Effect of defined point mutations in the pneumolysin gene on the virulence of Streptococcus pneumoniae.

The thiol-activated toxin pneumolysin is a known pneumococcal virulence factor, with both cytotoxic (hemolytic) and complement activation properties. Copies of the pneumolysin gene carrying defined point mutations affecting either or both of these properties were introduced into the chromosome of Streptococcus pneumoniae D39 by insertion-duplication mutagenesis. The virulences of these otherwise isogenic strains were then compared. There was no significant difference in either the median 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 complement activation property. However, mice challenged with strains carrying either His-367-->Arg or Trp-433-->Phe plus Cys-428-->Gly mutations, which reduce hemolytic activity to approximately 0.02 and 0.0001% of the wild-type level, respectively, had significantly greater median survival times and overall survival rates than mice challenged with D39 derivatives expressing a wild-type pneumolysin gene. No additional reduction in virulence was observed when mice were challenged with a D39 derivative carrying Trp-433-->Phe, Cys-428-->Gly, and Asp-385-->Asn, rather than Trp-433-->Phe and Cys-428-->Gly, mutations in the pneumolysin gene. Thus, it appears that in the intraperitoneal challenge model, the contribution of pneumolysin to virulence is largely attributable to its hemolytic (cytotoxic) properties rather than to its capacity to activate complement. Interestingly, however, the amount of pneumolysin required for full virulence may be very small, as D39 derivatives carrying the Trp-433-->Phe mutation (which reduces hemolytic activity to 0.1% of the wild-type level) had intermediate virulence.