Identification of Leptospira interrogans Phospholipase C as a Novel Virulence Factor Responsible for Intracellular Free Calcium Ion Elevation during Macrophage Death

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. Methodology/Principal Findings 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, P 2 X 7 , 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 P 2 X 7 was depleted. The LB361 gene product of the spirochete exhibited phosphatidylinositol phospholipase C (L-PI-PLC) activity to hydrolyze phosphatidylinositol-4,5-bisphosphate (PIP 2 ) into inositol-1,4,5-trisphosphate (IP 3 ), 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/Significance 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.