HYPERPRODUCTION OF ALPHA-TOXIN BY STAPHYLOCOCCUS-AUREUS RESULTS IN PARADOXICALLY REDUCED VIRULENCE IN EXPERIMENTAL ENDOCARDITIS - A HOST-DEFENSE ROLE FOR PLATELET MICROBICIDAL PROTEINS

Staphylococcal alpha-toxin targets several cell types which are import ant components of cardiac vegetations in endocarditis, including plate lets, erythrocytes, and endothelial cells. We evaluated the in vivo ro le of Staphylococcus aureus alpha-toxin in experimental endocarditis b y using isogenic strains differing in the capacity to produce function al alpha-toxin, including 8325-4 (wild-type strain), DU-1090 (a mutant strain with allelic replacement of the alpha-toxin gene [hla]), DU109 0(pH35L) (a mutant strain producing a target cell-binding but nonlytic toxin), DU1090(pDU1212) (a variant of DU1090 carrying the cloned hla gene on a multicopy plasmid), and DU1090(pCL84::hla) (a variant of DU1 090 with a single copy of the hla gene cloned into the chromosomal lip ase locus). In vitro, wild-type alpha-toxin (from parental strain 8325 -4) extensively lysed both erythrocytes and platelets. In contrast, mu tant alpha-toxin [from strain DU1090(pH35L)] lysed neither cell type. Following exposure to the wild-type alpha-toxin, platelet lysates were found to contain microbicidal activity against Bacillus subtilis (but not against Micrococcus luteus), as well as against the parental and alpha-toxin variant S. aureus strains noted above. Furthermore, lysate microbicidal activity was heat stable, neutralized by polyanionic fil ters or compounds, and recoverable from anionic filter membranes by hy pertonic saline elution. These characteristics are consistent with tho se of cationic platelet microbicidal proteins (PMPs). Reverse-phase hi gh-pressure liquid chromatography and polyacrylamide gel electrophores is confirmed the presence of three distinct PMPs (1, 2, and 3) in plat elet lysates. In experimental endocarditis, the two variant staphyloco ccal strains producing either minimal alpha-toxin or nonlytic alpha-to xin in vitro [strains DU1090 and DU1090(pH35L), respectively] exhibite d significantly lower virulence in vivo than the parental strain (decr eased intravegetation staphylococcal densities). Paradoxically, the tw o variant staphylococcal strains producing alpha-toxin at supraparenta l levels in vitro [strains DU1090(p1212) and DU1090(pCL84::hla)I also exhibited significantly decreased induction rates and intravegetation staphylococcal densities in experimental endocarditis versus the paren tal strain. The reduced in vivo virulence of the latter variant staphy lococcal strains could not be explained by differences in bacteremic c learance or initial adherence to sterile vegetations (compared to the parental strain). These findings suggest that the reduced virulence ex hibited by the variant staphylococcal strains in this model was relate d to pathogenetic events subsequent to bacterial adherence to the dama ged endocardium. Excess intravegetation secretion of alpha-toxin, lead ing to increased PMP release (secondary to either increased platelet s ecretion or lysis), may well explain the reduced virulence observed in experimental endocarditis.