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Marguerite Rinaudo - One of the best experts on this subject based on the ideXlab platform.
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on the gelling behaviour of nopal opuntia ficus indica low methoxyl pectin
Carbohydrate Polymers, 2008Co-Authors: Adriana Cardenas, Francisco M Goycoolea, Marguerite RinaudoAbstract:Abstract Fully de-esterified pectin with excellent gelling properties was isolated in the sodium-salt form from fresh ‘nopal’ cactus ( Opuntia ficus indica ) pads (0.6% w/w yield of fresh weight) using an alkaline extraction medium in the presence of a Sequestering Agent. Sugars composition of the cactus pectin alkaline extract (CPAE) was: 85.4% uronic acids, 7.0% galactose, 6.0% arabinose and minor quantities of rhamnose and xylose. Features of the Fourier-transformed infrared spectrum were nearly identical to those of commercial citrus pectin as well as to homogalacturan-rich pectin isolated from prickly pear, lime peel, and sugar beet pulp. The gelling behavior of this material was studied as a function of amount of Ca 2+ added and temperature by dynamic oscillatory rheology. Addition of Ca 2+ at 85 °C was adjusted at varying stoichiometric ratios, R (=2 [Ca 2+ ]/[COO − ]), namely 0.07, 0.21, 0.35 and 0.42 and fixed pectin concentration (16 g/L), and a temperature dependent behavior of the system on cooling was imposed. Evolution of the viscoelastic storage ( G ′) and loss ( G ′′ moduli on cooling revealed unequivocal set up of a gel network under a highly cooperative sol-gel transition at R ⩽ 0.21. At greater R values, the Ca 2+ -mediated dimeric association of pectin chains led to formation of a gel network even at 85 °C. On heating, pectin gels melted partially at temperatures notably greater than those at which they were formed. Thermal hysteresis observed between the cooling and heating temperature traces is explained in terms of helix–helix aggregation. The gelling behavior of this system is interpreted in terms of the formation of two distinct types of junctions mediated by the stoichiometric amount of calcium ( R ). Namely, short ‘egg-box’ type junctions formed directly at high temperature on addition of calcium (limited zones are related to high mobility of the chains) and highly cooperative 2 1 helix junctions followed by aggregation formed at lower temperature under a thermal conformational transition driven by charge neutralization and lower chain mobility (related to stabilization by H-bonds).
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on the gelling behaviour of nopal opuntia ficus indica low methoxyl pectin
Carbohydrate Polymers, 2008Co-Authors: Adriana Cardenas, Francisco M Goycoolea, Marguerite RinaudoAbstract:Abstract Fully de-esterified pectin with excellent gelling properties was isolated in the sodium-salt form from fresh ‘nopal’ cactus ( Opuntia ficus indica ) pads (0.6% w/w yield of fresh weight) using an alkaline extraction medium in the presence of a Sequestering Agent. Sugars composition of the cactus pectin alkaline extract (CPAE) was: 85.4% uronic acids, 7.0% galactose, 6.0% arabinose and minor quantities of rhamnose and xylose. Features of the Fourier-transformed infrared spectrum were nearly identical to those of commercial citrus pectin as well as to homogalacturan-rich pectin isolated from prickly pear, lime peel, and sugar beet pulp. The gelling behavior of this material was studied as a function of amount of Ca 2+ added and temperature by dynamic oscillatory rheology. Addition of Ca 2+ at 85 °C was adjusted at varying stoichiometric ratios, R (=2 [Ca 2+ ]/[COO − ]), namely 0.07, 0.21, 0.35 and 0.42 and fixed pectin concentration (16 g/L), and a temperature dependent behavior of the system on cooling was imposed. Evolution of the viscoelastic storage ( G ′) and loss ( G ′′ moduli on cooling revealed unequivocal set up of a gel network under a highly cooperative sol-gel transition at R ⩽ 0.21. At greater R values, the Ca 2+ -mediated dimeric association of pectin chains led to formation of a gel network even at 85 °C. On heating, pectin gels melted partially at temperatures notably greater than those at which they were formed. Thermal hysteresis observed between the cooling and heating temperature traces is explained in terms of helix–helix aggregation. The gelling behavior of this system is interpreted in terms of the formation of two distinct types of junctions mediated by the stoichiometric amount of calcium ( R ). Namely, short ‘egg-box’ type junctions formed directly at high temperature on addition of calcium (limited zones are related to high mobility of the chains) and highly cooperative 2 1 helix junctions followed by aggregation formed at lower temperature under a thermal conformational transition driven by charge neutralization and lower chain mobility (related to stabilization by H-bonds).
A T Adesogan - One of the best experts on this subject based on the ideXlab platform.
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biomarker of aflatoxin ingestion h nmr based plasma metabolomics of dairy cows fed aflatoxin b with or without Sequestering Agents
Toxins, 2018Co-Authors: Ibukun Ogunade, Yun Jiang, James Adeyemi, Diwakar Vyas, Andre Soares De Oliveira, A T AdesoganAbstract:The study applied ¹H NMR-based plasma metabolomics to identify candidate biomarkers of aflatoxin B1 (AFB₁) ingestion in dairy cows fed no Sequestering Agents and evaluate the effect of supplementing clay and/or a Saccharomyces cerevisiae fermentation product (SCFP) on such biomarkers. Eight lactating cows were randomly assigned to 1 of 4 treatments in a balanced 4 × 4 Latin square design with 2 squares. Treatments were: control, toxin (T; 1725 µg AFB₁/head/day), T with clay (CL; 200 g/head/day), and CL with SCFP (CL + SCFP; 35 g of SCFP/head/day). Cows in T, CL, and CL + SCFP were dosed with AFB₁ from d 26 to 30. The Sequestering Agents were top-dressed from d 1 to 33. On d 30 of each period, 15 mL of blood was taken from the coccygeal vessels and plasma samples were prepared by centrifugation. Compared to the control, T decreased plasma concentrations of alanine, acetic acid, leucine, arginine and valine. In contrast, T increased plasma ethanol concentration 3.56-fold compared to control. Treatment with CL tended to reduce sarcosine concentration, whereas treatment with CL + SCFP increased concentrations of mannose and 12 amino acids. Based on size of the area under the curve (AUC) of receiver operating characteristic and fold change (FC) analyses, ethanol was the most significantly altered metabolite in T (AUC = 0.88; FC = 3.56); hence, it was chosen as the candidate biomarker of aflatoxin ingestion in dairy cows fed no Sequestering Agent.
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Biomarker of Aflatoxin Ingestion: 1H NMR-Based Plasma Metabolomics of Dairy Cows Fed Aflatoxin B1 with or without Sequestering Agents
MDPI AG, 2018Co-Authors: Ibukun Ogunade, Yun Jiang, James Adeyemi, Andre Oliveira, Diwakar Vyas, A T AdesoganAbstract:The study applied 1H NMR-based plasma metabolomics to identify candidate biomarkers of aflatoxin B1 (AFB1) ingestion in dairy cows fed no Sequestering Agents and evaluate the effect of supplementing clay and/or a Saccharomyces cerevisiae fermentation product (SCFP) on such biomarkers. Eight lactating cows were randomly assigned to 1 of 4 treatments in a balanced 4 × 4 Latin square design with 2 squares. Treatments were: control, toxin (T; 1725 µg AFB1/head/day), T with clay (CL; 200 g/head/day), and CL with SCFP (CL + SCFP; 35 g of SCFP/head/day). Cows in T, CL, and CL + SCFP were dosed with AFB1 from d 26 to 30. The Sequestering Agents were top-dressed from d 1 to 33. On d 30 of each period, 15 mL of blood was taken from the coccygeal vessels and plasma samples were prepared by centrifugation. Compared to the control, T decreased plasma concentrations of alanine, acetic acid, leucine, arginine and valine. In contrast, T increased plasma ethanol concentration 3.56-fold compared to control. Treatment with CL tended to reduce sarcosine concentration, whereas treatment with CL + SCFP increased concentrations of mannose and 12 amino acids. Based on size of the area under the curve (AUC) of receiver operating characteristic and fold change (FC) analyses, ethanol was the most significantly altered metabolite in T (AUC = 0.88; FC = 3.56); hence, it was chosen as the candidate biomarker of aflatoxin ingestion in dairy cows fed no Sequestering Agent
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effect of adding a mycotoxin Sequestering Agent on milk aflatoxin m concentration and the performance and immune response of dairy cattle fed an aflatoxin b contaminated diet
Journal of Dairy Science, 2012Co-Authors: O.c.m. Queiroz, J H Han, C R Staples, A T AdesoganAbstract:This project aimed to examine the effects of adding 2 doses of a montmorillonite-based mycotoxin adsorbent on milk aflatoxin M(1) (AFM(1)) concentrations and the performance and innate immune response of dairy cows fed an aflatoxin B(1) (AFB(1))-contaminated diet. Eight lactating cows were used in a duplicated 4×4 Latin square design with 12-d periods. Treatments included the following: (1) control diet (C), (2) aflatoxin diet (T) containing C and 75 µg of AFB(1)/kg, 3) low-clay (LC) diet containing T and Calibrin A (Amlan International, Chicago, IL) added at 0.2% of the diet dry matter (DM), and 4) high-clay diet (HC) containing T and Calibrin A added at 1% of the diet DM. Milk production and DM intake were recorded daily and milk was sampled twice daily on d 5, 9, 10, 11, and 12 in each period. Blood samples were collected on d 5 and 9 of each period. Dietary treatments did not affect DM intake, milk yield, or feed efficiency. Even though cows were limit fed, feeding T instead of C reduced milk fat yield (0.67 vs. 0.74 kg/d) and milk protein concentration (3.28 vs. 3.36%). Concentrations of AFM(1) in milk of cows fed the T and LC diets were similar (0.57 and 0.64 µg/kg) and greater than those of cows fed the HC diet (0.46 µg/kg). Haptoglobin concentration was greater (22.0 vs. 14.4) and β(2)-integrin expression (220 vs. 131) tended to be greater in cows fed diet T instead of C, but values for cows fed LC, HC, and C did not differ. In comparison to C, feeding T increased the innate immune response and decreased milk fat yield and milk protein concentration, but feeding LC and HC did not affect these measures. Only the HC diet reduced milk AFM(1) concentration.
Adriana Cardenas - One of the best experts on this subject based on the ideXlab platform.
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on the gelling behaviour of nopal opuntia ficus indica low methoxyl pectin
Carbohydrate Polymers, 2008Co-Authors: Adriana Cardenas, Francisco M Goycoolea, Marguerite RinaudoAbstract:Abstract Fully de-esterified pectin with excellent gelling properties was isolated in the sodium-salt form from fresh ‘nopal’ cactus ( Opuntia ficus indica ) pads (0.6% w/w yield of fresh weight) using an alkaline extraction medium in the presence of a Sequestering Agent. Sugars composition of the cactus pectin alkaline extract (CPAE) was: 85.4% uronic acids, 7.0% galactose, 6.0% arabinose and minor quantities of rhamnose and xylose. Features of the Fourier-transformed infrared spectrum were nearly identical to those of commercial citrus pectin as well as to homogalacturan-rich pectin isolated from prickly pear, lime peel, and sugar beet pulp. The gelling behavior of this material was studied as a function of amount of Ca 2+ added and temperature by dynamic oscillatory rheology. Addition of Ca 2+ at 85 °C was adjusted at varying stoichiometric ratios, R (=2 [Ca 2+ ]/[COO − ]), namely 0.07, 0.21, 0.35 and 0.42 and fixed pectin concentration (16 g/L), and a temperature dependent behavior of the system on cooling was imposed. Evolution of the viscoelastic storage ( G ′) and loss ( G ′′ moduli on cooling revealed unequivocal set up of a gel network under a highly cooperative sol-gel transition at R ⩽ 0.21. At greater R values, the Ca 2+ -mediated dimeric association of pectin chains led to formation of a gel network even at 85 °C. On heating, pectin gels melted partially at temperatures notably greater than those at which they were formed. Thermal hysteresis observed between the cooling and heating temperature traces is explained in terms of helix–helix aggregation. The gelling behavior of this system is interpreted in terms of the formation of two distinct types of junctions mediated by the stoichiometric amount of calcium ( R ). Namely, short ‘egg-box’ type junctions formed directly at high temperature on addition of calcium (limited zones are related to high mobility of the chains) and highly cooperative 2 1 helix junctions followed by aggregation formed at lower temperature under a thermal conformational transition driven by charge neutralization and lower chain mobility (related to stabilization by H-bonds).
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on the gelling behaviour of nopal opuntia ficus indica low methoxyl pectin
Carbohydrate Polymers, 2008Co-Authors: Adriana Cardenas, Francisco M Goycoolea, Marguerite RinaudoAbstract:Abstract Fully de-esterified pectin with excellent gelling properties was isolated in the sodium-salt form from fresh ‘nopal’ cactus ( Opuntia ficus indica ) pads (0.6% w/w yield of fresh weight) using an alkaline extraction medium in the presence of a Sequestering Agent. Sugars composition of the cactus pectin alkaline extract (CPAE) was: 85.4% uronic acids, 7.0% galactose, 6.0% arabinose and minor quantities of rhamnose and xylose. Features of the Fourier-transformed infrared spectrum were nearly identical to those of commercial citrus pectin as well as to homogalacturan-rich pectin isolated from prickly pear, lime peel, and sugar beet pulp. The gelling behavior of this material was studied as a function of amount of Ca 2+ added and temperature by dynamic oscillatory rheology. Addition of Ca 2+ at 85 °C was adjusted at varying stoichiometric ratios, R (=2 [Ca 2+ ]/[COO − ]), namely 0.07, 0.21, 0.35 and 0.42 and fixed pectin concentration (16 g/L), and a temperature dependent behavior of the system on cooling was imposed. Evolution of the viscoelastic storage ( G ′) and loss ( G ′′ moduli on cooling revealed unequivocal set up of a gel network under a highly cooperative sol-gel transition at R ⩽ 0.21. At greater R values, the Ca 2+ -mediated dimeric association of pectin chains led to formation of a gel network even at 85 °C. On heating, pectin gels melted partially at temperatures notably greater than those at which they were formed. Thermal hysteresis observed between the cooling and heating temperature traces is explained in terms of helix–helix aggregation. The gelling behavior of this system is interpreted in terms of the formation of two distinct types of junctions mediated by the stoichiometric amount of calcium ( R ). Namely, short ‘egg-box’ type junctions formed directly at high temperature on addition of calcium (limited zones are related to high mobility of the chains) and highly cooperative 2 1 helix junctions followed by aggregation formed at lower temperature under a thermal conformational transition driven by charge neutralization and lower chain mobility (related to stabilization by H-bonds).
Francisco M Goycoolea - One of the best experts on this subject based on the ideXlab platform.
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on the gelling behaviour of nopal opuntia ficus indica low methoxyl pectin
Carbohydrate Polymers, 2008Co-Authors: Adriana Cardenas, Francisco M Goycoolea, Marguerite RinaudoAbstract:Abstract Fully de-esterified pectin with excellent gelling properties was isolated in the sodium-salt form from fresh ‘nopal’ cactus ( Opuntia ficus indica ) pads (0.6% w/w yield of fresh weight) using an alkaline extraction medium in the presence of a Sequestering Agent. Sugars composition of the cactus pectin alkaline extract (CPAE) was: 85.4% uronic acids, 7.0% galactose, 6.0% arabinose and minor quantities of rhamnose and xylose. Features of the Fourier-transformed infrared spectrum were nearly identical to those of commercial citrus pectin as well as to homogalacturan-rich pectin isolated from prickly pear, lime peel, and sugar beet pulp. The gelling behavior of this material was studied as a function of amount of Ca 2+ added and temperature by dynamic oscillatory rheology. Addition of Ca 2+ at 85 °C was adjusted at varying stoichiometric ratios, R (=2 [Ca 2+ ]/[COO − ]), namely 0.07, 0.21, 0.35 and 0.42 and fixed pectin concentration (16 g/L), and a temperature dependent behavior of the system on cooling was imposed. Evolution of the viscoelastic storage ( G ′) and loss ( G ′′ moduli on cooling revealed unequivocal set up of a gel network under a highly cooperative sol-gel transition at R ⩽ 0.21. At greater R values, the Ca 2+ -mediated dimeric association of pectin chains led to formation of a gel network even at 85 °C. On heating, pectin gels melted partially at temperatures notably greater than those at which they were formed. Thermal hysteresis observed between the cooling and heating temperature traces is explained in terms of helix–helix aggregation. The gelling behavior of this system is interpreted in terms of the formation of two distinct types of junctions mediated by the stoichiometric amount of calcium ( R ). Namely, short ‘egg-box’ type junctions formed directly at high temperature on addition of calcium (limited zones are related to high mobility of the chains) and highly cooperative 2 1 helix junctions followed by aggregation formed at lower temperature under a thermal conformational transition driven by charge neutralization and lower chain mobility (related to stabilization by H-bonds).
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on the gelling behaviour of nopal opuntia ficus indica low methoxyl pectin
Carbohydrate Polymers, 2008Co-Authors: Adriana Cardenas, Francisco M Goycoolea, Marguerite RinaudoAbstract:Abstract Fully de-esterified pectin with excellent gelling properties was isolated in the sodium-salt form from fresh ‘nopal’ cactus ( Opuntia ficus indica ) pads (0.6% w/w yield of fresh weight) using an alkaline extraction medium in the presence of a Sequestering Agent. Sugars composition of the cactus pectin alkaline extract (CPAE) was: 85.4% uronic acids, 7.0% galactose, 6.0% arabinose and minor quantities of rhamnose and xylose. Features of the Fourier-transformed infrared spectrum were nearly identical to those of commercial citrus pectin as well as to homogalacturan-rich pectin isolated from prickly pear, lime peel, and sugar beet pulp. The gelling behavior of this material was studied as a function of amount of Ca 2+ added and temperature by dynamic oscillatory rheology. Addition of Ca 2+ at 85 °C was adjusted at varying stoichiometric ratios, R (=2 [Ca 2+ ]/[COO − ]), namely 0.07, 0.21, 0.35 and 0.42 and fixed pectin concentration (16 g/L), and a temperature dependent behavior of the system on cooling was imposed. Evolution of the viscoelastic storage ( G ′) and loss ( G ′′ moduli on cooling revealed unequivocal set up of a gel network under a highly cooperative sol-gel transition at R ⩽ 0.21. At greater R values, the Ca 2+ -mediated dimeric association of pectin chains led to formation of a gel network even at 85 °C. On heating, pectin gels melted partially at temperatures notably greater than those at which they were formed. Thermal hysteresis observed between the cooling and heating temperature traces is explained in terms of helix–helix aggregation. The gelling behavior of this system is interpreted in terms of the formation of two distinct types of junctions mediated by the stoichiometric amount of calcium ( R ). Namely, short ‘egg-box’ type junctions formed directly at high temperature on addition of calcium (limited zones are related to high mobility of the chains) and highly cooperative 2 1 helix junctions followed by aggregation formed at lower temperature under a thermal conformational transition driven by charge neutralization and lower chain mobility (related to stabilization by H-bonds).
Weiyan Huang - One of the best experts on this subject based on the ideXlab platform.
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Infection by Anaplasma phagocytophilum Requires Recruitment of Low-Density Lipoprotein Cholesterol by Flotillins.
mBio, 2019Co-Authors: Qingming Xiong, Mingqun Lin, Weiyan HuangAbstract: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.
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Infection by Anaplasma phagocytophilum Requires Recruitment of Low-Density Lipoprotein Cholesterol by Flotillins
American Society for Microbiology, 2019Co-Authors: Qingming Xiong, Mingqun Lin, Weiyan Huang, Yasuko RikihisaAbstract: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.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
