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1.
Microbiology (Reading) ; 148(Pt 5): 1483-91, 2002 May.
Article in English | MEDLINE | ID: mdl-11988523

ABSTRACT

Differential fluorescence induction technology was used to identify promoters of Streptococcus pneumoniae genes that are expressed during lung infection of the mouse. Among the promoter clones that were identified multiple times was the psa promoter, which drives expression of the psaBCA operon. These genes have been identified previously and shown to encode a manganese permease system as well as play a role in the virulence of this organism. Mutations in psaB, psaC or psaA result in growth limitation in low manganese. The expression of the psa operon was examined in vivo and the virulence of deletion mutants of psaB, psaC, psaA and psaBCA was assessed in four different animal models of infection. The psa promoter was induced more than ten-fold in vivo using an intraperitoneal chamber implant model. The psaB, psaC and psaA mutants were completely attenuated in systemic, respiratory tract and otitis media infections. In addition, these mutants were unable to grow in an implanted peritoneal chamber, but growth was restored by the addition of manganese to the chambers.


Subject(s)
ATP-Binding Cassette Transporters/genetics , Bacterial Proteins , Disease Models, Animal , Membrane Transport Proteins , Otitis Media/microbiology , Promoter Regions, Genetic/genetics , Respiratory Tract Infections/microbiology , Streptococcus pneumoniae/genetics , Adhesins, Bacterial , Animals , Carrier Proteins/genetics , Diffusion Chambers, Culture , Gene Deletion , Gene Library , Green Fluorescent Proteins , Implants, Experimental , Lethal Dose 50 , Lipoproteins/genetics , Luminescent Proteins , Manganese/pharmacology , Membrane Proteins/genetics , Mice , Peritoneal Cavity/microbiology , Streptococcus pneumoniae/drug effects , Streptococcus pneumoniae/growth & development , Streptococcus pneumoniae/pathogenicity
2.
Infect Immun ; 70(3): 1422-33, 2002 Mar.
Article in English | MEDLINE | ID: mdl-11854229

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.


Subject(s)
Genes, Bacterial , Pneumococcal Infections/etiology , Streptococcus pneumoniae/genetics , Streptococcus pneumoniae/pathogenicity , Animals , Diffusion Chambers, Culture , Disease Models, Animal , Female , Fluorescence , Gene Expression Regulation, Bacterial , Gene Library , Genes, Reporter , Gerbillinae , Green Fluorescent Proteins , Luminescent Proteins , Male , Mice , Mutagenesis , Otitis Media/etiology , Peritoneal Cavity/microbiology , Promoter Regions, Genetic , Respiratory Tract Infections/etiology
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