In Vivo Gene Essentiality and Metabolism in Bordetella pertussis

Our study describes the first in vivo transposon sequencing (Tn-seq) analysis of B. pertussis and identifies genes predicted to be essential for in vivo growth in a murine model of intranasal infection, generating key resources for future investigations into B. pertussis pathogenesis and vaccine design. ABSTRACT Bordetella pertussis is the causative agent of whooping cough, a serious respiratory illness affecting children and adults, associated with prolonged cough and potential mortality. Whooping cough has reemerged in recent years, emphasizing a need for increased knowledge of basic mechanisms of B. pertussis growth and pathogenicity. While previous studies have provided insight into in vitro gene essentiality of this organism, very little is known about in vivo gene essentiality, a critical gap in knowledge, since B. pertussis has no previously identified environmental reservoir and is isolated from human respiratory tract samples. We hypothesize that the metabolic capabilities of B. pertussis are especially tailored to the respiratory tract and that many of the genes involved in B. pertussis metabolism would be required to establish infection in vivo. In this study, we generated a diverse library of transposon mutants and then used it to probe gene essentiality in vivo in a murine model of infection. Using the CON-ARTIST pipeline, 117 genes were identified as conditionally essential at 1 day postinfection, and 169 genes were identified as conditionally essential at 3 days postinfection. Most of the identified genes were associated with metabolism, and we utilized two existing genome-scale metabolic network reconstructions to probe the effects of individual essential genes on biomass synthesis. This analysis suggested a critical role for glucose metabolism and lipooligosaccharide biosynthesis in vivo. This is the first genome-wide evaluation of in vivo gene essentiality in B. pertussis and provides tools for future exploration. IMPORTANCE Our study describes the first in vivo transposon sequencing (Tn-seq) analysis of B. pertussis and identifies genes predicted to be essential for in vivo growth in a murine model of intranasal infection, generating key resources for future investigations into B. pertussis pathogenesis and vaccine design.

[1]  David S. Wishart,et al.  Circular genome visualization and exploration using CGView , 2005, Bioinform..

[2]  T. Merkel,et al.  Airborne transmission of Bordetella pertussis. , 2012, The Journal of infectious diseases.

[3]  S. Sood,et al.  Pertussis: still a cause of death, seven decades into vaccination , 2014, Current opinion in pediatrics.

[4]  S. Stibitz,et al.  Derivation of a physical map of the chromosome of Bordetella pertussis Tohama I , 1992, Journal of bacteriology.

[5]  S. Knapp,et al.  Two trans-acting regulatory genes (vir and mod) control antigenic modulation in Bordetella pertussis , 1988, Journal of bacteriology.

[6]  Jason A. Papin,et al.  Applications of genome-scale metabolic reconstructions , 2009, Molecular systems biology.

[7]  F. Mooi,et al.  Comparative genomics of prevaccination and modern Bordetella pertussis strains , 2010, BMC Genomics.

[8]  Jianjun Li,et al.  Identification of a Novel Lipopolysaccharide Core Biosynthesis Gene Cluster in Bordetella pertussis, and Influence of Core Structure and Lipid A Glucosamine Substitution on Endotoxic Activity , 2009, Infection and Immunity.

[9]  A. Melton,et al.  Use of the promoter fusion transposon Tn5 lac to identify mutations in Bordetella pertussis vir-regulated genes , 1989, Infection and immunity.

[10]  M. Waldor,et al.  The design and analysis of transposon insertion sequencing experiments , 2016, Nature Reviews Microbiology.

[11]  T. Merkel,et al.  Identification of a locus required for the regulation of bvg-repressed genes in Bordetella pertussis , 1995, Journal of bacteriology.

[12]  Adam D. Leaché,et al.  The Utility of Single Nucleotide Polymorphism (SNP) Data in Phylogenetics , 2017 .

[13]  J. Ireland,et al.  Functional single nucleotide polymorphism-based association studies , 2006, Human Genomics.

[14]  S. Elahi,et al.  The benefits of using diverse animal models for studying pertussis. , 2007, Trends in microbiology.

[15]  C. D. de Gooijer,et al.  Rational medium design for Bordetella pertussis: basic metabolism. , 1999, Journal of biotechnology.

[16]  L. Hurst,et al.  Genomic analysis of isolates from the United Kingdom 2012 pertussis outbreak reveals that vaccine antigen genes are unusually fast evolving. , 2015, The Journal of infectious diseases.

[17]  R. Fernandez,et al.  Protective activity of the Bordetella pertussis BrkA autotransporter in the murine lung colonization model. , 2008, Vaccine.

[18]  H. Schweizer,et al.  Versatile Dual-Technology System for Markerless Allele Replacement in Burkholderia pseudomallei , 2009, Applied and Environmental Microbiology.

[19]  R. Fernandez,et al.  Cloning and sequencing of a Bordetella pertussis serum resistance locus , 1994, Infection and immunity.

[20]  Jerry King,et al.  A curated genome-scale metabolic model of Bordetella pertussis metabolism , 2017, PLoS Comput. Biol..

[21]  M. Whiteley,et al.  Nutritional Cues Control Pseudomonas aeruginosa Multicellular Behavior in Cystic Fibrosis Sputum , 2007, Journal of bacteriology.

[22]  E. Harvill,et al.  Bordetella pertussis Acquires Resistance to Complement-Mediated Killing In Vivo , 2003, Infection and Immunity.

[23]  C. Locht,et al.  Role of ADP-Ribosyltransferase Activity of Pertussis Toxin in Toxin-Adhesin Redundancy with Filamentous Hemagglutinin duringBordetella pertussis Infection , 2001, Infection and Immunity.

[24]  A. Weiss,et al.  Adenylate cyclase toxin is critical for colonization and pertussis toxin is critical for lethal infection by Bordetella pertussis in infant mice , 1990, Infection and immunity.

[25]  K. Noel,et al.  A quinol oxidase, encoded by cyoABCD, is utilized to adapt to lower O2 concentrations in Rhizobium etli CFN42 , 2015, Microbiology.

[26]  V. de Lorenzo,et al.  Pseudomonas aeruginosa: the making of a pathogen. , 2015, Environmental microbiology.

[27]  V. Gerdts,et al.  Mouse and pig models for studies of natural and vaccine-induced immunity to Bordetella pertussis. , 2014, The Journal of infectious diseases.

[28]  Aldert L. Zomer,et al.  Adaptation of Bordetella pertussis to the Respiratory Tract , 2018, The Journal of infectious diseases.

[29]  N. Lindley,et al.  A Functional Tricarboxylic Acid Cycle Operates during Growth of Bordetella pertussis on Amino Acid Mixtures as Sole Carbon Substrates , 2015, PloS one.

[30]  Satoshi Kimura,et al.  The Nucleoid Binding Protein H-NS Biases Genome-Wide Transposon Insertion Landscapes , 2016, mBio.

[31]  B. Teusink,et al.  Probing the Genome-Scale Metabolic Landscape of Bordetella pertussis, the Causative Agent of Whooping Cough , 2017, Applied and Environmental Microbiology.

[32]  Matthew K. Waldor,et al.  ARTIST: High-Resolution Genome-Wide Assessment of Fitness Using Transposon-Insertion Sequencing , 2014, PLoS genetics.

[33]  T. J. Brickman,et al.  Differential expression of Bordetella pertussis iron transport system genes during infection , 2008, Molecular microbiology.

[34]  Thomas R. Ioerger,et al.  TRANSIT - A Software Tool for Himar1 TnSeq Analysis , 2015, PLoS Comput. Biol..

[35]  T. Merkel,et al.  Nonhuman Primate Model of Pertussis , 2012, Infection and Immunity.

[36]  D W Stainer,et al.  A simple chemically defined medium for the production of phase I Bordetella pertussis. , 1970, Journal of general microbiology.

[37]  Jason A. Papin,et al.  Reconstruction of the metabolic network of Pseudomonas aeruginosa to interrogate virulence factor synthesis , 2017, Nature Communications.

[38]  J. McArthur,et al.  Role of Phosphoglucomutase of Bordetella bronchiseptica in Lipopolysaccharide Biosynthesis and Virulence , 2000, Infection and Immunity.

[39]  Jason A. Papin,et al.  Biomedical applications of genome-scale metabolic network reconstructions of human pathogens. , 2018, Current opinion in biotechnology.

[40]  K. Edwards,et al.  Pertussis vaccines and the challenge of inducing durable immunity. , 2015, Current opinion in immunology.

[41]  Evan D. Brutinel,et al.  Anomalies of the anaerobic tricarboxylic acid cycle in Shewanella oneidensis revealed by Tn‐seq , 2012, Molecular microbiology.

[42]  C. K. Vanderpool,et al.  The Bordetella bhu Locus Is Required for Heme Iron Utilization , 2001, Journal of bacteriology.

[43]  J. Wade,et al.  The BvgAS Regulon of Bordetella pertussis , 2017, mBio.

[44]  Kelly D Elder,et al.  Strain-Dependent Role of BrkA during Bordetella pertussis Infection of the Murine Respiratory Tract , 2004, Infection and Immunity.

[45]  Thomas R. Ioerger,et al.  A Hidden Markov Model for identifying essential and growth-defect regions in bacterial genomes from transposon insertion sequencing data , 2013, BMC Bioinformatics.

[46]  S. Lory,et al.  A Comprehensive Analysis of In Vitro and In Vivo Genetic Fitness of Pseudomonas aeruginosa Using High-Throughput Sequencing of Transposon Libraries , 2013, PLoS pathogens.

[47]  B. Barrell,et al.  Comparative analysis of the genome sequences of Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica , 2003, Nature Genetics.