Isolation and characterization of a haemolysin from Trichophyton mentagrophytes.

Haemolytic activities of Trichophyton (T.) mentagrophytes were detected and characterized by qualitative and quantitative assays. On Columbia agar supplemented with blood from horses, cattle or sheep, T. mentagrophytes expressed a strong zone of complete haemolysis. No haemolytic activities could be detected in the closely related T. verrucosum var. ochraceum. The same results were obtained after cultivation of the fungi on sterile cellulose acetate filters placed on the surface on Columbia blood agar. After removal of the filter, complete haemolysis was detected below the colony of T. mentagrophytes. A soluble haemolysin from culture supernatant of this strain was isolated and partially purified. Specific haemolytic activity per mg protein was enriched 2.6-fold in filtrate F(1), a fraction obtained as filtrate after filtration through 3kDa cut-off membranes. The partially purified haemolysin was neither affected by proteinase K treatment, nor by high and low temperatures, suggesting that it represents a small peptide haemolysin. Accordingly, in a commercial enzymatic activity test only the crude culture filtrate, but none of the subsequent purification fractions showed reactivity. Evaluation of the specificity of the haemolysin using erythrocytes from different mammalian species revealed that sensitivity was highest to those of equines, followed by erythrocytes from sheep, cattle, swine, dogs and humans. None of the erythrocytes was lysed by filtrate F(1) from T. verrucosum var. ochraceum. Furthermore, different eukaryotic cell lines from different species were tested in their sensitivity to cytolytic activities of the haemolysin, but no membrane damage could be detected.

Dermatophytes can trigger cooperative (CAMP-like) haemolytic reactions.

BACKGROUND: Dermatophytes usually grow on skin that is also colonized by bacteria. Therefore, a possible interaction between dermatophytes and bacteria is of potential relevance for the pathogenesis of skin infections. Cell membranes contribute substantially to epidermal barrier function. Erythrocyte haemolysis is a model commonly used to study membrane-damaging effects of microbial factors. Cooperative (CAMP-like) haemolytic reactions are known from distinct bacteria, but not from dermatophytes and bacteria. OBJECTIVES: To investigate CAMP-like reactions of dermatophytes. METHODS: Species of dermatophytes representing the genera Microsporum, Trichophyton and Epidermophyton, erythrocytes from sheep, horse and cattle, cultures of Staphylococcus aureus, S. intermedius, Listeria ivanovii, S. hyicus and S. epidermidis, and cell-free supernatants of bacterial cultures were used to search for cooperative haemolytic effects in in vitro agar diffusion assays. RESULTS: A cooperative (CAMP-like) haemolytic reaction was identified for all dermatophytes tested. Using erythrocytes from sheep and cattle, pretreated with a sphingomyelinase from S. aureus, S. intermedius or L. ivanovii, the fungi induced a distinct zone of complete haemolysis on solid media and in a diffusion test. No CAMP reaction could be detected using S. hyicus or S. epidermidis as a first-step agent or with equine erythrocytes. The lytic reaction of the CAMP-cohaemolysin derived from dermatophytes was always separate from the zone of complete haemolysis, indicating two different cytolytic factors. CONCLUSIONS: Our results show, for the first time, that in principle a CAMP-like effect can occur with dermatophtyes. This is a new observation of potential relevance for the pathogenesis of skin infections. The membrane-damaging factors released by dermatophytes should be further analysed.

Haemolytic activities of Trichophyton species.

Dermatophytes are keratinophilic fungi able to invade the stratum corneum of the skin and other keratinized structures. The pathogenic interactions between host and fungus are poorly understood. Some enzymes, especially keratinases, have previously been taken into consideration as virulence factors. Haemolysins have not been evaluated in this regard, though they are known to play an important role in the host-parasite interaction in bacterial infections. We investigate the haemolytic activity of four Trichophyton species: T. rubrum, T. mentagrophytes complex, Tequinum and T. verrucosum. The strains were tested on Columbia agar with 5% blood from horses, cattle and sheep. They show different haemolytic activities. T. rubrum and T. equinum produce a zone of complete haemolysis followed by a small zone of incomplete haemolysis around the colony. T. mentagrophytes and T. verrucosum produce a zone of complete haemolysis. Haemolytic activity is pronounced in dermatophytes and may play an important role as a virulence factor.

Isolation and characterization of hyaluronidases from Streptococcus dysgalactiae, S. zooepidemicus and S. equi.

10 out of 10 cultures each of Streptococcus dysgalactiae and S. zooepidemicus and 6 out of 10 cultures of S. equi tested for hyaluronidase produced this enzyme. Hyaluronidase could be precipitated from the cell-free culture supernatant with ammonium sulphate and purified by chromatography on DEAE-cellulose, isoelectric focussing and preparative polyacrylamide gel electrophoresis. The isoelectric points of the hyaluronidases from S. dysgalactiae and S. equi were near pH 5, of that from S. zooepidemicus near pH 6. The hyaluronidases from S. dysgalactiae, S. zooepidemicus and S. equi had molecular weights of about 55,000 D. Maximal enzyme activities developed between 40 degrees C and 45 degrees C and pH 5.6 and 5.8. The Michaelis constants ranged from 7.5 x 10(-2) to 8.8 x 10(-2) mg/ml. Hyaluronidase activities were stimulated by Ca++, Mg++, Mn++, Co++, K+, and Li+ and inhibited by Zn++ and Cd++.

Isolation and characterization of an extracellular protease of Actinomyces pyogenes.

An extracellular protease from Actinomyces pyogenes ATCC 19411 could be isolated by ion exchange chromatography with DEAE cellulose and high performance gel filtration (FPLC) on Superose 12 prep grade. The purified enzyme had a relative molecular mass of approximately 37,000 Dalton, a pH optimum at 7.5, a temperature optimum at 50 degrees C, and a Km value of 2.2 mg/ml with azocasein as substrate. The enzyme activity was clearly inhibited by PMSF, EDTA, the metal ion Zn++ and only weakly by Cd++ and Co++. Preparative isoelectric focussing of the culture supernatant from A. pyogenes ATCC 19411 revealed one major point with protease activity at pH 5.2.

Characterization of extracellular neuraminidase produced by Actinomyces pyogenes.

Extracellular neuraminidase from Actinomyces pyogenes could be isolated by ammonium sulfate precipitation, ion exchange chromatography with DEAE cellulose and gel filtration on Ultrogel ACA 54. The purified enzyme had a molecular weight of approximately 50,000 Dalton, a pH optimum at pH 6.0, a temperature optimum at 55 degrees C and a Km value of 1.4 X 10(-4) mol/l with N-acetyl-neuraminlactose as substrate. Preparative isoelectric focussing of the culture supernatant revealed neuraminidase activity mainly at pH 6.5. The enzyme activity was not influenced by metalions or EDTA.

Isolation and characterization of hyaluronidase from Streptococcus uberis.

All tested cultures of Streptococcus uberis produced free hyaluronidase. Hyaluronidase could be isolated by ammonium sulfate precipitation and was further purified by chromatography on DEAE-cellulose, gelfiltration on ultragel ACA44 and isoelectric focusing. The purification factor was estimated to be 1689. The purified hyaluronidase had an isoelectric point at pH 4.9 and a molecular weight of approximately 54000 D. It showed maximal enzyme activity at pH 6.0 and 45 degrees C. The Michaelis constant was estimated to be 7.0 X 10(-2) mg/ml. Hyaluronidase activity was stimulated by Ca++, Mg++, Mn++, Co++, Li+, and K+ and inhibited by Zn++ and Cd++ at final concentrations of 10 mmol/l, respectively.

Bindings of plasma proteins to streptococci of serological group L with special reference to their immunoglobulin G Fc-receptor activity.

Of 33 streptococcal cultures belonging to serological group L, all bound human immunoglobulin (Ig) G, fibrinogen, and fibronectin; 32 bound bovine IgG; 31 bound alpha 2-macroglobulin; 5 bound albumin; and none bound either haptoglobin or IgA. The binding sites for IgG could be isolated from the L streptococci by trypsinization and purified by affinity chromatography on human IgG-Sepharose. The purified Fc receptors reacted with IgG subclasses 1, 2, 3, 4 of humans, 1 and 2 of bovines, ovines, and caprines as well as a, b, c, and T of equines. They had a molecular mass of approximately 49,000 Da. Thus, the Fc receptors from L streptococci corresponded to type III Fc receptors of Streptococcus dysgalactiae.

Properties of L-streptococci in comparison with those of A-streptococci.

Despite some similarities, L-streptococci could be clearly differentiated from A-streptococci. Formamide, autoclaved and nitrous acid extracts of all L-streptococcal cultures studied reacted only with their specific antisera and did not cross-react with any other group specific streptococcal antigens. All 33 L-streptococcal cultures, in contrast to A-streptococci, produced beta-D-glucuronidase and beta-D-galactosidase, hydrolyzed Na-hippurate, grew on 10% and 40% bile blood agar and were agglutinated by the lectin of Arachis hypogaea. Some differences between A- and L-streptococci were also observed in their sensitivity patterns to bacitracin and sulfamethoxazole-trimethoprim.

Rapid differentiation of streptococci isolated from cows with mastitis.

The demonstration of some species-specific streptococcal enzymes with 4-methylumbelliferyl-conjugated beta-D-glucuronide, N-acetyl-beta-D-glucosaminide, and beta-D-mannoside and agglutination with the lectin from Dolichos biflorus allowed rapid tentative identification of streptococci isolated from cows with mastitis.

Screening for bacterial Fc-receptor activity on nitrocellulose membranes.

Fc receptors released from staphylococci and streptococci could be readily detected by direct cultivation of the bacteria on nitrocellulose membranes or by microfiltration of their culture supernatants. The nitrocellulose membranes used in both assays were subsequently treated with human immunoglobulin (Ig) G. The reactions of the Fc receptor with IgG could be demonstrated by use of the respective peroxidase-labelled antibodies against IgG. The resulting color formation, which indicated Fc-receptor activity of the bacterial culture, closely corresponded to the 125I-labelled IgG-binding reactivity.

beta-D-galactosidase activity in streptococci of serological group B.

Group B streptococci isolated from humans differed significantly in beta-D-galactosidase-activity from those of bovine mastitis. This could be demonstrated in a relatively simple and rapid test using a fluorogenic 4-methylumbelliferyl-beta-D-galactoside conjugate. Only 10 (12%) of 82 group B streptococcal cultures from human produced beta-D-galactosidase. On the other hand, 74 (96%) of 77 "bovine" cultures formed this enzyme. Thus, beta-D-galactosidase activity could be used as an additional marker for the differentiation between group B streptococci of human and bovine origin.