Ehrlichia chaffeensis Uses Its Surface Protein EtpE to Bind GPI-Anchored Protein DNase X and Trigger Entry into Mammalian Cells

Abstract

Ehrlichia chaffeensis, an obligatory intracellular rickettsial pathogen, enters and replicates in monocytes/macrophages and several non-phagocytic cells. E. chaffeensis entry into mammalian cells is essential not only for causing the emerging zoonosis, human monocytic ehrlichiosis, but also for its survival. It remains unclear if E. chaffeensis has evolved a specific surface protein that functions as an ‘invasin’ to mediate its entry. We report a novel entry triggering protein of Ehrlichia, EtpE that functions as an invasin. EtpE is an outer membrane protein and an antibody against EtpE (the C-terminal fragment, EtpE-C) greatly inhibited E. chaffeensis binding, entry and infection of both phagocytes and non-phagocytes. EtpE-C-immunization of mice significantly inhibited E. chaffeensis infection. EtpE-C-coated latex beads, used to investigate whether EtpE-C can mediate cell invasion, entered both phagocytes and non-phagocytes and the entry was blocked by compounds that block E. chaffeensis entry. None of these compounds blocked uptake of non-coated beads by phagocytes. Yeast two-hybrid screening revealed that DNase X, a glycosylphosphatidyl inositol-anchored mammalian cell-surface protein binds EtpE-C. This was confirmed by far-Western blotting, affinity pull-down, co-immunoprecipitation, immunofluorescence labeling, and live-cell image analysis. EtpE-C-coated beads entered bone marrow-derived macrophages (BMDMs) from wild-type mice, whereas they neither bound nor entered BMDMs from DNase X ^-/- mice. Antibody against DNase X or DNase X knock-down by small interfering RNA impaired E. chaffeensis binding, entry, and infection. E. chaffeensis entry and infection rates of BMDMs from DNase X ^-/- mice and bacterial load in the peripheral blood in experimentally infected DNase X ^-/- mice, were significantly lower than those from wild-type mice. Thus this obligatory intracellular pathogen evolved a unique protein EtpE that binds DNase X to enter and infect eukaryotic cells. This study is the first to demonstrate the invasin and its mammalian receptor, and their in vivo relevance in any ehrlichial species.

Author Summary

Human monocytic ehrlichiosis (HME), discovered in 1986, was designated as a nationally notifiable disease by Centers for Disease Control and Prevention in 1998. HME is one of the most prevalent, life-threatening emerging infectious diseases in the United States. HME is caused by a bacterium, Ehrlichia chaffeensis and is transmitted by the bite of infected ticks. This bacterium has special ability to enter and replicate inside human white blood cells and this feature is very essential for the bacterial survival. How E. chaffeensis enters host cells has been a mystery. The present study revealed that E. chaffeensis outer-surface protein named EtpE binds a specific host cell-surface protein, DNase X, and this ligand-receptor interaction is required to induce bacterial entry into its host cells. In order to test whether E. chaffeensis infection can be prevented by EtpE immunization, mice were immunized with the recombinant EtpE protein, and challenged with live E. chaffeensis. Infection was significantly reduced in the EtpE protein-immunized mice compared to controls. Mice lacking DNase X were also resistant to infection. This study shows EtpE-mediated entry pathway of E. chaffeensis is important in infecting mammals and EtpE can be incorporated into a future HME vaccine design.