title: Identification of Leptospira interrogans phospholipase C as a novel virulence factor responsible for intracellular free calcium ion elevation during macrophage death.
abstract: BACKGROUND
  Leptospira-induced macrophage death has been confirmed to play a crucial role in pathogenesis of leptospirosis, a worldwide zoonotic infectious disease. Intracellular free Ca(2+) concentration ([Ca(2+)]i) elevation induced by infection can cause cell death, but [Ca(2+)]i changes and high [Ca(2+)]i-induced death of macrophages due to infection of Leptospira have not been previously reported.
  
  
  RESULTS
  We first used a Ca(2+)-specific fluorescence probe to confirm that the infection of L. interrogans strain Lai triggered a significant increase of [Ca(2+)]i in mouse J774A.1 or human THP-1 macrophages. Laser confocal microscopic examination showed that the [Ca(2+)]i elevation was caused by both extracellular Ca(2+) influx through the purinergic receptor, P2X7, and Ca(2+) release from the endoplasmic reticulum, as seen by suppression of [Ca(2+)]i elevation when receptor-gated calcium channels were blocked or P2X7 was depleted. The LB361 gene product of the spirochete exhibited phosphatidylinositol phospholipase C (L-PI-PLC) activity to hydrolyze phosphatidylinositol-4,5-bisphosphate (PIP2) into inositol-1,4,5-trisphosphate (IP3), which in turn induces intracellular Ca(2+) release from endoplasmic reticulum, with the Km of 199 µM and Kcat of 8.566E-5 S(-1). Secretion of L-PI-PLC from the spirochete into supernatants of leptospire-macrophage co-cultures and cytosol of infected macrophages was also observed by Western Blot assay. Lower [Ca(2+)]i elevation was induced by infection with a LB361-deficient leptospiral mutant, whereas transfection of the LB361 gene caused a mild increase in [Ca(2+)]i. Moreover, PI-PLCs (PI-PLC-β3 and PI-PLC-γ1) of the two macrophages were activated by phosphorylation during infection. Flow cytometric detection demonstrated that high [Ca(2+)]i increases induced apoptosis and necrosis of macrophages, while mild [Ca(2+)]i elevation only caused apoptosis.
  
  
  CONCLUSIONS
  This study demonstrated that L. interrogans infection induced [Ca(2+)]i elevation through extracellular Ca(2+) influx and intracellular Ca(2+) release cause macrophage apoptosis and necrosis, and the LB361 gene product was shown to be a novel PI-PLC of L. interrogans responsible for the [Ca(2+)]i elevation.
authors.affiliation: State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, P.R. China ; Department of Medical Microbiology and Parasitology, Zhejiang University School of Medicine, Hangzhou, Zhejiang, P.R. China ; Depatment of Clinical Laboratory, Zhejiang Provincial Hospital of Traditional Chinese Medicine, Hangzhou, Zhejiang, P.R. China.
authors.forename: Jing-Fang
authors.initials: JF
authors.lastname: Zhao
authors.forename: Hong-Hu
authors.initials: HH
authors.lastname: Chen
authors.forename: David M
authors.initials: DM
authors.lastname: Ojcius
authors.forename: Xin
authors.initials: X
authors.lastname: Zhao
authors.forename: Dexter
authors.initials: D
authors.lastname: Sun
authors.forename: Yu-Mei
authors.initials: YM
authors.lastname: Ge
authors.forename: Lin-Li
authors.initials: LL
authors.lastname: Zheng
authors.forename: Xu'ai
authors.initials: X
authors.lastname: Lin
authors.forename: Lan-Juan
authors.initials: LJ
authors.lastname: Li
authors.forename: Jie
authors.initials: J
authors.lastname: Yan
mesh_terms: D000818:Animals; D017209:Apoptosis; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D006801:Humans; D007921:Leptospira interrogans / Q000201:enzymology* / Q000472:pathogenicity*; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D010766:Phosphorylation; D010738:Type C Phospholipases / Q000378:metabolism*; D037521:Virulence Factors / Q000378:metabolism*
chemical_list: D037521:Virulence Factors; D010738:Type C Phospholipases; D002118:Calcium