Studies on the effect of divalent metal ions on exfoliative toxins from Staphylococcus hyicus: indications of ExhA and ExhB being metalloproteins

The exfoliative toxins ExhA and ExhB produced by Staphylococcus hyicus strains NCTC10350 and 1289D-88, respectively, were investigated with regard to the effect of divalent metal ions on toxin production as measured in indirect enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies. Data were obtained as endpoint titer values and used as semiquantitative measures for the amount of exfoliative toxin detected in culture supernatants. It was shown that the endpoint titers of ExhA in supernatants from cultures of strain NCTC10350 grown in the presence of 0.5 mM CaCl2, Cu(NO3)2 or ZnSO4 were higher compared to titers obtained by growth in medium supplemented with a number of other divalent metal salts. The titer of ExhB as determined in the indirect ELISA was increased by addition of 0.5 mM CoCl2, Cu(NO3)2 or CuSO4 to the growth medium. When ExhA or ExhB, prepared without addition of metal salt to the liquid growth medium, was subsequently incubated with 25 mM of Co2+, Cu2+ or Zn2+, the endpoint titers of the toxins were increased. Dialysis of ExhA and ExhB prepared with Zn2+ and Co2+, respectively, against certain metal chelators, resulted in a reduction of the titer determined in ELISA. Other metal chelators had varied effect in the detection of the toxins in ELISA. It was, however, not possible to restore the recognition of toxins by the monoclonal antibodies by incubation of EDDHA-dialyzed toxin preparations with Co2+, Cu2+ or Zn2+. The results of this study suggest that ExhA and ExhB are metalloproteins.