Plasma Membrane Cholesterol Modulates Cellular Vacuolation Induced by the Helicobacter pylori Vacuolating Cytotoxin

ABSTRACT The Helicobacter pylori vacuolating cytotoxin (VacA) induces the degenerative vacuolation of mammalian cells both in vitro and in vivo. Here, we demonstrate that plasma membrane cholesterol is essential for vacuolation of mammalian cells by VacA. Vacuole biogenesis in multiple cell lines was completely blocked when cholesterol was extracted selectively from the plasma membrane by using β-cyclodextrins. Moreover, increasing plasma membrane cholesterol levels strongly potentiated VacA-induced vacuolation. In contrast, inhibiting de novo biosynthesis of cholesterol with lovastatin or compactin had no detectable effect on vacuolation. While depletion of plasma membrane cholesterol has been shown to interfere with both clathrin-mediated endocytosis and caveola-dependent endocytosis, neither of these two internalization pathways was found to be essential for vacuolation of cells by VacA. Depleting plasma membrane cholesterol attenuated the entry of VacA into HeLa cells. In addition, β-cyclodextrin reagents blocked vacuolation of cells that were either preloaded with VacA or had VacA directly expressed within the cytosol. Collectively, our results suggest that plasma membrane cholesterol is important for both the intoxication mechanism of VacA and subsequent vacuole biogenesis.

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Direct Electrochemical Evaluation of Plasma Membrane Cholesterol in Live Mammalian Cells

Journal of the American Chemical Society ◽

10.1021/ja074373c ◽

2007 ◽

Vol 129 (37) ◽

pp. 11352-11353 ◽

Cited By ~ 27

Author(s):

Dechen Jiang ◽

Anando Devadoss ◽

M. Simona Palencsár ◽

Danjun Fang ◽

Nicole M. White ◽

...

Keyword(s):

Plasma Membrane ◽

Mammalian Cells ◽

Membrane Cholesterol ◽

Electrochemical Evaluation ◽

Plasma Membrane Cholesterol

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Reprogramming cholesterol metabolism in macrophages and its role in host defense against cholesterol-dependent cytolysins

Cellular and Molecular Immunology ◽

10.1038/s41423-021-00827-0 ◽

2022 ◽

Author(s):

Min-Sub Lee ◽

Steven J. Bensinger

Keyword(s):

Plasma Membrane ◽

Host Defense ◽

Mammalian Cells ◽

Cholesterol Metabolism ◽

Current Knowledge ◽

Membrane Integrity ◽

Cholesterol Homeostasis ◽

Metabolic Reprogramming ◽

Membrane Cholesterol ◽

Plasma Membrane Cholesterol

AbstractCholesterol is a critical lipid for all mammalian cells, ensuring proper membrane integrity, fluidity, and biochemical function. Accumulating evidence indicates that macrophages rapidly and profoundly reprogram their cholesterol metabolism in response to activation signals to support host defense processes. However, our understanding of the molecular details underlying how and why cholesterol homeostasis is specifically reshaped during immune responses remains less well understood. This review discusses our current knowledge of cellular cholesterol homeostatic machinery and introduces emerging concepts regarding how plasma membrane cholesterol is partitioned into distinct pools. We then discuss how proinflammatory signals can markedly reshape the cholesterol metabolism of macrophages, with a focus on the differences between MyD88-dependent pattern recognition receptors and the interferon signaling pathway. We also discuss recent work investigating the capacity of these proinflammatory signals to selectively reshape plasma membrane cholesterol homeostasis. We examine how these changes in plasma membrane cholesterol metabolism influence sensitivity to a set of microbial pore-forming toxins known as cholesterol-dependent cytolysins that specifically target cholesterol for their effector functions. We also discuss whether lipid metabolic reprogramming can be leveraged for therapy to mitigate tissue damage mediated by cholesterol-dependent cytolysins in necrotizing fasciitis and other related infections. We expect that advancing our understanding of the crosstalk between metabolism and innate immunity will help explain how inflammation underlies metabolic diseases and highlight pathways that could be targeted to normalize metabolic homeostasis in disease states.

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Direct Electrochemical Evaluation of Plasma Membrane Cholesterol in Living Mammalian Cells

ECS Meeting Abstracts ◽

10.1149/ma2007-01/10/496 ◽

2007 ◽

Keyword(s):

Plasma Membrane ◽

Mammalian Cells ◽

Membrane Cholesterol ◽

Electrochemical Evaluation ◽

Plasma Membrane Cholesterol

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Increased plasma membrane cholesterol in cystic fibrosis cells correlates with CFTR genotype and depends on de novo cholesterol synthesis

Respiratory Research ◽

10.1186/1465-9921-11-61 ◽

2010 ◽

Vol 11 (1) ◽

Cited By ~ 23

Author(s):

Danjun Fang ◽

Richard H West ◽

Mary E Manson ◽

Jennifer Ruddy ◽

Dechen Jiang ◽

...

Keyword(s):

Cystic Fibrosis ◽

Plasma Membrane ◽

Cholesterol Synthesis ◽

De Novo ◽

Membrane Cholesterol ◽

Plasma Membrane Cholesterol

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Comment on ‘Orthogonal lipid sensors identify transbilayer asymmetry of plasma membrane cholesterol’

eLife ◽

10.7554/elife.38493 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 7

Author(s):

Kevin C Courtney ◽

Karen YY Fung ◽

Frederick R Maxfield ◽

Gregory D Fairn ◽

Xiaohui Zha

Keyword(s):

Plasma Membrane ◽

Mammalian Cells ◽

Live Cells ◽

Current Evidence ◽

Dependent Manner ◽

Perfringolysin O ◽

Highly Sensitive ◽

Lipid Sensors ◽

Plasma Membrane Cholesterol

The plasma membrane in mammalian cells is rich in cholesterol, but how the cholesterol is partitioned between the two leaflets of the plasma membrane remains a matter of debate. Recently, Liu et al. used domain 4 (D4) of perfringolysin O as a cholesterol sensor to argue that cholesterol is mostly in the exofacial leaflet (<xref ref-type="bibr" rid="bib7">Liu et al., 2017</xref>). This conclusion was made by interpreting D4 binding in live cells using in vitro calibrations with liposomes. However, liposomes may be unfaithful in mimicking the plasma membrane, as we demonstrate here. Also, D4 binding is highly sensitive to the presence of cytosolic proteins. In addition, we find that a D4 variant, which requires >35 mol% cholesterol to bind to liposomes in vitro, does in fact bind to the cytoplasmic leaflet of the plasma membrane in a cholesterol-dependent manner. Thus, we believe, based on the current evidence, that it is unlikely that there is a significantly higher proportion of cholesterol in the exofacial leaflet of the plasma membrane compared to the cytosolic leaflet.

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Computational drug repurposing against SARS-CoV-2 reveals plasma membrane cholesterol depletion as key factor of antiviral drug activity

10.1101/2021.09.10.459786 ◽

2021 ◽

Author(s):

Szilvia Barsi ◽

Henrietta Papp ◽

Alberto Valdeolivas Urbelz ◽

Dániel J. Tóth ◽

Anett Kuczmog ◽

...

Keyword(s):

Gene Expression ◽

Plasma Membrane ◽

Drug Repurposing ◽

Antiviral Effect ◽

Membrane Cholesterol ◽

General Hypothesis ◽

Gene Expression Signatures ◽

Disease Signature ◽

Plasma Membrane Cholesterol

AbstractComparing SARS-CoV-2 infection-induced gene expression signatures to drug treatment-induced gene expression signatures is a promising bioinformatic tool to repurpose existing drugs against SARS-CoV-2. The general hypothesis of signature based drug repurposing is that drugs with inverse similarity to a disease signature can reverse disease phenotype and thus be effective against it. However, in the case of viral infection diseases, like SARS-CoV-2, infected cells also activate adaptive, antiviral pathways, so that the relationship between effective drug and disease signature can be more ambiguous.To address this question, we analysed gene expression data from in vitro SARS-CoV-2 infected cell lines, and gene expression signatures of drugs showing anti-SARS-CoV-2 activity. Our extensive functional genomic analysis showed that both infection and treatment with in vitro effective drugs leads to activation of antiviral pathways like NFkB and JAK-STAT. Based on the similarity - and not inverse similarity - between drug and infection-induced gene expression signatures, we were able to predict the in vitro antiviral activity of drugs. We also identified SREBF1/2, key regulators of lipid metabolising enzymes, as the most activated transcription factors by several in vitro effective antiviral drugs. Using a fluorescently labeled cholesterol sensor, we showed that these drugs decrease the cholesterol levels of plasma-membrane. Supplementing drug-treated cells with cholesterol reversed the in vitro antiviral effect, suggesting the depleting plasma-membrane cholesterol plays a key role in virus inhibitory mechanism.Our results can help to more effectively repurpose approved drugs against SARS-CoV-2, and also highlights key mechanisms behind their antiviral effect.Abstract Figure

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