Ehrlichia type IV secretion system effector Etf-2 binds to active RAB5 and delays endosome maturation

Proc Natl Acad Sci U S A. 2018 Sep 18;115(38):E8977-E8986. doi: 10.1073/pnas.1806904115. Epub 2018 Sep 4.

Abstract

Ehrlichia chaffeensis, an obligatory intracellular bacterium, infects monocytes/macrophages by sequestering a regulator of endosomal traffic, the small GTPase RAB5, on its membrane-bound inclusions to avoid routing to host-cell phagolysosomes. How RAB5 is sequestered on ehrlichial inclusions is poorly understood, however. We found that native Ehrlichia translocated factor-2 (Etf-2), a previously predicted effector of the Ehrlichia type IV secretion system, and recombinant Etf-2 (cloned into the Ehrlichia genome) are secreted into the host-cell cytoplasm and localize to ehrlichial inclusions. Ectopically expressed Etf-2-GFP also localized to inclusions and membranes of early endosomes marked with RAB5 and interacted with GTP-bound RAB5 but not with a GDP-bound RAB5. Etf-2, although lacking a RAB GTPase-activating protein (GAP) Tre2-Bub2-Cdc16 (TBC) domain, contains two conserved TBC domain motifs, namely an Arg finger and a Gln finger, and site-directed mutagenesis revealed that both Arg188 and Gln245 are required for Etf-2 localization to early endosomes. The yeast two-hybrid assay and microscale thermophoresis revealed that Etf-2 binds tightly to GTP-bound RAB5 but not to GDP-bound RAB5. However, Etf-2 lacks RAB5-specific GAP activity. Etf-2 localized to bead-containing phagosomes as well as endosomes containing beads coated with the C-terminal fragment of EtpE (entry-triggering protein of Ehrlichia), an Ehrlichia outer-membrane invasin, and significantly delayed RAB5 dissociation from and RAB7 localization to phagosomes/endosomes and RABGAP5 localization to endosomes. Thus, binding of Etf-2 to RAB5-GTP appears to delay RAB5 inactivation by impeding RABGAP5 localization to endosomes. This suggests a unique mechanism by which RAB5 is sequestered on ehrlichial inclusions to benefit bacterial survival and replication.

Keywords: Ehrlichia chaffeensis; RABGAP5; Rab5; T4SS effector; TBC motif.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Animals
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Cell Line
  • Ehrlichia chaffeensis / physiology*
  • Endosomes / immunology*
  • Endosomes / metabolism
  • GTP Phosphohydrolases / metabolism
  • Gene Knockdown Techniques
  • Host-Pathogen Interactions / immunology
  • Humans
  • Macaca mulatta
  • Phagosomes / immunology*
  • Phagosomes / metabolism
  • Protein Binding
  • Sequence Alignment
  • Type IV Secretion Systems / metabolism*
  • rab5 GTP-Binding Proteins / metabolism*

Substances

  • Bacterial Proteins
  • Type IV Secretion Systems
  • GTP Phosphohydrolases
  • rab5 GTP-Binding Proteins