Absence of a Cysteine Protease Effect on Bacterial Virulence in Two Murine Models of Human Invasive Group A Streptococcal Infection

The manifestations of group A streptococcus (GAS) infections in humans are diverse in both clinical presentation and morbidity. Pharyngitis and impetigo are common childhood illnesses with few complications. Infrequently, GAS causes invasive disease, of which the most serious presentation is necrotizing soft-tissue infection with associated shock and multisystem organ failure (24). The molecular details of the interaction between the bacterium and the host that determines the natural history of GAS infection remain poorly understood. GAS has the potential to produce a number of cell-associated and extracellular products that may contribute to pathogenesis. Virtually all GAS strains contain the speB gene that encodes a cysteine protease. Indirect support for a role of SpeB in invasive GAS infection includes the observations that the protease mediates degradation of the host tissue components vitronectin, fibronectin, and collagen; that the protease activates interleukin-1β, a proinflammatory cytokine; and that the protease may function as an adhesin by binding GAS to a variety of cell surface or ground substance molecules, including integrins, fibronectin, and laminectin (5, 7–9, 25). More direct evidence supporting an effect of SpeB in GAS infection is the demonstration that SpeB is directly toxic after intravenous injection and may enhance GAS virulence in animal models of human disease (10, 11, 22, 26). However, the interpretation of these experiments is confounded by the use of partially purified protein preparations and by uncertainty about the biologically relevant concentration of SpeB. A more rigorous method for evaluating SpeB's role in GAS pathogenesis is to compare the virulence of wild-type and isogenic speB mutants in animal models of human infection. Lukomski et al. challenged mice intraperitoneally with isogenic M3 and M49 wild-type or protease-deficient strains and found that the protease-deficient mutants were attenuated (16). It is of note that the attenuation was particularly pronounced in the M3 background but was of a much smaller magnitude in the M49 background. A follow-up study using the M3 strain AM3 extended the findings of the intraperitoneal challenge studies, demonstrating that the protease-deficient mutant also was less virulent than the wild-type strain in a murine model of invasive soft-tissue infection (15). Although the mechanism of the protease effect was not evaluated in these experiments, subsequent work suggested that the loss of the protease increased bacterial susceptibility to phagocytic killing, possibly due to diminished expression of the antiphagocytic hyaluronic acid capsule (14, 30). In contrast to these results, we previously found no contribution of the protease to invasive soft-tissue infection by using a different M type 3 GAS wild-type strain and an isogenic protease-deficient mutant, although in the same study, we observed a marked loss of virulence in mutants lacking either M3 protein or the hyaluronic acid capsule (4). Because SpeB is expressed predominantly during stationary-phase growth in vitro (6), it has been argued that the conflict between the results of our experiments and those of the previously cited studies is due to difference between the growth phases of the inocula used (14). Diminished virulence of protease-deficient GAS in vivo may only be seen when the bacterial challenge comprises organisms isolated during stationary-phase growth. To further investigate the role of the protease in GAS infection and to clarify both the potential effect of the loss of protease activity on capsule expression and the influence of the growth phase of the inoculum on the role of SpeB in invasive infection, we independently derived a protease-deficient mutant in the background of GAS M3 strain AM3 and determined its capsule expression, susceptibility to phagocytic killing, and virulence in mice. Our results indicate that the amount of cell-associated hyaluronic acid capsule is not influenced by the GAS cysteine protease. In addition, our findings demonstrate that, regardless of the growth phase of the bacterial inoculum, inactivation of speB does not significantly attenuate murine invasive infection after either an intraperitoneal or a subcutaneous challenge.