Tissue cages for study of experimental streptococcal infection in rabbits. I. Production of erythrogenic toxins in vivo

Tissue cages implanted subcutaneously were used to infect rabbits with erythrogenic toxin (ET) producing streptococci. The in-vivo production of ET was followed during the infection by immunoprecipitation analyses of the tissue cage fluid (TCF). ET types A and C were mainly detected during the first week of infection, but, as late as 4 weeks after the inoculation, ET was occasionally found in TCF. The nonspecific mitogenic activity of ET on human lymphocytes was also used as a biological marker to recognize ET in TCF. Mitogenic activity was detected in 90% of samples during the first week. In order to characterize the mitogenic material released by growing streptococci, TCF was electrofocused in polyacrylamide gel. The eluates of sliced gels were checked for mitogenic activity and compared with a purified ET preparation containing ET types A and C. It could be verified that ET type A was produced under in-vivo conditions by strains NY-5 and SF130, while ET type C was produced by strain T18. Differences between production of toxins in vitro and in vivo might be of significance for the understanding of the pathogenetic mechanisms in streptococcal infection.     

Tissue cages for study of experimental streptococcal infection in rabbits. II. Humoral and cell-mediated immune response to erythrogenic toxins

Infection of rabbits with erythrogenic toxin producing streptococcal strains caused a marked increase of humoral antibodies, which was detected by immunoprecipitation and ELISA. An antibody response directed towards the erythrogenic toxin type A was demonstrated by fused rocket immunoelectrophoresis. All toxinogenic reference strains produced ET type A under in vivo conditions despite that this toxin was not always demonstrated under in vitro conditions. The infection resulted in an increase of mitogenic response of peripheral lymphocytes to the initial nonspecific mitogenic erythrogenic toxins, whereas the Con A stimulation was depressed starting 14 days after infection and lasting during a period of 90 days. Since a normal antibody response was evoked, it seems likely that the T helper cell function was not affected.     

Antigenic similarity of toxins produced by Clostridium botulinum type C and D strains.

Antisera against purified type C1 toxin of Clostridium botulinum and its heavy-chain component cross-neutralized type D toxin. Antisera against partially purified type D toxin cross-neutralized type C1 toxin. From the latter serum, a component which neutralized only type D toxin and a component which equally neutralized both C1 and D toxins were obtained. We concluded that the cross-neutralization was not due to the fact that type C and D strains produce both C1 and D toxins but rather to the fact that the toxins have an antigen(s) common to their molecules. The results of the agar gel-double-diffusion test also supported this conclusion.     

Detection of toxins produced by vibrio fluvialis.

The results of studies with cell-free extracts and culture supernatant fluids of Vibrio fluvialis (a recently recognized, potential enteric pathogen for humans) grown in the absence and presence of lincomycin indicated that the bacterium could produce (i) a factor which causes CHO cell elongation (CEF) similar to that elicited by V. cholerae enterotoxin and by the heat-labile enterotoxin of Escherichia coli, (ii) cytolysin(s) active against erythrocytes, (iii) nonhemolytic, CHO cell-killing factor(s), and (iv) protease(s) active against azocasein. The CEF was heat labile and ammonium sulfate precipitable, and it had an isoelectric point (estimated by sucrose density gradient electrofocusing) and molecular weight (estimated by gel filtration) of about 5.1 and 135,000, respectively.     

Temporal production of streptococcal erythrogenic toxin B (streptococcal cysteine proteinase) in response to nutrient depletion.

The effects of various growth conditions on the production of streptococcal erythrogenic toxin B (streptococcal pyrogenic exotoxin B [SPE B]) by Streptococcus pyogenes were analyzed. SPE B was detected in broth culture supernatant fluid only during the stationary phase of growth when glucose and other potential carbon sources were depleted from the medium. Additionally, SPE B production was inhibited when the concentration of glucose in the medium was maintained. These results suggest that SPE B is secreted under conditions of starvation and may be involved in nutrient acquisition.     

Molecular epidemiologic analysis of the type A streptococcal exotoxin (erythrogenic toxin) gene (speA) in clinical Streptococcus pyogenes strains.

A molecular epidemiology analysis was performed with over 440 clinical isolates of Streptococcus pyogenes obtained from 11 different countries in order to determine the frequency of occurrence of the type A streptococcal exotoxin (erythrogenic toxin) gene (speA) among group A strains. The colony hybridization technique employing a specific internal fragment of the speA gene was used for initial screening, and all positive results were further confirmed by the Southern hybridization technique. Among over 300 general strains obtained from patients with a variety of diseases, except scarlet fever (such as tonsillitis, impetigo, cellulitis, pyoderma, abscess, rheumatic fever, and glomerulonephritis), 15% were found to contain the speA gene. Among a group of 146 strains obtained from individuals described as having scarlet fever, 45% were shown to contain the speA gene. Further analysis of the data indicated that strains with certain M- or T-type surface antigens showed a higher (such as M and T types 1 and 3/13) or lower (such as M2, M12, T4, T5, and T28) tendency to contain the speA gene. No correlation was found between speA content of a strain and the ability to cause a specific disease, although strains possessing the speA gene were more likely to be associated with scarlet fever and rheumatic fever than with other types of disease.     

Cross-neutralization of staphylococcal and streptococcal pyrogenic toxins by monoclonal and polyclonal antibodies.

We evaluated cross-reactivity of antibodies against staphylococcal and streptococcal pyrogenic toxins. Monoclonal antibodies against staphylococcal enterotoxin (ET) C1 and streptococcal pyrogenic exotoxin (SPE) A were tested for reactivity with homologous and heterologous pyrogenic toxins in vitro. Ten immunoglobulin G1 anti-ET C1 monoclonal antibodies showed little or no cross-reactivity in an enzyme-linked immunosorbent assay, but many of these could neutralize the mitogenic effect of ET B, SPE A, or both. Two immunoglobulin M anti-ET C1 monoclonal antibodies and eight immunoglobulin M anti-SPE A monoclonal antibodies showed extensive cross-reactivity in the enzyme-linked immunosorbent assay and the mitogenicity neutralization assay. No cross-reactivity was observed with SPE C or toxic shock syndrome toxin 1. Rabbits immunized against ET B, ET C1, or SPE A were resistant to challenge with the immunizing toxin. In addition, reciprocal immunity was stimulated by the two ETs, and immunity to SPE A provided protection against ET B but not ET C1. These results show that staphylococcal and streptococcal pyrogenic toxins which share sequence homology have common antigenic determinants which may not be detected in Ouchterlony immunodiffusion assays.     

Comparison of two toxins produced by Clostridium difficile.

Clostridium difficile was shown to produce a toxin which could be biochemically separated from the previously described cytotoxin of the same organism. The two proteins differ in biological activity and physical properties. Antiserum prepared to the second toxin does not neutralize the biological activity of the cytotoxin, and immunological cross-reactivity could not be demonstrated. However, some relationship may exist between the two toxins, since the newly described toxin degrades on polyacrylamide electrophoresis into two molecules, one of which appears to migrate with the band of purified cytotoxin. We suggest that this newly described toxin be designated toxin A until its primary biological activity and physical relationship to cytotoxin is determined. This toxin is active in biological assays of enteric disease and may play an important role in C. difficile-induced colitis.     

Inactivation of the streptococcal erythrogenic toxin B gene (speB) in Streptococcus pyogenes.

Streptococcal proteinase precursor (SPP) is a zymogen secreted by Streptococcus pyogenes that becomes activated to a cysteine proteinase. SPP has been shown to be immunologically identical to streptococcal erythrogenic toxin B (SPE B), and sequence comparison has shown a high degree of homology between the two proteins. In this study, we have constructed a speB mutant strain of S. pyogenes by insertional inactivation. An internal fragment of the cloned speB gene in plasmid pCR1000 was replaced with an erythromycin resistance determinant, and the recombinant plasmid was introduced into strain NZ131 by electrotransformation. Following the selection of erythromycin-resistant clones, Southern hybridization experiments confirmed the presence of the recombinant plasmid containing the erm gene in the chromosome of the resistant strains. Analysis of extracellular proteins produced by the wild-type and speB mutant strains by Ouchterlony immunodiffusion and isoelectric focusing revealed the presence of SPE B in the wild-type strain but not the speB mutant. Additionally, SPP, which has an isoelectric focusing pattern similar to that of SPE B and reacts with SPE B antiserum, was not detected among the extracellular proteins of the speB mutant strain. Proteinase activity as assayed by two different methods was present in the extracellular proteins produced by the wild-type strain, but the speB mutant strain had no extracellular proteinase activity. The mutant strain had a growth rate similar to that of the wild-type strain and produced normal levels of other extracellular products, suggesting that proteinase was not essential for viability as previously suggested. Our data are consistent with the view that a single gene (speB) produces a single protein that has been identified and/or assayed as either SPE B or SPP.     

Immunobiological relationships among new cholera toxins produced by CT gene-negative strains of Vibrio cholerae O1

The enterotoxic activities of partially purified new cholera toxins (NCT) prepared from the CT gene-negative strains of Vibrio cholerae serotype O1 isolated from cases of diarrhoea in man and from diverse environmental sources were assayed in the rabbit ileal-loop model and the toxin unit was calculated in micrograms of protein content. The secretory activities of one unit of homologous and heterologous enterotoxins were completely neutralised by 2.5 units of crude antiserum raised against one NCT preparation. In immunodiffusion tests, the NCT preparations from all strains tested gave precipitin bands against one antiserum showing reaction of complete identity. Thus, the present study clearly demonstrated that NCTs elaborated by CT gene-negative strains of V. cholerae O1 are immunobiologically similar.     

Immunologic cross-reactivity of type A streptococcal exotoxin (erythrogenic toxin) and staphylococcal enterotoxins B and C1.

Immunologic cross-reactivity between Streptococcus pyogenes type A exotoxin (erythrogenic toxin) and Staphylococcus aureus enterotoxins B and C1 was demonstrated by Ouchterlony double diffusion, Western immunoblot, and immunodot analyses. Specific antiserum to type A streptococcal exotoxin reacted more strongly with staphylococcal enterotoxin B than with enterotoxin C1. The reactivity of type A streptococcal exotoxin with antiserum to staphylococcal enterotoxin B was greater than that of antiserum to enterotoxin C1. These results suggest that a conserved domain is present in the three exotoxins, which most likely originated from a common evolutionary ancestor.     

Possible receptor for exfoliative toxins produced by Staphylococcus hyicus and Staphylococcus aureus.

Exfoliative toxin produced by Staphylococcus hyicus bound to the GM4-like glycolipid extracted from the skin of 1-day-old chickens but did not bind to glycolipid from adult chickens or suckling mice. Exfoliative toxin produced by Staphylococcus aureus bound to the GM4-like glycolipid extracted from the skin of suckling mice but not to glycolipid from 1-day-old or adult chickens. S. hyicus and S. aureus exfoliative toxins lost their toxicity by preincubation with GM4-like glycolipid from 1-day-old chickens and suckling mice, respectively.     

Production of Apx toxins by field strains of Actinobacillus pleuropneumoniae and Actinobacillus suis.

The three Apx toxins of Actinobacillus pleuropneumoniae have potential value for use in vaccines and diagnostic tests which will be species specific instead of serotype specific, provided that the Apx toxins are species specific and all field strains produce these toxins. We examined 114 A. pleuropneumoniae field strains and found that they secreted either ApxI, ApxII, ApxI and ApxII, or ApxII and ApxIII and secreted no other cytolytic activities. However, proteins similar to ApxI and ApxII were also produced by Actinobacillus suis.     

Comparative study of cytokine release by human peripheral blood mononuclear cells stimulated with Streptococcus pyogenes superantigenic erythrogenic toxins, heat-killed streptococci, and lipopolysaccharide.

The differences between toxic or septic shocks in humans during infections by streptococci and gram-negative bacteria remain to be fully characterized. For this purpose, a quantitative study of the cytokine-inducing capacity of Streptococcus pyogenes erythrogenic (pyrogenic) exotoxins (ETs) A and C, heat-killed S. pyogenes bacteria, and Neisseria meningitidis endotoxin (lipopolysaccharide [LPS]) on human peripheral blood mononuclear cells (PBMC) and monocytes has been undertaken. The levels of interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-6, IL-8, tumor necrosis factor alpha (TNF-alpha), and TNF-beta induced by these bacterial products and bacteria were determined by using cell supernatants. The capacity of ETs to elicit the monocyte-derived cytokines IL-1 alpha, IL-1 beta, IL-6, and TNF-alpha was found to depend on the presence of T lymphocytes, because of the failure of purified monocytes to produce significant amounts of these cytokines in response to ETs. PMBC elicited large amounts of these cytokines, as well as IL-8 and TNF-beta, with an optimal release after 48 to 96 h. The most abundant cytokine produced in response to ETA was IL-8. In contrast to the superantigens ETA and ETC, LPS and heat-killed streptococci stimulated the production of significant amounts of IL-1 alpha, IL-1 beta, IL-6, and TNF-alpha, with optimal production after 24 to 48 h in monocytes, indicating no significant involvement of T cells in the process. ETs, but neither LPS nor streptococci, were potent inducers of TNF-beta in PBMC. This study outlines the differences in the pathophysiological features of shock evoked by endotoxins and superantigens during infection by gram-negative bacteria and group A streptococci, respectively. The production of TNF-alpha was a common pathway for LPS, streptococcal cells, and ETs, although cell requirements and kinetics of cytokine release were different.     

Comparative study of hemolytic substances produced by coagulase-negative Staphylococcus strains.

Hemolytic substances H7, H62, and E56, produced by Staphylococcus haemolyticus 7 and 62 and S. epidermidis 56, respectively, were purified. H7 and H62 are probably similar on the basis of their isoelectric focusing profiles in 8 M urea and complete immunological identity as revealed by immunodiffusion with rabbit anti-H7 and anti-E56 sera. For E56, we observed seven bands instead of three in isoelectric focusing and only partial immunological identity with H7 and H62. However, H7 and E56 were similar with regard to the following characteristics: hemolytic spectra against different erythrocytes, kinetics of erythrocyte lysis, heat stability, and inhibition by phosphatidylcholine. E56 was not active at a temperature lower than or equal to 25 degrees C, and its activity increased more rapidly with increased temperature compared with H7. For both substances, the complexes obtained by molecular filtration on Ultrogel AcA54 and the purified peptides by reverse-phase high-pressure liquid chromatography showed some hemolytic activity. These results suggest that a particular association or the presence of a given peptide could enhance the activity.