Partial Characterisation of Escherichia Coli Haemolysin

PLATE XVIIISUMMARY. The influence of haemolysin production on virulence was studied in an experimental mouse model. Urinary strains of Escherichia coli can be divided into three virulence groups by determining their kinetics in the mouse kidney after intravenous injection. Virulent strains of groups I1 and I11 were more often haemolytic than avirulent group-I strains. Haemolytic virulent strains often caused haemoglobinuria in the mice, and killed the mice more rapidly than did non-haemolytic virulent strains. No relationship was found between a-haemolytic activity and virulence in wild-type haemolytic strains. When haemolysin production was reduced or eliminated by treatment with actinomycin-D or rifampicin, six out of seven group-I1 strains tested gave the same results as avirulent group-I strains. However, the kinetics in the mouse kidney of four haemolytic group-I11 strains tested was not changed after reduction or elimination of haemolysin production; only a small decrease in toxicity was observed. It is concluded that haemolysin production by E. coli is a decisive virulence factor in most of the mouse-nephropathogenic group-I1 strains, but not in the virulent group-I11 strains.

Section: Discussioncontrasting

confidence: 52%

“…The a haemolysin is a cell-free, filtrable protein, produced during log-phase growth by modification of a precursor molecule present in fresh meat and blood (Love11 and Rees, 1960; Smith, 1963;Snyder and Koch, 1966;Short and Kurtz, 1971;Zwadyk and Snyder, 1971; Rennie and Arbuthnott, 1974;Williams, 1979). The j?…”

Section: Introductionmentioning

confidence: 99%

Haemolysis by urinary Escherichia coli and virulence in mice

Bosch

Postma

Graaff

et al. 1981

“…Measurement of 100 of the structures seen in fig. 6b gave a normal distribution with a maximum at 15 nm, and this value was used to estimate the molecular weight of purified a-haemolysin (Rennie and Arbuthnott, 1974).…”

Section: Escherichia Coli Haemol Ysin Kinetics 193mentioning

confidence: 99%

“…Quantitative haemolysin titrations were performed as described previously (Rennie and Arbuthnott, 1974). Kinetic studies were carried out with silica cells of 1-cm light path in a controlled-temperature Unicam SP800 spectrophotometer, to which was attached a Unicam SP22 chart recorder (Pye-Unicam, Cambridge, England) set at a five-times multiplication factor.…”

Section: R P Rennie J H Freer and J P Arbuthnottmentioning

confidence: 99%

The Kinetics of Erythrocyte Lysis by Escherichia Coli Haemolysin

Rennie

Freer

Arbuthnott

1974

Self Cite

PLATE VITECHNIQUES for studying the mode of action of bacterial toxins at the molecular level are fast becoming valuable and necessary tools for the determination of the role of these agents as virulence factors in diseases of man and animals. The mechanism of action of Escherichia coli haemolysin is poorly understood. Although enough evidence now exists to conclude that calcium ions are required for haemolytic activity, only two groups of investigators have attempted to study the kinetics of erythrocyte lysis by E. coli a-haemolysin.The findings of Zwadyk and Snyder (1971) and Short and Kurtz (1971) indicated that lysis of sheep erythrocytes was dependent on haemolysin concentration, pH and temperature. Less haemolysis was observed as the concentration of red cells (RBC) was increased. It has been suggested that a complex consisting of haemolysin, calcium ions and RBC must be maintained until the lytic event (Short and Kurtz, 1971). These authors found that the addition of ethylenediaminetetraacetic acid (EDTA) at any time during the haemolytic reaction prevented subsequent haemolysis. They also noted that neither reducing agents nor lecithin or cholesterol affected a-haemolysin.Both groups of workers used methods that required centrifugation of reaction mixtures at different times, followed by spectrophotometric estimation of haemoglobin. In these processes there are unavoidable delays during which haemolysin-affected cells may be lysed.In this study, the turbidity of reaction mixtures was monitored continuously at 650 nm in a controlled-temperature spectrophotometer. By this technique it was possible to make rate measurements on haemolysis curves. MATERIALS AND METHODSHaemolysin production and purification. The preparations of E. coli u-haemolysin used in this study were produced in glucose-nutrient-broth medium and were purified by the three-stage method of Rennie and Arbuthnott (1974). Because it was more readily available, stage-I1 u-haemolysin was used for most assays. However, in each experiment, purified stage-III a-haemolysin was tested to confirm the results obtained with less pure preparations. (Rennie and Arbuthnott, 1974). Kinetic studies were carried out with silica cells of 1-cm light path in a controlled-temperature Unicam SP800 spectrophotometer, to which was attached a Unicam SP22 chart recorder (Pye-Unicam, Cambridge, England) set at a five-times multiplication factor. The spectrophotometer was adjusted so that extinction (E) was monitored at a constant wavelength of 650 nm.A 0.7% suspension of sheep erythrocytes (SRBC) was made in Veronal buffer, pH 7.3 (Cruickshank, 1969), containing lOmM CaCl2 (VC buffer). The spectrophotometer and chart recorder were then calibrated on a linear scale with the SRBC suspension. The contents of a 1-cm cell, containing 1-4 ml of VC buffer and 0.1 ml of 0.7% SRBC, were mixed by inversion and read at E 6 5 0 . The zero controls of the spectrophotometer and chart recorder were adjusted if necessary such that the E650 was 0.40. This represented 100% erythrocyte ...

“…The cod erythrocytes were washed four times in citrate-dextrosesaline buffer (CDS) (Hodgins and Ridgway, 1964) and a 1 % (vlv) suspension was made in CDS and standardised spectrophotometrically (Rennie and Arbuthnott, 1974). Erythrocytes from fish other than cod were not stored in liquid nitrogen, but were washed and suspended in phosphate buffered saline (PBSA) (Dulbecco and Vogt, 1954).…”

mentioning

confidence: 99%

Assay of Staphylococcal -Haemolysin with Fish Erythrocytes

Chao

Birkbeck

1978

Delta-haemolysin has a much lower specific activity than the other haemolysins, and in low dilutions lyses a wide range of erythrocytes; it is usually assayed with human or horse erythrocytes and these have indicated as many as 300 haemolytic units per mg (HU per mg) in purified preparations (Heatley, 1971 ; Kreger et al., 1971). Heatley (1971) found that ,8-haemolysin was capable of interfering with the assay of 8-haemolysin when human erythrocytes were used. Lysis of bacterial protoplasts (Bernheimer et al., 1972) provides a more sensitive assay (610 units per mg) but is less convenient when a large number of samples is screened. The release of radioactively labelled nucleotides from tissue-culture cells (Thelestam, Mollby and Wadstrom, 1973) also provides a sensitive assay (approximately 600 units per mg purified protein) in which interference by other haemolysins does not occur. The simple haemolytic assay is still routinely used for 8-haemolysin, but a more specific and sensitive assay would be desirable. The work described here resulted from a preliminary survey of the sensitivity of marine fish erythrocytes to staphylococcal haemolysins and we describe the use of fish erythrocytes for the assay of staphylococcal 8-haemoly sin. MATERIALS AND METHODSStaphylococcal strains. Staphylococcus aureus strains NCTC10345 and NCTC7121 (Wood 46) were obtained from Professor J. P. Arbuthnott. The remainder, obtained from Dr S. McKay, comprised S. uureus strain BB, a ,fl-toxinogenic bovine-mastitis isolate, and clinical isolates of S. aureus (strains SM9, SM10, SM14, JK21) and S. epidermidis (strain SM6). Cultures were maintained on nutrient agar slopes.