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.

The “Biological Weapons” of Ehrlichia chaffeensis: Novel Molecules and Mechanisms to Subjugate Host Cells

“…Moreover, in similar experiments with yeast, Etf-1 also localized to mitochondria and inhibited apoptosis induced by heterologous expression of human Bax (Liu et al, 2012). The N-terminal 24 amino-acid residues of Etf-1, especially residue K23, play a critical role in mitochondrial targeting of Etf-1, as deletion mutation of this residue significantly decreased Etf-1 localization to mitochondria (Zhang et al, 2021). The mitochondrial matrix protein manganese superoxide dismutase (MnSOD) maintains a basal level of ROS in cells by scavenging O − 2 and is essential for maintaining aerobic life (Holley et al, 2012).…”

“…To verify the functions of intracellular Etf-1 and investigate the possibility that Etf-1 could be used as a therapeutic target, Etf-1−specific nanobodies were developed by immunizing a llama (Zhang et al, 2021). One particular nanobody could form a stable complex with Etf-1 and thereby block the mitochondrial localization of Etf-1 (Zhang et al, 2021).…”

Preparation of a Nanobody Specific to Dectin 1 and Its Anti-inflammatory Effects on Fungal Keratitis

“…Extensive research has demonstrated that nanobodies play a significant role in several inflammatory diseases. [44][45][46] In related experiments, nanobodies specific to TLR4 treatment significantly decreased mRNA and protein levels of proinflammatory cytokines in NR8383 cells. 47 Consistently with that research, our in vitro tests showed that compared with negative nanobody treatment, specific nanobody treatment significantly suppressed mRNA and protein expression of IL1β and IL6 in HCECs stimulated by A. fumigatus.…”

Anaplasma phagocytophilum Hijacks Flotillin and NPC1 Complex To Acquire Intracellular Cholesterol for Proliferation, Which Can Be Inhibited with Ezetimibe

“…RF/6A monkey endothelial cells (ATCC) were used in immunofluorescence labeling for unambiguous localization analysis owing to their tight adherence and thinly spread morphology ( 5 ). Human embryonic kidney HEK293T cells (ATCC) were used for transfection and pulldown assays, as they can be transfected with high efficiency ( 41 ).…”