Ehrlichia chaffeensis, the agent of human monocytic ehrlichiosis (HME), is an obligatory intracellular rickettsial pathogen that replicates in the membrane-bound compartment (inclusion) in monocytes/macrophages. Entry into mammalian host cells is essential for an obligatory intracellular pathogen to sustain its life and consequently to manifest disease associated with the infection. Although some information regarding the host cell invasion of E. chaffeensis have been known, crucial information about the identity of its invasive ligand or cognate host cell receptor used for binding and entry was unknown. In this study, we report, a novel entry triggering protein of Ehrlichia named EtpE that functions as an invasin. Antibody against EtpE (the C-terminal fragment, EtpE-C) greatly inhibited E. chaffeensis binding, entry, and infection; EtpE-C-immunization of mice significantly inhibited E. chaffeensis infection. EtpE-C-coated beads entered both phagocytes and non-phagocytes, and the entry was blocked by compounds that block E. chaffeensis entry. Furthermore, yeast two-hybrid screening revealed that DNase X, a glycosylphosphatidyl inositol-anchored mammalian cell-surface protein binds EtpE-C. Antibody against DNase X or reduction of DNase X by small interfering RNA impaired E. chaffeensis binding, entry, and infection. In experimentally infected DNase X-/- mice, E. chaffeensis load in peripheral blood was significantly lower than in wild-type mice.
Using affinity pull-down of host cell lysates with rEtpE-C followed by LC-MS/MS analysis, we identified two additional interacting mammalian proteins: a transmembrane glycoprotein CD147 and a cytosolic protein hnRNP-K. Far-Western blot and immunofluorescence analysis showed that CD147 interacts with rEtpE-C and is recruited to E. chaffeensis entry foci. Functional neutralization of surface-exposed CD147 with monoclonal antibodies or knock-down of CD147 using shRNA inhibited E. chaffeensis internalization and infection, but not binding. Functional ablation of cytoplasmic hnRNP-K significantly hampered E. chaffeensis entry and subsequent infection.
We found that chemical inhibitors of actin dynamics drastically inhibited E. chaffeensis entry and infection of host cells. Actin and N-WASP was seen directly recruited to E. chaffeensis entry foci. By affinity column-based pull down and co-immunoprecipitation N-WASP was shown to be present in the protein complex, formed during E. chaffeensis invasion of host cells, along with EtpE, DNase X and CD147. EtpE-C was able to polymerize actin in an in vitro pyrenyl-actin polymerization assay. This activity was specific for EtpE-C but not for EtpE-N and was specifically mediated by DNase X in an N-WASP dependent manner. The phagosomes with rEtpE-C coated beads inhibited assembly of NADPH oxidase components on its phagosomal membrane and rEtpE-C coating of beads inhibited ROS generation by BMDM cells where as non-coated cells or rEtpE-N coated beads induced significant response. Thus, DNase X mediated entry pathway seemed to be evolutionarily selected by E. chaffeensis entry since this route of entry provided definitive survival advantage for the pathogen.
This study is the first to demonstrate the invasin, its mammalian receptor and their in vivo relevance in any ehrlichial species, a novel upstream mode of pathogen-induced potential actin cytoskeletal mobilization, establishes the unique advantage of this infectious entry route and multifunctional role of EtpE in E. chaffeensis entry and thus in HME pathogenesis.