high-throughput-measurement-plasma-membrane-resealing-efficiency

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...

The Ohio State University

Autophagy and inflammasome complex assembly are physiological processes that control homeostasis, inflammation, and immunity. Autophagy is a ubiquitous pathway that degrades cytosolic macromolecules or organelles, as well as intracellular pathogens. Inflammasomes are multi-protein complexes that assemble in the cytosol of cells upon detection of pathogen- or danger-associated molecular patterns. A critical outcome of inflammasome assembly is the activation of the cysteine protease caspase-1, which activates the pro-inflammatory cytokine precursors pro-IL-1β and pro-IL-18. Studies on chronic inflammatory diseases, heart diseases, Alzheimer's disease, and multiple sclerosis revealed that autophagy and inflammasomes intersect and regulate each other. In the context of infectious diseases, however, less is known about the interplay between autophagy and inflammasome assembly, although it is becoming evident that pathogens have evolved multiple strategies to inhibit and/or subvert these pathways and to take advantage of their intricate crosstalk. An improved appreciation of these pathways and their subversion by diverse pathogens is expected to help in the design of anti-infective therapeutic interventions.

Checks and Balances between Autophagy and Inflammasomes during Infection.

Host cell invasion is an indispensable step for a successful infection by intracellular pathogens. Recent studies identified pathogen-induced host cell plasma membrane perforation as a novel mechanism used by diverse pathogens (, , and adenovirus) to promote their internalization into target cells. It was concluded that  and adenovirus damage the host cell plasma membrane to hijack the endocytic-dependent membrane resealing machinery, thereby invading the host cell. We studied  and its secreted pore-forming toxin listeriolysin O (LLO) to identify key signaling events activated upon plasma membrane perforation that lead to bacterial internalization. Using various approaches, including fluorescence resonance energy transfer imaging, we found that the influx of extracellular Ca subsequent to LLO-mediated plasma membrane perforation is required for the activation of a conventional protein kinase C (cPKC). cPKC is positioned upstream of Rac1 and the Arp2/3 complex, which activation leads to F-actin--dependent bacterial internalization. Inhibition of this pathway did not prevent membrane resealing, revealing that perforation-dependent  endocytosis is distinct from the resealing machinery. These studies identified the LLO-dependent endocytic pathway of  and support a novel model for pathogen uptake promoted by plasma membrane injury that is independent of membrane resealing.

Host cell perforation by listeriolysin O (LLO) activates a Ca-dependent cPKC/Rac1/Arp2/3 signaling pathway that promotes  internalization independently of membrane resealing.

 is a facultative intracellular pathogen that infects a wide variety of cells, causing the life-threatening disease listeriosis.  virulence factors include two surface invasins, InlA and InlB, known to promote bacterial uptake by host cells, and the secreted pore-forming toxin listeriolysin O (LLO), which disrupts the phagosome to allow bacterial proliferation in the cytosol. In addition, plasma membrane perforation by LLO has been shown to facilitate  internalization into epithelial cells. In this work, we tested the host cell range and importance of LLO-mediated  internalization relative to the canonical invasins, InlA and InlB. We measured the efficiencies of  association with and internalization into several human cell types (hepatocytes, cytotrophoblasts, and endothelial cells) using wild-type bacteria and isogenic single, double, and triple deletion mutants for the genes encoding InlA, InlB and LLO. No role for InlB was detected in any tested cells unless the InlB expression level was substantially enhanced, which was achieved by introducing a mutation () in the gene encoding the transcription factor PrfA. In contrast, InlA and LLO were the most critical invasion factors, although they act in a different manner and in a cell-type-dependent fashion. As expected, InlA facilitates both bacterial attachment and internalization in cells that express its receptor, E-cadherin. LLO promotes  internalization into hepatocytes, but not into cytotrophoblasts and endothelial cells. Finally, LLO and InlA cooperate to increase the efficiency of host cell invasion by .

Stephanie Seveau

Department of Microbial Infection and Immunity,
Department of Microbiology,
Infectious Diseases Institute

The Ohio State University

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

Stephanie Seveau

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Microbial Infection and Immunity,Department of Microbiology,Infectious Diseases Institute

The Ohio State University

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

Department of Microbial Infection and Immunity,
Department of Microbiology,
Infectious Diseases Institute