Heterogeneity of group A streptococcal pyrogenic exotoxin type B

Abstract

Streptococcal pyrogenic exotoxin type B purified from culture filtrates of either the NY-5 or T-19 strain of group A streptococcus was found to be heterogeneous in charge. Three protein fractions with isoelectric points of 8.0, 8.4, and 9.0 were isolated by differential solubility in ethanol and acetate-buffered saline followed by isoelectric focusing and shown to be antigenically identical to streptococcal pyrogenic exotoxin type B. The molecular weights of all three fractions were approximately 17,500, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with aggregates forming in the presence of hyaluronic acid. Only the pI 8.4 fraction showed the characteristic activities of streptococcal pyrogenic exotoxin in rabbits: pyrogenicity and ability to enhance susceptibility to lethal endotoxin shock. The pI 8.0 and pl 9.0 fractions were not pyrogenic, but could be used to immunize against pyrogenicity. These two fractions failed either to enhance lethal endotoxin shock or to immunize against enhancement activity. When the isolated fractions were electrofocused again they appeared heterogeneous, suggesting an instability of the B toxin molecular forms.

Characterization of a fourth streptococcal pyrogenic exotoxin (SPE D)

“…It has been reported earlier that the pyrogenicity and enhancement of endotoxin capacity of the SPEs are separate properties [1]. SPED isolated from the Saint Louis Strain of group A streptococci was very pyrogenic, but not nearly as enhancing as SPE C. To assess the possible synergistic action of SPE C and SPED we chose a dose of SPED that was pyrogenic (1 #g/kg), but not enhancing, and added two different concentrations of SPE C. These concentrations of SPE C (0.05 and 0.005 #g/kg ) were not pyrogenic or enhancing.…”

“…Pyrogenic activity and ability to enhance the lethal effect of endotoxin were determined by methods previously described [1,3,9,10,[22][23][24]. Briefly, SPE was dissolved in pyrogen-free phosphate-buffered saline (PBS, 0.005 M phosphate, 0.15 M NaCl, pH 7.2) and injected (i.v.)…”

“…This dose was 21 times less than the lethal dose for 50% of rabbits injected. Enhanced animals usually succumbed within 24 h. The mitogenic capacity of SPED was assessed as previously described [1]. Various amounts of SPED were added to 2.0 x 105 rabbit spleen cells or human mononuclear leukocytes and mitogenicity was followed by incorporation of [3H]thymidine.…”

“…The resulting sera were dispensed into 1.0-ml aliquots and lyophilized. Antisera to SPE A, SPE B and SPE C were made in a similar manner [1,3,10].…”

“…Streptococcal pyrogenic exotoxins (SPEs) are extracellular proteins secreted by group A streptococci. Three antigenically distinct toxin types, designated SPE A, SPE B and SPE C, have been identified and characterized biologically and biochemically [1][2][3][4][5][6][7][8][9][10][11][12][13][14]. The exotoxins are readily distinguished biochemically based on their diverse isoelectric points and MrS, respectively: 5.1 and 8000 for SPE A [1,3], 8.0-9.0 and 17 500 for SPE B [1], and 6.7 and 13 200 for SPE C [10].…”