Werner Reichardt

Inactivation of Streptococcus pyogenes extracellular cysteine protease significantly decreases mouse lethality of serotype M3 and M49 strains.

Cysteine proteases have been implicated as important virulence factors in a wide range of prokaryotic and eukaryotic pathogens, but little direct evidence has been presented to support this notion. Virtually all strains of the human bacterial pathogen Streptococcus pyogenes express a highly conserved extracellular cysteine protease known as streptococcal pyrogenic exotoxin B (SpeB). Two sets of isogenic strains deficient in SpeB cysteine protease activity were constructed by integrational mutagenesis using nonreplicating recombinant plasmids containing a truncated segment of the speB gene. Immunoblot analyses and enzyme assays confirmed that the mutant derivatives were deficient in expression of enzymatically active SpeB cysteine protease. To test the hypothesis that the cysteine protease participates in host mortality, we assessed the ability of serotype M3 and M49 wild-type strains and isogenic protease-negative mutants to cause death in outbred mice after intraperitoneal inoculation. Compared to wild-type parental organisms, the serotype M3 speB mutant lost virtually all ability to cause mouse death (P < 0.00001), and similarly, the virulence of the M49 mutant was detrimentally altered (P < 0.005). The data unambiguously demonstrate that the streptococcal enzyme is a virulence factor, and thereby provide additional evidence that microbial cysteine proteases are critical in host-pathogen interactions.

Separation of mitogenic and pyrogenic activities from so-called erythrogenic toxin type B (Streptococcal Proteinase)*

It is well-established that three types of erythrogenic toxins (ETA, ETB, ETC) are produced by Streptococcus pyogenes (group A streptococci) strains. Culture filtrate concentrates from Streptococcus pyogenes strains T19P (T19, ETA+, ETB+, ETC-), 27337 (T12, B3264, ETA-, ETB+, ETC+), 27252 (T4, ETA-, ETB+, ETC+) and 27195 (T8, ETA-, ETB+, ETC-) were analyzed by preparative isoelectric focusing. These concentrates and the purified erythrogenic toxin type B (ETB) isolated by ion exchange chromatography had mitogenic and pyrogenic activity. Now, it has been found that the mitogenic activity and the pyrogenic activity of this ETB can be separated by preparative isoelectric focusing in Sephadex gels. This means that ETB is not a superantigen as described in literature. The mitogenic and biological activity is caused by traces of ETA (strain T19P), ETC (strains 27252 and 27337) and/or by unknown mitogen(s) (MX, strain 27195) which preferentially stimulate V beta 8+ T cells. The differentiation between ETA (stimulating V beta 12+ but not V beta 8+ or V beta 2+), ETC (stimulating V beta 2+ but not V beta 8+), and MX (stimulating V beta 8+) was done using established leukemic cell lines.

Purification and characterization of hyaluronidase from streptococcus agalactiae

Hyaluronidase from two different strains of Streptococcus agalactiae was purified and characterized. The purification was performed successively by chromatography and rechromatography on phenylsepharose, gel filtration with FPLC on Superdex G 200 and isoelectric focusing. The purified hyaluronidase had an isoelectric point of 8.75 and a molecular weight of approximately 116,000 D. It showed maximal enzyme activity at pH 6.30 and 40 degrees C. The Michaelis constant was estimated to be 8.17 x 10(-2) mg/ml. Hyaluronidase was stimulated only by Mg++ and inhibited by Zn++, Al , Cu++ and Fe++ at a final concentration of 10 mmol/l, respectively. The enzyme splitted hyaluronic acid and in low amounts dermatan sulphate and chondroitin sulphate A. Additionally, synthetic polyanions (like polymers of gentisic acid with formaldehyde and hydroxy sulphonic acid with formaldehyde) turned out to be also potent inhibitors of the enzyme.

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.

Mapping of Binding Sites for Human Serum Albumin and Fibrinogen on the M3-Protein

The emm3-gene and the emm78-gene encoding the M-protein type 3 (OF-) resp. type 78 (OF+) of Streptococcus pyogenes were completely sequenced (AC-No.: X80168) and the recombinant proteins expressed in E. coli. Concerning the type 3 the cell surface antigen as well as the isolated protein revealed a pronounced binding affinity towards the human plasma proteins human serum albumin (HSA) and fibrinogen (FBG). In comparison type 78 showed a reduced HSA-binding affinity. Several lines of evidence have led to the assumption that plasma protein binding is a general phenomenon and plays a significant role for survival and even expansion of pathogenic streptococcal strains having successfully invaded human patients (2).

Kinetics of growth and product formation in cultures from streptococci of groups a and c

During growth of streptococci of Lancefield groups A and C in a culture medium containing glucose, yeast extract and peptone, two main growth phases occur: growth phase I and growth phase II (diauxic growth). They are separated by a short stationary phase (1st stationary phase). The diauxic growth is caused by transient limitations as well as the availability of new sources of the amino acids L-serine and L-arginine. Growth phase I consists of an exponential and a nearly linear part. These growth kinetics are reflected by the kinetics of gas metabolism as well as by product formation. Hyaluronic acid is formed during the nearly linear phase whereas the enzyme alkaline phosphatase, is exclusively excreted in the 1st stationary phase. Also carbon dioxide and L-lactate are mainly produced in a growth phase-dependent mode. In the late stationary phase (2nd stationary phase) more oxygen is consumed whereas the demand for oxygen in the 1st stationary phase is nearly zero.

Virulence of Streptococcus pyogenes for chicken embryos after isogenic inactivation of different streptococcal pathogenicity factors.

M protein of Streptococcus pyogenes has been postulated as the major virulence factor of group A streptococci (GAS) based on its association with a phagocytosis-resistant phenotype (4). The antiphagocytic properties against human granulocytes of M proteins have been demonstrated by using defined isogeneic mutants lacking expression of M protein (1, 6). However, other proteins M-related proteins like Mrp, have also been described to contribute to streptococcal resistance against phagocytic attack (7). The genes encoding the proteins of the M-family are located in the vir-regulon and were regulated by the multigene regulator gene mga (3). But, hyaluronic acid capsule (HA), chemically completely different from streptococcal M proteins, also interferes in the phagocytic defense against streptococci (10) and it could be shown that both, M protein and HA, contribute to the virulence of one streptococcal strain (2, 9). In this study we investigated whether the chicken embryo model is worth to measure changes in virulence of streptococcal strains after isogeneic inactivation of genes encoding different M- and M-like proteins but also of genes encoding additional potential pathogenicity factors like streptococcal proteinase (SPEB), or the oligopeptide permease oppD.

Cases of streptococcal toxic shock syndrome in Germany since 1989, toxin (mitogen) expression, content of toxin genes and relation to M-serotypes.

A worldwide increase of severe invasive Streptococcus pyogenes infections beginning from the skin or soft tissue infections (cellulitis, fasciitis, myositis) has been reported during the last decade. This so-called Streptococcal Toxic Shock Syndrome (STSS) is considered as a resurgence of the historically known fulminant or toxic scarlet fever. It is characterized by fever, multiorgan failure, skin desquamation and occurs mainly in young/middle-aged, otherwise healthy adults within hours of the onset of symptoms. The onset of STSS seems to be related to the efficacy of bacterial products and host factors (particularly the immune state) of the patients. Bacterial products which are believed to be causally involved in these infections are the M-protein, different enzymes and the various erythrogenic toxins (ET or SPE), the streptococcal mitogenicity factor MF, the streptococcal superantigen (SSA), low molecular weight proteins (LMP) and the cell bound streptococcal membrane protein (CAP) (1). Our interest in this investigation has been focused on the examination of STSS strains isolated in Germany since 1989, when the first case was reported. More than 70 S. pyogenes strains isolated from cases of STSS in Germany and a group of control strains isolated from healthy people or from patients suffering from harmless streptococcal diseases were typed and investigated with regard to content of speA, speB, speC, and MF genes, toxin expression (ETA and ETC) and for mitogenic activity in culture supernatants. The results of this investigation are presented and discussed.

Group A Streptococcal M Protein Binds to Several Human Cell Types but not via MHC Class II Molecules

M protein is known as the major virulence factor of group A streptococci. Only a few years ago, M protein has been reported to be a superantigen like the streptococcal erythrogenic toxins ETA and ETC (3). Superantigens mediate the stimulation of T cells by antigen presenting cells via MHC class II molecules and the Vs T cell receptor. The aim of our contribution was to examine by immunoelectron microscopy the binding of M protein and M protein fragments to several human cell types including peripheral blood cells. Furthermore, some well characterized lymphoma cell lines were used to study whether M protein binds to MHC class II molecules.

Erythrogenic toxin type A (ETA): Epidemiological analysis of gene distribution and protein formation in clinical Streptococcus pyogenes strains causing scarlet fever and the streptococcal toxic shock-like syndrome (TSLS)

Erythrogenic toxin type A (ETA) is assumed to play a causative role in both scarlet fever and the streptococcal toxic shock-like syndrome (TSLS). For a molecular epidemiological analysis of the gene of erythrogenic toxin type A (speA) we used altogether 497 clinical isolates of Streptococcus pyogenes belonging to three groups: a) isolates from patients with scarlet fever, b) isolates from cases with TSLS, c) isolates from patients with other streptococcal infections (like otitis media, tonsillitis, impetigo) (general group). We found that less than 50% of the scarlet fever-associated strains possessed the speA gene as compared to 25% of the general group. Only 5 to 30% of these strains secreted the toxin under experimental conditions in very low quantities. Among strains isolated from TSLS, 67% appeared to contain the speA gene. The amount of ETA secreted into the medium was also extremely low. Southern hybridization patterns proved to be the same with an speA-specific probe in all three groups of streptococcal isolates (HaeIII, HindIII). Increased occurrence of the speA gene among scarlet fever and TSLS-associated strains does not seem to be sufficient to support the hypothesis that ETA may have a causative role in both diseases since a considerable number of strains in these groups did not possess the speA gene.