EtpE Binding to DNase X Induces Ehrlichial Entry via CD147 and hnRNP-K Recruitment, Followed by Mobilization of N-WASP and Actin
Authors: Mohan Kumar Dipu , Lin Mingqun , Xiong Qingming , Webber Mathew James , Kural Comert , Rikihisa Yasuko 
Published: November 2015

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
e
ntry-
t
riggering
p
rotein (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.