Role of Protein Kinase C During Listeria monocytogenes Infection
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Date
2025-05
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The Ohio State University
Abstract
Listeria monocytogenes (Lm) is the facultative intracellular bacterium responsible for the deadly foodborne illness listeriosis. Using an intracellular lifecycle, Lm effectively evades extracellular immune responses. Lm virulence factors including the secreted pore-forming toxin listeriolysin O (LLO) and phospholipases C mediate escape from the phagosome. Protein Kinase C (PKC) enzymes, which regulate cell signaling through phosphorylation, are activated during Lm infection. Conventional PKCs (cPKCs), a subfamily of PKCs, are activated by calcium (Ca2+), diacylglycerol, and phosphatidylserine. Our lab has previously shown that cPKCs are activated by extracellular Ca2+ influx following LLO-mediated plasma membrane perforation in HepG2 epithelial cells, promoting Lm internalization into epithelial cells. The goal of this work was to develop experimental tools to study the role of cPKC in Lm phagosomal escape in epithelial cells and macrophages.
To monitor the spatiotemporal dynamics of cPKC translocation and activation by live cell imaging, we engineered cell lines stably expressing a fluorescent chimera of the conventional PKCĪ± and cell lines expressing a C kinase activity reporter which uses fluorescence resonance energy transfer. We constructed lentiviral vectors using plasmid cloning techniques, generated and isolated lentiviruses, and transduced human HeLa epithelial cells and THP-1 monocytes. Antibiotic selection was used to select cells stably expressing each construct. We also validated the use of lentiviral vectors in differentiated murine Bone Marrow-Derived Macrophages (BMDMs) prior to Lm infection. For fluorescence imaging of Lm phagosomal escape, we developed wild type and isogenic Lm mutant strains with constitutive expression of a red fluorescent protein and phagocytic assays in BMDMs and THP-1 cells differentiated into macrophages. These cell lines and fluorescent Lm strains will be useful in studying cPKC during the intracellular Lm lifecycle in epithelial cells and macrophages. All tools required for establishing the role of cPKC were successfully generated and I am currently performing invasion assays using cPKC inhibitors. Future directions include conducting infection assays in the presence or absence of cPKC inhibitors, and wild type and isogenic Lm mutants defective in LLO and/or phospholipases C to determine the role of cPKCs. Understanding the role of cPKC in host cell infection by Lm is expected to facilitate the development of therapeutic interventions to alleviate the burden of listeriosis.
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Keywords
Phagocytosis, Listeria, Protein Kinase C, Microbiology