TY - JOUR
T1 - Development of Etf-3-specific nanobodies to prevent Ehrlichia infection and LNP-mRNA delivery in cellular and murine models
AU - Duan, Nan
AU - Lin, Mingqun
AU - Zhang, Wenqing
AU - Yan, Qi
AU - Chien, Rory C.
AU - Budachetri, Khemraj
AU - Denton, Stephen
AU - Kawahara, Jeffrey
AU - Lakritz, Jeffrey
AU - Zhong, Yichen
AU - Dong, Yizhou
AU - Rikihisa, Yasuko
N1 - Publisher Copyright:
© 2024
PY - 2025/3
Y1 - 2025/3
N2 - 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.
AB - 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.
KW - Ehrlichia chaffeensis
KW - Etf-3
KW - Ferritinophagy
KW - LNP-mRNA
KW - Type IV secretion system nanobody
KW - VHH
UR - http://www.scopus.com/inward/record.url?scp=85212330642&partnerID=8YFLogxK
U2 - 10.1016/j.micres.2024.128027
DO - 10.1016/j.micres.2024.128027
M3 - Article
AN - SCOPUS:85212330642
SN - 0944-5013
VL - 292
JO - Microbiological Research
JF - Microbiological Research
M1 - 128027
ER -