title: Cholesterol-dependent anaplasma phagocytophilum exploits the low-density lipoprotein uptake pathway.
abstract: In eukaryotes, intracellular cholesterol homeostasis and trafficking are tightly regulated. Certain bacteria, such as Anaplasma phagocytophilum, also require cholesterol; it is unknown, however, how this cholesterol-dependent obligatory intracellular bacterium of granulocytes interacts with the host cell cholesterol regulatory pathway to acquire cholesterol. Here, we report that total host cell cholesterol increased >2-fold during A. phagocytophilum infection in a human promyelocytic leukemia cell line. Cellular free cholesterol was enriched in A. phagocytophilum inclusions as detected by filipin staining. We determined that A. phagocytophilum requires cholesterol derived from low-density lipoprotein (LDL), because its replication was significantly inhibited by depleting the growth medium of cholesterol-containing lipoproteins, by blocking LDL uptake with a monoclonal antibody against LDL receptor (LDLR), or by treating the host cells with inhibitors that block LDL-derived cholesterol egress from late endosomes or lysosomes. However, de novo cholesterol biosynthesis is not required, since inhibition of the biosynthesis pathway did not inhibit A. phagocytophilum infection. The uptake of fluorescence-labeled LDL was enhanced in infected cells, and LDLR expression was up-regulated at both the mRNA and protein levels. A. phagocytophilum infection stabilized LDLR mRNA through the 3' UTR region, but not through activation of the sterol regulatory element binding proteins. Extracellular signal-regulated kinase (ERK) was up-regulated by A. phagocytophilum infection, and inhibition of its upstream kinase, MEK, by a specific inhibitor or siRNA knockdown, reduced A. phagocytophilum infection. Up-regulation of LDLR mRNA by A. phagocytophilum was also inhibited by the MEK inhibitor; however, it was unclear whether ERK activation is required for LDLR mRNA up-regulation by A. phagocytophilum. These data reveal that A. phagocytophilum exploits the host LDL uptake pathway and LDLR mRNA regulatory system to accumulate cholesterol in inclusions to facilitate its replication.
authors.affiliation: Department of Veterinary Biosciences, The Ohio State University, Columbus, Ohio, United States of America.
authors.forename: Qingming
authors.initials: Q
authors.lastname: Xiong
authors.forename: Mingqun
authors.initials: M
authors.lastname: Lin
authors.forename: Yasuko
authors.initials: Y
authors.lastname: Rikihisa
mesh_terms: D041081:Anaplasma phagocytophilum / Q000502:physiology*; D015153:Blotting, Western; D002784:Cholesterol / Q000378:metabolism*; D016873:Ehrlichiosis / Q000235:genetics / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D018922:HL-60 Cells; D006801:Humans; D008077:Lipoproteins, LDL / Q000378:metabolism*; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D011973:Receptors, LDL / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D008077:Lipoproteins, LDL; D012333:RNA, Messenger; D011973:Receptors, LDL; D002784:Cholesterol; D048049:Extracellular Signal-Regulated MAP Kinases; D020930:MAP Kinase Kinase Kinases