Host membrane lipids are trafficked to membranes of intravacuolar bacterium<i>Ehrlichia chaffeensis</i>

Abstract

<i>Ehrlichia chaffeensis</i>, a cholesterol-rich and cholesterol-dependent obligate intracellular bacterium, partially lacks genes for glycerophospholipid biosynthesis. We found here that <i>E. chaffeensis</i> is dependent on host glycerolipid biosynthesis, as an inhibitor of host long-chain acyl CoA synthetases, key enzymes for glycerolipid biosynthesis, significantly reduced bacterial proliferation. <i>E. chaffeensis</i> cannot synthesize phosphatidylcholine or cholesterol but encodes enzymes for phosphatidylethanolamine (PE) biosynthesis; however, exogenous NBD-phosphatidylcholine, Bodipy-PE, and TopFluor-cholesterol were rapidly trafficked to ehrlichiae in infected cells. DiI (3,3'-dioctadecylindocarbocyanine)-prelabeled host-cell membranes were unidirectionally trafficked to <i>Ehrlichia</i> inclusion and bacterial membranes, but DiI-prelabeled <i>Ehrlichia</i> membranes were not trafficked to host-cell membranes. The trafficking of host-cell membranes to <i>Ehrlichia</i> inclusions was dependent on both host endocytic and autophagic pathways, and bacterial protein synthesis, as the respective inhibitors blocked both infection and trafficking of DiI-labeled host membranes to <i>Ehrlichia</i> In addition, DiI-labeled host-cell membranes were trafficked to autophagosomes induced by the <i>E. chaffeensis</i> type IV secretion system effector Etf-1, which traffic to and fuse with <i>Ehrlichia</i> inclusions. Cryosections of infected cells revealed numerous membranous vesicles inside inclusions, as well as multivesicular bodies docked on the inclusion surface, both of which were immunogold-labeled by a GFP-tagged 2×FYVE protein that binds to phosphatidylinositol 3-phosphate. Focused ion-beam scanning electron microscopy of infected cells validated numerous membranous structures inside bacteria-containing inclusions. Our results support the notion that <i>Ehrlichia</i> inclusions are amphisomes formed through fusion of early endosomes, multivesicular bodies, and early autophagosomes induced by Etf-1, and they provide host-cell glycerophospholipids and cholesterol that are necessary for bacterial proliferation.