Elisabeth Günther

Expression of both M protein and hyaluronic acid capsule by group A streptococcal strains results in a high virulence for chicken embryos

The human pathogenic microorganismStreptococcus pyogenes can resist against phagocytic attack of human granulocytes. Streptococcal M protein and hyaluronic acid were identified as virulence factors involved in this protection. So far, no experiments have been reported which describe the contribution of both components together in one system. We used the chicken embryo as an in vivo phagocytosis model to investigate the role of both components on the virulence of streptococci. For this, isogeneic mutants of group A streptococcal strains (GAS) which lack hyaluronic acid capsule (cap−) or M protein (M−) expression were used for infection and their virulence was compared with laboratory strains which had lost their ability to produce one or both virulence factors after long-time laboratory passages on blood agar. The experiments revealed that strains producing both M protein and hyaluronic capsule were higly, virulent. Only 1–10 colonyforming units were enough to cause a 50% lethality of 12-day-old chicken embryos. Those strains lacking one of these components showed a significant decrease in virulence. Finally, strains which failed to express either hyaluronic acid or M protein showed an additional tenfold decrease in virulence. This indicates a partial contribution of both M protein and hyaluronic acid to the virulence of GAS in the chicken embryo.

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.

Occurrence of extracellular hyaluronic acid and hyaluronatlyase in streptococci of groups A, B, C, and G.

Streptococci of serological groups A (GAS), B (GBS), C (GCS) and G (GGS) were examined in vitro using an optimized medium in respect of their ability to produce hyaluronic acid (HA) and hyaluronatlyase (HY). In this study, 614 GAS (including 123 streptococcal toxic shock syndrome strains, STSS), 247 GBS, 225 GCS and 143 GGS were investigated in qualitative and quantitative tests. Only 4% of GAS and 2.7% of GCS were able to express HA. In contrast to GAS, isolates of GCS showed a highly specific HA formation (to 1 g HA/g dry biomass). In all strains of GBS and GGS, not even a single isolate was positive for HA. HY expression was detectable in all four serological groups. In GAS, only 12.5% of strains were positive; the most common types being 22 and 4, whereas in GBS, GCS and GGS, 72.1%, 84% and 85.3% of isolates, respectively, could be reported as positive. The data suggest that the HA capsule only plays a secondary role in infections caused by GAS strains pathogenic for humans.

Streptococcal Pyrogenic Exotoxin A, Streptolysin O, Exoenzymes, Serotype and Biotype Profiles of Streptococcus pyogenes Isolates from Patients with Toxic Shock Syndrome and other Severe Infections

The determination of protein M and T serotypes, biotypes and pyrogenic (erythrogenic) exotoxin A (SPE A), streptolysin O (SLO), streptokinase (SK), hyaluronidase (HA) and cysteine proteinase release by 212 S. pyogenes isolates from patients with severe invasive group A streptococcal (GAS) infections, among them 74 cases of streptococcal toxic shock syndrome (STSS) has been investigated. M1 or M3 serotypes were expressed by 25% of the isolates (53/212), whereas 59% (125/212) belonged to 15 other different serotypes and 16% (34/212) were untypeable. Of the 74 isolates from STSS patients, 42% (31/74) expressed M1 and to a lesser extent M3 serotypes versus 19% of the non STSS isolates (26/138). Among the ten different biotypes known, biotypes 1 and 3 were prevalent, particularly the former in the case of STSS isolates. SPE A was detectably produced by about 25% (54/212) of the strains. However, as high as 40.5% of the STSS isolates (30/74) versus 17.4% of non STSS isolates (24/138) released SPE A. Moreover, 67% of the SPE A producing strains were of serotype M1 or M3. SK and HA were released by 71% and 10% of the isolates respectively. All strains released SLO (4 to 256 HU/ml) and 85% cysteine proteinase. No relationship between toxin or enzyme titer and the type of disease or clinical origin of the strains was found. Culture supernatants of all isolates showed moderate to high lymphocyte transforming activity with index values ranging from 14.5 to 50.3 including those strains which did not release detectable amounts of SPE A suggesting that SPE C and other mitogenic factor(s) are released by the isolates investigated.

Isolation and characterization of a mitogen characteristic of group A streptococci (Streptococcus pyogenes)

It has been supposed for many years that group A streptococci may elaborate more than the three well known erythrogenic toxins A, B or C (ETA, ETB, ETC). The analysis of the culture supernatant of streptococcal strain 27297 carrying neither genes for ETA nor ETC revealed mitogenic activity at pH 7.3 in isoelectric focusing. This mitogen of strain 27297 was purified by hydrophobic adsorption to Phenyl-Sepharose following FPLC chromatography on a Mono S column resulting in two proteins with mitogenic activity called AX and BX, respectively. Both differed in only one aminoterminal residue. The mitogenic activity of BX lacking one aminoterminal arginine was found to be about 100 times higher than that of AX. The aminoterminus of BX does not correspond to a predictable cleavage site for signal peptidase. We assume that BX was produced after translation by cleavage of the mature protein or the AX molecule with streptococcal proteinase (ETB) or an arginylaminopeptidase which is detectable on whole cells. The purified proteins BX and AX showed molecular weights of about 27 kDa in SDS electrophoresis and isoelectric points of 8.3 (AX) and 7.3 (BX) in isoelectric focusing, respectively. Both proteins were produced by practically all group A strains tested but not by groups B, C, G or H streptococci. Therefore, AX or BX seem to be proteins characteristic of group A streptococci.

JM9 strains, a new type of group B Streptococci from Japan

JM9 strains isolated from human carriers and patients in several districts of Japan, represent a new serotype of group B streptococci (GBS, Streptococcus agalactiae). They were first detected in 1983 but are meanwhile prevailing among all other GBS serotypes in Japan. Outside of Japan, strains of this type have not been reported until now. In the present work, N-acetylneuraminic acid was detected in all strains investigated, by chemical analysis as well as by interaction with a sialic acid-binding lectin. This component is characteristic of all analyzed GBS type polysaccharides. In a chicken embryo model, all strains exhibited a very strong virulence. Examination of the antibiotic sensitivity revealed that all strains were susceptible to penicillin, cephalothin, clindamycin, mezlocillin, azlocillin, erythromycin, methicillin, chloramphenicol, tetracyclin, oxacillin and sulfamethoxazole/trimethoprim, whereas all strains were resistant against gentamicin, kanamycin and neomycin. Electron microscopic studies revealed for these strains relatively small capsules but unusually thick cell walls. By immunogold labelling, the type polysaccharide, the group polysaccharide, the lipoteichoic acid and in some strains, the protein R were localized.

Febrile lady with acute renal failure and desquamating erythema

A 63-year-old woman developed acute renal failure and streptococcal toxic shock syndrome caused by streptococcus group G. Initially, an erythema resembling vasculitis was misleading. The subsequent clinical course, however, was typical for streptococcal toxic shock syndrome and met the criteria put forward by The Working Group on Severe Streptococcal Infections. In patients infected with streptococcus group G, toxic shock syndrome is rare. The streptococcus group G strains isolated from this patient did not produce pyrogenic exotoxins. Instead they produced an M-like protein related to group C and G streptococci that do not act as superantigens.

Kinetics and Regulation of Erythrogenic Toxins Type A and C During Growth of Streptococcus Pyogenes

The production of erythrogenic toxins type A (ETA) and C (ETC) is described as a function of growth kinetics. Group A streptococcal strains C 203 S and NY 5 were cultivated in yeast-peptone extract, Todd-Hewitt medium and a synthetic medium. Two main growth phases occurred during growth: a first logarithmic phase and a second linear phase. These phases were separated by a short stationary interphase caused by limitation of the amino acids L-serine and L-leucine. Maximum production of ETC was observed during the logarithmic phase, it was correlated to a high level of viable cells. ETA was produced mainly during the short stationary interphase. The production of ETC is regulated by L-isoleucine. A stagnation or reduction of the concentration of viable cells was observed during the interphase. The phosphate limitation caused during streptococcal growth induced expression of the extracellular protein phosphatase and surprisingly, of a serine proteinase activity. The association between these results and the pathogenicity of streptococci is discussed.

Studies on Streptococcal NAD-Glycohydrolase

The cleavage by specific enzymes of nicotinamide adenine dinucleotide (NAD+, synonym diphosphopyridine nucleotide (DPN)) in animal tissues has been described first by HANDLER and KLEIN (3). One class of these enzymes are the ADP-ribosyl transferases of bacterial origin (Cory neb acterium diphtheriae, Vibrio cholerae, Bordetella pertussis, Clostridium ssp., and Pseudomonas ssp.) which catalyze the transfer of ADP-ribosyl moieties to proteins like EF2 (elongation factor 2) and also show a distinct NAD+ glycosidase activity.

Immunoelectron microscopic analysis of polysaccharide and protein surface antigens in wild strains of group B streptococci.

The ultrastructural location of the group polysaccharide, the type-specific polysaccharides and the protein antigens Ibc, R and X of freshly isolated strains of group B streptococci was studied by the direct immunoferritin technique. The results were compared with the findings in prototype strains representing the serological types of Streptococcus agalactiae. In some strains the group antigen was demonstrated over the entire cell surface, but in strains with a large type polysaccharide capsule labelling was confined to the equatorial zone of the cells. If the type polysaccharide was released by extraction, however, the group antigen of these strains was uncovered over the whole surface of the cell. The type polysaccharide capsule was not demonstrated by the conventional electron microscopic technique, but it was visualized after reacting with the homologous type-specific antibody. The extent and density of the polysaccharide capsule were found to differ in strains with the same type antigen. The type polysaccharide did not block demonstration of the simultaneous presence of protein antigens, implying that the two are arranged mosaic-wise inside the capsule.