title: Genome-Wide Identification of Klebsiella pneumoniae Fitness Genes during Lung Infection.
abstract: Klebsiella pneumoniae is an urgent public health threat because of resistance to carbapenems, antibiotics of last resort against Gram-negative bacterial infections. Despite the fact that K. pneumoniae is a leading cause of pneumonia in hospitalized patients, the bacterial factors required to cause disease are poorly understood. Insertion site sequencing combines transposon mutagenesis with high-throughput sequencing to simultaneously screen thousands of insertion mutants for fitness defects during infection. Using the recently sequenced K. pneumoniae strain KPPR1 in a well-established mouse model of pneumonia, insertion site sequencing was performed on a pool of >25,000 transposon mutants. The relative fitness requirement of each gene was ranked based on the ratio of lung to inoculum read counts and concordance between insertions in the same gene. This analysis revealed over 300 mutants with at least a 2-fold fitness defect and 69 with defects ranging from 10- to >2,000-fold. Construction of 6 isogenic mutants for use in competitive infections with the wild type confirmed their requirement for lung fitness. Critical fitness genes included those for the synthesis of branched-chain and aromatic amino acids that are essential in mice and humans, the transcriptional elongation factor RfaH, and the copper efflux pump CopA. The majority of fitness genes were conserved among reference strains representative of diverse pathotypes. These results indicate that regulation of outer membrane components and synthesis of amino acids that are essential to its host are critical for K. pneumoniae fitness in the lung.
  
  
  OBJECTIVE
  Klebsiella pneumoniae is a bacterium that commonly causes pneumonia in patients after they are admitted to the hospital. K. pneumoniae is becoming resistant to all available antibiotics, and when these infections spread to the bloodstream, over half of patients die. Since currently available antibiotics are failing, we must discover new ways to treat these infections. In this study, we asked what genes the bacterium needs to cause an infection, since the proteins encoded by these genes could be targets for new antibiotics. We identified over 300 genes that K. pneumoniae requires to grow in a mouse model of pneumonia. Many of the genes that we identified are found in K. pneumoniae isolates from throughout the world, including antibiotic-resistant forms. If new antibiotics could be made against the proteins that these genes encode, they may be broadly effective against K. pneumoniae.
authors.affiliation: Department of Pathology, School of Medicine, University of Michigan, Ann Arbor, Michigan, USA mikebach@umich.edu.
authors.forename: Michael A
authors.identifier: http://orcid.org/0000-0003-2507-6987
authors.initials: MA
authors.lastname: Bachman
authors.affiliation: Department of Pathology, School of Medicine, University of Michigan, Ann Arbor, Michigan, USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Breen
authors.affiliation: Department of Microbiology and Immunology, School of Medicine, University of Michigan, Ann Arbor, Michigan, USA.
authors.forename: Valerie
authors.initials: V
authors.lastname: Deornellas
authors.affiliation: Department of Pathology, School of Medicine, University of Michigan, Ann Arbor, Michigan, USA.
authors.forename: Qiao
authors.initials: Q
authors.lastname: Mu
authors.affiliation: Department of Biostatistics, School of Public Heath, University of Michigan, Ann Arbor, Michigan, USA.
authors.forename: Lili
authors.initials: L
authors.lastname: Zhao
authors.affiliation: BRCF Bioinformatics Core, School of Medicine, University of Michigan, Ann Arbor, Michigan, USA.
authors.forename: Weisheng
authors.initials: W
authors.lastname: Wu
authors.affiliation: Department of Computational Medicine and Bioinformatics, School of Medicine, University of Michigan, Ann Arbor, Michigan, USA.
authors.forename: James D
authors.initials: JD
authors.lastname: Cavalcoli
authors.affiliation: Department of Microbiology and Immunology, School of Medicine, University of Michigan, Ann Arbor, Michigan, USA.
authors.forename: Harry L T
authors.initials: HL
authors.lastname: Mobley
mesh_terms: D000818:Animals; D058491:Bacterial Load; D004252:DNA Mutational Analysis; D004251:DNA Transposable Elements; D020869:Gene Expression Profiling; D054884:Host-Pathogen Interactions; D007710:Klebsiella Infections / Q000382:microbiology*; D007711:Klebsiella pneumoniae / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D008168:Lung / Q000382:microbiology*; D051379:Mice; D016254:Mutagenesis, Insertional; D018410:Pneumonia, Bacterial / Q000382:microbiology*; D014774:Virulence
chemical_list: D004251:DNA Transposable Elements