Ehrlichia chaffeensis TRP120 moonlights as a HECT E3 ligase involved in selfand host ubiquitination to influence protein interactions and stability for intracellular survival

Bing Zhu, Seema Das, Shubhajit Mitra, Tierra R. Farris, Jere McBride

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Ehrlichia chaffeensis secretes tandem repeat protein (TRP) effectors that are involved in a diverse array of host cell interactions, some of which directly activate cell signaling pathways and reprogram host gene transcription to promote survival in the mononuclear phagocyte. However, the molecular details of these effectorhost interactions and roles in pathobiology are incompletely understood. In this study, we determined that the E. chaffeensis effector TRP120 is posttranslationally modified by ubiquitin (Ub) and that ubiquitination occurs through intrinsic and host-mediated HECT ligase activity. A functional HECT E3 ligase domain with a conserved catalytic site was identified in the C-terminal region of TRP120, and TRP120 autoubiquitination occurred in vitro in the presence of host UbcH5b/c E2 enzymes. TRP120 ubiquitination sites were mapped using a high-density microfluidic peptide array and confirmed by ectopic expression of TRP120 lysine mutants in cells. Moreover, we determined that the HECT E3 ubiquitin ligase, Nedd4L, interacts with TRP120 during infection and also mediates TRP120 ubiquitination. Nedd4L knockdown resulted in the reduction of TRP120-Ub, decreased ehrlichial infection, and reduced recruitment of a known TRP120-interacting host protein, PCGF5, to ehrlichial inclusions. TRP120- mediated PCGF5 polyubiquitination was associated with a reduction in PCGF5 levels. Inhibition of ubiquitination with small molecules also significantly decreased ehrlichial infection, indicating that the Ub pathway is critical for ehrlichial intracellular replication and survival. The current study identified a novel E. chaffeensis ubiquitin ligase and revealed an important role for the ubiquitin pathway in effector-host interactions and pathogen-mediated host protein stability in order to promote intracellular survival.

Original languageEnglish (US)
Article numbere00290-17
JournalInfection and Immunity
Volume85
Issue number9
DOIs
StatePublished - Sep 1 2017

Fingerprint

Ehrlichia chaffeensis
Ubiquitin-Protein Ligases
Protein Stability
Ubiquitination
Ubiquitin
Ligases
Infection
Host-Pathogen Interactions
Tandem Repeat Sequences
Critical Pathways
Microfluidics
Phagocytes
Cell Communication
Lysine
Catalytic Domain
Proteins
Peptides
Enzymes
Genes

Keywords

  • Effector
  • Ehrlichia
  • HECT ligase
  • SUMO
  • Tandem repeat protein
  • Ubiquitin

ASJC Scopus subject areas

  • Parasitology
  • Microbiology
  • Immunology
  • Infectious Diseases

Cite this

Ehrlichia chaffeensis TRP120 moonlights as a HECT E3 ligase involved in selfand host ubiquitination to influence protein interactions and stability for intracellular survival. / Zhu, Bing; Das, Seema; Mitra, Shubhajit; Farris, Tierra R.; McBride, Jere.

In: Infection and Immunity, Vol. 85, No. 9, e00290-17, 01.09.2017.

Research output: Contribution to journalArticle

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abstract = "Ehrlichia chaffeensis secretes tandem repeat protein (TRP) effectors that are involved in a diverse array of host cell interactions, some of which directly activate cell signaling pathways and reprogram host gene transcription to promote survival in the mononuclear phagocyte. However, the molecular details of these effectorhost interactions and roles in pathobiology are incompletely understood. In this study, we determined that the E. chaffeensis effector TRP120 is posttranslationally modified by ubiquitin (Ub) and that ubiquitination occurs through intrinsic and host-mediated HECT ligase activity. A functional HECT E3 ligase domain with a conserved catalytic site was identified in the C-terminal region of TRP120, and TRP120 autoubiquitination occurred in vitro in the presence of host UbcH5b/c E2 enzymes. TRP120 ubiquitination sites were mapped using a high-density microfluidic peptide array and confirmed by ectopic expression of TRP120 lysine mutants in cells. Moreover, we determined that the HECT E3 ubiquitin ligase, Nedd4L, interacts with TRP120 during infection and also mediates TRP120 ubiquitination. Nedd4L knockdown resulted in the reduction of TRP120-Ub, decreased ehrlichial infection, and reduced recruitment of a known TRP120-interacting host protein, PCGF5, to ehrlichial inclusions. TRP120- mediated PCGF5 polyubiquitination was associated with a reduction in PCGF5 levels. Inhibition of ubiquitination with small molecules also significantly decreased ehrlichial infection, indicating that the Ub pathway is critical for ehrlichial intracellular replication and survival. The current study identified a novel E. chaffeensis ubiquitin ligase and revealed an important role for the ubiquitin pathway in effector-host interactions and pathogen-mediated host protein stability in order to promote intracellular survival.",
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