EFFECT OF DEFINED POINT MUTATIONS IN THE PNEUMOLYSIN GENE ON THE VIRULENCE OF STREPTOCOCCUS-PNEUMONIAE

The thiol-activated toxin pneumolysin is a known pneumococcal virulenc e factor, with both cytotoxic (hemolytic) and complement activation pr operties, Copies of the pneumolysin gene carrying defined point mutati ons affecting either or both of these properties were introduced into the chromosome of Streptococcus pneumoniae D39 by insertion-duplicatio n mutagenesis, The virulences of these otherwise isogenic strains were then compared, There was no significant difference in either the medi an survival time or overall survival rate between mice challenged with D39 derivatives producing the wild-type toxin and those expressing a pneumolysin gene with an Asp-385-->Asn mutation, which abolishes the c omplement activation property. However, mice challenged with strains c arrying either His-367-->Arg or Trp-433-->Phe plus Cys-428-->Gly mutat ions, which reduce hemolytic activity to approximately 0.02 and 0.0001 % of the wild-type level, respectively, had significantly greater medi an survival times and overall survival rates than mice challenged with D39 derivatives expressing a Wild-type pneumolysin gene, No additiona l reduction in virulence was observed when mice were challenged with a D39 derivative carrying Trp-433-->Phe, Cys-428-->Gly, and Asp-385-->A sn, rather than Trp-433-->Phe and Cys-428-->Gly, mutations in the pneu molysin gene, Thus, it appears that in the intraperitoneal challenge m odel, the contribution of pneumolysin to virulence is largely attribut able to its hemolytic (cytotoxic) properties rather than to its capaci ty to activate complement. Interestingly, however, the amount of pneum olysin required for full virulence may be very small, as D39 derivativ es carrying the Trp-433-->Phe mutation (which reduces hemolytic activi ty to 0.1% of the wild-type level) had intermediate virulence.