Virulence gene identification by differential fluorescence induction analysis of Staphylococcus aureus gene expression during infection-simulating culture

Abstract

We have employed a strategy utilizing differential fluorescence induction (DFI) in an effort to identify Staphylococcus aureus genes whose products can be targeted for antimicrobial drug development. DFI allows identification of promoters preferentially active under given growth conditions on the basis of their ability to drive expression of a promoterless green fluorescent protein gene (gfp). A plasmid-based promoter trap library was constructed of 200- to 1,000-bp fragments of S. aureus genomic DNA fused to gfp, and clones with active promoters were isolated under seven different in vitro growth conditions simulating infection. Six thousand two hundred sixty-seven clones with active promoters were screened to identify those that exhibited differential promoter activity. Bioinformatic analysis allowed the identification of 42 unique operons, containing a total of 61 genes, immediately downstream of the differentially active putative promoters. Replacement mutations were generated for most of these operons, and the abilities of the resulting mutants to cause infection were assessed in two different murine infection models. Approximately 40% of the mutants were attenuated in at least one infection model.

FIG. 1.

S. aureus promoter trap library. The promoter trap shuttle vector pSE1 gfpm2 is shown. The portion of the vector derived from pBR322 is white, the portion from RN5543 is shaded gray, and gfp is black. The SmaI site into which the chromosomal DNA fragments were inserted is indicated.

FIG. 2.

Scheme for identification and isolation of clones showing differential gfp expression. Cells carrying the promoter trap library and cultured under an inducing condition were sorted by fluorescence-activated cell sorter in two rounds. The left histogram shows a comparison of the negative control cells with the vector only (shaded gray) with a cell population after a first sorting (black line). The right histogram shows a comparison of cell populations following a first sorting (black line) and a second sorting (shaded gray). Following the second sorting, cells were plated for single-colony isolation. Single colonies were then used to inoculate individual cultures for screening.

FIG. 3.

Fluorescence of selected library clones showing differential gfp expression. The histograms show the fluorescence of cells cultured under inducing (black) and noninducing (gray) conditions. Cells are strain 8325 carrying pSE1 gfpm2 with a fragment containing the DCS06/STS01 (A) or the LFe 06 (B) promoter fragment upstream of gfp.

FIG. 4.

Generation of S. aureus replacement mutants. In this example, mutants of a bicistronic operon are made by selecting for integration of a mutagenic plasmid into the chromosome. The double-crossover product replacement mutant is identified by screening for an Em^r Cm^s phenotype (see Materials and Methods).

FIG. 5.

Kidney abscess infections with selected S. aureus mutants. Infections were performed as described in Materials and Methods. Each data point represents the logarithm of the number of bacterial CFU recovered from the kidneys of a single mouse at 5 days postinfection. The horizontal bars represent the geometric means. The limit of detection in the experiment is 40 CFU.