Large-scale identification of genes required for full virulence of Staphylococcus aureus

Abstract

Gene products required for in vivo growth and survival of microbial pathogens comprise a unique functional class and may represent new targets for antimicrobial chemotherapy, vaccine construction, or diagnostics. Although some factors governing Staphylococcus aureus pathogenicity have been identified and studied, a comprehensive genomic analysis of virulence functions will be a prerequisite for developing a global understanding of interactions between this pathogen and its human host. In this study, we describe a genetic screening strategy and demonstrate its use in screening a collection of 6,300 S. aureus insertion mutants for virulence attenuation in a murine model of systemic infection. Ninety-five attenuated mutants were identified, reassembled into new pools, and rescreened using the same murine model. This effort identified 24 highly attenuated mutants, each of which was further characterized for virulence attenuation in vivo and for growth phenotypes in vitro. Mutants were recovered in numbers up to 1,200-fold less than wild type in the spleens of systemically infected animals and up to 4,000-fold less than wild type in localized abscess infections. Genetic analysis of the mutants identified insertions in 23 unique genes. The largest gene classes represented by these mutants encoded enzymes involved in small-molecule biosynthesis and cell surface transmembrane proteins involved in small-molecule binding and transport. Additionally, three insertions defined two histidine kinase sensor-response regulator gene pairs important for S. aureus in vivo survival. Our findings extend the understanding of pathogenic mechanisms employed by S. aureus to ensure its successful growth and survival in vivo. Many of the gene products we have identified represent attractive new targets for antibacterial chemotherapy.

FIG. 1.

Isolation of DNA tags, strain construction, mutagenesis, and screening strategy. (A) Isolation of DNA SMIT tags and integration into the S. aureus chromosome. Tags were generated by digestion of salmon sperm DNA with Sau3AI, size fractionated by agarose gel electrophoresis, and isolated by cloning into the BamHI site of pCL84 (see Materials and Methods). Twenty-five unique tag clones were selected, and the tags were individually subcloned into the BamHI site of pMP820. Schematic maps of S. aureus integrative plasmids pCL84 and pMP820 are shown. The 50 tag clones, 25 constructed in pCL84 and 25 constructed in pMP820, were introduced individually into the S. aureus 8325-4 chromosome at the geh locus (see Materials and Methods), creating 50 isogenic SMIT-tagged S. aureus strains. (B) Generation of S. aureus insertion mutant banks and pools. Transposon delivery plasmids pI258repA36 Ω1(Tn551) and pTV32ts, introduced individually into each of the 50 tagged S. aureus strains, were used to create banks of 50 Tn551 and 50 Tn917lac insertion mutants, respectively. Pools of up to 50 S. aureus mutants were generated by combining 25 pCL84::SMIT-tagged mutants with 25 pMP820::SMIT-tagged mutants. (C) In vivo screening strategy. Inocula were prepared from subcultured mutant pools and used to establish systemic infections in mice. At 48 h postchallenge, animals were sacrificed and spleens were dissected and homogenized. Viable bacterial cells were isolated by plating aliquots of inoculum (input) and spleen (recovered) samples from each pool of mutant cells. Extracted chromosomal DNA was used as template for PCR amplification of SMIT tags, which were subsequently resolved by polyacrylamide gel electrophoresis and visualized by staining with silver.

FIG. 2.

Limiting dilution SMIT PCR amplification and detection. Genomic DNA was isolated from 25 pooled pCL84::SMIT-tagged S. aureus strains grown individually and combined in equal portions (lane 1) or combined so that 24 strains were represented equally and one strain (arrow) was represented at 50% (lane 2), 25% (lane 3), 12.5% (lane 4), 6.25% (lane 5), or 3.12% (lane 6) relative to each of the other 24 strains. Products of tag amplification reactions were resolved by polyacrylamide gel electrophoresis and visualized by staining with silver. Positions of migration of double-stranded 100-bp-ladder DNA size markers are indicated (in base pairs).

FIG. 3.

Identification of virulence-attenuated mutants. Products of tag amplification reactions were resolved by polyacrylamide gel electrophoresis and visualized by staining with silver. Shown are input tags (lane I) and recovered tags (lane R) derived from a pool (same pool for both panels) plated on TSA medium containing Tc (A) or containing Cm (B). Attenuated mutants in panel B are indicated by arrows. Positions of migration of double-stranded 100-bp-ladder DNA size markers are indicated (in base pairs).