Staphylococcus aureus virulence genes identified by bursa aurealis mutagenesis and nematode killing

Abstract

Staphylococcus aureus is the leading cause of wound and hospital-acquired infections worldwide. The emergence of S. aureus strains with resistance to multiple antibiotics requires the identification of bacterial virulence genes and the development of novel therapeutic strategies. Herein, bursa aurealis, a mariner-based transposon, was used for random mutagenesis and for the isolation of 10,325 S. aureus variants with defined insertion sites. By screening for loss-of-function mutants in a Caenorhabditis elegans killing assay, 71 S. aureus virulence genes were identified. Some of these genes are also required for S. aureus abscess formation in a murine infection model.

Fig. 1.

Plasmids used for transposon mutagenesis in S. aureus. (A) Bursa aurealis, a minimariner transposable element, was cloned into pTS2, with a temperature-sensitive plasmid replicon (rep_ts) and chloramphenicol resistance gene (cat) to generate pBursa; the 3.2-kb bursa aurealis encompasses the mariner terminal inverted repeats (TIR), the green fluorescent protein gene (gfp), the R6K replication origin (oriV), and the erythromycin-resistance determinant (ermB). The position of restriction enzyme recognition sites (AciI and BamHI) is indicated. (B) Plasmid pFA545 encodes the mariner transposase (tnp), the origin of replication (repC), and the tetracycline- and ampicillin-resistance markers (tetBD and bla, respectively). (C) The structure of the 5.3-kb transposon Tn917 with the ermB erythromycin-resistance determinant, tnpR resolvase, and tnpA transposase is shown.

Fig. 2.

Mapping insertion sites by inverse PCR. Fifteen bursa aurealis transposon mutants of S. aureus strain Newman were subjected to DNA purification, AciI restriction, fragment ligation, inverse PCR, and agarose gel electrophoresis. M indicates the molecular weight marker (1-kb DNA ladder).

Fig. 3.

Comparison of the transposon insertion sites of bursa aurealis (A) and Tn917 (B). S. aureus strain Newman was subjected to mutagenesis, and 585 insertion mutants of each transposon were mapped by DNA sequencing and aligned according to their insertion site on the genome of S. aureus strain Mu50.

Fig. 4.

Bursa aurealis transposon insertions in the genome of S. aureus strain Newman. Four regions of the genome, in which very few transposon mutants were isolated, are indicated by blue bars with the names of the genes residing in each of the four regions. The first (outer) circle represents ORFs that were disrupted (black) or not disrupted (white) by bursa aurealis. The second and third circles represent ORFs in the genome of S. aureus strain Mu50, in which different colors were assigned according to their functions. Green and red arrows indicate tRNA and rRNA, respectively. The fourth circle shows the scale of the map (in bp). The fifth (inner) circle is a circular bar graph showing the number of transposon mutants per ORF (scale bar with each ladder step representing 10 mutants). Sccmec, staphylococcal cassette chromosome mec, and SaPIn1, staphylococcal pathogenicity island 1, are indicated. Bursa aurealis insertions (116) were isolated at the same position within thiD, but these insertions are likely to represent siblings, because the isolated mutants arose from the same batch of transposon insertion mutants.

Fig. 5.

Identification of S. aureus virulence genes in a mouse model of infection. S. aureus strain Newman or bursa aurealis insertion mutants ΦNΞ12478 (A) or ΦNΞ02863 (B) were used to infect 10 mice each via retroorbital injection [≈1.3 × 10⁷ colony-forming units (cfu)]. After 4 days, mice were killed and organs (kidney and liver) were removed. Homogenized tissues were incubated on agar medium for S. aureus colony formation and enumeration. Each circle indicates an animal experiment. The dashed line represents the limit of detection, and horizontal bars indicate the mean.