Tracking insertion mutants within libraries by deep sequencing and a genome-wide screen for Haemophilus genes required in the lung
UMass Chan AffiliationsDepartment of Molecular Genetics and Microbiology
MetadataShow full item record
AbstractRapid 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.
Proc Natl Acad Sci U S A. 2009 Sep 22;106(38):16422-7. Epub 2009 Sep 4. Link to article on publisher's site
Permanent Link to this Itemhttp://hdl.handle.net/20.500.14038/37359
See Additional Files below to download the informational figures (.pdf), the raw data (.txt), and the processed database data (.xls).
Related ResourcesLink to Article in PubMed
Publisher PDF posted as allowed by the publisher's author rights policy at http://www.pnas.org/site/misc/authorfaq.shtml.