title: Differential fluorescence induction analysis of Streptococcus pneumoniae identifies genes involved in pathogenesis.
abstract: Differential fluorescence induction (DFI) technology was used to identify promoters of Streptococcus pneumoniae induced under various in vitro and in vivo conditions. A promoter-trap library using green fluorescent protein as the reporter was constructed in S. pneumoniae, and the entire library was screened for clones exhibiting increased gfp expression under the chosen conditions. The in vitro conditions used were chosen to mimic aspects of the in vivo environment encountered by the pathogen once it enters a host: changes in temperature, osmolarity, oxygen, and iron concentration, as well as blood. In addition, the library was used to infect animals in three different models, and clones induced in these environments were identified. Several promoters were identified in multiple screens, and genes whose promoters were induced twofold or greater under the inducing condition were mutated to assess their roles in virulence. A total of 25 genes were mutated, and the effects of the mutations were assessed in at least two different infection models. Over 50% of these mutants were attenuated in at least one infection model. We show that DFI is a useful tool for identifying bacterial virulence factors as well as a means of elucidating the microenvironment encountered by pathogens upon infection.
authors.affiliation: Protein Design Labs, Inc., Fremont, California 94555, USA. andrea_marra@groton.pfizer.com
authors.forename: Andrea
authors.initials: A
authors.lastname: Marra
authors.forename: Jyoti
authors.initials: J
authors.lastname: Asundi
authors.forename: Magdalena
authors.initials: M
authors.lastname: Bartilson
authors.forename: Stacey
authors.initials: S
authors.lastname: Lawson
authors.forename: Flora
authors.initials: F
authors.lastname: Fang
authors.forename: Jillian
authors.initials: J
authors.lastname: Christine
authors.forename: Cedric
authors.initials: C
authors.lastname: Wiesner
authors.forename: Daniel
authors.initials: D
authors.lastname: Brigham
authors.forename: William P
authors.initials: WP
authors.lastname: Schneider
authors.forename: Alexander E
authors.initials: AE
authors.lastname: Hromockyj
mesh_terms: D000818:Animals; D015194:Diffusion Chambers, Culture; D004195:Disease Models, Animal; D005260:Female; D005453:Fluorescence; D015964:Gene Expression Regulation, Bacterial; D015723:Gene Library; D005798:Genes, Bacterial; D017930:Genes, Reporter; D005849:Gerbillinae; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008297:Male; D051379:Mice; D016296:Mutagenesis; D010033:Otitis Media / Q000209:etiology; D010529:Peritoneal Cavity / Q000382:microbiology; D011008:Pneumococcal Infections / Q000209:etiology*; D011401:Promoter Regions, Genetic; D012141:Respiratory Tract Infections / Q000209:etiology; D013296:Streptococcus pneumoniae / Q000235:genetics* / Q000472:pathogenicity*
chemical_list: D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins