First Streptococcus pyogenes signature-tagged mutagenesis screen identifies novel virulence determinants

Abstract

The virulence of bacterial pathogens is a complex process that requires the dynamic expression of many genes for the pathogens to invade and circumvent host defenses, as well as to proliferate in vivo. In this study, we employed a large-scale screen, signature-tagged mutagenesis (STM), to identify Streptococcus pyogenes virulence genes important for pathogenesis within the host. Approximately 1,200 STM mutants were created and screened using the zebrafish infectious disease model. The transposon insertion site was identified for 29 of the 150 mutants that were considered attenuated for virulence. Previously reported streptococcal virulence genes, such as mga, hasA, amrA, smeZ, and two genes in the sil locus, were identified, confirming the utility of the model for revealing genes important for virulence. Multiple genes not previously implicated in virulence were also identified, including genes encoding putative transporters, hypothetical cytosolic proteins, and macrolide efflux pumps. The STM mutant strains display various levels of attenuation, and multiple separate insertions were identified in either the same gene or the same locus, suggesting that these factors are important for this type of acute, invasive infection. We further examined two such genes, silB and silC of a putative quorum-sensing regulon, and determined that they are significant virulence factors in our model of necrotizing fasciitis. sil locus promoter expression was examined under various in vitro conditions, as well as in zebrafish tissues, and was found to be differentially induced. This study was a unique investigation of S. pyogenes factors required for successful invasive infection.

FIG. 1.

Diagram of STM. Mutant strains were pooled, and a mixed inoculum containing 10⁵ CFU was injected into the dorsal muscle of four zebrafish. At 24 h, zebrafish were euthanized, and bacterial DNA was isolated from the homogenized spleens and muscles of three fish. Both input and output pools were subjected to PCR analysis with a specific primer for each of the 12 tags (lanes 1 to 12). The gel shows that the STM-2 clone was missing from the output pools of both organs of all three fish but was present in the input pool. Also missing were STM-9 and STM-11, but since they were also missing from the input (in vitro) pool, they were not considered further. Lanes L1 and L2, DNA ladder; lane C, positive control amplified from an STM plasmid. M, muscle; S, spleen.

FIG. 2.

Zebrafish histology after S. pyogenes infection. Tissue sections were prepared from the dorsal muscle 24 h postinfection with 10⁵ CFU wild-type bacteria (A), a silB mutant (B), and a silC mutant (C) and stained with hematoxylin and eosin. The arrows in panel A point to aggregates of bacteria, and the arrowheads in panels B and C point to inflammatory cells at the site of infection. Magnification, ×1,000.

FIG. 3.

Analysis of sil promoter expression. (A) Response to temperature. Overnight cultures of the wild-type strain carrying the sil promoter construct were grown at 37°C and 30°C (*, P < 0.0001). (B) Response to oxygen tension and agitation. The HSC5 wild-type strain carrying the sil promoter construct was cultured in 10 ml of TP under the following conditions: in a 15-ml conical tube without shaking, in a 15-ml conical tube with shaking, or in a 50-ml flask with shaking overnight (*, P < 0.0001). (C) Response to H₂O₂ treatment. The wild-type strain carrying the sil promoter construct was grown to mid-log phase and then treated with 5 mM or 7 mM H₂O₂ for 2 h at 37°C (*, P < 0.001). (D) Response to growth phase. The wild-type strain (dark gray bars), the silC mutant strain (light gray bars), and the silB mutant strain (black bars) carrying the sil promoter construct were assayed for activity at different times during growth.

FIG. 4.

Analysis of sil promoter expression in vivo. Following 5 h of intramuscular infection with 10⁵ CFU of a strain carrying the sil promoter construct, zebrafish sections were prepared and stained with antibodies against S. pyogenes (A and C) and alkaline phosphatase (B and D), as described in Materials and Methods. Magnification, ×400.

FIG. 5.

Analysis of sil promoter expression in vivo. Following 24 h of intramuscular infection with 10⁵ CFU of a strain carrying the sil promoter construct, zebrafish sections were prepared and stained with antibodies against S. pyogenes (A and C) and alkaline phosphatase (B and D), as described in Materials and Methods. Magnification, ×400.