Staphylococcal enterotoxin type E: isolation, purification, identification

Homogeneous protein of staphylococcal enterotoxin type E has been isolated. The technique of isolation, permitting 48% yield of active material, includes concentration by ammonium sulfate precipitation, ion exchange chromatography on DEAE-cellulose and gel-filtration on sephacryl S-200. The molecular mass of the isolated protein is 32 Kd. Antigenic affinity of staphylococcal toxins types A and E has been established by immunochemical analysis.     

The comparative characteristics of the immunomodulating properties of protein A from Staphylococcus aureus of different origins

The immunomodulating properties of highly purified staphylococcal protein A and its analog obtained by gene engineering techniques have been compared with those of commercial preparations. The comparison has shown that the differences observed in this investigation may be explained by the presence of admixtures of staphylococcal nature in commercial preparations. The preparations of highly purified staphylococcal and recombinant protein A stimulate humoral immune response and the processes of phagocytosis and do not show mitogenic activity with respect to T cells. The conclusion on the identity of the immunomodulating activity of the preparations of natural and recombinant protein A has been made.     

Choice of the method for isolating conjugates of staphylococcal protein A with peroxidase for immunoenzyme analysis

An attempt to obtain the preparations of peroxidase-labeled staphylococcal protein A, intended for use in the enzyme immunoassay, by the glutaraldehyde method has failed. The modified periodate method permitting the preparation of active conjugates of staphylococcal protein A with peroxidase has been developed.     

The modification of human immunoglobulin binding to staphylococcal protein A using diethylpyrocarbonate

Human IgG subclasses 1, 2, and 4, as well as proteins of the IgG3 subclass that are allotype G3m (s+t+), bind avidly to staphylococcal protein A by means of their Fc portion. Proteins of the IgG3 subclass that are allotype G3m (s-t-) do not bind. The importance of a histidine residue at position 435 has been implicated from comparison of amino acid sequences of immunoglobulins that bind with those that do not bind to staphylococcal protein A, as well as from crystallographic data. Modification of histidines at a low concentration of diethylpyrocarbonate successfully and reversibly alters the binding of immunoglobulins to staphylococcal protein A with only minimal change in the antigenic properties. This method provides strong evidence for the critical importance of histidine in the binding of immunoglobulins to staphylococcal protein A.     

Rapid colorimetric detection of in vitro amplified DNA sequences

A colorimetric assay to detect immobilized amplified nucleic acids has been designed. This approach provides a rapid assay, suitable for clinical diagnosis, to analyze DNA sequences amplified by the polymerase chain reaction. The specific DNA sequences are captured on a solid support by the use of a recombinant fusion protein consisting of the Escherichia coli lac repressor and staphylococcal protein A. The biotin streptavidin system is used to detect the immobilized material. Positive samples can be analyzed by direct solid-phase sequencing. Here, we show that this nonradioactive concept can be used for analysis of Staphylococci and Streptococci and for specific detection of the protozoa Plasmodium falciparum in clinical samples.     

Substrate-specific inactivation of staphylococcal penicillinase

1. The rate of hydrolysis of methicillin, cloxacillin and quinacillin by staphylococcal extracellular penicillinase decreases progressively with time. 2. The inactivation is prevented but not reversed by benzylpenicillin. 3. The rate of inactivation produced by quinacillin is minimal when the rate of hydrolysis is at a maximum. 4. Under certain conditions, partially inactivated enzyme can be reactivated. 5. Combination of the enzyme with antiserum, while permitting hydrolysis, prevents inactivation. 6. No evidence for a stable enzyme-substrate complex has been found.     

Region X, the cell-wall-attachment part of staphylococcal protein A

The sequence of region X of staphylococcal protein A has been determined. The hypothesis has been put forward that this region spans the Staphylococcus aureus cell wall and is responsible for the binding to the peptidoglycan. The primary amino acid sequence of region X was determined for two strains exhibiting cell-wall-bound protein A, Cowan I and 8325-4. The sequence determination of the Cowan I material is partial and was performed by Edman degradation, in contrast to the sequence of the 8325-4 material which was completely analyzed by nucleotide sequencing of the corresponding gene. The region consists of two structurally different domains, a highly repetitive region (Xr), with an octapeptide structure repeated approximately 12 times, and a C-terminal domain (Xc) with an unique sequence. A comparison between the two strains reveals a high mutual homology as well as a high internal homology between the octapeptide structures. Six out of eight amino acids are identical in the repetition of this structure throughout region Xr in both proteins and the other two are changed in a rather regular pattern.     

Escherichia coli ghost production by expression of lysis gene E and Staphylococcal nuclease

The production of bacterial ghosts from Escherichia coli is accomplished by the controlled expression of phage phiX174 lysis gene E and, in contrast to other gram-negative bacterial species, is accompanied by the rare detection of nonlysed, reproductive cells within the ghost preparation. To overcome this problem, the expression of a secondary killing gene was suggested to give rise to the complete genetic inactivation of the bacterial samples. The expression of staphylococcal nuclease A in E. coli resulted in intracellular accumulation of the protein and degradation of the host DNA into fragments shorter than 100 bp. Two expression systems for the nuclease are presented and were combined with the protein E-mediated lysis system. Under optimized conditions for the coexpression of gene E and the staphylococcal nuclease, the concentration of viable cells fell below the lower limit of detection, whereas the rates of ghost formation were not affected. With regard to the absence of reproductive cells from the ghost fractions, the reduction of viability could be determined as being at least 7 to 8 orders of magnitude. The lysis process was characterized by electrophoretic analysis and absolute quantification of the genetic material within the cells and the culture supernatant via real-time PCR. The ongoing degradation of the bacterial nucleic acids resulted in a continuous quantitative clearance of the genetic material associated with the lysing cells until the concentrations fell below the detection limits of either assay. No functional, released genetic units (genes) were detected within the supernatant during the lysis process, including nuclease expression.     

衣壳蛋白靶向灭活策略应用于抗登革病毒感染的研究

衣壳蛋白靶向病毒灭活是近年来新兴的抗病毒策略。为探索该策略在抗登革病毒感染中的应用 ,首先建立了稳定表达登革 2型病毒衣壳蛋白 (D2C)与葡萄球菌核酸酶 (SN)融合蛋白D2C_SN的哺乳动物细胞系 ,然后以登革病毒攻击上述细胞系 ,研究表达的融合蛋白D2C_SN对产生的子代病毒颗粒感染性的影响。结果表明融合蛋白D2C_SN能够在病毒装配过程中与野生型衣壳蛋白共组装入子代病毒颗粒内部 ,并导致病毒基因组的降解。与正常BHK细胞相比较 ,融合蛋白D2C_SN可导致产生的子代病毒感染性滴度降低 10 3～ 10 4 ,显示出很强的抗病毒效果     

Variability of the pathogenicity traits of populations of staphylococci in a surgical hospital

The results of the study of heterogeneity of staphylococcal populations at a surgical ward are presented. The study deals with qualitative and quantitative characteristics of three groups of pathogenicity factors: protease (the penetration factor), protein A (the function of protection from phagocytosis) and alpha-hemolysin (the toxic function). The study shows that the greatest number of S. aureus strains with a high content of protein A has been isolated from patients with postoperative and wound infections. On the basis of the data obtained in this study the groups of strains have been defined in accordance with the association of the signs of pathogenicity. These groups reflect pronounced heterogeneity of staphylococcal strains at a surgical ward.     

Formalin Inactivation of Japanese Encephalitis Virus Vaccine Alters the Antigenicity and Immunogenicity of a Neutralization Epitope in Envelope Protein Domain III

Formalin-inactivated Japanese encephalitis virus (JEV) vaccines are widely available, but the effects of formalin inactivation on the antigenic structure of JEV and the profile of antibodies elicited after vaccination are not well understood. We used a panel of monoclonal antibodies (MAbs) to map the antigenic structure of live JEV virus, untreated control virus (UCV), formalin-inactivated commercial vaccine (FICV), and formalin-inactivated virus (FIV). The binding activity of T16 MAb against Nakayama-derived FICV and several strains of FIV was significantly lower compared to live virus and UCV. T16 MAb, a weakly neutralizing JEV serocomplex antibody, was found to inhibit JEV infection at the post-attachment step. The T16 epitope was mapped to amino acids 329, 331, and 389 within domain III (EDIII) of the envelope (E) glycoprotein. When we explored the effect of formalin inactivation on the immunogenicity of JEV, we found that Nakayama-derived FICV, FIV, and UCV all exhibited similar immunogenicity in a mouse model, inducing anti-JEV and anti-EDII 101/106/107 epitope-specific antibodies. However, the EDIII 329/331/389 epitope-specific IgG antibody and neutralizing antibody titers were significantly lower for FICV-immunized and FIV-immunized mouse serum than for UCV-immunized. Formalin inactivation seems to alter the antigenic structure of the E protein, which may reduce the potency of commercially available JEV vaccines. Virus inactivation by H₂O₂, but not by UV or by short-duration and higher temperature formalin treatment, is able to maintain the antigenic structure of the JEV E protein. Thus, an alternative inactivation method, such as H₂O₂, which is able to maintain the integrity of the E protein may be essential to improving the potency of inactivated JEV vaccines.     

Some improved versions of methods for the indirect detection of staphylococcal protein A gene expressed in Escherichia coli.

Several versions of methods for the indirect detection of expression of staphylococcal protein A gene (spa) in Escherichia coli (E. coli) were devised by making use of biological properties of staphylococcal protein A (SpA). i) Hemagglutination of sheep red blood cells (SRBC) sensitized with anti-SRBC-antibodies using heat-treated spa-transformed E. coli organisms; Native spa-transformed E. coli organisms did not agglutinate the sensitized SRBC. The heat-treatment (60 C, 4 hr) of the transformants, however, caused positive hemagglutination like SpA-positive Staphylococcus aureus (S. aureus) organisms. ii) Halo formation around colonies on agar plates containing normal dog serum, which is originally used for the detection of SpA of S. aureus. A mutant strain NMJ was isolated, which showed formation of the halo of precipitate due to interaction between immunoglobulin and SpA. iii) A new version of immunodetection; After lysis of the transformants grown on a nitrocellulose membrane by alkali, SpA could be directly detected by immuno-detection procedures after inactivation of endogenous peroxidase in bacteria by phenylhydrazine and hydrogen peroxide.     

Preparation and properties of the staphylococcal toxin causing the toxic shock syndrome

A method is proposed for isolation and purification of the staphylococcal toxin causing the toxic shock syndrome (TSS). The method includes three steps: aggregation of protein from the cultural filtrate of Staphylococcus aureus strain 1169 in the presence of 0.025% sodium hexametaphosphate at pH 3.0; gel filtration of the concentrated material on the sephadex G75; ion-exchange chromatography on DEAE 32 cellulose. The proposed method permits to obtain the purified biologically active preparation of toxin with the yield about 40%. The obtained preparations are homogeneous in polyacrylamide electrophoresis and as analyzed by immunochemical methods. The mol mass of the isolated protein is 24 kD, it is not immunologically identical to staphylococcal toxins A-D and is lethal for New Zealand white rabbits and chinchilla rabbits. Interferon inducing activity of the protein is identical to the one of staphylococcal enterotoxin type A.     

Primary structure of histone H2A from nucleated erythrocyte of the marine worm Sipunculus nudus. Presence of two forms of H2A in the sipunculid chromatin

The complete amino acid sequence (123 residues) of histone H2A from erythrocytes of the marine worm Sipunculus nudus, has been established from data provided by automated sequence analysis of large fragments generated by V8 staphylococcal protease digestion of histone H2A and by limited hydrolysis of the protein with alpha-chymotrypsin and from structural studies of tryptic peptides of the protein. By comparison with calf homologous histone, the sipunculid histone H2A shows 6 deletions and 13 substitutions. Six of the substitutions are non-conservative. Most of the evolutionary changes are mainly observed in the basic amino-terminal and carboxy-terminal regions of the molecule, which are the primary DNA-binding sites. Few conservative point changes are observed in the central region (residues 18-118) which interacts strongly with histone H2B to form the dimer H2A-H2B. 60% of the H2A molecules were found phosphorylated on the amino-terminal residue, N-acetyl-serine. The high content of phosphorylated histone H2A in the sipunculid erythrocyte chromatin could probably be related to smaller repeat length (177 +/- 5 base pairs) of nucleosomal DNA and to nuclear inactivation and chromatin condensation.     

Mutagenic activity of native and inactivated herpes simplex virus

The mutagenic activity of native and inactivated herpes simplex virus (HSV-1) has been studied. The native HSV-1 strain has been shown to induce the gene mutations of 6-mercaptopurine resistance in Chinese hamster cells. The 1.5- to 2-fold decrease of mutagenic activity is due to HSV inactivation by thiophosphamide and formalin treatment. These data indicate the possibility of the given agents application for decreasing of the genetic danger of antiviral vaccine.     

Escherichia coli Ghost Production by Expression of Lysis Gene E and Staphylococcal Nuclease

The production of bacterial ghosts from Escherichia coli is accomplished by the controlled expression of phage X174 lysis gene E and, in contrast to other gram-negative bacterial species, is accompanied by the rare detection of nonlysed, reproductive cells within the ghost preparation. To overcome this problem, the expression of a secondary killing gene was suggested to give rise to the complete genetic inactivation of the bacterial samples. The expression of staphylococcal nuclease A in E. coli resulted in intracellular accumulation of the protein and degradation of the host DNA into fragments shorter than 100 bp. Two expression systems for the nuclease are presented and were combined with the protein E-mediated lysis system. Under optimized conditions for the coexpression of gene E and the staphylococcal nuclease, the concentration of viable cells fell below the lower limit of detection, whereas the rates of ghost formation were not affected. With regard to the absence of reproductive cells from the ghost fractions, the reduction of viability could be determined as being at least 7 to 8 orders of magnitude. The lysis process was characterized by electrophoretic analysis and absolute quantification of the genetic material within the cells and the culture supernatant via real-time PCR. The ongoing degradation of the bacterial nucleic acids resulted in a continuous quantitative clearance of the genetic material associated with the lysing cells until the concentrations fell below the detection limits of either assay. No functional, released genetic units (genes) were detected within the supernatant during the lysis process, including nuclease expression.     

Enzyme-linked immunosorbent assay for detection of staphylococcal enterotoxins in foods.

An enzyme-linked immunosorbent assay (ELISA) was developed for detection of staphylococcal enterotoxins in foods. The "double-antibody sandwich" protocol combines parts of several procedures reported previously. Horseradish peroxidase was conjugated to antibody specific for an enterotoxin, and the antibody-enzyme conjugate was assayed with a 2,2'-azino-di-(3-ethylbenzthiazoline sulfonic acid)-H2O2 substrate solution. Enterotoxins were added to a variety of foods that were representative of those implicated in staphylococcal food poisoning outbreaks. Extracts of the foods were assayed by the ELISA and radioimmunoassay. Enterotoxin levels below 1 ng/g of food were consistently detectable by the ELISA. These results compared favorably with those of the radioimmunoassay. Experiments confirmed the interference of protein A in double-antibody sandwich ELISAs. Although protein A interference has not been demonstrated to be a problem in food extracts, we suggest a screen for protein A interference in which immunoglobulin G from nonimmunized rabbits is used. All of the known staphylococcal enterotoxins could be detected by this method. Analysis of a food product for entertoxin by the ELISA can be completed in an 8-h working day.     

Inactivated Eastern Equine Encephalomyelitis Vaccine Propagated in Rolling-Bottle Cultures of Chick Embryo Cells

A method was developed for the production of Eastern equine encephalomyelitis vaccine from virus grown in rolling-bottle cultures (840 cm(2) growth area) of chick embryo cells. The PE-6 strain of virus was propagated in chick embryo cell roller cultures maintained on serum-free medium 199 containing 0.25% human serum albumin and antibiotics (MM). A multiplicity of inoculum of 0.005 yielded acceptable titers of virus at a convenient harvest time of 18 to 24 hr and reduced the carry-over of extraneous material from the virus seed. Growth studies in which 100, 200, or 300 ml of MM was used showed that use of 300 ml of MM offered two advantages: (i) cytopathic effects were less at the 18- to 24-hr harvest time, thereby decreasing cellular material in the final product, and (ii) total virus yield was not substantially reduced, thus permitting large-scale production of virus for further processing. Studies on formalin inactivation at 37 C indicated that the virus was inactivated by 0.05% formalin within 12 to 16 hr and with 0.1% formalin within 6 to 8 hr. Antigen extinction tests in hamsters revealed excellent potency (e.g., median-effective-dose values of 0.069 to 0.012 ml) for both fluid and freeze-dried products. The advantages of the roller-bottle technique in vaccine production are discussed.     

Fibronectin binding to a Streptococcus pyogenes strain.

In previous studies, Staphylococcus aureus has been shown to bind fibronectin (P. Kuusela, Nature (London) 276:718-720, 1978), an interaction that may be important in bacterial attachment and opsonization. Recently some strains of streptococci of serological groups A, C, and G were also found to bind fibronectin. The binding to one selected strain of Streptococcus pyogenes has been characterized here. The binding of [125I]fibronectin to streptococcal cells resembles that to staphylococcal cells and was found to be time dependent, functionally irreversible, and specific in the sense that unlabeled proteins other than fibronectin did not block binding. Bacteria incubated with proteases largely lost their ability to bind fibronectin, and material released from the streptococci by a brief trypsin digestion contained active fibronectin receptors. This material inhibited the binding of [125I]fibronectin to the streptococci. The inhibitory activity was adsorbed on a column of fibronectin-Sepharose but not on a column of unsubstituted Sepharose 4B or egg albumin Sepharose. The receptor appeared to be a protein nature since the inhibitory activity of the trypsinate was destroyed by papain and was not absorbed on a column containing monoclonal antibodies directed against lipoteichoic acid bound to protein A-Sepharose. Binding sites in fibronectin for streptococci and staphylococci, respectively, were localized by analyzing the ability of isolated fragments to inhibit [125I]fibronectin binding to bacteria and by adsorbing 125I-labeled tryptic fragments with staphylococcal and streptococcal cells. Both species of bacteria appeared to preferentially bind a fragment (Mr = approximately 25,000) originating from the N-terminal region of the protein. In addition, streptococci also bound a slightly smaller fragment (Mr = approximately 23,000). Fibronectin receptors solubilized from either streptococci or staphylococci inhibited the binding of fibronectin to both species of bacteria.     

The destruction of methicillin by soil bacteria

Summary The stability in the soil of a new penicillin (methicillin) which is resistant to staphylococcal penicillinase, has been investigated. The results revealed its inactivation in both sterile and non-sterile soils of p _H 7.4–7.6, with indication of biological inactivation in the latter.Three strains identified as Pseudomonas spp., were isolated by enrichment technique from the soil, and were found able to inactivate methicillin through production of an exocellular enzyme destructable at 90°C. Such an enzyme proved to be a type of penicillinase that inactivated benzyl penicillin more actively than methicillin.