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. 2002 Sep;70(9):4818-25.
doi: 10.1128/IAI.70.9.4818-4825.2002.

Macrophage plasma membrane cholesterol contributes to Brucella abortus infection of mice

Masahisa Watarai  1 , Sou-ichi Makino, Makoto Michikawa, Katsuhiko Yanagisawa, Shigeru Murakami, Toshikazu Shirahata
Affiliations

Macrophage plasma membrane cholesterol contributes to Brucella abortus infection of mice

Masahisa Watarai et al. Infect Immun. 2002 Sep.

Abstract

Brucella abortus is a facultative intracellular bacterium capable of surviving inside macrophages. Intracellular replication of B. abortus requires the VirB complex, which is highly similar to conjugative DNA transfer systems. In this study, we show that plasma membrane cholesterol of macrophages is required for the VirB-dependent internalization of B. abortus and also contributes to the establishment of bacterial infection in mice. The internalization of B. abortus was accelerated by treating macrophages with acetylated low-density lipoprotein (acLDL). Treatment of acyl coenzyme A:cholesterol acyltransferase inhibitor, HL-004, to macrophages preloaded with acLDL accelerated the internalization of B. abortus. Ketoconazole, which inhibits cholesterol transport from lysosomes to the cell surface, inhibited the internalization and intracellular replication of B. abortus in macrophages. The Niemann-Pick C1 gene (NPC1), the gene for Niemann-Pick type C disease, characterized by an accumulation of cholesterol in most tissues, promoted B. abortus infection. NPC1-deficient mice were resistant to the bacterial infection. Molecules associated with cholesterol-rich microdomains, "lipid rafts," accumulate in intracellular vesicles of macrophages isolated from NPC1-deficient mice, and the macrophages yielded no intracellular replication of B. abortus. Thus, trafficking of cholesterol-associated microdomains controlled by NPC1 is critical for the establishment of B. abortus infection.

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Figures

FIG. 1.
FIG. 1.
Schema of cholesterol trafficking in macrophages. The effect of each pharmacological and genetic treatment examined in this study is shown.
FIG. 2.
FIG. 2.
Internalization of B. abortus accelerated by acLDL. Ba600 (wild type) (A) or Ba604 (ΔvirB4) (B) was deposited onto bone marrow-derived macrophages with (white bars) or without (black bars) acLDL treatment and incubated at 37°C for the periods of time indicated. One hundred macrophages were examined per coverslip. Data are the average of triplicate samples from three identical experiments, and the error bars represent the standard deviation.
FIG. 3.
FIG. 3.
Internalization of B. abortus into macrophages was influenced by plasma membrane cholesterol. Ba600 (wild-type) (A and B) or Ba604 (ΔvirB4) (C and D) was deposited onto bone marrow-derived macrophages with ketoconazole (white bars) or with ACAT inhibitor HL-004 (gray bars), or without drug treatment (black bars) in the presence (B and D) or absence (A and C) of acLDL and incubated at 37°C for the periods of time indicated. One hundred macrophages were examined per coverslip. Data are the average of triplicate samples from three identical experiments, and the error bars represent the standard deviation. (E) Ketoconazole inhibited intracellular replication of B. abortus in macrophages. Macrophages in the presence or absence of ketoconazole, ACAT inhibitor HL-004, or acLDL were infected with Ba600 (wild type) as described in Materials and Methods. Data points and error bars represent the mean CFU of triplicate samples from a typical experiment (performed at least four times) and their standard deviation, respectively.
FIG. 4.
FIG. 4.
Intracellular distribution of lipid raft-associated molecules in NPC1-deficient macrophages. Macrophages of wild-type (upper panels) or NPC1-deficient (lower panels) mice were labeled with indicated molecules.
FIG. 5.
FIG. 5.
NPC1-influenced B. abortus infection. (A and B) Internalization of B. abortus. Macrophages from wild-type (black bars) or NPC1-deficient (white bars) mice were infected with virulent Ba600 (wild type) (A) or Ba604 (ΔvirB4) (B) for the periods of time indicated. Data are the average of triplicate samples from three identical experiments, and the error bars represent the standard deviation. (C) Intracellular replication of B. abortus. Macrophages from wild-type or NPC1-deficient mice were infected with Ba600 (wild type). Data points and error bars represent the mean CFU of triplicate samples from a typical experiment (performed at least four times) and their standard deviation, respectively. (D) Proliferation in mice. Wild-type (black bar) or NPC1-deficient (white bars) mice were infected with virulent B. abortus. Recovery of viable bacteria from the spleen and the weights of spleens of infected mice at 10 days postinfection are shown. Error bars indicate standard deviations.
FIG. 6.
FIG. 6.
Colocalization of B. abortus with late endosomal and lysosomal marker LAMP-1 in macrophages from NPC1-deficient mice by immunofluorescence microscopy. Macrophages from wild-type mice (A and C) or NPC1-deficient mice (B and D) were infected with Ba600 (wild type) for 1 h, fixed, and stained for LAMP-1 colocalization (A and B) and intracellular bacteria (C and D). Arrows point to bacteria.
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