title: Use of a Dictyostelium model for isolation of genetic loci associated with phagocytosis and virulence in Klebsiella pneumoniae.
abstract: Phagocytosis resistance is an important virulence factor in Klebsiella pneumoniae. Dictyostelium has been used to study the interaction between phagocytes and bacteria because of its similarity to mammalian macrophages. In this study, we used a Dictyostelium model to investigate genes for resistance to phagocytosis in NTUH-K2044, a strain of K. pneumoniae causing pyogenic liver abscess that is highly resistant to phagocytosis. A total of 2,500 transposon mutants were screened by plaque assay, and 29 of them permitted phagocytosis by Dictyostelium. In the 29 mutants, six loci were identified; three were capsular synthesis genes. Of the other three, one was related to carnitine metabolism, one encoded a subunit of protease (clpX), and one encoded a lipopolysaccharide O-antigen transporter (wzm). Deletion and complementation of these genes showed that only ΔclpX and Δwzm mutants became susceptible to Dictyostelium phagocytosis, and their complementation restored the phagocytosis resistance phenotype. These two mutants were also susceptible to phagocytosis by human neutrophils and revealed attenuated virulence in a mouse model, implying that they play important roles in the pathogenesis of K. pneumoniae. Furthermore, we demonstrated that clpP, which exists in an operon with clpX, was also involved in resistance to phagocytosis. The transcriptional profile of ΔclpX was examined by microarray analysis and revealed a 3-fold lower level of expression of capsular synthesis genes. Therefore, we have identified genes involved in resistance to phagocytosis in K. pneumoniae using Dictyostelium, and this model is useful to explore genes associated with resistance to phagocytosis in heavily encapsulated bacteria.
authors.affiliation: Department of Microbiology, National Taiwan University College of Medicine, 1, Sec 1, Jen-Ai Rd., Taipei 10051, Taiwan.
authors.forename: Yi-Jiun
authors.initials: YJ
authors.lastname: Pan
authors.forename: Tzu-Lung
authors.initials: TL
authors.lastname: Lin
authors.forename: Chun-Ru
authors.initials: CR
authors.lastname: Hsu
authors.forename: Jin-Town
authors.initials: JT
authors.lastname: Wang
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics* / Q000276:immunology; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000276:immunology; D004251:DNA Transposable Elements; D004023:Dictyostelium / Q000276:immunology*; D049071:Endopeptidase Clp / Q000235:genetics* / Q000276:immunology; D005260:Female; D056426:Genetic Loci; D006801:Humans; D015151:Immunoblotting; D007711:Klebsiella pneumoniae / Q000235:genetics* / Q000276:immunology / Q000472:pathogenicity; D051379:Mice; D008807:Mice, Inbred BALB C; D046228:Microarray Analysis; D018613:Microscopy, Confocal; D010587:Phagocytosis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014774:Virulence
chemical_list: D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D004251:DNA Transposable Elements; C472370:Wzm protein, Klebsiella pneumoniae; D049071:Endopeptidase Clp