Activation of host phospholipases C and D in macrophages after infection with Listeria monocytogenes
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Updated: Jul 21, 2026
Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes
Published on: September 20, 2013
H Goldfine1, S J Wadsworth, N C Johnston
1Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6076, USA. goldfinh@mail.med.upenn.edu
Abstract:
Infection of the J774 murine macrophage-derived cell line with Listeria monocytogenes results in several elevations of intracellular calcium during the first 15 min of infection. These appear to result from the actions of secreted bacterial proteins, including phosphatidylinositol-specific phospholipase C (PI-PLC), a broad-range phospholipase C, and listeriolysin O (LLO) (S. J. Wadsworth and H. Goldfine, Infect. Immun. 67:1770-1778, 1999). We have measured hydrolysis of host PI and the activation of host polyphosphoinositide-specific PLC and host phospholipase D (PLD) during infection with wild-type and mutant L. monocytogenes. Elevated hydrolysis of host PI occurred within the first 10 min of infection and was dependent on both bacterial PI-PLC and LLO, both of which were required for the earliest elevations of intracellular calcium in the host cell. A more rapid hydrolysis of host PI was observed at 30 min after infection, at the time when wild-type bacteria have been internalized. Activation of host PLC, also occurred in the first 10 min of infection but was not dependent on the presence of bacterial PI-PLC. Similar observations were made in murine bone marrow-derived macrophages. In J774 cells, activation of host PLD was observed after 20 min of infection and was dependent on bacterial LLO. Mutants in the bacterial phospholipases produced levels of PLD activation similar to those produced by the wild type. Phorbol myristate acetate (PMA) also activated host PLD, while long-term treatment with PMA resulted in loss of the ability of L. monocytogenes to activate host PLD, suggesting an involvement of protein kinase C (PKC) in the activation of PLD. Rottlerin, an inhibitor of PKC delta in J774 cells, also inhibited the activation of PLD, but hispidin, an inhibitor of PKC betaI and betaII, did not. Pretreatment of J774 cells with the PLD inhibitor, 2, 3-diphosphoglycerate partially inhibited escape of the bacteria from the primary phagocytic vacuole.
Insights
Listeria monocytogenes infection elevates host cell calcium via bacterial phospholipase C and listeriolysin O. These bacterial proteins trigger host PI hydrolysis and PLC activation, crucial for early calcium spikes and bacterial survival.
Area of Science:
- Microbiology
- Cell Biology
- Immunology
Background:
- Listeria monocytogenes infection causes intracellular calcium elevations in macrophages.
- Bacterial secreted proteins, including phosphatidylinositol-specific phospholipase C (PI-PLC) and listeriolysin O (LLO), are implicated in these calcium changes.
- Host cell signaling pathways involving phospholipases are activated during infection.
Purpose of the Study:
- To investigate the roles of host PI hydrolysis and phospholipase C (PLC) and phospholipase D (PLD) activation during Listeria monocytogenes infection.
- To determine the contribution of bacterial PI-PLC and LLO to host cell calcium signaling and phospholipase activation.
- To elucidate the involvement of protein kinase C (PKC) in host PLD activation during infection.
Main Methods:
- Infection of J774 murine macrophage cell line and murine bone marrow-derived macrophages with wild-type and mutant Listeria monocytogenes.
- Measurement of host phosphatidylinositol (PI) hydrolysis and activation of host PLC and PLD.
- Utilized phorbol myristate acetate (PMA) and specific PKC inhibitors (rottlerin, hispidin) to investigate PLD activation pathways.
- Assessed the impact of a PLD inhibitor on bacterial escape from phagocytic vacuoles.
Main Results:
- Elevated host PI hydrolysis and PLC activation occurred within the first 10 minutes of infection, dependent on bacterial PI-PLC and LLO for earliest calcium elevations.
- Host PLD activation was observed at 20 minutes post-infection and was dependent on bacterial LLO.
- PKC delta was implicated in LLO-dependent PLD activation, while PKC beta isoforms were not.
- Inhibition of PLD partially impaired bacterial escape from the phagocytic vacuole.
Conclusions:
- Bacterial PI-PLC and LLO are critical for early host cell calcium signaling and PI hydrolysis during Listeria monocytogenes infection.
- Host PLD activation is LLO-dependent and involves PKC delta, contributing to bacterial intracellular survival.
- Understanding these host-pathogen interactions provides insights into bacterial pathogenesis and host defense mechanisms.
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