An intracellular nanobody targeting T4SS effector inhibits Ehrlichia infection.

Autor:	Zhang W; Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210., Lin M; Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210., Yan Q; Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210., Budachetri K; Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210., Hou L; Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210., Sahni A; Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210., Liu H; Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210., Han NC; Department of Microbiology, The Ohio State University, Columbus, OH 43210., Lakritz J; Department of Veterinary Preventive Medicine, The Ohio State University, Columbus, OH 43210., Pei D; Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210., Rikihisa Y; Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210; Rikihisa.1@osu.edu.; Department of Microbiology, The Ohio State University, Columbus, OH 43210.
Jazyk:	angličtina
Zdroj:	Proceedings of the National Academy of Sciences of the United States of America [Proc Natl Acad Sci U S A] 2021 May 04; Vol. 118 (18).
DOI:	10.1073/pnas.2024102118
Abstrakt:	Infection with obligatory intracellular bacteria is difficult to treat, as intracellular targets and delivery methods of therapeutics are not well known. Ehrlichia translocated factor-1 (Etf-1), a type IV secretion system (T4SS) effector, is a primary virulence factor for an obligatory intracellular bacterium, Ehrlichia chaffeensis In this study, we developed Etf-1-specific nanobodies (Nbs) by immunizing a llama to determine if intracellular Nbs block Etf-1 functions and Ehrlichia infection. Of 24 distinct anti-Etf-1 Nbs, NbD7 blocked mitochondrial localization of Etf-1-GFP in cotransfected cells. NbD7 and control Nb (NbD3) bound to different regions of Etf-1. Size-exclusion chromatography showed that the NbD7 and Etf-1 complex was more stable than the NbD3 and Etf-1 complex. Intracellular expression of NbD7 inhibited three activities of Etf-1 and E. chaffeensis : up-regulation of mitochondrial manganese superoxide dismutase, reduction of intracellular reactive oxygen species, and inhibition of cellular apoptosis. Consequently, intracellular NbD7 inhibited Ehrlichia infection, whereas NbD3 did not. To safely and effectively deliver Nbs into the host cell cytoplasm, NbD7 was conjugated to cyclized cell-permeable peptide 12 (CPP12-NbD7). CPP12-NbD7 effectively entered mammalian cells and abrogated the blockade of cellular apoptosis caused by E. chaffeensis and inhibited infection by E. chaffeensis in cell culture and in a severe combined-immunodeficiency mouse model. Our results demonstrate the development of an Nb that interferes with T4SS effector functions and intracellular pathogen infection, along with an intracellular delivery method for this Nb. This strategy should overcome current barriers to advance mechanistic research and develop therapies complementary or alternative to the current broad-spectrum antibiotic. Competing Interests: Competing interest statement: D.P. is a cofounder and shareholder of Entrada Therapeutics. (Copyright © 2021 the Author(s). Published by PNAS.)
Databáze:	MEDLINE
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