Virulence gene identification by differential fluorescence induction analysis of Staphylococcus aureus gene expression during infection-simulating culture.
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.
Schneider
William P
WP
Protein Design Labs, Inc., Fremont, California 94555, USA. bschneider@pdl.com
Ho
Sun K
SK
Christine
Jillian
J
Yao
Monique
M
Marra
Andrea
A
Hromockyj
Alexander E
AE
D000038:Abscess / Q000382:microbiology; D000818:Animals; D001431:Bacteriological Techniques; D005260:Female; D005453:Fluorescence; D020869:Gene Expression Profiling / Q000379:methods*; D005798:Genes, Bacterial; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D007674:Kidney Diseases / Q000382:microbiology; D008164:Luminescent Proteins; D051379:Mice; D011401:Promoter Regions, Genetic; D013203:Staphylococcal Infections / Q000382:microbiology; D013211:Staphylococcus aureus / Q000235:genetics* / Q000472:pathogenicity*; D014774:Virulence / Q000235:genetics
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins