Obligate intracellular bacteria, such as is the etiological agent of human monocytic ehrlichiosis, an emerging infectious disease. inhibited entry. Upon incubation with host cell lysate, EtpE-C but not an EtpE N-terminal fragment stimulated 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 entry by engaging CD147 and hnRNP-K and activating N-WASP-dependent actin polymerization. IMPORTANCE bacteria, and hence, their ability to cause disease, depends on their specific mode of entry into eukaryotic host cells. Understanding the mechanism by which 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 ARRY334543 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. INTRODUCTION Human monocytic ehrlichiosis (HME) is one of the most prevalent life-threatening emerging tick-borne zoonoses in the United States (1). The disease was discovered in 1986 and was designated ARRY334543 a nationally notifiable disease in 1998 by the U. S Centers for Disease Control and Prevention. HME is caused by infection with replicates within human monocytes and macrophages and causes severe flulike symptoms accompanied by hematologic abnormalities and signs of hepatitis. No vaccines exist for HME. The broad-spectrum antibiotic doxycycline is the only drug effective for treating HME but is contraindicated for pregnant women and children. Delayed initiation of therapy, the presence of underlying illness, or immunosuppression often lead to severe complications or even death (2). The incidence of tick-borne zoonoses has risen continuously and dramatically in the past 2 decades (3). The Centers for Disease Control and Prevention confirmed 1,404 HME cases in 2014 and 1,509 in 2013 (1), which is more than a 10-fold increase in HME incidence over a 15-year period. The 2011 U.S. Institute of Medicine report Critical Needs and Gaps in understanding prevention, amelioration, and resolution of Lyme and Other Tick-Borne Diseases (4) pointed to the urgent need for research on HME. entry into the human acute leukemia cell line THP-1 leads to productive infection and is dependent on host cell surface lipid rafts and glycosylphosphatidyl inositol (GPI)-anchored proteins (5). After entry, replicates in an early endosome-like compartment which contains early endosome antigen 1 (EEA1), Rab5, transferrin receptor, and vacuolar-type H+ ATPase ARRY334543 (6) but does not contain lysosomal membrane-associated protein 1, CD63, or NADPH oxidase components (6,C8). cells die if they fail to enter an appropriate host cell. Previous studies using pharmacological inhibitors have suggested that the mechanism of entry is distinct from that of classic antimicrobial phagocytosis (5, 9). outer membrane protein ECH1038, with highly strain-conserved N- and C-terminal segments, is highly expressed at the stress-resistant and infectious stage of the intracellular developmental cycle (10). We previously named ECH1038 of surface and serves as an invasin for direct binding of its mammalian receptor DNase X (DNase I-like protein 1), a GPI-anchored ubiquitous cell surface protein, to trigger infectious entry (11). In patient blood specimens, is primarily seen in monocytes and not in neutrophils (12); hence, human monocytic ehrlichiosis is so named Rabbit polyclonal to ATL1 to distinguish it from other human ehrlichioses caused by granulocytotropic specieshas been stably cultivated only in canine macrophage DH82 cells (13) or THP-1 cells (14). To apply transfection and mouse mutagenesis techniques and distinguish ehrlichial infectious entry from classic phagocytosis, we developed an effective ARRY334543 culture system in nonphagocytic cell lines (human embryonic kidney HEK293 cells and monkey endothelial RF/6A cells), as well as primary phagocytic cells (human and canine peripheral blood monocytes and mouse bone marrow-derived macrophages [BMDMs]) (11). An antibody (Ab) against the strain-conserved C-terminal segment of EtpE (EtpE-C) greatly inhibits binding, entry, and infection of both phagocytic and nonphagocytic host cells entry (11). An antibody against DNase X or small interfering.