title: Pullulanase Is Necessary for the Efficient Intracellular Growth of Francisella tularensis.
abstract: Pullulanase, an enzyme that catalyzes the hydrolysis of polysaccharides, has been identified in a broad range of organisms, including bacteria, yeasts, fungi, and animals. The pullulanase (pulB; FTT_0412c) of F. tularensis subspecies tularensis Schu S4 is considered to be a homologue of the type I pullulanase (pulA) of the other Francisella subspecies. The significance of Francisella pullulanase has been obscure until now. In the present study, we characterized a recombinant PulB of F. tularensis SCHU P9, which was expressed as a his-tagged protein in Escherichia coli. The recombinant PulB was confirmed to be a type I pullulanase by its enzymatic activity in vitro. A pulB gene knockout mutant of F. tularensis SCHU P9 (ΔpulB) was constructed using the TargeTron Knockout system and plasmid pKEK1140 to clarify the function of PulB during the growth of F. tularensis in macrophages. The intracellular growth of the ΔpulB mutant in murine macrophage J774.1 cells was significantly reduced compared with that of the parental strain SCHU P9. Expression of PulB in ΔpulB, using an expression plasmid, resulted in the complementation of the reduced growth in macrophages, suggesting that PulB is necessary for the efficient growth of F. tularensis in macrophages. To assess the role of PulB in virulence, the knockout and parent bacterial strains were used to infect C57BL/6J mice. Histopathological analyses showed that tissues from ΔpulB-infected mice showed milder lesions compared to those from SCHU P9-infected mice. However, all mice infected with SCHU P9 and ΔpulB showed the similar levels of bacterial loads in their tissues. The results suggest that PulB plays a significant role in bacterial growth within murine macrophage but does not contribute to bacterial virulence in vivo.
authors.affiliation: Department of Veterinary Science, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan.
authors.forename: Akihiko
authors.initials: A
authors.lastname: Uda
authors.affiliation: Department of Veterinary Science, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan.
authors.forename: Neekun
authors.initials: N
authors.lastname: Sharma
authors.affiliation: Division of Experimental Animal Research, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Takimoto
authors.affiliation: Department of Veterinary Science, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan.|Laboratory of Veterinary Public Health, Graduate School of Agricultural and Life Science, The University of Tokyo, Bunkyo, Tokyo, Japan.
authors.forename: Tian
authors.initials: T
authors.lastname: Deyu
authors.affiliation: Department of Veterinary Science, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan.|United Graduate School of Veterinary Science, Gifu University, Gifu, Japan.
authors.forename: Yuuki
authors.initials: Y
authors.lastname: Koyama
authors.affiliation: Department of Veterinary Science, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan.
authors.forename: Eun-Sil
authors.initials: ES
authors.lastname: Park
authors.affiliation: Department of Veterinary Science, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan.
authors.forename: Osamu
authors.initials: O
authors.lastname: Fujita
authors.affiliation: Department of Veterinary Science, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan.
authors.forename: Akitoyo
authors.initials: A
authors.lastname: Hotta
authors.affiliation: Department of Veterinary Science, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan.|United Graduate School of Veterinary Science, Gifu University, Gifu, Japan.
authors.forename: Shigeru
authors.initials: S
authors.lastname: Morikawa
mesh_terms: D000818:Animals; D004195:Disease Models, Animal; D004789:Enzyme Activation; D005260:Female; D005604:Francisella tularensis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development* / Q000472:pathogenicity; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008264:Macrophages / Q000276:immunology / Q000378:metabolism / Q000382:microbiology; D051379:Mice; D009154:Mutation; D013696:Temperature; D014406:Tularemia / Q000276:immunology / Q000378:metabolism / Q000382:microbiology* / Q000473:pathology; D014774:Virulence
chemical_list: D006026:Glycoside Hydrolases; C100181:pullulanase