An intracellular nanobody targeting T4SS effector inhibits Ehrlichia infection.

Abstract

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.

• Authors: Wenqing Zhang, Mingqun Lin, Qi Yan, Khemraj Budachetri, Libo Hou, Ashweta Sahni, Hongyan Liu, Nien-Ching Han, Jeffrey Lakritz, Dehua Pei, Yasuko Rikihisa
• Journal: Proceedings of the National Academy of Sciences of the United States of America (Proc Natl Acad Sci U S A) Vol. 118 Issue 18 (05 04 2021) ISSN: 1091-6490 [Electronic] United States
• PMID: 33903242 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)
• Copyright: Copyright © 2021 the Author(s). Published by PNAS.

Chemical References

• Reactive Oxygen Species
• Single-Domain Antibodies
• Type IV Secretion Systems
• Virulence Factors

Topics

• Animals
• Apoptosis (genetics)
• B-Lymphocyte Subsets (immunology)
• Ehrlichia chaffeensis (drug effects, genetics, immunology, pathogenicity)
• Ehrlichiosis (drug therapy, genetics, immunology, pathology)
• Host-Pathogen Interactions (genetics, immunology)
• Humans
• Mice
• Reactive Oxygen Species (metabolism)
• Single-Domain Antibodies (immunology, pharmacology)
• Type IV Secretion Systems (antagonists & inhibitors, genetics, immunology)
• Virulence Factors