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Infection by Anaplasma phagocytophilum Requires Recruitment of Low-Density Lipoprotein Cholesterol by Flotillins

Anaplasma phagocytophilum is an obligatory intracellular bacterium that proliferates in membrane-bound inclusions. A. phagocytophilum is dependent on cholesterol and acquire cholesterol from low-density lipoprotein (LDL) endocytosed by mammalian host cells. The mechanism of cholesterol transport to...

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Autores principales: Xiong, Qingming, Lin, Mingqun, Huang, Weiyan, Rikihisa, Yasuko
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Microbiology 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6437059/
https://www.ncbi.nlm.nih.gov/pubmed/30914515
http://dx.doi.org/10.1128/mBio.02783-18
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author Xiong, Qingming
Lin, Mingqun
Huang, Weiyan
Rikihisa, Yasuko
author_facet Xiong, Qingming
Lin, Mingqun
Huang, Weiyan
Rikihisa, Yasuko
author_sort Xiong, Qingming
collection PubMed
description Anaplasma phagocytophilum is an obligatory intracellular bacterium that proliferates in membrane-bound inclusions. A. phagocytophilum is dependent on cholesterol and acquire cholesterol from low-density lipoprotein (LDL) endocytosed by mammalian host cells. The mechanism of cholesterol transport to Anaplasma inclusions, however, is not fully understood. Flotillin-1 (FLOT1) and FLOT2 are cholesterol-associated membrane proteins that form a heterodimer and/or oligomer complex. Here, we found that Anaplasma infection was significantly reduced by small interfering RNA (siRNA) knockdown of FLOT1 or FLOT2. Anaplasma inclusions were encircled with small vesicles containing endogenous FLOT1 or FLOT2 or with ectopically expressed FLOT1-mCherry and FLOT2-green fluorescent protein (FLOT2-GFP). FLOT1- and FLOT2-containing vesicles were enriched with unesterified cholesterol, as indicated by labeling with filipin and aminomethyl coumarin acetic acid-conjugated theonellamide. Localization of FLOT2 to Anaplasma inclusions was dependent on cholesterol, as FLOT2-GFP bearing two mutations in the cholesterol recognition/interaction motif could not target the inclusions. The cholesterol-sequestering agent methyl-β-cyclodextrin abrogated FLOT1 localization to Anaplasma inclusions and cleared infection. FLOT2-GFP also localized to fluorescent 3,3′-dioctadecylindocarbocyanine (DiI)-LDL-containing vesicles, including those surrounding Anaplasma inclusions. FLOT2 siRNA knockdown blocked DiI-LDL trafficking to Anaplasma inclusions and reduced bacteria-associated cholesterol amount, and therefore inhibiting Anaplasma infection. Vesicles containing acid lipase, which hydrolyzes LDL cholesterol esters to free cholesterol, colocalized with FLOT2 and encircled Anaplasma inclusions, while the acid lipase inhibitor orlistat significantly inhibited Anaplasma replication. Together, the data revealed that FLOTs are crucial for Anaplasma replication in host cells, likely by aiding vesicular traffic of LDL-derived free cholesterol to Anaplasma inclusions, and suggest a new way of inhibiting Anaplasma infection.
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spelling pubmed-64370592019-04-03 Infection by Anaplasma phagocytophilum Requires Recruitment of Low-Density Lipoprotein Cholesterol by Flotillins Xiong, Qingming Lin, Mingqun Huang, Weiyan Rikihisa, Yasuko mBio Research Article Anaplasma phagocytophilum is an obligatory intracellular bacterium that proliferates in membrane-bound inclusions. A. phagocytophilum is dependent on cholesterol and acquire cholesterol from low-density lipoprotein (LDL) endocytosed by mammalian host cells. The mechanism of cholesterol transport to Anaplasma inclusions, however, is not fully understood. Flotillin-1 (FLOT1) and FLOT2 are cholesterol-associated membrane proteins that form a heterodimer and/or oligomer complex. Here, we found that Anaplasma infection was significantly reduced by small interfering RNA (siRNA) knockdown of FLOT1 or FLOT2. Anaplasma inclusions were encircled with small vesicles containing endogenous FLOT1 or FLOT2 or with ectopically expressed FLOT1-mCherry and FLOT2-green fluorescent protein (FLOT2-GFP). FLOT1- and FLOT2-containing vesicles were enriched with unesterified cholesterol, as indicated by labeling with filipin and aminomethyl coumarin acetic acid-conjugated theonellamide. Localization of FLOT2 to Anaplasma inclusions was dependent on cholesterol, as FLOT2-GFP bearing two mutations in the cholesterol recognition/interaction motif could not target the inclusions. The cholesterol-sequestering agent methyl-β-cyclodextrin abrogated FLOT1 localization to Anaplasma inclusions and cleared infection. FLOT2-GFP also localized to fluorescent 3,3′-dioctadecylindocarbocyanine (DiI)-LDL-containing vesicles, including those surrounding Anaplasma inclusions. FLOT2 siRNA knockdown blocked DiI-LDL trafficking to Anaplasma inclusions and reduced bacteria-associated cholesterol amount, and therefore inhibiting Anaplasma infection. Vesicles containing acid lipase, which hydrolyzes LDL cholesterol esters to free cholesterol, colocalized with FLOT2 and encircled Anaplasma inclusions, while the acid lipase inhibitor orlistat significantly inhibited Anaplasma replication. Together, the data revealed that FLOTs are crucial for Anaplasma replication in host cells, likely by aiding vesicular traffic of LDL-derived free cholesterol to Anaplasma inclusions, and suggest a new way of inhibiting Anaplasma infection. American Society for Microbiology 2019-03-26 /pmc/articles/PMC6437059/ /pubmed/30914515 http://dx.doi.org/10.1128/mBio.02783-18 Text en Copyright © 2019 Xiong et al. https://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Research Article
Xiong, Qingming
Lin, Mingqun
Huang, Weiyan
Rikihisa, Yasuko
Infection by Anaplasma phagocytophilum Requires Recruitment of Low-Density Lipoprotein Cholesterol by Flotillins
title Infection by Anaplasma phagocytophilum Requires Recruitment of Low-Density Lipoprotein Cholesterol by Flotillins
title_full Infection by Anaplasma phagocytophilum Requires Recruitment of Low-Density Lipoprotein Cholesterol by Flotillins
title_fullStr Infection by Anaplasma phagocytophilum Requires Recruitment of Low-Density Lipoprotein Cholesterol by Flotillins
title_full_unstemmed Infection by Anaplasma phagocytophilum Requires Recruitment of Low-Density Lipoprotein Cholesterol by Flotillins
title_short Infection by Anaplasma phagocytophilum Requires Recruitment of Low-Density Lipoprotein Cholesterol by Flotillins
title_sort infection by anaplasma phagocytophilum requires recruitment of low-density lipoprotein cholesterol by flotillins
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6437059/
https://www.ncbi.nlm.nih.gov/pubmed/30914515
http://dx.doi.org/10.1128/mBio.02783-18
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