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.