title: Functional Genomic Screen Identifies Klebsiella pneumoniae Factors Implicated in Blocking Nuclear Factor κB (NF-κB) Signaling.
abstract: Klebsiella pneumoniae is an etiologic agent of community-acquired and nosocomial pneumonia. It has been shown that K. pneumoniae infections are characterized by reduced early inflammatory response. Recently our group has shown that K. pneumoniae dampens the activation of inflammatory responses by antagonizing the activation of the NF-κB canonical pathway. Our results revealed that K. pneumoniae capsule polysaccharide (CPS) was necessary but not sufficient to attenuate inflammation. To identify additional Klebsiella factors required to dampen inflammation, we standardized and applied a high-throughput gain-of-function screen to examine a Klebsiella transposon mutant library. We identified 114 mutants that triggered the activation of NF-κB. Two gene ontology categories accounted for half of the loci identified in the screening: metabolism and transport genes (32% of the mutants) and envelope-related genes (17%). Characterization of the mutants revealed that the lack of the enterobactin siderophore was linked to a reduced CPS expression, which in turn underlined the NF-κB activation induced by the mutant. The lipopolysaccharide (LPS) O-polysaccharide and the pullulanase (PulA) type 2 secretion system (T2SS) are required for full effectiveness of the immune evasion. Importantly, these factors do not play a redundant role. The fact that LPS O-polysaccharide and T2SS mutant-induced responses were dependent on TLR2-TLR4-MyD88 activation suggested that LPS O-polysaccharide and PulA perturbed Toll-like receptor (TLR)-dependent recognition of K. pneumoniae. Finally, we demonstrate that LPS O-polysaccharide and pulA mutants are attenuated in the pneumonia mouse model. We propose that LPS O-polysaccharide and PulA T2SS could be new targets for the design of new antimicrobials. Increasing TLR-governed defense responses might provide also selective alternatives for the management of K. pneumoniae pneumonia.
authors.affiliation: From the Infection and Immunity Program, Fundación de Investigación Sanitaria de las Islas Baleares (FISIB), 07110 Mallorca, Spain, the Instituto de Investigación Sanitaria de Palma (IdisPa), 07120 Mallorca, Spain, the Centro de Investigación Biomédica en Red Enfermedades Respiratorias (CIBERES), 28029 Madrid, Spain.
authors.forename: Anna
authors.initials: A
authors.lastname: Tomás
authors.affiliation: the Unité de Pathogénie Microbienne Moléculaire, Institut Pasteur, 75724 Paris, France, INSERM U786, 75724 Paris, France.
authors.forename: Leticia
authors.initials: L
authors.lastname: Lery
authors.affiliation: From the Infection and Immunity Program, Fundación de Investigación Sanitaria de las Islas Baleares (FISIB), 07110 Mallorca, Spain, the Instituto de Investigación Sanitaria de Palma (IdisPa), 07120 Mallorca, Spain, the Centro de Investigación Biomédica en Red Enfermedades Respiratorias (CIBERES), 28029 Madrid, Spain.
authors.forename: Verónica
authors.initials: V
authors.lastname: Regueiro
authors.affiliation: From the Infection and Immunity Program, Fundación de Investigación Sanitaria de las Islas Baleares (FISIB), 07110 Mallorca, Spain, the Instituto de Investigación Sanitaria de Palma (IdisPa), 07120 Mallorca, Spain, the Centro de Investigación Biomédica en Red Enfermedades Respiratorias (CIBERES), 28029 Madrid, Spain.
authors.forename: Camino
authors.initials: C
authors.lastname: Pérez-Gutiérrez
authors.affiliation: From the Infection and Immunity Program, Fundación de Investigación Sanitaria de las Islas Baleares (FISIB), 07110 Mallorca, Spain, the Instituto de Investigación Sanitaria de Palma (IdisPa), 07120 Mallorca, Spain, the Centro de Investigación Biomédica en Red Enfermedades Respiratorias (CIBERES), 28029 Madrid, Spain.
authors.forename: Verónica
authors.initials: V
authors.lastname: Martínez
authors.affiliation: From the Infection and Immunity Program, Fundación de Investigación Sanitaria de las Islas Baleares (FISIB), 07110 Mallorca, Spain, the Instituto de Investigación Sanitaria de Palma (IdisPa), 07120 Mallorca, Spain, the Centro de Investigación Biomédica en Red Enfermedades Respiratorias (CIBERES), 28029 Madrid, Spain.
authors.forename: David
authors.initials: D
authors.lastname: Moranta
authors.affiliation: From the Infection and Immunity Program, Fundación de Investigación Sanitaria de las Islas Baleares (FISIB), 07110 Mallorca, Spain, the Instituto de Investigación Sanitaria de Palma (IdisPa), 07120 Mallorca, Spain, the Centro de Investigación Biomédica en Red Enfermedades Respiratorias (CIBERES), 28029 Madrid, Spain.
authors.forename: Enrique
authors.initials: E
authors.lastname: Llobet
authors.affiliation: From the Infection and Immunity Program, Fundación de Investigación Sanitaria de las Islas Baleares (FISIB), 07110 Mallorca, Spain, the Instituto de Investigación Sanitaria de Palma (IdisPa), 07120 Mallorca, Spain, the Centro de Investigación Biomédica en Red Enfermedades Respiratorias (CIBERES), 28029 Madrid, Spain.
authors.forename: Mar
authors.initials: M
authors.lastname: González-Nicolau
authors.affiliation: the Centre for Infection and Immunity, Queen's University Belfast, Belfast BT9 7AE, United Kingdom.
authors.forename: Jose L
authors.initials: JL
authors.lastname: Insua
authors.affiliation: the Departamento de Microbiología, Facultad de Biología, Universidad de Barcelona, 08028 Barcelona, Spain.
authors.forename: Juan M
authors.initials: JM
authors.lastname: Tomas
authors.affiliation: the Unité de Pathogénie Microbienne Moléculaire, Institut Pasteur, 75724 Paris, France, INSERM U786, 75724 Paris, France, Chaire de Microbiologie et Maladies Infectieuses, Collège de France, 75231 Paris, France.
authors.forename: Philippe J
authors.initials: PJ
authors.lastname: Sansonetti
authors.affiliation: the Unité de Pathogénie Microbienne Moléculaire, Institut Pasteur, 75724 Paris, France, INSERM U786, 75724 Paris, France, Imagopole, Plateforme d'Imagerie Dynamique, Institut Pasteur, 75724 Paris, France, and.
authors.forename: Régis
authors.initials: R
authors.lastname: Tournebize
authors.affiliation: the Centre for Infection and Immunity, Queen's University Belfast, Belfast BT9 7AE, United Kingdom, the Consejo Superior de Investigaciones Científicas (CSIC), 28008 Madrid, Spain j.bengoechea@qub.ac.uk.
authors.forename: José A
authors.initials: JA
authors.lastname: Bengoechea
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000276:immunology; D005260:Female; D023281:Genomics; D006801:Humans; D057131:Immune Evasion; D007710:Klebsiella Infections / Q000235:genetics / Q000276:immunology* / Q000382:microbiology; D007711:Klebsiella pneumoniae / Q000235:genetics* / Q000276:immunology; D008070:Lipopolysaccharides / Q000276:immunology; D051379:Mice; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology*; D015398:Signal Transduction
chemical_list: D001426:Bacterial Proteins; D008070:Lipopolysaccharides; D016328:NF-kappa B
keywords: Klebsiella pneumonia; NF-κ B (NF-κB); iron; lipopolysaccharide (LPS); pullulanase; toll-like receptor (TLR)