Ehrlichia chaffeensis and Anaplasma phagocytophilum lack genes for lipid A biosynthesis and incorporate cholesterol for their survival

Abstract

Ehrlichia chaffeensis and Anaplasma phagocytophilum are agents of human monocytic and granulocytic ehrlichioses, respectively. They are extremely sensitive to mechanical stress and are pleomorphic gram-negative bacteria. Membrane incorporation of cholesterol from the eukaryotic host is known to be essential for other fragile and pleomorphic bacteria and mycoplasmas that lack a cell wall. Thus, we tested whether cholesterol is required for E. chaffeensis and A. phagocytophilum. Using a freeze fracture technique and biochemical analysis, these bacteria were found to contain significant levels of membrane cholesterol. These bacteria lack genes for cholesterol biosynthesis or modification. However, host cell-free bacteria had the ability to take up directly exogenous cholesterol or NBD-cholesterol, a fluorescent cholesterol derivative. Treatment of the bacteria with cholesterol extraction reagent methyl-beta-cyclodextrin caused their ultrastructural changes. Furthermore, pretreatment of the bacteria with methyl-beta-cyclodextrin or NBD-cholesterol deprived these bacteria of the ability to infect leukocytes, thus killing these obligate intracellular bacteria. Analysis of E. chaffeensis and A. phagocytophilum genome sequences revealed that these bacteria lack all genes for the biosynthesis of lipid A and most genes for the biosynthesis of peptidoglycan, which confer structural strength to gram-negative bacteria. Taken together, these results suggest that human ehrlichiosis agents became cholesterol dependent due to the loss of these genes. As the first report of gram-negative bacteria incorporating cholesterol for survival, these findings offer insight into the unique nature of their parasitism and imply that cholesterol is important in the control of human ehrlichioses.

FIG. 1.

Freeze fracture of filipin-labeled host cell-free E. chaffeensis and A. phagocytophilum. Freeze-fractured replicas of filipin-labeled E. chaffeensis (EC) and A. phagocytophilum (AP) were examined by TEM. Intramembranous protuberances of filipin-cholesterol complexes in diameters of around 20 to 25 nm can be seen in the outer membranes (arrow 1) of filipin-treated EC and AP but are absent from the inner membrane (arrow 2) and the cytosol (arrow 3) of the bacteria. The bottom arrow indicates the direction of shadowing. Bar, 0.2 μm.

FIG. 2.

Fluorescence microscopy of filipin-labeled E. chaffeensis and A. phagocytophilum (A) and E. coli (B). (A) Fluorescence emitted by filipin-labeled E. chaffeensis (EC) and A. phagocytophilum (AP) was localized in the bacterial surface labeled with bacterium-specific antibodies. (B) Fluorescence emitted by filipin is undetectable in E. coli (E. coli was visualized by phase contrast). For easier viewing, the blue fluorescence of filipin was converted to green pseudo-color in Adobe Photoshop. Bars, 5 μm.

FIG. 3.

NBD-cholesterol was directly incorporated into E. chaffeensis (EC) and A. phagocytophilum (AP). Fluorescence microscopy showed that the green fluorescence emitted by NBD-cholesterol was localized in the bacterial surface labeled with bacterium-specific antibodies, indicating the direct uptake of NBD-cholesterol by these bacteria. Bar, 5 μm.

FIG. 4.

The ultrastructure of E. chaffeensis was impaired by MβCD treatment. Host cell-free E. chaffeensis was treated with 10 mM MβCD at 37°C for 5 or 15 min, and the control group was incubated with RPMI medium, a solvent of MβCD, in the same conditions for 15 min. The arrow in the middle panel indicates irregular dilations of the periplasmic space in E. chaffeensis treated with MβCD for 5 min. The area between the two arrowheads in the right panel shows the discontinuity of the inner membrane in E. chaffeensis treated with MβCD for 15 min. More than 50% of the treated bacteria showed changes (indicated by arrow and arrowheads) compared to bacteria in the control group. Bar, 0.2 μm.

FIG. 5.

MβCD (A) and NBD-cholesterol (B) blocked the infection of E. chaffeensis and A. phagocytophilum in host cells. Host cell-free E. chaffeensis (EC) and A. phagocytophilum (AP) were incubated with 10 mM MβCD, 20 μg of water-soluble cholesterol (CHO)/ml, or 10 μg of NBD-cholesterol (NBD-Cho)/ml at 37°C for 30 min. Infectivities were determined at day 3 postinfection. CTL1, bacteria treated with same volume of RPMI as the control groups for MβCD and cholesterol; CTL2, bacteria treated with same volume of methanol (0.05% final concentration in RPMI) as a control for NBD-cholesterol. One asterisk (*) indicates a statistical difference (P < 0.05) from the control groups and two asterisks (**) indicate a statistical difference (P < 0.05) from the MβCD-treated groups, as determined by Student's t test. Data are representative of three experiments.