Anaplasma phagocytophilum Hijacks Flotillin and NPC1 Complex To Acquire Intracellular Cholesterol for Proliferation, Which Can Be Inhibited with Ezetimibe.

Weiyan Huang,Mingqun Lin,Qingming Xiong,Yasuko Rikihisa

doi:10.1128/mbio.02299-21

Abstract

ABSTRACTThe intracellular cholesterol transport protein Niemann-Pick type C1 (NPC1) and lipid-raft protein flotillin (FLOT) are required for cholesterol uptake by the obligatory intracellular bacterium Anaplasma phagocytophilum and for infection, and each protein localizes to membrane-bound inclusions containing replicating bacteria. Here, we found striking localization of FLOT2 in NPC1-lined vesicles and a physical interaction between FLOT2 and NPC1. This interaction was cholesterol dependent, as a CRAC (cholesterol recognition/interaction amino acid cholesterol-binding) domain mutant of FLOT2 did not interact with NPC1, and the cholesterol-sequestering agent methyl-β-cyclodextrin reduced the interaction. The stomatin-prohibitin-flotillin-HflC/K domain of FLOT2, FLOT21–183, was sufficient for the unique FLOT2 localization and interaction with NPC1. NPC1, FLOT2, and FLOT21–183 trafficked to the lumen of Anaplasma inclusions. A loss-of-function mutant, NPC1P691S (mutation in the sterol-sensing domain), did not colocalize or interact with FLOT2 or with Anaplasma inclusions and inhibited infection. Ezetimibe is a drug that blocks cholesterol absorption in the small intestine by inhibiting plasma membrane Niemann-Pick C1-like 1 interaction with FLOTs. Ezetimibe blocked the interaction between NPC1 and FLOT2 and inhibited Anaplasma infection. Ezetimibe did not directly inhibit Anaplasma proliferation but inhibited host membrane lipid and cholesterol traffic to the bacteria in the inclusion. These data suggest that Anaplasma hijacks NPC1 vesicles containing cholesterol bound to FLOT2 to deliver cholesterol into Anaplasma inclusions to assimilate cholesterol for its proliferation. These results provide insights into mechanisms of intracellular cholesterol transport and a potential approach to inhibit Anaplasma infection by blocking cholesterol delivery into the lumen of bacterial inclusions.

Highlights

The intracellular cholesterol transport protein Niemann-Pick type C1 (NPC1) and lipid-raft protein flotillin (FLOT) are required for cholesterol uptake by the obligatory intracellular bacterium Anaplasma phagocytophilum and for infection, and each protein localizes to membrane-bound inclusions containing replicating bacteria
Immunoprecipitation with anti-GFP affinity gel resulted in pulldown of endogenous FLOT2 from the lysate of HEK293T cells transfected with NPC1-GFP but not cells transfected with the green fluorescent protein (GFP) negative control (Fig. 1K)
Because NPC1 and FLOT2 are required for A. phagocytophilum infection [6, 16] and we found that ezetimibe blocks NPC1-FLOT2 colocalization and interaction (Fig. 6), we examined whether ezetimibe could block infection of cells with A. phagocytophilum

Summary

Introduction

The intracellular cholesterol transport protein Niemann-Pick type C1 (NPC1) and lipid-raft protein flotillin (FLOT) are required for cholesterol uptake by the obligatory intracellular bacterium Anaplasma phagocytophilum and for infection, and each protein localizes to membrane-bound inclusions containing replicating bacteria. The cholesterol-binding membrane glycoprotein Niemann-Pick type C1 (NPC1) is critical for managing cellular cholesterol level and its intracellular transport, and mutation of the gene encoding NPC1 causes the fatal cholesterol storage disease, Niemann-Pick disease, type C Both FLOT and NPC1 are trafficked to inclusions created by the cholesterol-dependent bacterium Anaplasma phagocytophilum and required for cholesterol uptake by this bacterium for replication. Large NPC1-bearing vesicles devoid of lysosomal markers were found to be increased in the human promyelocytic leukemia cell line HL-60 infected with A. phagocytophilum, and this subset trafficked to the bacterial inclusions [6] This localization was abolished by the LDL-derived cholesterol-trafficking inhibitor U18666A, which, when administered to cells, mimics the molecular aspects of Niemann-Pick Type C disease [6]. Studies using an NPC1-specific short interfering RNA (siRNA) and a cell line with dysfunctional NPC1 demonstrated that NPC1 function is required for cholesterol acquisition by A. phagocytophilum and infection [6]

Methods

Results

Conclusion