Whole-Genome Sequencing and Phenotypic Analysis of Streptococcus equi subsp. zooepidemicus Sequence Type 147 Isolated from China.

Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is one of the important zoonotic and opportunistic pathogens. In recent years, there has been growing evidence that supports the potential role of S. zooepidemicus in severe diseases in horses and other animals, including humans. Furthermore, the clinical isolation and drug resistance rates of S. zooepidemicus have been increasing yearly, leading to interest in its in-depth genomic analysis. In order to deepen the understanding of the S. zooepidemicus characteristics and genomic features, we investigated the genomic islands, mobile genetic elements, virulence and resistance genes, and phenotype of S. zooepidemicus strain ZHZ 211 (ST147), isolated from an equine farm in China. We obtained a 2.18 Mb, high-quality chromosome and found eight genomic islands. According to a comparative genomic investigation with other reference strains, ZHZ 211 has more virulence factors, like an iron uptake system, adherence, exoenzymes, and antiphagocytosis. More interestingly, ZHZ 211 has acquired a mobile genetic element (MGE), prophage Ph01, which was found to be in the chromosome of this strain and included two hyaluronidase (hyl) genes, important virulence factors of the strain. Moreover, two transposons and two virulence (virD4) genes were found to be located in the same genome island of ZHZ 211. In vitro phenotypic results showed that ZHZ 211 grows faster and is resistant to clarithromycin, enrofloxacin, and sulfonamides. The higher biofilm-forming capabilities of ZHZ 211 may provide a competitive advantage for survival in its niche. The results expand our understanding of the genomic, pathogenicity, and resistance characterization of Streptococcus zooepidemicus and facilitate further exploration of its molecular pathogenic mechanism.

Complete genome sequencing and comparative genomic analysis of three donkey Streptococcus equi subsp. equi isolates.

Introduction: Streptococcus equi subspecies equi (S. equi) is the causative agent of strangles, which is one of the most common and highly contagious respiratory infectious illnesses in horses. Streptococcus equi (S. equi) is a horse-specific pathogen that originated from the closely related zoonotic pathogen Streptococcus equi subspecies zooepidemicus (S. zooepidemicus). Despite decades of research, the movement of genetic material across host-restricted diseases remains a mystery. Methods: Three S. equi donkey isolates (HTP133, HTP232, and HT1112) were recently isolated from a strangles epidemic on donkey farms in China's Xinjiang Province. In this study, we performed a comprehensive comparative analysis of these isolates using whole genome sequencing and compared them to the published genomic sequences of equine strain S. equi 4047 to uncover evidence of genetic events that shaped the evolution of these donkey S. equi isolates' genomes. Results: Whole genome sequencing indicated that both strains were closely related, with comparable gene compositions and a high rate of shared core genomes (1788-2004). Our comparative genomic study indicated that the genome structure is substantially conserved across three donkey strains; however, there are several rearrangements and inversions when compared to the horse isolate S. equi 4047. The virulence factors conveyed by genomic islands and prophages, in particular, played a key role in shaping the pathogenic capacity and genetic diversity of these S. equi strains. Furthermore, we discovered that the HT133 isolate had a strong colonization ability and increased motility; the HT1112 isolates had a significantly higher ability for antimicrobial resistance and biofilm formation, and the HT232 isolate gained pathogenic specialization by acquiring a bacteriophage encoding hyaluronate lyase. Discussion: In summary, our findings show that genetic exchange across S. equi strains influences the development of the donkey S. equi genome, offering important genetic insights for future epidemiological studies of S. equi infection.

[Prokaryotic expression of the GapC protein of Streptococcus uberis and prediction, identification of its B-cell epitopes].

The GapC protein of Streptococcus uberis located on the surface of bacteria is a protein with glyceraldehyde-3-phosphate dehydrogenase activity. It participates in cellular processes and exhibits a variety of biological activities. In addition, it has good antigenicity. The aim of this study was to predict the possible B-cell epitopes of the GapC protein and verify the immunogenicity of candidate epitope peptides. The gapC gene of S. uberis isolate RF5-1 was cloned into a recombinant expression plasmid pET-28a-GapC and inducibly expressed. The purified protein was used to immunize experimental rabbits to produce anti-GapC polyclonal antibodies. The three-dimensional structure and three-dimensional location of the GapC B-cell epitopes and the homology comparison of the GapC protein and its B-cell epitopes were carried out using bioinformatics softwares. The results showed that the 44-kDa GapC protein had a good immunological reactivity. Six linear and 3 conformational dominant B-cell epitopes against the GapC protein were selected and synthesized. Three dimensional analysis indicated that the selected peptides have better antigen epitope formation potential. Rabbit anti-GapC polyclonal antibodies were generated after immunized with the purified GapC protein, and the polyclonal antibodies were used to identify the epitope peptide by an indirect ELISA. The ELISA results showed that all of the 9 epitope peptides could react with anti-GapC polyclonal antibodies with varying titers. Among them, the epitope polypeptide 266AANDSYGYTEDPIVSSD282 reacted with the polyclonal antibodies significantly stronger than with other epitope peptides. This study laid an experimental foundation for in-depth understanding of the immunological properties and utilizing effective epitopes of the GapC protein of S. uberis.

Enhanced immune effects and protection conferred by simultaneously targeting GAPDH, SeM, and EAG of S. equi via TLR4.

Strangles, which is caused by Streptococcus equi subspecies equi, is one of the most prevalent equine infectious diseases and poses heavy economic losses worldwide. Although various vaccines have been used for decades, they seemed to be sub-optimal to demonstrate effective protection, and the antigen component of vaccines against S. equi remains to be optimized. In the present study, three target antigens (M-like protein, α2-macroglobulin and IgG-binding protein, and glyceraldehyde-3-phosphate dehydrogenase) were selected and expressed. Mice were immunized and challenged, and their immune response and efficacy were evaluated. The results revealed that this optimized multi-antigen treatment elicited a high expression level of T-cell receptor, major histocompatibility complex I, toll-like receptor TLR-4, and increased specific antibody. In addition, the challenge experiment showed an evidently improved protection efficacy. The present work demonstrated that these three proteins might be used as a promising multicomponent subunit vaccine candidate against S. equi infection.

Optimized GAPDH-truncated immunogen of Streptococcus equi elicits an enhanced immune response and provides effective protection in a mouse model.

Strangles is an acute and frequently diagnosed infectious disease caused by Streptococcus equi subsp. equi. Infection with this pathogen can cause grave losses to the equine industry. The present work investigates glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an important surface-localized virulence factor of S. equi, to determine whether it could be developed into an efficacious and suitable subunit vaccine against strangles. Two different recombinant fragments of S. equi GAPDH, namely, GAPDH-L and GAPDH-S, were constructed and expressed. Further, the antigenicity and immunogenicity of these two recombinant proteins were compared and evaluated in a mouse model. Our results revealed that immune responses were efficiently induced by the proteins in immunized mice. Remarkably, higher survival rates and significantly lower bacterial loads in the lung, liver, kidney, and spleen were observed in the GAPDH-S group compared with the GAPDH-L group after challenge with S. equi. High levels of specific antibodies, elevated antibody titers, and increased proportions of CD8 + T cells further indicated that GAPDH-S elicited better humoral and cellular immune responses than GAPDH-L. Furthermore, the induction of TCR, TLR-2, TLR-3, and TLR-4 significantly increased in the GAPDH-S group compared with those in the GAPDH-L and negative control groups. In summary, our results indicate that the optimized recombinant protein GAPDH-S is a promising candidate construct that may be further developed into a multivalent subunit vaccine for strangles.

A comparative investigation on the role and interaction of EsxA and EsxB in host immune response.

Staphylococcus aureus (S. aureus) is a frequent and major cause of bovine mastitis; it poses a tremendous economic burden to dairy industries of numerous countries. Early-secretion antigen-6 secretion system (ESS) has been viewed as an essential virulence and pathogenic factor of S. aureus. EsxA and EsxB are small acidic proteins secreted by ESS and identified as potential T-cell antigens of S. aureus. Unlike those of Mycobacterium tuberculosis (M. tuberculosis), the EsxA and EsxB of S. aureus do not form a dimer. Instead, EsxA dimerizes with itself or EsaC. Therefore, the interaction of EsxA and EsxB remains incompletely understood. In this study, to explore their interactions, EsxA and EsxB were expressed and used for immunization, alone or in combination, of murine infection models. Both components can interact with each other. Through the analysis of the immune response by immunological method, EsxB could significantly enhance the EsxA-specific IgG2a antibody level and increase the proliferation proportion of CD8+ T cells. These results indicate that when vaccinated with EsxA, EsxB can play a critical role in stimulating T helper 1 immunity by activating IgG2a and CD8+ T cells. We further show that vaccination with the combination of EsxA and EsxB resulted in enhanced stimulation of TLR-4 and improved protection against S. aureus. The findings may help us better understand the role of EsxB in the virulence and pathogenesis of S. aureus.

Effects of srtA variation on phagocytosis resistance and immune response of Streptococcus equi.

Strangles, which is caused by Streptococcus equi subspecies equi (S. equi), is one of the most prevalent equine infectious diseases with worldwide distribution and leads to serious economic loss in the horse industry. Sortase A (srtA) is a transpeptidase that anchors multiple virulence-associated surface proteins to the cell surface of S. equi. srtA plays a major role in S. equi infection and colonization of the host cell. In this study, we aimed to investigate the effects of srtA mutation on the phagocytic activity and immunogenicity of S. equi. The point-mutated recombinant sortases, including srtA-HT1112 (I88V), srtA-5012 (R147G), and srtA-ZZM17 (control), were expressed, purified, and used to immunize the mouse models. Phagocytic activity was assessed using equine polymorphonuclear cells, whereas opsonophagocytic function and adherence inhibition were measured using the antiserum of these mutants. Mouse serum antibody, bacterial load, and weight gain were also measured. The srtA-HT1112 (I88V) mutant showed significantly enhanced antiphagocytic capability, and its antiserum exhibited increased adherence inhibition activity. In addition, the srtA-HT1112 (I88V) mutant presented the highest lung bacterial load and lowest protection rate (50%) after the challenge with S. equi ZZM17. The srtA-5012 (R147G) mutant exhibited a high IgG2a level and protection rate (62.5%-75%) and the lowest lung bacterial load. These results indicate that the I88V mutation is associated with a high antiphagocytic activity, whereas R147G mutation is associated with the decreased lung bacterial load. Our findings may be useful for the evaluation and development of vaccines.

Antibody kinetics and immune profile analysis of a Streptococcus equi DNA vaccine expressing the FljB and SeM fusion protein in murine and equine models.

Strangles is a highly prevalent, extremely contagious, and occasionally lethal infectious disease affecting horses worldwide. Prophylactic antibiotics are ineffective in prevention of disease but are recommended for exposed horses at the first sign of fever and any horse obviously ill from strangles or with complications and there is an urgent need of a cost-effective, safe, efficacious vaccine. In the present study, we sought to develop effective vaccines by fusing the Streptococcus equi subspecies equi (S. equi) antigen SeM with the flagellin of Salmonella abortus equi FljB. We also explored the immunogenicity and efficacy of this candidate vaccine in mice and horses by intramuscular injection. Mice and horses immunized with FljB-SeM DNA vaccine showed high levels of specific antibody and increased production of IFN-γ and IL-4. This confirmed that both Th1 and Th2 type responses were induced. The mice survival rate was significantly higher after immunization with FljB-SeM than with SeM alone. The FljB-SeM DNA could strengthen both the Th1 and Th2 immune responses compared to SeM and could provide better protection against S. equi. This technique could help develop a candidate vaccine for S. equi infection.

TLR-5 agonist Salmonella abortus equi flagellin FliC enhances FliC-gD-based DNA vaccination against equine herpesvirus 1 infection.

Equine herpesvirus 1 (EHV-1) induces serious respiratory infections, viral abortion, neurological signs, and neonatal mortality in horses. Despite the use of vaccines, EHV-1 infection also causes a high annual economic burden to the equine industry. The poor immunogenicity of and protection conferred by EHV-1 vaccines are the major factors responsible for the spread of EHV-1 infection. The present study examined the immunogenicity of a novel DNA vaccine co-expressing FliC, a flagellin protein, in Salmonella abortus equi and the gD protein of EHV-1. Mice and horses were immunized intramuscularly with the vaccine, and mice were challenged with EHV-1. Immunofluorescence and western blotting revealed that FliC and gD can be efficiently expressed in cells. This novel vaccine significantly increased gD-specific antibody and interferon gamma (IFN-γ) levels in immunized mice and horses. Compared with controls, the viral load and morbidity were markedly reduced in FliC-gD-immunized mice after they were challenged with EHV-1. Furthermore, the immunogenicity of FliC-gD in a natural host was tested. Our results indicate that vaccinated mice and horses exhibit increased humoral and improved cellular immune responses.

Analysis of the Genetic Diversity in Methicillin-Resistant Staphylococcus aureus Isolates from Bovine Subclinical Mastitis Case in Xinjiang, China.

BACKGROUND: Diseases caused by livestock-associated methicillin-resistant Staphylococcus aureus (MRSA) are an important global public health concern, and MRSA is increasingly being isolated in bovine milk. However, information on the genotype and antimicrobial resistance of MRSA in bovine milk in Xinjiang is limited. The objective of this study was to determine the antimicrobial-susceptible phenotypes and genotypes of the circulating MRSA clone isolated in bovine mastits milk samples in Xinjiang, China. METHODS: Fifty six MRSA isolates collected from milk of bovine mastitis were investigated by multilocus sequence typing (MLST), staphylococcal protein A (spa) typing, and a minimum inhibitory concentration test with 21 antimicrobial agents. RESULTS: Antibiotic resistance results showed that 47.4% of the isolates were resistant to 16 or more antibiotics. Twelve MLST types were defined in this study, and ST398 (n = 7) and ST2393 (n = 2) were found to be the most prevalent types. Seven spa types (t034, t269, t4030, t114, t35, t189, and t7589) were identified, of which t034 (n = 7), t189 (n = 3), and t4030 (n = 3) were predominant. Here, 3 MRSA ST188 is reported among human MRSA isolates in China, and this is the first time that it is reported in bovine MRSA strains. CONCLUSIONS: The antimicrobial susceptibility of MRSA in this area exhibited multidrug resistance, and clonal complexes CC398 and CC188, which have been reported among human MRSA isolates, do occur in Xinjiang dairy cows. This study provides a foundation for further MRSA monitoring.

[Immunological characteristic comparison of different genetic polymorphism recombinant of FnBPA-A of bovine Staphylococcus aureus strain].

Objective: To understand the impact of genetic polymorphism of FnBPA-A on the immune biological characteristics of Staphylococcus aureus. Methods: Sequence of FnBPA-A of Staphylococcus aureus isolated from bovine in Xinjiang was analyzed and 8 different genetic polymorphism eukaryotic recombinants of FnBPA-A were constructed. C57BL/6 mice were immunized with these recombinant plasmids and mice sera were collected. Level of the immune protection of immunized mice was compared. Results: GS801, GS819 and GS856 were on the same branch; GW10-1, GW20-2, GY288 and GY309 belong to the same branch; GY278 was on a different branch. For the challenge experiment, GW20-2, GS801, GS819, GS856 and GY288 showed better protection. Conclusion: The genetic polymorphism of FnBPA-A could significantly affect the immune protection of immunized mice.

[Prokaryotic expression and immunogenicity of IgG-binding protein of Streptococcus equi subspecies equi].

To analyze the immunogenicity and protective ability of recombinant IgG-binding protein (EAG) of Streptococcus equi subspecies equi and to evaluate its value when used as equine vaccine antigen, EAG gene was amplified by PCR and inserted into pET-28a vector. The EAG recombinant proteins were expressed and purified to immune mice. The serum antibody and challenge protection were tested. The purified recombinant protein of EAG was 26 kDa, and the protein reacted specifically with positive serum of Streptococcus equi subspecies equi. The mice antibody level for EAG immunization group was 1∶8 100. The immunological protection result showed that the protection rate of the EAG recombinant protein was 90%. The results suggested that the EAG protein has good immunogenicity and immunological protection, and it can effectively increase the humoral immune response and immunological protection of mice.

[Immunological comparison of Efb and ClfA of Staphylococcus aureus isolated from bovine].

To compare immunological characteristics of Extracellular fibrinogen-binding protein (Efb) and Clumping factor A (CfA) of Staphylococcus aureus, we constructed two prokaryotic expression vector pET28a-Efb and pET28a-ClfA. After prokaryotical expression and purification, Efb and ClfA were used to immunize experimental animal. After the second immunization the antisera were collected and the antibody titers, the bacteria binding activity and adhesion inhibition activity of these antisera were detected by enzyme linked immunosorbent assay, adhesion inhibition assay and challenge. Both Efb and ClfA had Fibrinogen binding activity whereas the former had better Fibronectin binding activity. The bacteria binding capability of antisera of rabbits immunized with ClfA was better than that with Efb (P < 0.01). Both antisera of Efb and ClfA could inhibit adherence activity of Staphylococcus aureus to Fibrinogen and Fibronectin adherence compare to the control group (P < 0.01), and Efb had better adhesion inhibition activity than ClfA. The antibody titer of immunized group could reach 1:40 500. After the second immunization, both Efb and ClfA had good protective efficacy. This result constitutes a good foundation for Staphylococcus aureus subunit vaccine development.

[Effect of Kozak sequence on mice DNA vaccine immunization of Staphylococcus aureus adhesion fibronectin-binding protein FnBPA-A].

Fibronectin-binding protein (FnBPA) is a protein that expresses on cell surface of Staphylococcus aureus during early stage of infection. FnBPA was capable of promoting Staphylococcus aureus to invade cells and was viewed as a potential immune target. Based on the FnBPA-A gene two recombinant expression vectors with or without Kozak sequence were constructed. After identified and confirmed by restriction enzyme digestion and sequencing they were used to immunize C57BL/6 mice. Then induced antibody titer, T lymphocyte proliferative response and experiment mice challenge test were measured. Our result indicates that humoral immune responses and challenge experiment induced by recombinant DNA with Kozak sequence were better than those without Kozak sequence (P < 0.05). For T lymphocyte proliferative response the induced effect of recombinant DNA with Kozak sequence was higher than that without Kozak sequence, but there was no significant difference (P > 0.05). We conclude that Kozak sequence could play an important role in immune response induced by FnBPA-A recombinant DNA.

CD4 detected from Lactobacillus helps understand the interaction between Lactobacillus and HIV.

Human immunodeficiency virus (HIV) preferentially infects and destroys CD4+ cells and leads to a gradual decline in the number of CD4 cells. Despite evidence that probiotics increase CD4+ T lymphocytes in patients with HIV/acquired immunodeficiency syndrome (AIDS) and lower the risk of HIV transmission, little is known about the detailed mechanism underlying these effects. In this study, we investigated the cell surface protein of Lactobacillus and its role in blocking HIV-1 transmission by lactobacilli. Using reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence, and flow cytometry (fluorescence-activated cell sorting, FACS), we detected the CD4 receptor on the surface of Lactobacillus. Monoclonal antibody (mAb) for the CD4 receptor could partially inhibit HIV-1 binding to Lactobacillus. In addition, Lactobacillus could decrease HIV-1 pseudovirus infection of TZM-bl cells in vitro by 60-70%. Our data suggest that Lactobacillus can use this receptor to bind HIV and block HIV infection. This may in turn increase the CD4 T lymphocyte count in patients with HIV. These data provide direct evidence that Lactobacillus expresses the CD4 receptor and utilizes it to block HIV transmission.

[Using reporter gene to compare infection efficiency of HIV-1 pseudovirus to suspended and adherent cells].

OBJECTIVE: In order to compare the infection of HIV-1 pseudovirus to suspended and adherent cells, Tzmbl cells containing beta-gal (beta-galactosidase) reporter gene were used here to do the analysis. pseudoviruses were generated by co-transfection of 293T cells with the plasmid pNL43R-E- and HIV envelope expressing plasmid. Supernatant of co-transfected 293T cells was collected and used to infect Tzmbl cells with or without trypsin treatment. Forty-eight hours after infection, beta-gal positive Tzmbl cells and virus infection were determined using X-gal staining and beta-glo (beta-galactosidase) assay. RESULTS: The efficiency of HIV pseudoviruses infection of suspended Tzmbl cell was higher than that of adherent cells and the increase of infection correlated with the pseudoviral subtype. CONCLUSION: This study may provide a useful method for HIV biological study and neutralization assays using a single-round replicative pseudovirus in the future.

[Effects of signal peptide sequence of hemagglutinin on the expression of green fluorescent protein and hemagglutinin fusion gene].

OBJECTIVE: Purified avian influenza virus (AIV) hemagglutinin (HA) gene fragment was inserted into green fluorescent protein (GFP) expression vector, pEGFP-C1. The role of signal peptide in the HA-GFP expression in 293T cells was investigated by cutting the signal peptide or placing it in different locations of HA-GFP fusion gene. METHODS: The expression of GFP in 293T cells was examined directly with fluorescence microscope and flow cytometry analysis. RESULTS: After transfected with pEGFP-C1, M1, M2 and M3 plasmid, under fluorescent microscope the HA-GFP fusion protein was successfully expressed. Fluorescence was seen homogeneously distributed in the entire cell body of the cells transfected by the empty vector pEGFP-C1, whereas signal peptide could obviously reduced the expression of GFP-HA compared with recombinant plasmid without signal peptide. In addition, we found that there was no significant effect of the location of signal peptide on the expression of HA-GFP fusion protein.

Analysis of a point mutation in H5N1 avian influenza virus hemagglutinin in relation to virus entry into live mammalian cells.

Binding to and infection of human cells is essential for avian influenza virus transmission. Since virus binding is not always predictive for efficient infection of the cells, here we wished to investigate how hemagglutinin (HA) mutations of avian influenza virus H5N1 influence virus post-binding events in a single cycle of replication. One mutation observed in H5 HA of avian and natural human isolates from mainland China, Hong Kong, Vietnam and Thailand was identified and analyzed. The effects of the mutation on receptor binding, fusion and virus entry into cultured cells were investigated using hemadsorption, polykaryon formation and pseudotyped virus that express luciferase in the cytoplasm of transduced cell. Our results revealed that replacing aspartic acid at residue 94 with asparagine enhanced virus fusion activity and increased the binding of HA to sialic acid alpha2,6 galactose, while it decreased pseudotyped virus entry into cells expressing the avian type receptor, sialic acid alpha2,3 galactose. Our result may have implications for the understanding of the role of HA mutations in virus entry into live cells that exclusively display one type of receptor.

A dual reporter gene based system to quantitate the cell fusion of avian influenza virus H5N1.

Membrane fusion is central to the entry of influenza virus into host cells. To quantitatively determine the fusion activity of hemagglutinin (HA) of avian influenza virus H5N1, we established a cell fusion assay based on a dual luciferase reporter gene. The HA fusion activity was assayed by measuring luciferase expression in fused cells, allowing a rapid, sensitive, and quantitative comparison of HA fusion activities at various pHs and in different cells types. The simplicity and the quantitative nature of this novel assay are ideally suited for identifying viral receptors or screening for inhibitors of viral entry in the future.