A locus of group A streptococcus involved in invasive disease and DNA transfer

Abstract

SummaryGroup A streptococcus (GAS) causes diseases ranging from benign to severe infections such as necrotizing fasciitis (NF). The reasons for the differences in severity of streptococcal infections are unexplained. We developed the polymorphic-tag-lengthstransposon-mutagenesis (PTTM) method to identify virulence genes in vivo . We applied PTTM on an emm 14 strain isolated from a patient with NF and screened for mutants of decreased virulence, using a mouse model of human soft-tissue infection. A mutant that survived in the skin but was attenuated in its ability to reach the spleen and to cause a lethal infection was identified. The transposon was inserted into a small open reading frame (ORF) in a locus termed sil , s treptococcal i nvasion l ocus. sil contains at least five genes ( sil A-E) and is highly homologous to the quorum-sensing competence regulons of Streptococcus pneumoniae . sil A and sil B encode a putative two-component system whereas sil D and sil E encode two putative ABC transporters. sil C is a small ORF of unknown function preceded by a combox promoter. Insertion and deletion mutants of sil had a diminished lethality in the animal model. Virulence of a deletion mutant of sil C was restored when injected together with the avirulent emm 14-deletion mutant, but not when these mutants were injected into opposite flanks of a mouse. DNA transfer between these mutants occurred in vivo but could not account for the complementation of virulence. DNA exchange between the emm 14-deletion mutant and mutants of sil occurred also in vitro, at a frequency of ~ 10 ----8 for a single antibiotic marker. Whereas sil C and sil D mutants exchanged markers with the emm 14 mutant, sil B mutant did not. Thus, we identified a novel locus, which controls GAS spreading into deeper tissues and could be involved in DNA transfer.

Recruited Macrophages Control Dissemination of Group A Streptococcus from Infected Soft Tissues

“…inoculation with serotypes causing invasive GAS diseases in human (13,21,24,25,29,30,43,44). Studies based on the above-mentioned murine models demonstrated that PMNs constitute an essential defense component preventing the transition of GAS from local to disseminated infection and, hence, GAS produces a plethora of virulence factors that hamper PMN responses (11)(12)(13)(14).…”

“…The experiments were conducted using GAS strains JS95 of the M14 serotype (21) and 5448 of the M1T1 serotype (22) GAS strains were cultured in Todd-Hewitt medium supplemented with 0.2% yeast extract (Difco, BD Diagnostics) or on blood agar plates (Novamed, Jerusalem, Israel).…”

“…The murine model of human soft tissue infection was performed, as detailed previously (21,24,25). Briefly, GAS strains were cultured at 37˚C in Todd-Hewitt medium supplemented with 0.2% yeast extract and grown to OD 600 = 0.3-0.4.…”

“…The ability of GAS to survive in mouse blood was tested by the direct bactericidal test of Lancefield, as we described previously (21).…”

Molecular Epidemiology of sil Locus in Clinical Streptococcus pyogenes Strains

“…Hidalgo-Grass et al identified the streptococcal invasion locus (sil) using the polymorphic tag-length transposon mutagenesis (PTTM) method on JS95, an emm14 GAS strain isolated from a patient with NF (13). In this strain, the sil locus was shown to control GAS spread into deeper tissues in a mouse model of human soft tissue infection and to be involved in DNA transfer between two emm14 strains (13).…”