title: Toll-like receptor 2-mediated signaling requirements for Francisella tularensis live vaccine strain infection of murine macrophages.
abstract: Francisella tularensis, an aerobic, non-spore-forming, gram-negative coccobacillus, is the causative agent of tularemia. We reported previously that F. tularensis live vaccine strain (LVS) elicited strong, dose-dependent NF-kappaB reporter activity in Toll-like receptor 2 (TLR2)-expressing HEK293T cells and proinflammatory gene expression in primary murine macrophages. Herein, we report that F. tularensis LVS-induced murine macrophage proinflammatory cytokine gene and protein expression are overwhelmingly TLR2 dependent, as evidenced by the abrogated responses of TLR2(-/-) macrophages. F. tularensis LVS infection also increased expression of TLR2 both in vitro, in mouse macrophages, and in vivo, in livers from F. tularensis LVS-infected mice. Colocalization of intracellular F. tularensis LVS, TLR2, and MyD88 was visualized by confocal microscopy. Signaling was abrogated if the F. tularensis LVS organisms were heat or formalin killed or treated with chloramphenicol, indicating that the TLR2 agonist activity is dependent on new bacterial protein synthesis. F. tularensis LVS replicates in macrophages; however, bacterial replication was not required for TLR2 signaling because LVSDeltaguaA, an F. tularensis LVS guanine auxotroph that fails to replicate in the absence of exogenous guanine, activated NF-kappaB in TLR2-transfected HEK293T cells and induced cytokine expression in wild-type macrophages comparably to wild-type F. tularensis LVS. Collectively, these data indicate that the primary macrophage response to F. tularensis LVS is overwhelmingly TLR2 dependent, requires de novo bacterial protein synthesis, and is independent of intracellular F. tularensis replication.
authors.affiliation: Department of Microbiology and Immunology, University of Maryland, Baltimore, 660 West Redwood Street, Room 324, Baltimore, MD 21201, USA.
authors.forename: Leah E
authors.initials: LE
authors.lastname: Cole
authors.forename: Kari Ann
authors.initials: KA
authors.lastname: Shirey
authors.forename: Eileen
authors.initials: E
authors.lastname: Barry
authors.forename: Araceli
authors.initials: A
authors.lastname: Santiago
authors.forename: Prasad
authors.initials: P
authors.lastname: Rallabhandi
authors.forename: Karen L
authors.initials: KL
authors.lastname: Elkins
authors.forename: Adam C
authors.initials: AC
authors.lastname: Puche
authors.forename: Suzanne M
authors.initials: SM
authors.lastname: Michalek
authors.forename: Stefanie N
authors.initials: SN
authors.lastname: Vogel
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis; D001428:Bacterial Vaccines / Q000276:immunology*; D002460:Cell Line; D016207:Cytokines / Q000096:biosynthesis; D004195:Disease Models, Animal; D005604:Francisella tularensis / Q000276:immunology* / Q000502:physiology; D015870:Gene Expression; D006801:Humans; D008099:Liver / Q000473:pathology; D008264:Macrophages / Q000737:chemistry / Q000382:microbiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D053594:Myeloid Differentiation Factor 88 / Q000032:analysis; D052002:NF-kappa B p50 Subunit / Q000032:analysis; D010588:Phagosomes / Q000382:microbiology; D012333:RNA, Messenger / Q000032:analysis; D015398:Signal Transduction / Q000276:immunology*; D051195:Toll-Like Receptor 2 / Q000032:analysis / Q000235:genetics / Q000276:immunology*
chemical_list: D001426:Bacterial Proteins; D001428:Bacterial Vaccines; D016207:Cytokines; D053594:Myeloid Differentiation Factor 88; D052002:NF-kappa B p50 Subunit; C489548:NFKB1 protein, human; D012333:RNA, Messenger; D051195:Toll-Like Receptor 2