Effect of divalent cations on hemolysin synthesis by Serpulina (Treponema) hyodysenteriae: inhibition induced by zinc and copper

Abstract

Serpulina hyodysenteriae produces an oxygen-stable heat-labile hemolysin that may be an important virulence factor in the pathogenesis of swine dysentery. We examine the effect of Ca 2+, Co 2+, Cu 2+, Fe 2+, Mg 2+, Mn 2+, Ni 2+, and Zn 2+ on the hemolytic activity of cell-free supernatant (CFS) from S. hyodysenteriae, isolate B204. Cells harvested from late logarithmic phase cultures were incubated in phosphate-buffered saline containing glucose and RNA-core (PBS-GR) with or without cations and the hemolytic activity of CFS obtained after successive 30 min incubation and washing cycles was determined. The addition of either ZnSO 4 or CuSO 4 to the PBS-GR caused complete inhibition of hemolytic activity after 3 cycles; other cations gave results similar to control extracts. Reduction in the concentration of Zn 2+ in CFS by 60 to 80% after each incubation cycle and binding of Zn 2+ by EDTA indicated that Zn 2+ was associated with the cell fraction, and inhibition of hemolysin activity was specifically mediated by Zn 2+. When the spirochetes were washed after incubation in the presence of ZnSO 4 for 2 cycles and incubated in fresh PBS-GR without Zn 2+, inhibition of hemolysin activity remained unchanged, indicating that the inhibitory effect of ZnSO 4 was due to a direct action of ZnSO 4 on the spirochetes. Since neither the viability of the spirochetes nor the activity of pre-formed hemolysin were affected by the presence of ZnSO 4, the inhibitory effect of Zn 2+ cations was attributed to reduced biosynthesis by viable S. hyodysenteriae cells rather than interference of Zn 2+ cations with lysis of erythrocytes by the hemolysin. Transmission electron microscopic examination of spirochetes after incubation in PBS-GR containing ZnSO 4 revealed clumping of ribosomes and clearing of cell cytoplasm.