EtpE Binding to DNase X Induces Ehrlichial Entry via CD147 and hnRNP-K Recruitment, Followed by Mobilization of N-WASP and Actin

ABSTRACT 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 invasi...

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Autores principales:	Dipu Mohan Kumar, Mingqun Lin, Qingming Xiong, Mathew James Webber, Comert Kural, Yasuko Rikihisa
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2015
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/cb75451bbc0c48209c951e81935703db
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spelling	oai:doaj.org-article:cb75451bbc0c48209c951e81935703db2021-11-15T15:41:24ZEtpE Binding to DNase X Induces Ehrlichial Entry via CD147 and hnRNP-K Recruitment, Followed by Mobilization of N-WASP and Actin10.1128/mBio.01541-152150-7511https://doaj.org/article/cb75451bbc0c48209c951e81935703db2015-12-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01541-15https://doaj.org/toc/2150-7511ABSTRACT 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.Dipu Mohan KumarMingqun LinQingming XiongMathew James WebberComert KuralYasuko RikihisaAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 6, Iss 6 (2015)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Dipu Mohan Kumar Mingqun Lin Qingming Xiong Mathew James Webber Comert Kural Yasuko Rikihisa EtpE Binding to DNase X Induces Ehrlichial Entry via CD147 and hnRNP-K Recruitment, Followed by Mobilization of N-WASP and Actin
description	ABSTRACT 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.
format	article
author	Dipu Mohan Kumar Mingqun Lin Qingming Xiong Mathew James Webber Comert Kural Yasuko Rikihisa
author_facet	Dipu Mohan Kumar Mingqun Lin Qingming Xiong Mathew James Webber Comert Kural Yasuko Rikihisa
author_sort	Dipu Mohan Kumar
title	EtpE Binding to DNase X Induces Ehrlichial Entry via CD147 and hnRNP-K Recruitment, Followed by Mobilization of N-WASP and Actin
title_short	EtpE Binding to DNase X Induces Ehrlichial Entry via CD147 and hnRNP-K Recruitment, Followed by Mobilization of N-WASP and Actin
title_full	EtpE Binding to DNase X Induces Ehrlichial Entry via CD147 and hnRNP-K Recruitment, Followed by Mobilization of N-WASP and Actin
title_fullStr	EtpE Binding to DNase X Induces Ehrlichial Entry via CD147 and hnRNP-K Recruitment, Followed by Mobilization of N-WASP and Actin
title_full_unstemmed	EtpE Binding to DNase X Induces Ehrlichial Entry via CD147 and hnRNP-K Recruitment, Followed by Mobilization of N-WASP and Actin
title_sort	etpe binding to dnase x induces ehrlichial entry via cd147 and hnrnp-k recruitment, followed by mobilization of n-wasp and actin
publisher	American Society for Microbiology
publishDate	2015
url	https://doaj.org/article/cb75451bbc0c48209c951e81935703db
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EtpE Binding to DNase X Induces Ehrlichial Entry via CD147 and hnRNP-K Recruitment, Followed by Mobilization of N-WASP and Actin

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