Absence of a cysteine protease effect on bacterial virulence in two murine models of human invasive group A streptococcal infection

Abstract

The cysteine protease of group A streptococci has been suggested to contribute to the pathogenesis of invasive infection through degradation of host tissue, activation of the host inflammatory response, release of protective molecules from the bacterial cell surface, or other mechanisms. However, studies of the effects on virulence of inactivating the cysteine protease gene speB have yielded conflicting results. In some reports, a speB mutant was relatively avirulent in mouse models of invasive infection whereas little or no attenuation of virulence was observed in other studies of similar mutant strains. Possible reasons for these discordant results include differences in the streptococcal strains from which the speB mutants were derived, differences in the infection models employed, or unintended effects on another virulence determinant(s) that arose during the derivation of a speB mutant. We attempted to clarify these issues by characterizing the phenotypic properties and relative virulence in mice of two speB mutant strains, both derived from wild-type strain AM3: speB mutant AM3speB, which has been shown to be markedly attenuated in virulence in mice after intraperitoneal or subcutaneous challenge, and AM3speBOmega, a new mutant strain derived for this investigation. Both mutant strains were negative for protease activity, as expected, and both produced wild-type amounts of type 3 M protein and streptolysin O. However, AM3speB produced significantly less cell-associated hyaluronic acid capsule than did parent strain AM3 or strain AM3speBOmega. Compared to wild-type strain AM3, AM3speB was more sensitive to opsonophagocytic killing in vitro and was significantly less virulent in mice after intraperitoneal challenge. By contrast, AM3speBOmega was fully resistant to phagocytosis and did not differ significantly from the wild-type strain in mouse virulence after an intraperitoneal or subcutaneous challenge. We concluded that previous reports attributing loss of virulence in strain AM3speB to inactivation of speB are in error. Within the limitations of the models used, we found no effect of cysteine protease on invasive streptococcal infection.

FIG. 1

Growth of GAS in whole blood. Values represent the mean log fold increase in CFU after 3 h of rotation in fresh human blood. Each point represents a single experiment. The differences in net growth between AM3 and AM3speB and between AM3speBΩ and AM3speB were significant (P < 0.05).

FIG. 2

Mouse survival after an intraperitoneal challenge with GAS. The curves represent Kaplan-Meier survival function estimates after an intraperitoneal challenge with either 10⁴ exponential-phase (A) or 10⁷ stationary-phase (B) GAS bacteria. Each curve represents combined data from two experiments with 10 mice per strain. Survival was significantly longer in animals challenged with AM3speB than in animals challenged with either AM3 or AM3speBΩ, and this effect was independent of the growth phase of the inoculum (exponential phase, P = 0.003; stationary phase, P < 0.001).

FIG. 3

Mouse survival after a subcutaneous challenge with exponential-phase GAS. The curves represent Kaplan-Meier survival function estimates after a mouse challenge with 10⁷ exponential-phase GAS bacteria. Each curve represents combined data from two experiments with 10 mice per strain. The survival of animals challenged with AM3 was not significantly different from that of animals challenged with AM3speBΩ.

FIG. 4

Mouse survival after a subcutaneous challenge with stationary-phase GAS. The curves represent Kaplan-Meier survival function estimates after a mouse challenge with 10⁸ stationary-phase GAS bacteria. Each curve represents combined data from three experiments with 10 mice per strain. For clarity, the survival curve for animals challenged with AM3 is compared with the survival curve for animals challenged with AM3speBΩ (A) or with the survival curve for animals challenged with AM3RV (B). The survival of animals challenged with AM3 was not significantly different from that of animals challenged with AM3speBΩ or AM3RV.