EFFECT OF DIVALENT-CATIONS ON HEMOLYSIN SYNTHESIS BY SERPULINA (TREPONEMA) HYODYSENTERIAE - INHIBITION INDUCED BY ZINC AND COPPER

Serpulina hyodysenteriae produces an oxygen-stable heat-labile hemolys in that may be an important virulence factor in the pathogenesis of sw ine dysentery. We examined the effect of Ca2+, Co2+ Cu2+, Fe2+, Mg2+ , Mn2+ , Ni2+, and Zn2+ on the hemolytic activity of cell-free supernat ant (CFS) from S. hyodysenteriae, isolate B204. Cells harvested from l ate logarithmic phase cultures were incubated in phosphate-buffered sa line containing glucose and RNA-core (PBS-GR) with or without cations and the hemolytic activity of CFS obtained after successive 30 min inc ubation and washing cycles was determined. The addition of either ZnSO 4 or CuSO4 to the PBS-GR caused complete inhibition of hemolytic activ ity after 3 cycles; other cations gave results similar to control extr acts. Reduction in the concentration of Zn2+ in CFS by 60 to 80% after each incubation cycle and binding of Zn2+ by EDTA indicated that Zn2 was associated with the cell fraction, and inhibition of hemolysin ac tivity was specifically mediated by Zn2+. When the spirochetes were wa shed after incubation in the presence of ZnSO4 for 2 cycles and incuba ted in fresh PBS-GR without Zn2+ inhibition of hemolysin activity rema ined unchanged, indicating that the inhibitory effect of ZnSO4 was due to a direct action of ZnSO4 on the spirochetes. Since neither the via bility of the spirochetes nor the activity of pre-formed hemolysin wer e affected by the presence of ZnSO4, the inhibitory effect of Zn2+ cat ions was attributed to reduced biosynthesis by viable S. hyodysenteria e cells rather than interference of Zn2+ cations with lysis of erythro cytes by the hemolysin. Transmission electron microscopic examination of spirochetes after incubation in PBS-GR containing ZnSO4 revealed cl umping of ribosomes and clearing of cell cytoplasm.