The hemolysins produced by Serpulina hyodysenteriae as putative virulence determinants.

Abstract

Serpulina hyodysenteriae is the etiological agent of swine dysentery (SD). The hemolysin produced by this spirochete is believed to be a virulence factor of the bacteria. Muir et aI. (78) have recently cloned a gene (tlyA) from S. hyodysenteriae responsible for the expression of a hemolysin protein. The B204 RNA-core extract and recombinant hemolysins were active at temperatures ranging from 27-42 °C and pH ranges of three to nine. There was no inhibitory effect by calcium, magnesium, BSA, sucrose or 50 mM EDTA while both zinc and copper were inhibitory. The activity of the two hemolysins could be partially blocked by components with a diameter of 2.0-2.3 nm. Neutralization of the hemolysins by newborn but not fetal bovine serum was observed. Cholesterol and cholesterol derivatives were able to partially block the hemolytic activity. To test the mode of activity of the hemolysins, sheep erythrocytes were internally labeled with ⁸⁶rubidium. The release of both hemoglobin and ⁸⁶rubidium from the erythrocytes were compared after exposure to the B204 RNA-core hemolysin. It was found that ⁸⁶rubidium was released after 2 minute exposure to the hemolysin. Hemoglobin was released at 4 minutes of exposure, indicating that the hemolysin lyses the erythrocytes using a porin mechanism. T/yA-minus mutants of two S. hyodysenteriae strains (B204 and C5) were tested for virulence in pigs. None of the animals inoculated with the mutant strains developed SD. Inoculation of pigs with the wild-type B204 or C5 strain produced SD in 100% and 60% respectively. Pigs infected with the mutant strains were further tested for susceptibility to challenge with the wild-type strain B204. After challenge, 50% of the pigs previously inoculated with the B204 tlyA -minus mutant were protected, whereas all of the control pigs contracted SD. None of the pigs previously inoculated with the C5 tlyA-minus mutant, developed SD upon challenge with the B204 wild-type strain, whereas 40% of the control pigs developed SD in this experiment. Thus the tlyA encoded hemolysin of S. hyodysenteriae appears to be an important virulence factor in SD and previous colonization with tlyA -minus mutants provides partial protection to challenge with a virulent strain.