Group A Streptococcus (GAS), a Gram-positive bacterium, is the causative agent of a wide spectrum of diseases in its human host. Whereas most GAS diseases are limited to superficial site infections, such as the pharyngeal mucosa (“strep throat”) and skin (impetigo), the organism is also a leading agent of invasive diseases, such as the life-threatening toxic shock syndrome (TSS) and necrotizing soft-tissue infections. The latter consist of a range of rapidly progressive diseases including necrotizing fasciitis (NF), a deep-seated infection of subcutaneous tissue that can occur in healthy children and adults and that results in progressive and rapid destruction of fascia and fat. Because of these devastating symptoms, NF is known to the public as “flesh-eating disease.” Despite prompt antibiotic treatment and surgical debridement, GAS TSS and NF are associated with high death rates, ranging from 20 to 60 percent. The worldwide burden of invasive GAS infections is estimated to be more than 650,000 cases annually, with 150,000 deaths.
GAS strains of many serotypes are capable of producing invasive infections; however, M1 and M3 are the most prevalent sterile-site isolates worldwide. In a prospective population-based study of invasive GAS infections in Israel, we found that M14 strains cause a high proportion of NF. These clusters of M14 NF-causing isolates suggested a gain of enhanced virulence due to either acquisition of novel virulence genes or a change in gene expression profiles. To identify the genes involved in the highly virulent phenotype, we conducted an in vivo screen of transposon-tagged mutants in a mixed population generated for one of the M14 NF-causing strains. We used a murine model mimicking human NF with respect to rapid spread of necrosis of soft tissues from the subcutaneous site of inoculation, which is characterized by a paucity of neutrophil infiltration.
Thein vivoscreening of a transposon-tagged mutant library identified a mutant that was attenuated in its ability to spread from the skin to the spleen and to cause a lethal infection. Mapping of the insertion site of the transposon implicated a genetic locus that we termed sil, for streptococcal invasion locus, which shares homology with the quorum-sensing competence (com)andbacteriocin-like peptide(blp) regulons of Streptococcus pneumoniae. These quorum-sensing regulons enable S. pneumoniae and several other streptococcal species to interchange their genetic material, develop tolerance to acid, and form a biofilm. These complex processes are primarily initiated by a 17-residue unmodified peptide termed competence-stimulating peptide (CSP), which is matured and secreted by a dedicated ATP-binding cassette (ABC) transporter. Upon interaction with the sensor of a two-component system (TCS), theCSP of S. pneumoniae activates the expression of both the ABC transporter and the TCS, forming an autocatalytic circuit.
The sil locus consists of the TCS SilA/B and the ABC transporter SilD/E. Situated between these entities is a unique small open reading frame, silC. Overlapping silC on the complementary strand by more than 70 percent, silCR encodes a predicted CSP consisting of a mature peptide of 17 residues (SilCR). However, in the M14 strain, a missense mutation changes the AUG start codon of silCR to AUA, suggesting that SilCR is not translated.
The location of the transposon in the sil-attenuating mutation is in silC, disrupting both silC and silCR (see figure). A deletion mutant lacking silC (ΔsilC) exhibits less virulence than the original transposon-attenuated mutant. SilCR is probably not produced by the M14 type strain because of the start codon mutation (see figure), which implies that silC promotes virulence. Although silC might promote virulence, synthetic SilCR obliterates virulence when coinjected into mice with the M14 strain. A key molecular mechanism for this effect is the production of a serine-type protease, termed ScpC, that specifically cleaves the C terminus of CXC chemokines, impairing neutrophil functions. SilCR treatment downregulates ScpC activity, increasing neutrophil-mediated killing of bacteria. Loss of ScpC expression dramatically reduces GAS virulence in the mouse NF model as a result of an increased neutrophil influx to the site of infection.
To unravel the mechanism by which silC and SilCR exert opposite effects on GAS virulence, we studied the transcriptional regulation of the sil locus and found that silC and silCR form a novel regulatory circuit that is controlled by the SilCR peptide. Addition of SilCR autocatalytically upregulates the transcription of silE/D/CR from the P3 promoter (see figure), obliterating silC transcription. We found that 18 percent of clinically relevant GAS strains possess the sil locus and produce SilCR.
We plan to fully characterize the mode by which sil regulates GAS virulence through SilCR and to characterize the CXC chemokine protease ScpC, which is highly conserved among all GAS clinical isolates. We hope to achieve these goals by combining genetic and biochemical techniques, a mouse model of human invasive soft-tissue infections, and genome-wide transcriptional profiling. Our studies will elucidate novel aspects of the regulation of GAS virulence and generate new targets for the prevention and therapy of a life-threatening human disease.
Last updated October 2008
