IdeE, an IgG-endopeptidase of Streptococcus equi ssp. equi

Streptococcus equi ssp. equi is the causative agent of strangles, a highly contagious and serious disease in the upper respiratory tract of horses. The present study describes the characterization of IdeE, a homolog of the secreted IgG-specific protease IdeS/Mac of Streptococcus pyogenes. The activity of IdeE is compared with the activity of IdeZ, the corresponding enzyme of the closely related S. equi ssp. zooepidemicus. A study of the proteolytic activity of recombinant IdeE and IdeZ on IgG from a selection of mammals shows that only antibodies containing the substrate site of IdeS/Mac are cleaved, indicating that the specificities of these enzymes are similar. Interestingly, IgG from horse is less effectively cleaved than IgG from e.g. dog or humans, as the dominating IgG isotype in horse sera (IgG4) lacks a distinct substrate site for IdeE/IdeZ. IgG-degradation is observed when S. equi ssp. equi is grown in the presence of horse serum, but not when grown with purified IgG. As the fraction of degraded IgG contains IgG4, the observed activity might be due to the expression of an unknown enzyme rather than IdeE. In a similar assay, no proteolysis of IgG was detected in the growth media of S. equi ssp. zooepidemicus.     

马疫链球菌SH-5生产透明质酸营养条件的研究

对Streptococcus equiSH-5生产透明质酸的营养条件进行了研究,摇瓶发酵实验表明该菌株的适宜氮源为酵母膏和牛肉膏以2∶1组成的混合氮源,在碳氮比为2∶1时有利于透明质酸的合成和菌体的生长;通过正交实验摸索出营养培养基的最佳配比为葡萄糖5%,酵母粉6.67%,牛肉膏3.33%,MgSO4.7H2O 0.1%,MnSO4.4H2O 0.02%,NaHCO30.3%,Na2HPO40.5‰,尿嘧啶0.08‰;添加0.05‰异甘草素对产酸有利。     

Neither the A- nor B-repeat regions of the fibrinogen-binding protein of Streptococcus equi subsp. equi are essential for fibrinogen binding

The major cell wall-associated protein (FgBP) of Streptococcus equi subsp. equi possesses two internal blocks of repeated sequence (A and B) and binds horse fibrinogen (Fg) avidly through residues located in the N-terminal half of the molecule. In the present study, we investigated the roles of the two repeats blocks in Fg binding through construction of recombinant FgBP proteins containing defined internal deletions of sequence. Ligand binding experiments clearly showed that neither repeat is essential for Fg binding. However, residues within the B repeats seem to play a major role in the aberrant mobility observed for FgBP following sodium dodecyl sulfate polyacrylamide gel electrophoresis.     

Identification and characterization of a novel fibronectin-binding protein gene from Streptococcus equi subspecies zooepidemicus strain VTU211

This work describes the cloning and sequencing of genes encoding fibronectin-binding proteins from Streptococcus equi subspecies zooepidemicus strain VTU211. A gene encoding a cell-wall protein FNZ was amplified and sequenced. In the same bacterial strain, a second gene termed fnz2 was now discovered, encoding another fibronectin-binding protein (FNZ2). The complete amino acid sequence encoded by fnz2 was deduced and compared to that deduced from fnz. The sequence comparison of the fnz and fnz2 predicted that fibronectin-binding activity is localizing a domain in the C terminal part of FNZ2, since this domain is composed of three repeats, which contain a motif similar to what has earlier been found in other fibronectin-binding proteins in streptococci. Three parts of fnz2 [fnz2(1-8), fnz2(2-4), and fnz2(4-3)] were amplified using polymerase chain reaction and ligated into an expression vector, and recombinant FNZ2 proteins were produced in Escherichia coli. Fibronectin bound to the FNZ2(1-8) [amino acids 212-396] and FNZ2(2-4) (amino acids 36-448) but not to the FNZ2(4-3) (amino acids 36-191) in a Western ligand blot, showing that repeat domain of FNZ2 protein was sufficient for binding of fibronectin. Purified FNZ2(2-4) protein was also shown to display collagen-binding activity to collagen-coated microtiter wells. These results show that recombinant FNZ2 has fibronectin- and collagen-binding activities.     

透明质酸产生菌SH-2发酵条件的优化

对经紫外诱变,筛选出的突变菌株马疫链球菌SH-2,进行发酵条件的优化实验,通过单因素实验确定最佳摇瓶条件是种龄14-16h,初始pH7.6,发酵温度37℃,发酵时间44h;通过多因素正交实验摸索出营养培养基的最佳配比为:葡萄糖5%,酵母粉6.67%,牛肉膏3.33%,MgSO4·7H2O0.1%,MnSO4·4H2O0.02%,NaHCO30.3%,Na2HPO40.15%,NaCl0.3%,UTP0.05%,异甘草素0.08‰。     

Outbreak of Streptococcus equi subsp. zooepidemicus infection in a group of rhesus monkeys (Macaca mulatta)

Background  A severe upper respiratory tract infection occurred in a breeding group of rhesus monkeys housed together in one of six indoor/outdoor corals of the German Primate Center. The clinical signs of the disease included severe purulent conjunctivitis, rhinitis, pharyngitis, respiratory distress and lethargy. Six of 45 animals died within a few days after developing signs of infection.
Methods and results  Histopathologic and microbiologic examinations of the dead animals were consistent with a severe fibrinopurulent bronchopneumonia. Microbiology revealed a Lancefield group C streptococcus identified as Streptococcus equi subsp . zooepidemicus as the causative agent of infection.
Conclusions  The infection was passed on from animal to animal but did not spread to the other five breeding groups nearby. Extensive diagnostic testing failed to reveal the consisting presence of copathogens in individual cases. A visitor with upper respiratory disease was suspected as source of infection.     

Immunoproteomic assay of extracellular proteins in Streptococcus equi ssp. zooepidemicus

A proteomic approach combining two-dimensional electrophoresis, Western blot and matrix-assisted laser desorption tandem time-of-flight mass spectrometry has been used to map the extracellular proteins of Streptococcus equi ssp. zooepidemicus ( S . zooepidemicus ) strain ATCC 35246. These bioinformatic technologies facilitated the identification of novel S . zooepidemicus vaccine candidate antigens and therapeutic agents. Despite the limitations posed by the unavailability of complete genome and proteome data for S . zooepidemicus , seven of 15 chosen immunogenic spots were successfully identified as streptococcal proteins (AE1 and AE4 c . 10) from homologous Streptococcus species. Among these, AE6 and AE7 were identified as S . zooepidemicus UDP- N -acetyl-glucosamine pyrophosphorylase and UDP-glucose pyrophosphorylase proteins. In addition, AE4 was determined to be glyceraldehyde-3-phosphate dehydrogenase from Enterococcus faecalis . Following signalip 3.0 ( http://www.cbs.dtu.dk/servicess/SignalIP ) prediction, data suggested that AE5, AE7 and AE9 contained signal peptides. blast ( http://www.sanger.ac.uk ) results found that nucleotide sequences of all identified proteins shared high homology (≥65%) with S. zooepidemicus . The majority of proteins identified in our study remain formally unreported in S. zooepidemicus . However, these proteins serve a vital role in the immune system and reproduction of host species. Therefore, we further evaluated the proteins as vaccine candidates in this study.     

Production of Monoclonal Anti-Idiotype Antibodies Which Mimic an M-Like Protein of Streptococcus equi

Rat monoclonal anti-idiotype antibodies (mAb2) were raised against two mouse monoclonal antibodies (mAb1), 1D10 and 2A6, with specificity for the M-like protein of Streptococcus equi. The capacity of the mAb2 to inhibit the binding between the corresponding mouse mAbl against which the mAb2 were raised and the M-like protein was investigated in an inhibition EIA. One of the ten mAb2 examined, namely 5D1 (anti-mAb1 1D10), was able to inhibit this binding. The mAb2 5D1 bound to the mAb1 1D10 in such a way as to completely inhibit the subsequent binding of the M-like protein antigen to the paratope of the mAb1 1D10. The mAb2 5D1 is likely to represent a true image of the M-like protein antigen and may thus be described as an Ab2β anti-idiotype antibody.     

Two new cysteine endopeptidases obtained from the latex of Araujia hortorum fruits

Two new endopeptidases were purified to homogeneity from the latex of Araujia hortorum fruits by a simple purification procedure involving ultracentrifugation and ion exchange chromatography. Molecular weights of araujiain h II and araujiain h III were 23,718 and 23546 (mass spectrometry), respectively. The isoelectric point of araujiain h II was 8.9, whereas araujiain h III had a pI higher than 9.3. Maximum proteolytic activity on caseine was reached at pH 8.0-9.0 for both endopeptidases, which were irreversibly inhibited by iodoacetate and E-64, suggesting they belong to the cysteine protease family. Esterolytic activity was determined on N--CBZ-amino acid-p-nitrophenyl esters, and the highest k _cat/K _m values for the both enzymes were obtained with the glutamine derivative. The N-terminal sequences of araujiain h II and araujiain h III showed a high degree of homology with other plant cysteine endopeptidases.     

Bivalent Ligation of the Collagen-binding Modules of Fibronectin by SFS,a Non-anchored Bacterial Protein of Streptococcus equi

SFS is a non-anchored protein of Streptococcus equi subspecies equi that causes upper respiratory infection in horses. SFS has been shown to bind to fibronectin (FN) and block interaction of FN with type I collagen. We have characterized interactions of a recombinant 60-mer polypeptide, R1R2, with FN. R1R2 contains two copies of collagen-like 19-residue repeats. Experiments utilizing various FN fragments and epitope-mapped anti-FN monoclonal antibodies located the binding site to ^8-9FNI modules of the gelatin-binding domain. Fluorescence polarization and competitive enzyme-linked assays demonstrated that R1R2 binds preferentially to compact dimeric FN rather than monomeric constructs containing ^8-9FNI or a large dimeric FN construct that is constitutively in an extended conformation. In contrast to bacterial peptides that bind ^2–5FNI in addition to ^8-9FNI, R1R2 did not cause conformational extension of FN as assessed by a conformationally sensitive antibody. Equilibrium and stopped-flow binding assays and size exclusion chromatography were compatible with a two-step binding reaction in which each of the repeats of R1R2 interacts with one of the subunits of dimeric FN, resulting in a stable complex with a slow k_off. In addition to not binding to type I collagen, the R1R2·FN complex incorporated less efficiently into extracellular matrix than free FN. Thus, R1R2 binds to FN utilizing features of compact soluble FN and in doing so interferes with the organization of the extracellular matrix. A similar bivalent binding strategy may underlie the collagen-FN interaction.     

检测人血清中肺炎球菌10A型荚膜多糖IgG抗体的包被抗原适用性研究

目的 确定用于23价肺炎多糖疫苗免疫后临床血清样本检测的包被用10A型肺炎球菌荚膜多糖(Pn10A)。方法 根据WHO推荐的检测人血清中肺炎球菌荚膜多糖IgG抗体含量的ELISA(PnPSELISA),包被不同来源(ATCC、A公司、B公司、5个公司混合)的10A多糖[Pn10A(ATCC)、Pn10A(A)、Pn10A(B)、Pn10A(mix)],检测38份血清中Pn10AIgG抗体的几何平均浓度(GMC)和相同样本免疫前、后的阳转率(免疫后/免疫前≥2为阳转),确定用于临床血清检测的包被Pn10A。结果 用Pn10A(ATCC)、Pn10A(A)、Pn10A(B)包被检测38份相同样本免疫前、后血清中Pn10AIgG抗体的GMC和阳转率,Pn10A(A)与Pn10A(ATCC)包被的检测结果差异无统计学意义(P>0.05),数据一致性好(r>0.9);Pn10A(B)与Pn10A(ATCC)包被的检测结果差异有统计学意义(P<0.05),数据一致性差(r<0.8);再以Pn10A(A)、Pn10A(ATCC)、Pn10A(mix)包被检测另46份相同样本免疫前、后血清中Pn10AIgG抗体的GMC和阳转率,Pn10A(A)、Pn10A(mix)与Pn10A(ATCC)包被的检测结果差异无统计学意义(P>0.05),数据一致性好(r>0.9),免疫前、后GMC值相近,阳转率相近,差异无统计学意义(P>0.05)。结论 Pn10A(A)、Pn10A(mix)与Pn10A(ATCC)均可以作为包被多糖用于检测人血清中肺炎球菌Pn10AIgG抗体;但从长久使用相同抗原检测大批量临床样本的需求考虑,Pn10A(mix)更具有足量、经济的优势。     

Identification and Characterization of IgdE,a Novel IgG-degrading Protease of Streptococcus suis with Unique Specificity for Porcine IgG

Streptococcus suis is a major endemic pathogen of pigs causing meningitis, arthritis, and other diseases. Zoonotic S. suis infections are emerging in humans causing similar pathologies as well as severe conditions such as toxic shock-like syndrome. Recently, we discovered an IdeS family protease of S. suis that exclusively cleaves porcine IgM and represents the first virulence factor described, linking S. suis to pigs as their natural host. Here we report the identification and characterization of a novel, unrelated protease of S. suis that exclusively targets porcine IgG. This enzyme, designated IgdE for immunoglobulin G-degrading enzyme of S. suis, is a cysteine protease distinct from previous characterized streptococcal immunoglobulin degrading proteases of the IdeS family and mediates efficient cleavage of the hinge region of porcine IgG with a high degree of specificity. The findings that all S. suis strains investigated possess the IgG proteolytic activity and that piglet serum samples contain specific antibodies against IgdE strongly indicate that the protease is expressed in vivo during infection and represents a novel and putative important bacterial virulence/colonization determinant, and a thus potential therapeutic target.     

Development of a New Selective Plating Medium for Rhodococcus equi

A new selective plating medium for Rhodococcus equi containing ceftazidime (20 mg/l) and novobiocin (25 mg/l) on a Mueller-Hinton agar basis is described. It proved to be less inhibitory for R. equi than selective plating media devised earlier and grew only very few other nocardioform bacteria.     

Experimental infection of rhesus macaques with Streptococcus pneumoniae: a possible model for vaccine assessment

BACKGROUND: We explored the possibility of using normal adult rhesus macaques for the preclinical assessment of safety, immunogenicity, and efficacy of newly developed vaccines against Streptococcus pneumoniae infection of the lung. METHODS: Our primary objective was to determine whether an intra-bronchial inoculum of at least 10(6)S. pneumoniae colony-forming units, or one as high as 10(8)-10(9) organisms, could detectably survive in rhesus macaques for a period longer than 1-2 weeks. If so, we hypothesized, it would be possible to observe signs of pneumonia commonly observed in humans, and discriminate between vaccinated/protected animals and controls. Infection was detectable in bronchoalveolar lavage fluids 3-5 weeks post-inoculation. RESULTS: The clinical course of disease mimicked aspects of that of human pneumococcal pneumonia. Signs of inflammation typical of the disease in humans, such as elevated concentrations of neutrophils and of pro-inflammatory cytokines in bronchoalveolar lavage fluids were also observed. CONCLUSIONS: These findings underscore the utility of this model to assess the safety, immunogenicity, and efficacy of newly developed S. pneumoniae vaccines.     

Diversity of isolates of Rhodococcus equi from Australian Thoroughbred horse farms

Pulsed field gel electrophoresis of restriction endonuclease digested genomic DNA from a collection of clinical isolates of Rhodococcus equi was used to compare strain diversity on different Thoroughbred horse farms over time. Restricted diversity was found among the isolates tested, as the same strains were detected on multiple farms and in multiple years. Marked variation occurred in strain prevalence with some strains being represented by single isolates, and the most prevalent by 26 isolates. There were dominant strains on some farms and the prevalence of some strains differed between farms. Infection with multiple strains was noted in some cases where multiple isolates from a single foal were examined.     

猪链球菌2型基因工程疫苗研究进展

猪链球菌2型引起的链球菌病是一种重要的人兽共患传染病。目前对于猪链球菌病的预防主要依靠灭活疫苗,而灭活疫苗对于同源菌的攻击保护率仅在70%左右,对异源菌的攻击保护率更低。目前对于猪链球菌2型基因工程疫苗的研究主要有两个方面,一是对某些毒力因子缺失的基因缺失疫苗的研究,由于尚未发现猪链球菌2型的标志性毒力因子,因此这方面的研究尚不太多;二是对基因重组亚单位疫苗的研究。随着分子生物学技术的发展,许多学者对猪链球菌2型的具有免疫原性的一些蛋白片段进行分析、重组、表达,以期制造出可以对猪链球2型进行有效预防的基因工程疫苗。由于猪链球菌2型的毒力因子复杂,对其毒力因子及其基因组成的研究尚不彻底,随着研究的深入,如在基因工程疫苗方面取得突破性进展,对于猪链球菌2型引起的链球菌病的预防与控制必将有着重要意义。     

Feeding Mechanisms of Babesia equi

SYNOPSIS. Electron microscope and time lapse, phase contrast cinematography studies on Babesia equi organisms within equine red blood cells revealed that this hemoprotozoon has two organelles possibly involved with ingestion of nutrients: a cytostome that takes in hemoglobin from the host cell and a tubule that extends from the main body of the parasite through the erythrocyte to the blood plasma and appears to ingest plasma during periods of rapid growth and development.     

DNA vaccines based on genetically detoxified derivatives of pneumolysin fail to protect mice against challenge with Streptococcus pneumoniae

The 7-valent polysaccharide conjugate vaccine currently administered against Streptococcus pneumoniae has been shown to be highly effective in high risk-groups, but its use in developing countries will probably not be possible due to high costs. The use of conserved protein antigens using the genetic vaccination strategy is an interesting alternative for the development of a cost-effective vaccine. We have analyzed the potential of DNA vaccines expressing genetically detoxified derivatives of pneumolysin (pneumolysoids) against pneumococcal infections, and compared this with immunization using recombinant protein. The purified recombinant pneumolysoid with the highest residual cytolytic activity was able to confer partial protection against a lethal intraperitoneal challenge, with the induction of high antibody levels. Immunization with DNA vaccines expressing pneumolysoids, on the other hand, induced a significantly lower antibody response and no protection was observed.     

Expression of Virulence-Associated Antigens of Rhodococcus equi Is Regulated by Temperature and pH

We recently reported that there are two different virulence-associated antigens correlated with virulence levels in Rhodococcus equi isolates from AIDS patients: virulent R. equi that kills mice with 10⁶ cells expresses 15- to 17-kDa antigens and intermediately virulent R. equi that kills mice with 10⁷ cells expresses a 20-kDa antigen. Environmental parameters were evaluated for their effects on the expression of these virulence-associated antigens in virulent R. equi strains by immunoblotting using monoclonal antibodies in this study. Expression of these two virulence-associated antigens of R. equi was regulated by pH and temperature; the antigens were produced maximally when the isolates were grown at 38 C and pH 6.5, but were not produced when grown at 38 C and pH 8, nor at temperatures below 30 C. The 20-kDa antigen was found to be located on the cell surface, as were the 15- to 17-kDa antigens, and showed susceptibility to proteolysis by trypsin. These results indicate that expression of the virulence-associated antigens of R. equi is dependent on the environmental conditions.     

Interaction of virulent and non-virulent Rhodococcus equi human isolates with phagocytes, fibroblast- and epithelial-derived cells

Abstract Rhodococcus equi is a facultative, intracellular, Gram-positive coccobacillus, increasingly reported in pneumonia of AIDS-infected patients. We investigated killing resistance properties of human R. equi virulent and avirulent human strains. Avirulent β-lactam-susceptible strains had lower intracellular colony forming units after 45 min incubation in murine macrophages J774 and human monocyte-macrophage TPH-1 than those of virulent strains. Only virulent β-lactam-resistant strains persisted within macrophages for at least 18 min only. A β-lactam-resistant mutant was obtained from a β-lactam-susceptible strain after selection in a penicillin G-containing culture medium. This mutant strain, like the natural virulent strains, persisted within macrophages, harboured cell-associated appendages, produced phage-like particles and induced, after its intravenous inoculation, a chronic infection in BALB/c nude mice. Supernatant culture of virulent strains transferred partial macrophage-killing resistance properties to avirulent strains. The same supernatant was toxic for L-929, HeLa and Vero cell cultures. These supernatant effects were heat-inactivated, trypsin-inactivated and did not seem to be linked to phage-like particle presence. These data argue that virulence, β-lactam-resistance, and macrophage-killing resistance are associated in human R. equi isolates. Moreover, only virulent strains produced uncharacterized toxic factors.