Identification of Leptospira interrogans phospholipase C as a novel virulence factor responsible for intracellular free calcium ion elevation during macrophage death.

<h4>Background</h4>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+)...

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Autores principales:	Jing-Fang Zhao, Hong-Hu Chen, David M Ojcius, Xin Zhao, Dexter Sun, Yu-Mei Ge, Lin-Li Zheng, Xu'ai Lin, Lan-Juan Li, Jie Yan
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Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/b9ab9383faef4f35b2ba4b330b7095db
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spelling	oai:doaj.org-article:b9ab9383faef4f35b2ba4b330b7095db2021-11-18T08:52:29ZIdentification of Leptospira interrogans phospholipase C as a novel virulence factor responsible for intracellular free calcium ion elevation during macrophage death.1932-620310.1371/journal.pone.0075652https://doaj.org/article/b9ab9383faef4f35b2ba4b330b7095db2013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24124502/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>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.<h4>Methodology/principal findings</h4>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.<h4>Conclusions/significance</h4>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.Jing-Fang ZhaoHong-Hu ChenDavid M OjciusXin ZhaoDexter SunYu-Mei GeLin-Li ZhengXu'ai LinLan-Juan LiJie YanPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 10, p e75652 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Jing-Fang Zhao Hong-Hu Chen David M Ojcius Xin Zhao Dexter Sun Yu-Mei Ge Lin-Li Zheng Xu'ai Lin Lan-Juan Li Jie Yan Identification of Leptospira interrogans phospholipase C as a novel virulence factor responsible for intracellular free calcium ion elevation during macrophage death.
description	<h4>Background</h4>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.<h4>Methodology/principal findings</h4>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.<h4>Conclusions/significance</h4>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.
format	article
author	Jing-Fang Zhao Hong-Hu Chen David M Ojcius Xin Zhao Dexter Sun Yu-Mei Ge Lin-Li Zheng Xu'ai Lin Lan-Juan Li Jie Yan
author_facet	Jing-Fang Zhao Hong-Hu Chen David M Ojcius Xin Zhao Dexter Sun Yu-Mei Ge Lin-Li Zheng Xu'ai Lin Lan-Juan Li Jie Yan
author_sort	Jing-Fang Zhao
title	Identification of Leptospira interrogans phospholipase C as a novel virulence factor responsible for intracellular free calcium ion elevation during macrophage death.
title_short	Identification of Leptospira interrogans phospholipase C as a novel virulence factor responsible for intracellular free calcium ion elevation during macrophage death.
title_full	Identification of Leptospira interrogans phospholipase C as a novel virulence factor responsible for intracellular free calcium ion elevation during macrophage death.
title_fullStr	Identification of Leptospira interrogans phospholipase C as a novel virulence factor responsible for intracellular free calcium ion elevation during macrophage death.
title_full_unstemmed	Identification of Leptospira interrogans phospholipase C as a novel virulence factor responsible for intracellular free calcium ion elevation during macrophage death.
title_sort	identification of leptospira interrogans phospholipase c as a novel virulence factor responsible for intracellular free calcium ion elevation during macrophage death.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/b9ab9383faef4f35b2ba4b330b7095db
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Identification of Leptospira interrogans phospholipase C as a novel virulence factor responsible for intracellular free calcium ion elevation during macrophage death.

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