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. IMPORTANCE 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. 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.

[1]  W. Vogel,et al.  Levels of amino acids and related compounds in bronchoalveolar lavage fluids of asthmatic patients. , 1997, American journal of respiratory and critical care medicine.

[2]  J. Hacker,et al.  Loss of Regulatory Protein RfaH Attenuates Virulence of Uropathogenic Escherichia coli , 2002, Infection and Immunity.

[3]  Kael F. Fischer,et al.  Combining Quantitative Genetic Footprinting and Trait Enrichment Analysis to Identify Fitness Determinants of a Bacterial Pathogen , 2013, PLoS genetics.

[4]  M. Kiupel,et al.  Branched-Chain Amino Acids Are Required for the Survival and Virulence of Actinobacillus pleuropneumoniae in Swine , 2009, Infection and Immunity.

[5]  Q. Gao,et al.  RfaH Promotes the Ability of the Avian Pathogenic Escherichia coli O2 Strain E058 To Cause Avian Colibacillosis , 2013, Journal of bacteriology.

[6]  Ling-Hui Li,et al.  Genome Sequencing and Comparative Analysis of Klebsiella pneumoniae NTUH-K2044, a Strain Causing Liver Abscess and Meningitis , 2009, Journal of bacteriology.

[7]  C. Dozois,et al.  Klebsiella pneumoniae Yersiniabactin Promotes Respiratory Tract Infection through Evasion of Lipocalin 2 , 2011, Infection and Immunity.

[8]  B. Wanner,et al.  One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[9]  R. Olsen,et al.  Molecular dissection of the evolution of carbapenem-resistant multilocus sequence type 258 Klebsiella pneumoniae , 2014, Proceedings of the National Academy of Sciences.

[10]  John Carbon,et al.  A colony bank containing synthetic CoI EI hybrid plasmids representative of the entire E. coli genome , 1976, Cell.

[11]  D. Balestrino,et al.  Identification of Klebsiella pneumoniae Genes Involved in Intestinal Colonization and Adhesion Using Signature-Tagged Mutagenesis , 2002, Infection and Immunity.

[12]  M. Pallen,et al.  Down-Regulation of Key Virulence Factors Makes the Salmonella enterica Serovar Typhimurium rfaH Mutant a Promising Live-Attenuated Vaccine Candidate , 2006, Infection and Immunity.

[13]  J. M. Bell,et al.  Amino acid requirements of the growing mouse. , 1976, Journal of NutriLife.

[14]  V. L. Miller,et al.  Identification of Klebsiella pneumoniae virulence determinants using an intranasal infection model , 2005, Molecular microbiology.

[15]  Weisheng Wu,et al.  Complete Genome Sequence of Klebsiella pneumoniae Strain ATCC 43816 KPPR1, a Rifampin-Resistant Mutant Commonly Used in Animal, Genetic, and Molecular Biology Studies , 2014, Genome Announcements.

[16]  Chia-Tung Shun,et al.  A Novel Virulence Gene in Klebsiella pneumoniae Strains Causing Primary Liver Abscess and Septic Metastatic Complications , 2004, The Journal of experimental medicine.

[17]  C. Struve,et al.  Application of a novel multi-screening signature-tagged mutagenesis assay for identification of Klebsiella pneumoniae genes essential in colonization and infection. , 2003, Microbiology.

[18]  V. L. Miller,et al.  Mucosal Lipocalin 2 Has Pro-Inflammatory and Iron-Sequestering Effects in Response to Bacterial Enterobactin , 2009, PLoS pathogens.

[19]  Leopold Parts,et al.  Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants. , 2009, Genome research.

[20]  Lloyd H. Michael,et al.  The Guide for the Care and Use of Laboratory Animals. , 2016, ILAR journal.

[21]  Chuan-Yun Li,et al.  KOBAS 2.0: a web server for annotation and identification of enriched pathways and diseases , 2011, Nucleic Acids Res..

[22]  Allan Matte,et al.  Structures of Shikimate Dehydrogenase AroE and Its Paralog YdiB , 2003, Journal of Biological Chemistry.

[23]  A. Yang,et al.  Amino Acid Substitutions of MagA in Klebsiella pneumoniae Affect the Biosynthesis of the Capsular Polysaccharide , 2012, PloS one.

[24]  Irina Artsimovitch,et al.  An α Helix to β Barrel Domain Switch Transforms the Transcription Factor RfaH into a Translation Factor , 2012, Cell.

[25]  R. Podschun,et al.  Klebsiella spp. as Nosocomial Pathogens: Epidemiology, Taxonomy, Typing Methods, and Pathogenicity Factors , 1998, Clinical Microbiology Reviews.

[26]  D. Wei,et al.  Genetics and Molecular Biology of Industrial Organisms Red Recombinase Assisted Gene Replacement in Klebsiella Pneumoniae , 2022 .

[27]  J. Gordon,et al.  Identifying microbial fitness determinants by insertion sequencing using genome-wide transposon mutant libraries , 2011, Nature Protocols.

[28]  Lin Wan,et al.  CEDER: Accurate Detection of Differentially Expressed Genes by Combining Significance of Exons Using RNA-Seq , 2012, IEEE/ACM Transactions on Computational Biology and Bioinformatics.

[29]  Shih-Feng Tsai,et al.  Serum-induced iron-acquisition systems and TonB contribute to virulence in Klebsiella pneumoniae causing primary pyogenic liver abscess. , 2008, The Journal of infectious diseases.

[30]  Julie C. Sung,et al.  Growth Inhibition of Pathogenic Bacteria by Sulfonylurea Herbicides , 2012, Antimicrobial Agents and Chemotherapy.

[31]  C. Rensing,et al.  CopA: An Escherichia coli Cu(I)-translocating P-type ATPase. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[32]  S. Solomon,et al.  Antibiotic resistance threats in the United States: stepping back from the brink. , 2014, American family physician.

[33]  Udo Albus,et al.  Book Review: Guide for the Care and use of Laboratory Animals , 1998 .

[34]  A. Rowan.  Guide for the Care and Use of Laboratory Animals , 1979 .

[35]  E. Budinská,et al.  aro Mutations in Salmonella enterica Cause Defects in Cell Wall and Outer Membrane Integrity , 2008, Journal of bacteriology.

[36]  P. Andrew,et al.  The cop operon is required for copper homeostasis and contributes to virulence in Streptococcus pneumoniae , 2011, Molecular microbiology.

[37]  S. Merino,et al.  Mechanisms of Klebsiella pneumoniae resistance to complement-mediated killing , 1992, Infection and immunity.

[38]  J. Imlay,et al.  The iron-sulfur clusters of dehydratases are primary intracellular targets of copper toxicity , 2009, Proceedings of the National Academy of Sciences.

[39]  J. Badger,et al.  Complete Genome Sequence of the N2-Fixing Broad Host Range Endophyte Klebsiella pneumoniae 342 and Virulence Predictions Verified in Mice , 2008, PLoS genetics.

[40]  Rob Knight,et al.  Identifying genetic determinants needed to establish a human gut symbiont in its habitat. , 2009, Cell host & microbe.

[41]  B. Palsson,et al.  Capsule deletion via a λ-Red knockout system perturbs biofilm formation and fimbriae expression in Klebsiella pneumoniae MGH 78578 , 2014, BMC Research Notes.