Streptococcus pyogenes with null mutations in the csrRS regulatory locus are highly virulent in mice due to derepression of hyaluronic acid capsule synthesis and exotoxins, e.g., streptolysin S (SLS) and pyrogenic exotoxin B (SpeB). We generated derivatives of a ⌬csrRS strain that also carry deletions in hasAB (leading to an acapsular phenotype) or in sagA (phenotypically SLS ؊ ) or an interruption of speB (SpeB ؊ ) to test the relative contributions of these factors to the development of necrotic skin lesions. Inoculation of 2 ؋ 10 6 to 4 ؋ 10 6 CFU of either acapsular or SLS ؊ strains into hairless mice resulted in lesions ϳ70% smaller than those of the ⌬csrRS parent strain. Elimination of SLS also reduced lethality from 100% to 0% at this inoculum (P < 10 ؊7 ; Fisher exact test). In contrast, SLS ؉ SpeB ؊ mutants yielded lesions that were only 41% smaller than the parent strain (t ‫؍‬ 2.2; P ‫؍‬ 0.04), but only 3 the 17 lesions had dermal sloughing (P ‫؍‬ 10 ؊5 ). The nonulcerative lesions associated with SpeB ؊ strains appeared pale with surrounding erythema. We conclude that capsule and SLS contribute to the subcutaneous spread of S. pyogenes and to a fatal outcome of infection. SpeB facilitates early dermal ulceration but has minor influence on lesion size and mortality. Large ulcerative lesions are observed only when both toxins are present.Enhanced virulence of Streptococcus pyogenes csrR mutants is associated with increased expression of the hyaluronic acid capsule and several exotoxins, most notably streptolysin S (SLS) and pyrogenic exotoxin B (SpeB, or cysteine proteinase) (12). In the hairless mouse model of streptococcal skin infection, csrR mutants produce rapidly expanding, necrotic lesions, whereas the CsrR ϩ wild-type M1 strain from which they are derived induces only small abscesses or self-limited erythema.There is little disagreement about the importance of the hyaluronic acid capsule in streptococcal pathogenesis; however, significant controversy exists concerning the importance of many of the other CsrRS-regulated gene products in virulence. Several research groups have characterized SLS as a key factor contributing to virulence (4,17,28). In contrast, discrepancies have been reported concerning the importance of cysteine proteinase in studies with speB mutants (2,3,21,24,32,34). We hypothesized that the incremental pathogenicity associated with the mutations in csrR depends on the expression of these regulated virulence factors. Accordingly, we characterized here the impact of these toxins on murine infection by deleting them individually or in combination in S. pyogenes strains lacking CsrR. We show an additive effect of hyaluronic acid capsule, SLS, and SpeB on mouse virulence, and we observe that these factors contribute in different ways to the character, extent, and lethality of the skin lesions. MATERIALS AND METHODSAll experiments used derivatives of a type M1 isolate, MGAS166 (27). Streptococci were stored at Ϫ70°C and passaged minimally before and after genetic manipulations. Tod...