Obligate intracellular bacteria, such as Ehrlichia chaffeensis, perish unless they can enter eukaryotic cells. E. chaffeensis is the etiological agent of human monocytic
ehrlichiosis, an
emerging infectious disease. To infect cells, Ehrlichia uses the C terminus of the outer membrane invasin entry-triggering
protein (EtpE) of Ehrlichia (EtpE-C), which directly binds the mammalian cell surface glycosylphosphatidyl
inositol-anchored
protein,
DNase X. How this binding drives Ehrlichia entry is unknown. Here, using affinity pulldown of host cell lysates with recombinant EtpE-C (rEtpE-C), we identified two new human
proteins that interact with EtpE-C: CD147 and
heterogeneous nuclear ribonucleoprotein K (
hnRNP-K). The interaction of CD147 with rEtpE-C was validated by far-Western blotting and coimmunoprecipitation of native EtpE with endogenous CD147. CD147 was ubiquitous on the cell surface and also present around foci of rEtpE-C-coated-bead entry. Functional neutralization of surface-exposed CD147 with a specific antibody inhibited Ehrlichia internalization and
infection but not binding. Downregulation of CD147 by
short hairpin RNA (
shRNA) impaired E. chaffeensis
infection. Functional ablation of cytoplasmic
hnRNP-K by a nanoscale intracellular antibody markedly attenuated bacterial entry and
infection but not binding. EtpE-C also interacted with
neuronal Wiskott-Aldrich syndrome protein (N-WASP), which is activated by
hnRNP-K.
Wiskostatin, which inhibits N-WASP activation, and
cytochalasin D, which inhibits actin polymerization, inhibited Ehrlichia entry. Upon incubation with host cell lysate, EtpE-C but not an EtpE N-terminal fragment stimulated in vitro actin polymerization in an N-WASP- and
DNase X-dependent manner. Time-lapse video images revealed N-WASP recruitment at EtpE-C-coated bead entry foci. Thus, EtpE-C binding to
DNase X drives Ehrlichia entry by engaging CD147 and
hnRNP-K and activating N-WASP-dependent actin polymerization.
IMPORTANCE: Ehrlichia chaffeensis, an obligate intracellular bacterium, causes a
blood-borne disease called human monocytic
ehrlichiosis, one of the most prevalent life-threatening emerging tick-transmitted
infectious diseases in the United States. The survival of Ehrlichia bacteria, and hence, their ability to cause disease, depends on their specific mode of entry into eukaryotic host cells. Understanding the mechanism by which E. chaffeensis enters cells will create new opportunities for developing effective
therapies to prevent bacterial entry and disease in humans. Our findings reveal a novel cellular signaling pathway triggered by an ehrlichial
surface protein called EtpE to induce its infectious entry. The results are also important from the viewpoint of human cell physiology because three EtpE-interacting human
proteins,
DNase X, CD147, and
hnRNP-K, are hitherto unknown partners that drive the uptake of small particles, including bacteria, into human cells.