The Kinetics of Erythrocyte Lysis by Escherichia Coli Haemolysin

Abstract

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 ...

Cytotoxic Activity of Partially Purified Escherichia Coli Alpha Haemolysin

“…Similar data were reported for the cytotoxic effects of other haemolysins (Snyder and Hamilton, 1963;Wiseman, 1968;Thelestam, Mollby and Wadstrom, 1973;Mollby, Thelestam and Wadstrom, 1974). In addition, the dose-response kinetics for 3T3-cell cytotoxicity were similar to those reported for erythrocyte lysis by Rennie, Freer, and Arbuthnott (1974). As little as 10 HU50 (23 ng of protein) of PH killed 90% or 4.3 x lo5 3T3 cells and even 2 HU50 showed significant killing.…”

E. coli <FONT FACE=Symbol>a</FONT>-hemolysin: a membrane-active protein toxin

“…Other divalent cations (e.g., Mg 2+ , Mn 2+ , Zn 2+ , Cd 2+ ) do not support HlyA activity. The data are in accordance with early observations of Ca 2+ requirements (57)(58)(59). However, other reports appeared to suggest that Ca 2+ was not required for the hemolytic action (60)(61)(62).…”

Pore formation in artificial membranes by the secreted hemolysins of Proteus vulgaris and Morganella morganii

“…Further studies on the E. coli hemolysin have revealed that two other regions may also be involved in pore formation, the N-terminus and a hydrophobic domain at amino acid 177-411 [15, 161. Extensive similarity has been demonstrated between the four different hly genes of E. coli and those of the Proteacae [17, 181, and also to the hemolysins and leukotoxins of Actinobacillus Erythrocyte lysis by E. coli HlyA is characterised by three phases, an initial lag, an accelerated rate of hemolysis followed by a plateau, then a decline in rate [33]. An initial adsorption step (due to the repeat domain [l -31) may determine the lag step of erythrocyte lysis.…”

“…A high incidence of hemolytic strains has been reported amongst extraintestinal M. morganii isolates, and a markedly higher virulence in three animal models was observed for hemolytic strains [32]. Erythrocyte lysis by E. coli HlyA is characterised by three phases, an initial lag, an accelerated rate of hemolysis followed by a plateau, then a decline in rate [33]. An initial adsorption step (due to the repeat domain [l -31) may determine the lag step of erythrocyte lysis.…”