Development of Etf-3-specific nanobodies to prevent Ehrlichia infection and LNP-mRNA delivery in cellular and murine models

Nan Duan, Mingqun Lin, Wenqing Zhang, Qi Yan, Rory C. Chien, Khemraj Budachetri, Stephen Denton, Jeffrey Kawahara, Jeffrey Lakritz, Yichen Zhong, Yizhou Dong, Yasuko Rikihisa

Research output: Contribution to journalArticlepeer-review

Abstract

Ehrlichia chaffeensis is an obligatory intracellular bacterium that infects monocytes and macrophages and causes human monocytic ehrlichiosis. Ehrlichia translocated factor-3 (Etf-3) is a type IV secretion system effector that binds host-cell ferritin light chain and induces ferritinophagy, thus increasing cellular labile iron pool for Ehrlichia proliferation. To further characterize roles of Etf-3 in Ehrlichia infection, we produced immune libraries of Etf-3−specific nanobodies (Nbs). Based on distinct complementarity-determining region 3 sequences, we identified 16 and 15 families of anti-Etf-3 Nbs that could specifically bind the N- and C-terminal halves of Etf-3, respectively. Transfection with plasmids encoding the anti-Etf-3 Nbs N48 and N51, but not N59, significantly inhibited E. chaffeensis infection in HEK293 cells. All three Nbs colocalized with Etf-3-GFP in co-transfected RF/6A cells, but N48 and N51 had significantly higher binding affinities for recombinant Etf-3. Etf-3-GFP transfection–induced ferritinophagy and endogenous ferritin degradation was abrogated in HEK293 cells co-transfected with N48 or N51, but not with N59. To efficiently express Nbs in the infected host-cell cytoplasm, lipid nanoparticles-encapsulated mRNAs (LNP-mRNAs) encoding N48, N51, or N59 were created for delivery into cells or mice. Incubation of HEK293 cells or inoculation of mice with LNP-mRNA-N48 or LNP-mRNA-N51 significantly inhibited E. chaffeensis infection compared to those with LNP-mRNA-N59 or without LNP-mRNA. Our results demonstrate that Etf-3–specific Nbs delivered via LNP-mRNAs can inhibit Etf-3 functions and Ehrlichia infection.

Original languageEnglish
Article number128027
JournalMicrobiological Research
Volume292
DOIs
StatePublished - Mar 2025

Keywords

  • Ehrlichia chaffeensis
  • Etf-3
  • Ferritinophagy
  • LNP-mRNA
  • Type IV secretion system nanobody
  • VHH

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