Infection by <named-content content-type="genus-species">Anaplasma phagocytophilum</named-content> Requires Recruitment of Low-Density Lipoprotein Cholesterol by Flotillins

ABSTRACT 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 tran...

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Autores principales:	Qingming Xiong, Mingqun Lin, Weiyan Huang, Yasuko Rikihisa
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	Anaplasma phagocytophilum cholesterol flotillin-1 flotillin-2 LDL acid lipase Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/ec9217fa150944b9b7f29af6dd4a331e
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spelling	oai:doaj.org-article:ec9217fa150944b9b7f29af6dd4a331e2021-11-15T15:55:24ZInfection by <named-content content-type="genus-species">Anaplasma phagocytophilum</named-content> Requires Recruitment of Low-Density Lipoprotein Cholesterol by Flotillins10.1128/mBio.02783-182150-7511https://doaj.org/article/ec9217fa150944b9b7f29af6dd4a331e2019-04-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.02783-18https://doaj.org/toc/2150-7511ABSTRACT 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. IMPORTANCE Cholesterol is essential for animal cells, but most bacteria do not depend on cholesterol and instead lack cholesterol. However, the intracellular Gram-negative bacterium Anaplasma phagocytophilum that causes human granulocytic anaplasmosis (HGA) is unusual, as it contains significant amount of cholesterol and depends on cholesterol for survival and infection. A. phagocytophilum lacks genes for cholesterol biosynthesis or modification but acquire cholesterol from host cells exclusively from the LDL uptake pathway by a yet-to-be defined mechanism. Here, we uncovered a role of cholesterol-binding proteins FLOT1 and FLOT2 in LDL-derived cholesterol trafficking to Anaplasma inclusions and cholesterol acquisition by Anaplasma species. Importantly, we found that FLOTs localize to A. phagocytophilum-containing inclusions and the compartments containing LDL, and the acid lipase inhibitor orlistat significantly inhibits Anaplasma replication. Our data suggest a fundamental role of FLOTs in intracellular vesicular transport of LDL-derived free cholesterol and may provide insight regarding a new therapeutic target for HGA treatment.Qingming XiongMingqun LinWeiyan HuangYasuko RikihisaAmerican Society for MicrobiologyarticleAnaplasma phagocytophilumcholesterolflotillin-1flotillin-2LDLacid lipaseMicrobiologyQR1-502ENmBio, Vol 10, Iss 2 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	Anaplasma phagocytophilum cholesterol flotillin-1 flotillin-2 LDL acid lipase Microbiology QR1-502
spellingShingle	Anaplasma phagocytophilum cholesterol flotillin-1 flotillin-2 LDL acid lipase Microbiology QR1-502 Qingming Xiong Mingqun Lin Weiyan Huang Yasuko Rikihisa Infection by <named-content content-type="genus-species">Anaplasma phagocytophilum</named-content> Requires Recruitment of Low-Density Lipoprotein Cholesterol by Flotillins
description	ABSTRACT 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. IMPORTANCE Cholesterol is essential for animal cells, but most bacteria do not depend on cholesterol and instead lack cholesterol. However, the intracellular Gram-negative bacterium Anaplasma phagocytophilum that causes human granulocytic anaplasmosis (HGA) is unusual, as it contains significant amount of cholesterol and depends on cholesterol for survival and infection. A. phagocytophilum lacks genes for cholesterol biosynthesis or modification but acquire cholesterol from host cells exclusively from the LDL uptake pathway by a yet-to-be defined mechanism. Here, we uncovered a role of cholesterol-binding proteins FLOT1 and FLOT2 in LDL-derived cholesterol trafficking to Anaplasma inclusions and cholesterol acquisition by Anaplasma species. Importantly, we found that FLOTs localize to A. phagocytophilum-containing inclusions and the compartments containing LDL, and the acid lipase inhibitor orlistat significantly inhibits Anaplasma replication. Our data suggest a fundamental role of FLOTs in intracellular vesicular transport of LDL-derived free cholesterol and may provide insight regarding a new therapeutic target for HGA treatment.
format	article
author	Qingming Xiong Mingqun Lin Weiyan Huang Yasuko Rikihisa
author_facet	Qingming Xiong Mingqun Lin Weiyan Huang Yasuko Rikihisa
author_sort	Qingming Xiong
title	Infection by <named-content content-type="genus-species">Anaplasma phagocytophilum</named-content> Requires Recruitment of Low-Density Lipoprotein Cholesterol by Flotillins
title_short	Infection by <named-content content-type="genus-species">Anaplasma phagocytophilum</named-content> Requires Recruitment of Low-Density Lipoprotein Cholesterol by Flotillins
title_full	Infection by <named-content content-type="genus-species">Anaplasma phagocytophilum</named-content> Requires Recruitment of Low-Density Lipoprotein Cholesterol by Flotillins
title_fullStr	Infection by <named-content content-type="genus-species">Anaplasma phagocytophilum</named-content> Requires Recruitment of Low-Density Lipoprotein Cholesterol by Flotillins
title_full_unstemmed	Infection by <named-content content-type="genus-species">Anaplasma phagocytophilum</named-content> Requires Recruitment of Low-Density Lipoprotein Cholesterol by Flotillins
title_sort	infection by <named-content content-type="genus-species">anaplasma phagocytophilum</named-content> requires recruitment of low-density lipoprotein cholesterol by flotillins
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/ec9217fa150944b9b7f29af6dd4a331e
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_version_	1718427189012070400

Infection by <named-content content-type="genus-species">Anaplasma phagocytophilum</named-content> Requires Recruitment of Low-Density Lipoprotein Cholesterol by Flotillins

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