Host membrane lipids are trafficked to membranes of intravacuolar bacterium Ehrlichia chaffeensis.

Ehrlichia chaffeensis, a cholesterol-rich and cholesterol-dependent obligate intracellular bacterium, partially lacks genes for glycerophospholipid biosynthesis. We found here that E. chaffeensis 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. E. chaffeensis 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 Ehrlichia inclusion and bacterial membranes, but DiI-prelabeled Ehrlichia membranes were not trafficked to host-cell membranes. The trafficking of host-cell membranes to Ehrlichia 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 Ehrlichia In addition, DiI-labeled host-cell membranes were trafficked to autophagosomes induced by the E. chaffeensis type IV secretion system effector Etf-1, which traffic to and fuse with Ehrlichia 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 Ehrlichia 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.

Laboratory maintenance of Ehrlichia chaffeensis and Ehrlichia canis and recovery of organisms for molecular biology and proteomics studies.

Tick-borne illnesses are emerging as a major concern for human health in recent years. These include the human monocytic ehrlichiosis caused by the Amblyomma americanum tick-transmitted bacterium, Ehrlichia chaffeensis; human ewingii ehrlichiosis caused by Ehrlichia ewingii (also transmitted by A. americanum ticks); and human granulocytic anaplasmosis caused by the Ixodes scapularis tick-transmitted pathogen, Anaplasma phagocytophilum. Likewise, tick-borne rickettsial pathogens are also a major concern to the health of various vertebrates including dogs, cattle, and several wild animals. In vitro-cultured pathogens grown in a vertebrate host cell and a tick cell culture system will be useful in studies to understand the pathogenic differences as well as to perform experimental infection studies and to generate large quantities of purified antigens. In this unit, methods for culturing E. chaffeensis and Ehrlichia canis (a canine monocytic ehrlichiosis pathogen) in cell lines to represent vertebrate and tick hosts are described. The unit also includes methods useful in purifying bacteria from the host cells and to evaluate proteins by 2-D gel electrophoresis and western blotting.

Survival of Ehrlichia chaffeensis in refrigerated, ADSOL-treated RBCs.

BACKGROUND: The purpose of this study was to investigate the persistence of viable Ehrlichia chaffeensis in ADSOL-treated RBCs stored at 4 to 6 degrees C. STUDY DESIGN AND METHODS: The continuous monocytic cell lines THP-1 and DH82 were infected with E. chaffeensis (St. Vincent isolate). Packed RBC units were inoculated in separate experiments with E. chaffeensis-infected cells as final concentrations of 8.02 x 10(4) (DH82) and 1.43 x 10(4) (THP-1) infected cells per mL. Aliquots were stored at 4 to 6 degrees C for 1 to 42 days. At selected intervals, nucleated cells from the RBC aliquots were obtained by using a ficoll-isopaque separation procedure. Uninfected DH82 cell cultures were inoculated with the harvested nucleated cells or supernatant. The cell cultures were evaluated for infection by weekly examination of Wright's (Diff-Quik) stained cytocentrifuged slides. PCR amplification was also used to test the harvested nucleated cells or supernatant for the presence of E. chaffeensis DNA. RESULTS: In both types of infected cell lines, E. chaffeensis was reisolated in DH82 cells for as long as 11 days from the cellular fraction and for up to 5 days from the supernatant fraction. PCR results were positive throughout the 42-day testing period. CONCLUSION: Cell-associated E. chaffeensis remains viable in ADSOL-treated RBCs stored at 4 to 6 degrees C for at least 11 days. These data suggest that transfusion-acquired infection is possible. Successful reisolation was achieved from the supernatant fraction, which suggests that RBC products treated with a WBC-reduction procedure may still present a risk for transfusion transmission. No correlation between PCR positivity and viability of bacteria was noted.