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.     

Mitogenicity of M5 protein extracted from Streptococcus pyogenes cells is due to streptococcal pyrogenic exotoxin C and mitogenic factor MF.

M proteins of Streptococcus pyogenes are virulence factors which impede phagocytosis, bind to many plasma proteins, and induce formation of cross-reactive autoimmune antibodies. Recently, it has been reported that some M proteins, extracted with pepsin from streptococci (pep M), are superantigens. One of these, pep M5, was investigated in detail and was shown to stimulate human T cells bearing V beta 2, V beta 4, and V beta 8. In the present study, we extracted and purified M5 protein by different biochemical methods from two M type 5 group A streptococcal strains. The crude extracts were fractionated by affinity chromatography and ion-exchange chromatography. All fractions were tested in parallel for M protein by immunoblotting and for T-cell-stimulating activity. Although several crude preparations of M5 protein were associated with mitogenicity for V beta 2 and V beta 8 T cells, the M5 proteins, irrespective of the extraction method, could be purified to the extent that they were no longer mitogenic. The mitogenic activity was not destroyed during the purification procedures but was found in fractions separated from M protein. In these fractions, streptococcal pyrogenic exotoxin C and mitogenic factor MF could be detected by protein blotting and enzyme-linked immunosorbent assay. Moreover, anti-M protein sera did not inhibit the mitogenic activity of crude extracts, but antisera which contained anti-streptococcal pyrogenic exotoxin C antibodies showed inhibition. The inability of M5 protein to stimulate T cells was confirmed with recombinant pep M5 produced in Escherichia coli. Our data strongly suggest that the mitogenic activity in M protein preparations is caused by traces of streptococcal superantigens different from M protein.     

Role of proteinase in the formation of inhibitory levels of hematin by group A streptococcus cultures on blood-containing media.

Group A streptococci were tested for proteinase production and for the possible relationship of this production to the generation of bacteriocinlike inhibitor activity. Of 126 strains tested, 83% were positive for proteinase, and a similar distribution was found among strains isolated in association with rheumatic fever (89%) and nephritis (94%) and from uncomplicated acute infections (78%). Although application of an inhibitor production (P) typing scheme demonstrated a variety of P types, all of the proteinase-positive strains produced inhibitory activity and over 65% of these strains were P type 204. It was shown that hematin was responsible for this P type 204 activity and that it was produced only by actively proteolytic strains when grown on a hemoglobin-containing medium. Conditions optimizing proteinase production (anaerobic incubation at 37 degrees C on a test medium prepared from Columbia agar base [GIBCO Laboratories, Grand Island, N.Y.]) increased P type 204 activity. Interference with proteinase activity either by growth of the cultures at an alkaline pH or by incorporation of sub-growth inhibitory concentrations of either iodoacetic acid or lincomycin into the medium prevented production of P type 204 activity. Whether significant conversion of hemoglobin to hematin occurs in vivo and the possible implications of this conversion with regard to the pathogenesis of group A streptococcal infections remain to be determined.     

Scarlet fever and types of erythrogenic toxins produced by the infecting streptococcal strains.

Group A streptococcal strains were isolated from the throats of 46 children suffering from scarlet fever. For detection of erythrogenic toxins (ETs), the culture supernatants were concentrated 100 times by ethanol precipitation and solubilisation in acetate buffer. ELISA was used to identify ETA and double immunodiffusion to identify ETB and ETC. The presence of the ETA gene was detected by a specific DNA probe. ETA (alone or in combination with ETB and/or ETC) was found in 51.9% of the strains, ETB (alone or in combination with ETA and/or ETC) in 76.9% and ETC (in combination with ETA and ETB) in 28.9%. Only 5.8% of strains did not produce any detectable ET. In SDS-PAGE, supernatants of ETB-producing strains showed a pronounced band in either the region of the proteinase zymogen or the active proteinase. There was no correlation between the type of erythrogenic toxin and the serological M or T type of the producing strain. The mitogenic potency of culture supernatants did not differ significantly irrespective of the toxin type(s) present. Culture supernatants of strains without a detectable amount of the known ETs were highly mitogenic, indicating the production of other streptococcal mitogens. A correlation with clinical symptoms was determined with regard to exanthema and fever. Strains producing two or three toxins caused a more intense exanthema. Patient temperature was higher (greater than or equal to 38 degrees C) when the infecting strain produced ETB. The toxin-producing patterns of the strains of this study were compared with those isolated during the last epidemic outbreak of scarlet fever in East Germany.     

"Fingerprinting" beta-haemolytic streptococci by their production of and sensitivity to bacteriocine-like inhibitors.

A scheme for the "fingerprinting" of streptococci according to their production of (P typing) and sensitivity to (S typing) bacteriocine-like inhibitory substances has been developed. P typing of 450 beta-haemolytic streptococci by their action on a set of nine standard indicator strains revealed that 80% of strains produced one or more detectable inhibitors, and that 17 different P types could be recognised. Production of some inhibitors seemed to be a property of strains of a particular serological group or type. Bacteriocine-like substances were produced by streptococci of serological groups, A, B, C, D, E, F and G. Nine strains were selected as standard producers for S typing. These strains differed in their spectra of inhibition, but all seemed to be active only against gram-positive bacteria. One producer, a group-F streptococcus, specifically inhibited group-A streptococci. The conditions of incubation were critical for demonstration of inhibitor production. A requirement for blood and for incubation at 32 degrees C were important factors. None of the inhibitors was induced by ultraviolet irradiation. The observed inhibitory effects were not attributable to either hydrogen peroxide or low pH, but to the production of a variety of substances having diverse physicochemical properties and production requirements. Most of the inhibitors do not seem to be produced in liquid media. The "fingerprinting" procedure is simple and inexpensive, and provides a reliable means of subdividing streptococcal strains that may find application as a supplement to the existing serological typing schemes.     

Standardization and evaluation of the CAMP reaction for the prompt, presumptive identification of Streptococcus agalactiae (Lancefield group B) in clinical material.

Primary cultures of clinical material were screened for the presence of colonies suspected of being Streptococcus agalactiae (Lancefield group B). Sixty-three such cultures and 108 other isolates of beta-hemolytic streptococci (groups A, C, and G), encountered during the first 3 months of the investigation, were studied by Lancefield grouping, sodium hippurate hydrolysis, and a standardized CAMP test. All streptococci were inoculated perpendicularly to streaks of a beta-toxin-producing staphylococcus on sheep blood agar plates and incubated aerobically in a candle jar and anaerobically at 37 C. Plates were examined after 5 to 6 and 18 h of incubation. The production of a distinct "arrowhead" of hemolysis was indicative of a positive CAMP reaction. All group B streptococci produced a positive CAMP reaction in the candle jar or anaerobically, usually within 5 to 6 h, and aerobically after 18 h of incubation. All group A streptococci produced a positive reaction only under anaerobic conditions. Groups C and G streptococci were negative under all atmospheres. The CAMP reaction is a prompt and reliable procedure for the presumptive identification of group B streptococci when a candle jar atmosphere is used during incubation.     

Virulence properties of erysipelas-associated group A streptococci

Group A streptococcal isolates (n=53) recovered from 38 erysipelas patients in 1988 and 1990 in Sweden were analysed with respect to serotype, erythrogenic toxin production and polymorphism in theemm gene region. Serotype determination showed a dominance of type T1M1 (28.6 % of the strains), but T type 8 was also prevalent (14.3 %). In the majority of the strains only a low production of erythrogenic toxin A was demonstrated, while both toxin B and C production were high. Polymorphism was detected in theemm gene region of T1M1 strains at a frequency of 64 %. These erysipelas associated group A streptococci were more heterogenic with respect to serotype distribution and polymorphism in theemm gene region compared to previously studied group A streptococci isolated during an outbreak of serious streptococcal infections in Sweden in 1988/1989. The material included isolates from two cases of recurrence, and typing of the isolates indicated that the patients had been infected by the same serotype as in the primary infection.     

Human fibroblast interferon in tears of patients with picornavirus epidemic conjunctivitis.

We report the levels of coxsackievirus type A24 (CA24) and the levels and type of interferon produced early during naturally acquired picornavirus epidemic conjunctivitis. Virus levels ranging from 10(1.8) to 10(5.8) 50% tissue culture infective doses per ml were detected in 29 of 37 acute (collected 1 to 4 days after onset of conjunctivitis) tear samples. Interferon (10(1.5) to 10(3.3) U/ml) was detected in 12 of 29 tear samples collected on day 1, in 2 of 6 tear samples collected on day 2, and in 1 tear sample collected on day 3 after onset of conjunctivitis. The interferon activity in pooled tear samples was completely neutralized by antiserum against human fibroblast interferon, stable at pH 2.0, and active against different viruses. In addition, the interferon activity in tears, like human fibroblast and leukocyte interferons produced in vitro, protected human and rabbit, but not mouse, cells. This is the first report of production and identification of the antigenic type of interferon induced by an enterovirus during natural infection. The early appearance of fibroblast interferon suggests that it may be an important host defense at the local site of implantation against this and possibly other enterovirus infections.     

Differential induction of helper T cell subsets during blood-stage Plasmodium chabaudi AS infection in resistant and susceptible mice.

The induction of T helper cell subsets during the course of non-lethal or lethal blood-stage Plasmodium chabaudi AS infection was investigated using inbred strains of mice which differ in the level of resistance to this intraerythrocytic parasite. Resistant C57Bl/6 mice experience a non-lethal course of infection characterized by moderate levels of both parasitaemia and anaemia and resolution of primary acute infection by 4 weeks, while susceptible A/J mice experience lethal infection with fulminant parasitaemia and severe anaemia. T helper subset function was assessed during infection by determining the kinetics of spleen cell production in vitro of the Th1-derived cytokine, interferon-gamma (IFN-gamma), and of the Th2-derived cytokine, IL-5, using sandwich ELISAs. Spleen cells from resistant C57Bl/6 mice were found to produce high levels of IFN-gamma within 1 week of infection in response to both the mitogen concanavalin A (Con A) and malaria antigen. Furthermore, CD4+ T cells were found to be the source of IFN-gamma while both CD4+ and CD8+ T cells were found to produce IL-5. Decreased IFN-gamma production after day 10 was concomitant with significant production of IL-5 between 2 and 3 weeks post infection. In contrast, spleen cells from susceptible A/J mice produced high levels of IL-5 within the first week of infection. In addition, these animals were found to have high serum levels of IL-5. These results, thus, confirm previous observations that resolution of primary blood-stage P. chabaudi infection occurs by sequential activation of Th1 CD4+ T cells followed by activation of the Th2 subset, and in addition, suggest that induction of a strong Th2 response early in infection may lead to a severe and lethal course of malaria.     

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.     

The gene encoding a new mitogenic factor in a Streptococcus pyogenes strain is distributed only in group A streptococci.

We recently cloned a gene encoding a new mitogenic factor (MF) from Streptococcus pyogenes NY-5. In the present study, we determined the distribution of this MF gene (mf) by PCR based upon its sequence. Of 371 streptococcal group A strains isolated from clinical specimens, 370 (99.7%) were positive for mf. The strain that was negative for the MF gene was also negative for the streptolysin O gene (slo). Some streptococcal strains belonging to groups C and G were negative for mf but positive for slo. Group B strains were negative for both. Furthermore, we examined the presence of mf in 54 strains belonging to 28 families and found mf only in group A streptococci. These results indicate that mf is distributed specifically in group A streptococci and the presence of mf in clinical samples strongly suggests infection with group A streptococci.     

Antigens of group A streptococci involved in passive hemagglutination reactions.

Antibodies to streptococcal extracellular products were detected in rabbit sera by passive hemagglutination tests as early as 1 week after intravenous injection of live group A streptococci (strain C203S). Using these antibodies in immunoadsorbent columns, we prepared an antigenic fraction from crude concentrates of streptococcal extracellular products. The specific activity of this fraction in sensitizing glutaraldehyde-treated erythrocytes for passive hemagglutination tests and in absorbing passive hemagglutination antibodies from immune sera was increased by 80-fold and at least 24-fold, respectively, as compared with crude extracellular products. The fraction has been found to contain at least three serologically active antigens, which are of considerable interest because: (i) they gave rise to early and consistent immune responses both in humans and in experimental animals; (ii) they appear so far to be produced only by beta-hemolytic streptococci; and (iii) antibodies to these antigens are present in high titers in patients with acute rheumatic fever. Our data suggest that passive hemagglutination antigens may be distinct from those extracellular enzymes and hemolysins ordinarily employed in streptococcal serological studies.     

Streptococcal growth and toxin production in vivo

Streptococcal growth in vivo was studied with inoculated micropore filter chambers which were implanted into the peritoneal cavities of mice. Eight of nine group A strains and one group B strain grew in vivo, achieving concentrations of 10(7) to 10(9) CFU/ml in the chambers. Experiments with the Richards strain showed that the number of viable organisms remained high at 5 and 8 weeks after infection. The use of specific inhibitors and appropriate toxin-negative strains demonstrated that both cytolytic toxins produced by group A streptococci, streptolysin S and streptolysin O, were present in the culture fluids harvested from the chambers. This finding represents the first evidence that streptolysin S is produced in vivo. The host response to streptococci growing in vivo was examined by following the increase in serum antistreptolysin O levels. The response was first detected 2 weeks after chamber implantation and appeared to be maximal after 5 weeks. In addition, the production of antibody to streptococcal cell surface antigens was demonstrated indirectly with fluorescein-labeled anti-mouse immunoglobulin G.     

Serum antibody response to Pseudomonas aeruginosa antigens during corneal infection.

Previous studies in our laboratory have indicated that naturally resistant, inbred DBA/2J mice mount a greater serum antibody response to Pseudomonas aeruginosa 19660 than susceptible C57BL/6J mice. However, the specificity of the antibody produced was not known. The present study examines the specificity and kinetics of the humoral response of these mouse strains to potential virulence factors produced by the organism during both a primary and a secondary corneal infection administered 4 weeks after the primary infection. Serum antibody levels specific for lipopolysaccharide (LPS), exotoxin A, phospholipase C (PLC), alkaline protease, elastase, and flagella were measured by enzyme-linked immunosorbent assay. Little or no antibody to either alkaline protease or elastase was detected during either primary or secondary infection. Immunoglobulin G (IgG) antibodies specific to exotoxin A, PLC, and flagella were detected 2 weeks after primary infection, and a rapid response to these antigens was measured 1 week after secondary infection. During primary infection, detectable LPS-specific antibody was only IgM, while IgG appeared only after secondary infection. The kinetics of the humoral response in susceptible C57BL/6J mice were similar to those in resistant DBA/2J mice, although the magnitude of the response varied according to the antigen tested. These results indicate that LPS, exotoxin A, PLC, and flagella are present or produced in amounts that are immunogenic during corneal infection by P. aeruginosa 19660 in the mouse strains tested.     

Quantification and toxicity of group A streptococcal pyrogenic exotoxins in an animal model of toxic shock syndrome-like illness.

Toxic shock-like syndrome isolates of group A streptococci were evaluated for production of pyrogenic exotoxins (also called SPEs, scarlet fever toxins, and erythrogenic toxins). The isolates were consecutively obtained during 1987 and 1988. Of these isolates, 23 of 26 made SPE type A, 10 of 26 made SPE B, and 8 of 26 made SPE C. SPE A was produced in significantly greater amounts than SPEs B and C (3.2 micrograms/ml of culture fluid compared with 0.7 and 0.6 microgram/ml, respectively). SPE A, administered in miniosmotic pumps implanted subcutaneously in rabbits, was significantly more toxic than SPE C; seven of eight rabbits succumbed after challenge with 150 or 300 micrograms of SPE A, compared with one of six after challenge with SPE C.     

C5a peptidase alters clearance and trafficking of group A streptococci by infected mice.

Group A streptococcal C5a peptidase (SCPA) specifically cleaves the human serum chemotaxin C5a at the polymorphonuclear leukocyte (PMNL) binding site. This study tested the proposal that SCPA contributes to virulence by retarding the influx of inflammatory cells and clearance of streptococci during the first few hours after infection. To investigate the specific contribution of SCPA to the virulence of group A streptococci, scpA insertion and deletion mutants were created by directed plasmid insertion into scpA and gene replacement. The precise locations of insertion and deletion mutations were confirmed by PCR and DNA sequence analysis. The impact of mutation on virulence was investigated with a mouse air sac model of inflammation. Experiments evaluated clearance of streptococci from the air sac within 4 h after infection. SCPA- streptococci were cleared more efficiently than wild-type bacteria. Localization of streptococci in lymph nodes and spleens of infected mice revealed a significant difference between mutant and wild-type streptococci. PMNLs and other granulocytes that infiltrated the air sac were quantitated by single-color flow cytometry. The total cellular infiltrate was greater and PMNLs dominated the granulocytic infiltrates of air sacs inoculated with SCPA- mutant bacteria. The data obtained are consistent with the possibility that SCPA- streptococci are initially cleared from the site of infection primarily by PMNLs. Moreover, mutant and wild-type streptococci followed different paths of dissemination. SCPA- bacteria were transported to lymph nodes, whereas wild-type streptococci avoided transport to the lymph nodes and rapidly spread to the spleen.     

Molecular analysis of pyrogenic exotoxins from Streptococcus pyogenes isolates associated with toxic shock-like syndrome.

Toxic shock-like syndrome (TSLS) is characterized by hypotension or shock, fever, multiorgan system involvement, and a concurrent group A streptococcal infection. We analyzed 34 streptococcal strains isolated from patients with clinically well-documented TSLS for their pyrogenic toxin profiles and M-protein types. Although strains of nine different M types were represented in the sample, 74% of the isolates were of either M type 1 or 3. It was determined that 53% produced streptococcal pyrogenic exotoxin type A under in vitro growth conditions and that 85% contained the gene encoding this toxin. These values are in contrast to the published value of 15% for the incidence of this gene in a sample of general group A streptococcal isolates. As has been found with all group A streptococci examined to date, regardless of disease association, 100% of TSLS-associated isolates contained the gene encoding pyrogenic exotoxin type B. This toxin was detectably produced by 59% of isolates. The gene encoding pyrogenic toxin type C was found in only 21% of isolates. We conclude that the pyrogenic exotoxin type A gene is associated with group A streptococcal strains isolated from patients with TSLS and may play a causative role in this illness. However, other factors are also likely to be important, since not all strains from patients with TSLS contained the A toxin gene.     

Demonstration of a B-lymphocyte mitogen produced by the Lyme disease pathogen, Borrelia burgdorferi.

Lyme disease refers to the multisymptomatic illness in humans which results from infection with the tick-borne spirochete Borrelia burgdorferi. The white-footed mouse is the major reservoir for B. burgdorferi and, upon infection, certain inbred mice develop symptoms similar to those reported in human disease. Sonicated preparations of washed spirochetes were found to have potent mitogenic activity when cultured with lymphocytes from naive C57BL/6, C3H/HeJ, or BALB/c mice. The activity of the B. burgdorferi sonicate was approximately fourfold greater than that of a similarly prepared Escherichia coli sonicate. Polymyxin B efficiently inhibited the mitogenic activity of the E. coli sonicate but only slightly inhibited that of the B. burgdorferi sonicate, suggesting that a lipid A-containing lipopolysaccharide was not responsible for the B. burgdorferi activity. Kinetic analysis indicated peak proliferation at 2 to 3 days of culturing, suggesting polyclonal activation. B- and T-lymphocyte depletion experiments indicated that the major cell type responding to the B. burgdorferi mitogen was the B lymphocyte. This mitogen stimulated murine B cells not only to proliferate but also to differentiate into antibody-secreting cells, as demonstrated by the production of immunoglobulin by stimulated splenocytes. Furthermore, the sonicated preparation stimulated the B-cell tumor line CH12.LX to secrete immunoglobulin in the absence of accessory cells. B. burgdorferi also stimulated interleukin-6 production in splenocyte cultures. The observation that B. burgdorferi can stimulate activation of and immunoglobulin production by normal B lymphocytes may directly reflect on the development of arthritis associated with persistent infection by this organism.