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Identification of Leptospira interrogans phospholipase C as a novel virulence factor responsible for intracellular
Jing-Fang Zhao1, Hong-Hu Chen, David M Ojcius
1State 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.
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.
Insights
Leptospira interrogans infection increases intracellular calcium levels in macrophages, leading to cell death. A novel bacterial enzyme, L-PI-PLC, is identified as the cause of this calcium dysregulation.
Area of Science:
- Microbiology
- Cell Biology
- Immunology
Background:
- Leptospira-induced macrophage death is critical in leptospirosis pathogenesis.
- Elevated intracellular free calcium concentration ([Ca(2+)]i) is a known cause of cell death, but its role in Leptospira infection was uncharacterized.
Purpose of the Study:
- To investigate the changes in intracellular calcium concentration ([Ca(2+)]i) during Leptospira infection.
- To elucidate the mechanisms of [Ca(2+)]i elevation and its role in macrophage death.
- To identify bacterial factors responsible for inducing [Ca(2+)]i changes.
Main Methods:
- Utilized Ca(2+)-specific fluorescence probes and laser confocal microscopy to monitor [Ca(2+)]i in infected macrophages (J774A.1 and THP-1).
- Investigated calcium sources through receptor blockade (P2X7) and endoplasmic reticulum Ca(2+) release.
- Analyzed the role of the Leptospira LB361 gene product (L-PI-PLC) using gene deletion and transfection mutants, confirmed by Western Blot and enzymatic assays (Km, Kcat).
- Assessed macrophage apoptosis and necrosis via flow cytometry.
Main Results:
- L. interrogans infection significantly increased [Ca(2+)]i in macrophages via extracellular Ca(2+) influx through P2X7 and intracellular release from the endoplasmic reticulum.
- The Leptospira LB361 gene product, a novel phosphatidylinositol phospholipase C (L-PI-PLC), hydrolyzes PIP2 to IP3, inducing Ca(2+) release.
- A LB361-deficient mutant caused lower [Ca(2+)]i elevation, while LB361 gene transfection increased it.
- Macrophage PI-PLCs (PI-PLC-β3 and PI-PLC-γ1) were activated by phosphorylation during infection.
- High [Ca(2+)]i increases led to macrophage apoptosis and necrosis; mild increases caused only apoptosis.
Conclusions:
- L. interrogans infection elevates macrophage [Ca(2+)]i through extracellular influx and intracellular release, inducing apoptosis and necrosis.
- The LB361 gene product is identified as a novel L. interrogans PI-PLC responsible for the observed [Ca(2+)]i elevation.
