Display Settings:

Format

Send to:

Choose Destination
See comment in PubMed Commons below
Proc Natl Acad Sci U S A. 2009 Sep 22;106(38):16422-7. doi: 10.1073/pnas.0906627106. Epub 2009 Sep 4.

Tracking insertion mutants within libraries by deep sequencing and a genome-wide screen for Haemophilus genes required in the lung.

Author information

  • 1Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, 55 Lake Avenue North, S6-242, Worcester, MA 01655, USA.

Abstract

Rapid genome-wide identification of genes required for infection would expedite studies of bacterial pathogens. We developed genome-scale "negative selection" technology that combines high-density transposon mutagenesis and massively parallel sequencing of transposon/chromosome junctions in a mutant library to identify mutants lost from the library after exposure to a selective condition of interest. This approach was applied to comprehensively identify Haemophilus influenzae genes required to delay bacterial clearance in a murine pulmonary model. Mutations in 136 genes resulted in defects in vivo, and quantitative estimates of fitness generated by this technique were in agreement with independent validation experiments using individual mutant strains. Genes required in the lung included those with characterized functions in other models of H. influenzae pathogenesis and genes not previously implicated in infection. Genes implicated in vivo have reported or potential roles in survival during nutrient limitation, oxidative stress, and exposure to antimicrobial membrane perturbations, suggesting that these conditions are encountered by H. influenzae during pulmonary infection. The results demonstrate an efficient means to identify genes required for bacterial survival in experimental models of pathogenesis, and this approach should function similarly well in selections conducted in vitro and in vivo with any organism amenable to insertional mutagenesis.

PMID:
19805314
[PubMed - indexed for MEDLINE]
PMCID:
PMC2752563
Free PMC Article
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for HighWire Icon for PubMed Central
    Loading ...
    Write to the Help Desk