Single-nucleotide changes are the most common cause of natural genetic variation among members of the same species, but there is remarkably little information bearing on how they alter bacterial virulence. We recently discovered a single-nucleotide mutation in the group A Streptococcus genome that is epidemiologically associated with decreased human necrotizing fasciitis ("flesh-eating disease"). Working from this clinical observation, we find that wild-type mtsR function is required for group A Streptococcus to cause necrotizing fasciitis in mice and nonhuman primates. Expression microarray analysis revealed that mtsR inactivation results in overexpression of PrsA, a chaperonin involved in posttranslational maturation of SpeB, an extracellular cysteine protease. Isogenic mutant strains that overexpress prsA or lack speB had decreased secreted protease activity in vivo and recapitulated the necrotizing fasciitis-negative phenotype of the ΔmtsR mutant strain in mice and monkeys. mtsR inactivation results in increased PrsA expression, which in turn causes decreased SpeB secreted protease activity and reduced necrotizing fasciitis capacity. Thus, a naturally occurring single-nucleotide mutation dramatically alters virulence by dysregulating a multiple gene virulence axis. Our discovery has broad implications for the confluence of population genomics and molecular pathogenesis research.group A streptococcus | invasive infection | molecular epidemiology of strain genotype patient phenotype relationships | nonhuman primate S ingle-nucleotide mutations are the most abundant cause of genetic variation among members of the same species (1, 2). However, in striking contrast to humans, who have been studied extensively, our understanding of how naturally occurring singlenucleotide mutations alter bacterial phenotypes is rudimentary. Most prokaryotic pathogenesis research efforts have focused intensively on large regions of genetic difference, such as pathogenicity islands and prophages. Thus, there is little information that directly bears on the relationship between particular singlenucleotide changes, their direct or indirect effect on virulence factor expression, and the manifestation of medically important traits such as strain virulence and infection specificity.Recently, we have investigated the molecular genomic landscape of infection phenotype-strain genotype relationships in human patients at the nucleotide level in group A Streptococcus (GAS), a bacterial pathogen that is a major cause of human morbidity and mortality worldwide (1, 3-5). These studies were made possible by the availability of the 1.9-Mb genome sequences of 12 GAS strains cultured from patients with welldefined clinical syndromes such as pharyngitis, acute rheumatic fever, and necrotizing fasciitis (also known as "flesh-eating" disease) (1, 5). The core genome of strains of distinct M protein serotype differed, on average, by 14,475 SNPs (1, 3). In contrast, strains with the same M protein serotype were far less variable, differing overall by less...
