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

被引：20

作者：

Zhao, Jing-Fang ^{[1
,2
,3
]}

Chen, Hong-Hu ^{[2
]}

Ojcius, David M. ^{[4
,5
]}

Zhao, Xin ^{[2
]}

Sun, Dexter ^{[6
,7
,8
]}

Ge, Yu-Mei ^{[1
,2
]}

Zheng, Lin-Li ^{[2
]}

Lin, Xu'ai ^{[1
,2
]}

Li, Lan-Juan ^{[1
]}

Yan, Jie ^{[1
,2
]}

机构：

[1] Zhejiang Univ, Sch Med, Affiliated Hosp 1, State Key Lab Diag & Treatment Infect Dis, Hangzhou 310003, Zhejiang, Peoples R China

[2] Zhejiang Univ, Sch Med, Dept Med Microbiol & Parasitol, Hangzhou 310003, Zhejiang, Peoples R China

[3] Zhejiang Prov Hosp Tradit Chinese Med, Dept Clin Lab, Hangzhou, Zhejiang, Peoples R China

[4] Univ Calif Merced, Hlth Sci Res Inst, Merced, CA USA

[5] Univ Calif Merced, Dept Mol Cell Biol, Merced, CA USA

[6] Cornell Univ, Weill Med Coll, New York Presbyterian Hosp, Dept Neurol, New York, NY 10021 USA

[7] Cornell Univ, Weill Med Coll, New York Presbyterian Hosp, Dept Neurosci, New York, NY 10021 USA

[8] Cornell Univ, Weill Med Coll, Hosp Special Surg, New York, NY 10021 USA

来源：

PLOS ONE | 2013年 / 8卷 / 10期

基金：

中国国家自然科学基金;

关键词：

CELL-DEATH; APOPTOSIS; ACTIVATION; INFECTION; PROTEIN; ENTRY; PATHOGENESIS; EXPRESSION; IMMUNITY; SUBTYPES;

D O I：

10.1371/journal.pone.0075652

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

Background: Leptospira-induced macrophage death has been confirmed to play a crucial role in pathogenesis of leptospirosis, a worldwide zoonotic infectious disease. Intracellular free Ca2+ concentration ([Ca2+](i)) elevation induced by infection can cause cell death, but [Ca2+](i) changes and high [Ca2+](i)-induced death of macrophages due to infection of Leptospira have not been previously reported. Methodology/Principal Findings: We first used a Ca2+-specific fluorescence probe to confirm that the infection of L. interrogans strain Lai triggered a significant increase of [Ca2+](i) in mouse J774A.1 or human THP-1 macrophages. Laser confocal microscopic examination showed that the [Ca2+](i) elevation was caused by both extracellular Ca2+ influx through the purinergic receptor, P2X7, and Ca2+ release from the endoplasmic reticulum, as seen by suppression of [Ca2+](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 Ca2+ release from endoplasmic reticulum, with the Km of 199 mu M 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 [Ca2+](i) elevation was induced by infection with a LB361-deficient leptospiral mutant, whereas transfection of the LB361 gene caused a mild increase in [Ca2+](i). Moreover, PI-PLCs (PI-PLC-beta 3 and PI-PLC-gamma 1) of the two macrophages were activated by phosphorylation during infection. Flow cytometric detection demonstrated that high [Ca2+](i) increases induced apoptosis and necrosis of macrophages, while mild [Ca2+](i) elevation only caused apoptosis. Conclusions/Significance: This study demonstrated that L. interrogans infection induced [Ca2+](i) elevation through extracellular Ca2+ influx and intracellular Ca2+ 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 [Ca2+](i) elevation.

引用

页数：15