Identification of Leptospira interrogans Phospholipase C as a Novel Virulence Factor Responsible for Intracellular Free Calcium Ion Elevation during Macrophage Death
Authors: Jing-Fang Zhao
Dexter Sun
Jie Yan
Hong-Hu Chen
David M Ojcius
Lan-Juan Li
Lin-Li Zheng
Xu&#x27;ai Lin
Xin Zhao
Yu-Mei Ge
Journal: PLoS ONE (2013)
Discipline: Multidisciplinary
Keywords: Leptospira; macrophage death; pathogenesis; leptospirosis; Ca(2+)
Affiliations: 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.

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. 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, 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/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. 

Retracted: No

PubPeer comments:
 Figure 6B. There are similar features between different panels. ![file] ![file] ![file] ![file] , 
 A couple more. P2X1-depleted Wild-Type 0 and 0.5: ![file] BATPA-AM treated wild-type 0.5 and 2: ![file] , 
 #1 Dear Edentulina Martensi, Thank you for your comment. We will check the data and contact the journal for advice. All the best! Jie Yan , 
 There is a panel very similar to one in [Shijun Li et al, 2010] Figure 4 from Shijun Li s paper: ![file] Figure 2 from this paper ![file] ,