia is increasing seriously. But commonly used antibiotic drugs often lead to resistance. N. seriolae dUTPase (NsdUTPase) plays a key role in the proliferation of Nocardia, and was regarded as a potent drug target. However, there was little report about the NsdUTPase inhibitors. In this study, we discovered a series of novel NsdUTPase inhibitors to fight against Nocardia. The first crystal structure of NsdUTPase was released, and a structure-based computational design was performed. Compounds 4b and 12b exhibited promising activities towards NsdUTPase (IC50 = 0.99 µM and 0.7 µM). In addition, they showed satisfied anti-Nocardia activity (MIC value ranges from 0.5 to 2 mg/L) and low cytotoxicity, which were better than approved drugs oxytetracycline and florfenicol. Molecular modelling study indicated that hydrophobic interaction might be the main contribution for ligand binding. Our results suggested that NsdUTPase inhibitors might be a useful way to repress Nocardia.

The protective effect of nigeglanine on dextran sulfate sodium-induced experimental colitis in mice and Caco-2 cells.

Ulcerative colitis (UC) was a nonspecific inflammatory disease. The treatment of UC is imperative. The present study aimed to investigate the effect of nigeglanine on dextran sulfate sodium-induced UC in experimental mice and Caco-2 cells and define the underlying mechanism. The nigeglanine was shown a significant protective effect on the colon, significantly reduced the weight and colon length loss and inhibited intestinal epithelial cell damage. Nigeglanine also reduced proinflammatory factors and increased anti-inflammatory factor production. The results indicate that nigeglanine suppresses the nuclear factor kappa B and mitogen-activated protein kinases pathways in addition to NLRP3 inflammasome action, inhibiting colon epithelial cell pyroptosis. Surprisingly, ZO-1 and occludin protein levels increased with nigeglanine treatment, suggesting that nigeglanine plays a protective role in barrier integrity, reducing colitis progression. The present study suggests that dietary therapy with nigeglanine may be a useful treatment for prophylaxis and palliative UC.

Selenium deficiency induced an inflammatory response by the HSP60 - TLR2-MAPKs signalling pathway in the liver of carp.

Selenium (Se) is one of the essential trace elements for immune regulation and antioxidant systems in fish growth. The dietary Se plays an important role in immune regulation and inflammation by regulating HSPs and TLRs in liver of many animals. The liver is an important digestive organ in carp. Liver damage can seriously affect the growth and survival of carp. This study was conducted to determine whether Se regulated liver inflammation by affecting HSPs-TLR2 signalling and the potential mechanisms of action in common carp. The gene was analysed by qPCR. The proteins of inflammatory factors were detected by ELISA. The others proteins were analysed by Western blot. The results indicated the Se concentrations in blood and liver tissues were significantly influenced by dietary Se. The Se deficiency increased the expression of HSP60 and TLR2 and the secretion of the proinflammatory factor TNF-α, IL-1ß and IL-6, induced a low secretion of the anti-inflammatory TGF-ß, but the Se supplements could transform these events. Further research showed that with the dose-dependently decrease of Se, the HSP60 expressions were increased, and the MAPKs pathway were significantly activated by the phosphorylation of p38, JNK and ERK in liver tissue and cell. The results provide evidence that Se deficiency induced and exacerbated inflammatory injury to the liver through the HSP60 and TLR2-MAPKs signalling pathways in carp.

Selenium deficiency inhibits micRNA-146a to promote ROS-induced inflammation via regulation of the MAPK pathway in the head kidney of carp.

Selenium (Se) is a necessity in multiple species of fish. Se plays an important role in immunoregulation, inflammation, and antioxidant systems in fish and other animals. The head kidney is the major immune organ in adult carp, and it produces white blood cells and destroys old red blood cells. The present study aimed to explore the effects and regulatory molecular mechanisms of Se on ROS and micRNA-146a as part of the inflammatory response in fancy carp. Adult fancy carp were fed different concentrations of Se in their diets. The Se content of the head kidney changed in a pattern consistent with the dietary content of Se. Se deficiency induced a significant increase in ROS, restrained the activities of GPx, SOD and CAT and increased MDA content. qPCR analysis showed a reduction in micRNA-146a with Se deficiency. The Se content, miRNA-146a expression and ROS levels were correlated. H2O2 cell stimulation assays found that ROS could activate the MAPK pathway, and ELISA results showed p38, JNK and ERK phosphorylation significantly increased with H2O2 stimulation. TNF-α, IL-1ß, and IL-6 were appreciably increased. At same time, miRNA-146a, which should have increased to regulate the inflammatory response, was reduced with Se deficiency. Therefore, with Se deficiency, the head kidney was inflamed. All these results indicated that Se deficiency inhibits micRNA-146a to promote ROS-induced inflammation via regulating the MAPK pathway in the head kidney of carp. The present study revealed that supplementing the diet of carp with selenium is beneficial for growth and disease prevention.

De novo design of a protein binder against Staphylococcus enterotoxin B.

Staphylococcus enterotoxin B (SEB) interacts with MHC-II molecules to overactivate immune cells and thereby to produce excessive pro-inflammatory cytokines. Disrupting the interactions between SEB and MHC-II helps eliminate the lethal threat posed by SEB. In this study, a de novo computational approach was used to design protein binders targeting SEB. The MHC-II binding domain of SEB was selected as the target, and the possible promising binding mode was broadly explored. The obtained original binder was folded into triple-helix bundles and contained 56 amino acids with molecular weight 5.9 kDa. The interface of SEB and the binder was highly hydrophobic. ProteinMPNN optimization further enlarged the hydrophobic region of the binder and improved the stability of the binder-SEB complex. In vitro study demonstrated that the optimized binder significantly inhibited the inflammatory response induced by SEB. Overall, our research demonstrated the applicability of this approach in de novo designing protein binders against SEB, and thereby providing potential therapeutics for SEB induced diseases.

Development of nanobodies specific to clumping factors A of Staphylococcus aureus by yeast surface display.

The Staphylococcus aureus clumping factor A (ClfA) is a fibrinogen (Fg) binding protein that plays an important role in the clumping of S. aureus in blood plasma. The current anti-infective approaches targeting ClfA are mainly based on monoclonal antibodies but showed less impressive efficacy for clinical applications. Nanobodies offer advantages in enhanced tissue penetration and a propensity to bind small epitopes. However, there is no report on generating specific nanobodies for ClfA. Here, we constructed a synthetic nanobody library based on yeast surface display to isolate nanobodies against the Fg binding domain ClfA221-550. We firstly obtained a primary nanobody directed to ClfA221-550, and then employed error-prone mutagenesis to enhance its binding affinity. Finally, 18 variants were isolated with high affinities (EC50, 1.1 ± 0.1 nM to 4.8 ± 0.3 nM), in which CNb1 presented the highest inhibition efficiency in the adhesion of S. aureus to fibrinogen. Moreover, structural simulation analysis indicated that the epitope for CNb1 partially overlapped with the binding sites for fibrinogen, thus inhibiting ClfA binding to Fg. Overall, these results indicated that the specific nanobodies generated here could prevent the adhesion of S. aureus to fibrinogen, suggesting their potential capacities in the control of S. aureus infections.

De novo design of mini-binder proteins against IL-2 receptor ß chain.

IL-2 regulates the immune response by interacting with different IL-2 receptor (IL-2R) subunits. High dose of IL-2 binds to IL-2Rßγc heterodimer, which induce various side effects while activating immune function. Disrupting IL-2 and IL-2R interactions can block IL-2 mediated immune response. Here, we used a computational approach to de novo design mini-binder proteins against IL-2R ß chain (IL-2Rß) to block IL-2 signaling. The hydrophobic region where IL-2 binds to IL-2Rß was selected and the promising binding mode was broadly explored. Three mini-binders with amino acid numbers ranging from 55 to 65 were obtained and binder 1 showed the best effects in inhibiting CTLL-2 cells proliferation and STAT5 phosphorylation. Molecular dynamics simulation showed that the binding of binder 1 to IL-2Rß was stable; the free energy of binder1/IL-2Rß complex was lower, indicating that the affinity of binder 1 to IL-2Rß was higher than that of IL-2. Free energy decomposition suggested that the ARG35 and ARG131 of IL-2Rß might be the key to improve the affinity of binder. Our efforts provided new insights in developing of IL-2R blocker, offering a potential strategy for ameliorating the side effects of IL-2 treatment.

Development of nanobodies against Staphylococcus enterotoxin B through yeast surface display.

Staphylococcus enterotoxin B (SEB) is one of the primary virulence factors of Staphylococcus aureus but there is still a lack of targeted drugs. SEB activates immune cells via interacting with MHC-II on antigen-presenting cells, leading to the production of large amounts of pro-inflammatory cytokines. Blocking the interaction between SEB and MHC-II can avert the overactivation of immune cells. Nanobodies are the smallest functional antibodies that can bind stably to antigens. In this study, an ideal approach to obtain specific nanobodies without immunizing camelids was introduced. We constructed a library containing up to 5 × 108 nanobodies, and then screened those targeting SEB by using yeast surface display (YSD) technique and fluorescence-activated cell sorting (FACS). A total of 8 nanobodies with divergent complementarity-determining regions (CDRs) sequences were identified and one candidate Nb8 with high affinity to SEB was isolated. In vitro study demonstrated that Nb8 significantly inhibited SEB-induced inflammatory response. Molecular docking simulation indicated that the unique CDR3 sequence contributed to the binding of Nb8 to the MHC-II binding domain of SEB and accordingly cut off the connection between SEB and MHC-II. Our efforts contributed to the development of specific nanobodies for eliminating the threats of SEB.

Characterization of Streptococcus suis isolates from the diseased pigs in China between 2003 and 2007.

The aim of this study was to illustrate the characteristics of 407 strains of Streptococcus suis (S. suis) isolated from diseased pigs in China. The results revealed that S. suis, with 56.6% of the Streptococci isolates, had replaced Streptococcus equi subsp. zooepidemicus as the predominant agent. Among the strains investigated, serotype 2 (43.2%) was most prevalent, followed by serotypes 3 (14.7%) and 4, 8, 5, 7, 1/2 (3.2-6.4%). Serotype 2 was more frequently isolated from swine with systemic infection, while serotype 3 was significantly associated with pneumonia. A high percentage of S. suis serotype 2 strains (54%) belonged to the genotype sly+ mrp+ epf+, which is a highly virulent strain, as confirmed by mice infection test. Biofilm producers only had lowly virulence compared to strains with the same genotype, indicating that biofilm could be associated with virulence but be not the character of virulent strains. The present study contributes to understanding the characteristics of Streptococcus suis and controlling Streptococcal disease in China.

Identification of a surface protective antigen, MAP of Streptococcus equi subspecies zooepidemicus.

Streptococcus equi subspecies zooepidemicus (SEZ) is a zoonotic pathogen with adhesive and invasive properties. Due to the shortcomings of antibiotics and traditional inactivated vaccine, identifying protective antigens against SEZ would be helpful to the development of novel vaccines. MAP has been identified as a membrane anchored protein with a typical LPXTG-like cell wall-anchored motif. In present study, the objective was to evaluate the effects of MAP as a subunit vaccine with mouse model. The Western blot analysis shown that the purified recombinant MAP presented good immunoreactive to convalescent porcine sera against SEZ. The protein could elicit a remarkable humoral antibody response and protect 80% of mice against lethal dose challenge of SEZ in mouse model. Moreover, the hyperimmune sera against MAP could efficiently kill the bacteria in whole blood killing assay and conferred significant protection against SEZ in passive immunization experiments. This study suggests with good reasons that MAP could be a novel and effective vaccine candidate for SEZ.

Characterization of SeseC_01411 as a surface protective antigen of Streptococcus equi ssp. zooepidemicus.

Streptococcus equi ssp. zooepidemicus (Streptococcus zooepidemicus, SEZ) is a commensal bacterium related to opportunistic infections of many species, including humans, dogs, cats, and pigs. SeseC_01411 has been proven to be immunogenic. However, its protective efficacy remained to be evaluated. In the present study, the purified recombinant SeseC_01411 could elicit a strong humoral antibody response and protect against lethal challenge with virulent SEZ in mice. Our finding confirmed that SeseC_01411 distributes on the surface of SEZ. In addition, the hyperimmune sera against SeseC_01411 could efficiently kill the bacteria in the phagocytosis test. The present study identified the immunogenic protein, SeseC_01411, as a novel surface protective antigen of SEZ.

Identification and characterization of a novel protective antigen, Sec_205 of Streptococcus equi ssp. Zooepidemicus.

Streptococcus equi ssp. zooepidemicus (SEZ) is an important pathogen of swine streptococcal diseases and can infect a wide range of animals as well as human beings. The absence of effective vaccine confounds the control of SEZ infection. Sec_205, a novel protein identified in the previous study, was inducibly over-expressed in Escherichia coli in the present study. The purified recombinant protein could elicit a significant humoral antibody response and provide efficient protection against lethal challenge of SEZ C55138 in mouse model. The protection against SEZ infection was mediated by specific antibodies to Sec_205 to some extent and was identified by the passive protection assay. The Sec_205 was an in vivo-induced antigen confirmed by the real-time PCR and could adhere to the Hep-2 cells by the inhibition assay. These suggest that Sec_205 may play a vital role in pathogenicity and serve as a new vaccine candidate against SEZ infection.

miR-194b-3p partially inhibits Streptococcus equi subsp. zooepidemicus adherence to PK15 cells.

MicroRNAs are increasingly reported implicated in the host cell response to bacterial pathogens. In order to investigate whether miR-194b-3p regulates the adherence of Streptococcus equi subsp. Zooepidemicus (SeZ) to porcine kidney cell line PK15, the miR-194b-3p agomir and antagomir were transfected into PK15 cells respectively and the adherence rate of SeZ to each was determined. Adherence rate of SeZ C55138 was significantly decreased when miR-194b-3p agomir was transfected in PK15, while that of miR-194b-3p antagomir evaluated. These results confirmed that miR-194b-3p markedly inhibit the adherence of SeZ C55138 to PK15 cells. In addition, miR-194b-3p indeed regulated the expression level of CD44 in PK15 cells by targeting CD44 3' UTR, and this interaction was involved in adhesion process. This study contributes to understanding the mechanism of the crosstalk between SeZ and PK15 cells.

CD44 enhances macrophage phagocytosis and plays a protective role in Streptococcus equi subsp. zooepidemicus infection.

Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is an important pathogen associated with a wide range of diseases in many mammalian species. CD44 is a transmembrane adhesion molecule involved in innate and adaptive immune responses. The aim of this study was to determine the protective role of CD44 during S. zooepidemicus infection. CD44-deficient mice exhibited reduced macrophage accumulation in the bronchoalveolar space and enhanced bacterial outgrowth and dissemination, which resulted in reduced mouse survival. An in vitro analysis revealed that CD44 can directly bind to S. zooepidemicus. Additionally, S. zooepidemicus interacted with macrophage-associated CD44, as reflected by the reduced uptake of S. zooepidemicus by CD44-deficient macrophages. These data suggest that CD44 contributes to effective antibacterial defense during S. zooepidemicus infection, thereby limiting the accompanying injury and death.

CD44 deficiency enhanced Streptococcus equi ssp. zooepidemicus dissemination and inflammation response in a mouse model.

Streptococcus equi ssp. zooepidemicus (S. zooepidemicus) is responsible for peritonitis, septicemia, meningitis, arthritis and several other serious diseases in various species. Recent studies have demonstrated that CD44 is implicated in the process of host defense against pathogenic microorganisms. In the present study, the role of CD44 in the host response to S. zooepidemicus infection was investigated in a mouse model. Upon intraperitoneal infection with S. zooepidemicus, the expression of CD44 on the peritoneal exudate cells from wild-type (WT) mice was increased. CD44 deficiency accelerated mortality, which was accompanied by increased peritoneal bacterial growth and dissemination to distant body sites. CD44 knock-out (KO) mice showed enhanced early inflammatory cell recruitment into the peritoneal fluid on S. zooepidemicus infection. In line with this, the expression of proinflammatory cytokines, chemokines in peritoneal exudate cells and peritoneal macrophages of CD44 KO mice were increased compared with those of WT mice. In addition, CD44 deficiency was associated with reduced expression of A20, a negative regulator in TLR signaling. Overall, the present study suggests that CD44 plays a protective role in antibacterial defense against S. zooepidemicus in mice.

Streptococcus equi ssp. zooepidemicus C5a peptidase, a putative invasin, induces protective immune response in mice.

Streptococcus equi ssp. zooepidemicus (Streptococcus zooepidemicus, SEZ) is responsible for septicemia, meningitis, arthritis and several other serious diseases in various species with adhesive and invasive properties. The absence of suitable vaccine confounds the control of SEZ infection. The highly conserved C5a peptidase was served as an invasin to host epithelial cells and involved in the pathogenicity in other streptococcus species. In the present study, the purified recombinant SEZ C5a peptidase (rSCPZ) could adhere to Hep-2 cells and interfere with the invasion of SEZ into Hep-2 cells confirmed by adherence and invasion assay. Immunization of BALB/c mice with rSCPZ could elicit a significant humoral antibody response and could confer significant protection against challenge with a lethal dose of SEZ. In addition, the hyperimmune sera against rSCPZ could efficiently inhibit bacterial growth in a whole blood assay and confer significant protection against SEZ infection in the experiment of passive immunization. The present study suggests that SCPZ could be useful for development of subunit vaccine against SEZ.

Glyceraldehyde-3-phosphate dehydrogenase, an immunogenic Streptococcus equi ssp. zooepidemicus adhesion protein and protective antigen.

Streptococcus equi ssp. zooepidemicus (Streptococcus zooepidemicus, SEZ) is an important pathogen associated with opportunistic infections of a wide range of species, including pigs and humans. The absence of a suitable vaccine makes it difficult to control SEZ infection. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been previously identified as an immunogenic protein using immunoproteomic techniques. In the present study, we confirmed that the sequence of GAPDH was highly conserved with other Streptococcus spp. The purified recombinant GAPDH could elicit a significant humoral antibody response in mice and confer significant protection against challenge with a lethal dose of SEZ. GAPDH could adhere to the Hep-2 cells, confirmed by flow cytometry, and inhibit adherence of SEZ to Hep-2 cells in an adherence inhibition assay. In addition, real-time PCR demonstrated that GAPDH was induced in vivo following infection of mice with SEZ. These suggest that GAPDH could play an important role in the pathogenesis of SEZ infection and could be a target for vaccination against SEZ.

Antimicrobial susceptibility, tetracycline and erythromycin resistance genes, and multilocus sequence typing of Streptococcus suis isolates from diseased pigs in China.

Streptococcus suis (S. suis) is an emerging zoonotic pathogen causing significant economic losses in the swine industry. Here, we investigated the antimicrobial susceptibility, associated antibiotic-resistant determinants and sequence type (ST) of S. suis isolates from diseased pigs in China from 2008 to 2010. Serotype 2 was the most frequently observed strain (n=95) among the 106 S. suis strains collected, followed by serotypes 3 (n=3), 5 (n=3), 4 (n=2), 7 (n=1), 11 (n=1) and 28 (n=1). Multilocus sequence typing analysis revealed that ST1 (n=21) and ST7 (n=74) were the predominant STs, and serotype 2 was found to be significantly correlated with ST7 (P=0.017, Fisher's exact test) and CC1 (P=0.024, Fisher's exact test). The antimicrobial susceptibility results indicated that the antibiotic resistance rate was highest for tetracycline (99.1%), followed by azithromycin (68.9%), erythromycin (67.9%), clindamycin (67.9%), trimethoprim/sulfamethoxazole (16%), levofloxacin (2.8%), chloramphenicol (1.9%), cefaclor (0.9%) and ceftriaxone (0.9%). Antibiotic-resistant genes tet(M), tet(O), tet(O/W/32/O), tet(O/32/O), tet(S), tet(W), tet(L), tet(40), erm(B), mef(A/E) and msr(D) could be detected, and several tandem organizations of antibiotic resistance genes were also found in this study. In conclusion, S. suis strains isolated from diseased pigs in China were less diverse and multi-drug resistant.

Identification of a surface protective antigen, CSP of Streptococcus equi ssp. zooepidemicus.

Streptococcus equi ssp. zooepidemicus (Streptococcus zooepidemicus, SEZ) is an important pathogen associated with opportunistic infections of a wide range of species, including horses, pigs and humans. The absence of suitable vaccine confounds the control of SEZ infection. Cell surface protein (CSP) has been identified as an immunogenic protein in the previous study but its protective efficacy is not clear. In the present study, the purified recombinant CSP could elicit a significant humoral antibody response and could confer significant protection against challenge with lethal dose of SEZ in mice model. CSP could adhere to the HEp-2 cells confirmed by flow cytometry and inhibit adherence of SEZ to HEp-2 cells in an adherence inhibition assay. In addition, real-time PCR demonstrated that CSP was induced in vivo following infection of mice with SEZ. Our findings suggest that CSP may play a potential role in the pathogenesis of SEZ and could be a target for the development of a novel subunit vaccine against SEZ infection.

Attenuated Streptococcus equi ssp. zooepidemicus as a bacterial vector for expression of porcine circovirus type 2 capsid protein.

Porcine circovirus type 2 (PCV2) infection and other concurrent factors is associated with post-weaning multisystemic wasting syndrome, which is becoming a major problem for the swine industry worldwide. Coinfection of Streptococcus equi ssp. zooepidemicus (SEZ) and PCV2 in swine has necessitated demand for a recombinant vaccine against these two pathogens. A recombinant SEZ-Cap strain expressing the major immunogenic capsid protein of PCV2 in place of the szp gene of acapsular SEZ C55138 ΔhasB was constructed. Fluorescence-activated cell sorting and immunofluorescence microscopy analyses indicated that the capsid protein is expressed on the surface of the recombinant strain. Experiments in mice demonstrated that strain SEZ-Cap was less virulent than the parental strain and that it induced significant anti-PCV2 antibodies when administered intraperitoneally, which is worthy of further investigation in swine.