title: Identification of Klebsiella pneumoniae virulence determinants using an intranasal infection model.
abstract: Klebsiella pneumoniae is a Gram-negative enterobacterium that has historically been, and currently remains, a significant cause of human disease. It is a frequent cause of urinary tract infections and pneumonia, and subsequent systemic infections can have mortality rates as high as 60%. Despite its clinical significance, few virulence factors of K. pneumoniae have been identified or characterized. In this study we present a mouse model of acute K. pneumoniae respiratory infection using an intranasal inoculation method, and examine the progression of both pulmonary and systemic disease. Wild-type infection recapitulates many aspects of clinical disease, including significant bacterial growth in both the trachea and lungs, an inflammatory immune response characterized by dramatic neutrophil influx, and a steady progression to systemic disease with ensuing mortality. These observations are contrasted with an infection by an isogenic capsule-deficient strain that shows an inability to cause disease in either pulmonary or systemic tissues. The consistency and clinical accuracy of the intranasal mouse model proved to be a useful tool as we conducted a genetic screen to identify novel virulence factors of K. pneumoniae. A total of 4800 independent insertional mutants were evaluated using a signature-tagged mutagenesis protocol. A total of 106 independent mutants failed to be recovered from either the lungs or spleens of infected mice. Small scale independent infections proved to be helpful as a secondary screening method, as opposed to the more traditional competitive index assay. Those mutants showing verified attenuation contained insertions in loci with a variety of putative functions, including a large number of hypothetical open reading frames. Subsequent experiments support the premise that the central mechanism of K. pneumoniae pathogenesis is the production of a polysaccharide-rich cell surface that provides protection from the inflammatory response.
authors.affiliation: Department of Molecular Microbiology, Washington University School of Medicine, St Louis, MO 63110, USA.
authors.forename: Matthew S
authors.initials: MS
authors.lastname: Lawlor
authors.forename: James
authors.initials: J
authors.lastname: Hsu
authors.forename: Paul D
authors.initials: PD
authors.lastname: Rick
authors.forename: Virginia L
authors.initials: VL
authors.lastname: Miller
mesh_terms: D000818:Animals; D016470:Bacteremia / Q000382:microbiology / Q000473:pathology; D016667:Bacterial Capsules / Q000276:immunology; D015169:Colony Count, Microbial; D004195:Disease Models, Animal; D005260:Female; D005798:Genes, Bacterial; D007710:Klebsiella Infections / Q000382:microbiology* / Q000473:pathology; D007711:Klebsiella pneumoniae / Q000235:genetics / Q000472:pathogenicity*; D008168:Lung / Q000382:microbiology / Q000473:pathology; D051379:Mice; D016254:Mutagenesis, Insertional; D016366:Open Reading Frames / Q000235:genetics; D018410:Pneumonia, Bacterial / Q000382:microbiology / Q000473:pathology; D012141:Respiratory Tract Infections / Q000382:microbiology* / Q000473:pathology; D013154:Spleen / Q000382:microbiology; D014132:Trachea / Q000382:microbiology / Q000473:pathology; D014574:Uronic Acids / Q000032:analysis; D037521:Virulence Factors / Q000032:analysis*
chemical_list: D014574:Uronic Acids; D037521:Virulence Factors