Anti-atherosclerotic effect of microbial hyaluronate lyase from group B streptococci.

The effect of the microbial hyaluronic acid splitting enzyme hyaluronate lyase produced by Streptococcus agalactiae was investigated in vitro in human atherosclerotic plaque specimens and in vivo on Watanabe heritable hyperlipidaemic rabbits (WHHL) as an animal model for familiar hypercholesteraemia. The in vitro presence of the enzyme caused a partial destruction of the atherosclerotic plaque surfaces as well as releasing of glucuronic acid and solid calcium-containing materials from pieces of atherosclerotic plaques in human arteries. Accordingly hyaluronic acid seems to be the main component for anchoring of calcium deposits on the plaque surfaces. Repeated intravenous injections of hyaluronate lyase in WHHL rabbits resulted in a tendency of decreased formation of atherosclerotic plaques. The observed effects are discussed to be primary the result of the splitting of hyaluronic acid in the vessels.

Complementary characterization of a hyaluronic acid splitting enzyme from Streptococcus agalactiae.

A hyaluronic acid splitting enzyme of Streptococcus agalactiae was characterized by splitting mechanism, Michaelis-constant and inhibition type for sulfated hyaluronic acid: The enzyme splits hyaluronic acid as a hyaluronate lyase [EC 4.2.2.1]. The Km = 8 x 10(-4) mg ml-1 was determined with the influence of substrate inhibition constant Kiu = 2 x 10(-6) mg ml-1. Sulfated hyaluronic acid inhibits the enzyme in a partially non-competitive way. The inhibition constant is Ki = 5.47 x 10(-4) mg ml-1. The GBS-hyaluronate lyase cleaves hyaluronic acid as an endoglycosidase. The work is related with the intention to establish a hyaluronate lyase of microbial origin as a therapeutical enzyme replacing bovine hyaluronidase.

Quantification of hyaluronan in pharmaceutical formulations using high performance capillary electrophoresis and the modified uronic acid carbazole reaction.

The amount of hyaluronan (HA) in pharmaceutical formulations was determined by high-performance capillary electrophoresis (HPCE) and the results were compared with the carbazole reaction established by Bitter and Muir (T. Bitter, H.M. Muir, Anal. Biochem. 4 (1962) 330-334), HA analysis was performed in less than 10 min by using an untreated fused silica capillary with bubble detection cell. The influence of several buffers and pH values was examined. Calibration curve shows good linearity from 0.01 to 5.0 mg/ml. The lower limit of detection by monitoring the absorbance at 195 nm was 10 microg/ml at a signal to noise ratio of 5.

Influence of the phosphorylation state on the biological activity of a low-molecular mitogen from group A streptococci.

A low molecular weight mitogen (LMP) from Streptococcus pyogenes strain NY 5 was successively purified by adsorption on phenylsepharose, chromatography on Resource S and Superdex G 30 and finally by affinity chromatography on antiphosphothreonine agarose. The N-terminal protein sequence of the mitogen was determined. The occurrence of phosphoamino acids was investigated by immunoassay using monoclonal antibodies. The LMP is a threonine-phosphorylated protein different of HPR protein of PTS-system, its mitogenic activity was lost after treatment with streptococcal protein phosphatase or alkaline phosphatase. The inactivated LMP was activated by phosphorylation with phosphokinase and ATP. The active LMP was also inactivated in streptococcal cultures secreting acid protein phosphatase during the phase of phosphate limitation.

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.

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.

Human pro- and anti-inflammatory cytokine patterns induced by Streptococcus pyogenes erythrogenic (pyrogenic) exotoxin A and C superantigens.

The superantigenic streptococcal erythrogenic toxins A and C (ETA/SPEA and ETC/SPEC) elicit the production by human peripheral blood mononuclear cells of substantial amounts of Th1-derived cytokines (interleukin-2 [IL-2] and gamma interferon) as well as anti-inflammatory cytokines (IL-10 and IL-1 receptor antagonist). In contrast, very low levels of IL-4 and no alpha interferon were induced. The production of these cytokines after stimulation with Streptococcus pyogenes heat-killed bacteria and lipopolysaccharide from gram negative bacteria differed qualitatively and quantitatively from that elicited by the 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.

Purification and some properties of streptococcal NAD-glycohydrolase.

NAD-glycohydrolase (NADase) was purified from culture supernatant fluids of group C streptococci by adsorption on silica gel, chromatography on hydroxyapatite and ion exchange on Mono S column. After inactivation of a chymotrypsin-like protease, a homogeneous enzyme was isolated with an N-terminal sequence of VSGKEGKKSDVKYEMTKVMEANATSSKEDKHVMHTLDKVM. According to serological methods, the purified enzyme of group C streptococci was identical to the group A enzyme showing a specific activity of 10 000 000 U mg-1. It did not attack NADH, NADP or NADPH. In addition, a streptodornase was isolated having an N-terminal sequence of KTVSVNQTYGE.

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.

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.

Production and partial characterization of monoclonal antibodies against erythrogenic toxins type A and C from Streptococcus pyogenes.

Hybridoma cell lines producing monoclonal antibodies against streptococcal erythrogenic toxins type A and C were established from fusions of immunized BALB/c mice splenocytes with P3X63-Ag8.653 myeloma cells. Six MAbs recognize ETA and 11 MAbs bind to ETC. Two MAbs (designated ETA-2 and ETC-10) were produced in ascitic fluid and further characterized. ETA-2 (IgG2a) binds to ETA with an affinity constant of 1.8 x 10(10) M-1 and ETC-10 (IgG1) binds to ETC with an affinity constant of 3.5 x 10(9) M-1. The specificities of the MAbs were evaluated by ELISA and immunoblotting. Both MAbs ETA-2 and ETC-10 are useful in developing specific double-sandwich ELISAs, in which the MAbs were used as solid-phase capture antibodies for the quantitative determinations of ETA and ETC.

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.

Streptococcal erythrogenic toxin type C is not a phosphorylated protein. Description of two different purification procedures and investigation of its phosphorylation state.

Erythrogenic toxin type C (ETC) from different streptococcal group A strains was successively purified by absorption on phenylsepharose, acidic dialysis of the eluate at 40% saturated ammonium sulphate solution, CM-Sepharose chromatography, finally by immunoaffinity chromatography on monoclonal antibodies. Second, after growing of bacteria in the presence of [32P]orthophosphate to phosphorylate ETC, the ETC was purified with phenylsepharose following immunoaffinity chromatography. The occurrence of phosphoamino acids in the purified ETC was investigated by an immunoassay. No phosphoamino acids could be detected in the ETC molecule. Also after radiolabelling with 32P it was not possible to demonstrate a radioactive signal. The treatment with alkaline phosphatase has no influence on the mitogenicity or position of ETC in isoelectric focusing. The results obtained led to the conclusion that in contrast to the literature, ETC is not a phosphorylated protein.

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.