An intracellular nanobody targeting T4SS effector inhibits Ehrlichia infection

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.

Human monocytic ehrlichiosis (HME), one of the most prevalent, life-threatening, and emerging tick-borne diseases in the United States (1, 2) is caused by infection with Ehrlichia chaffeensis, an obligatory intracellular bacterium in the order Rickettsiales. E. chaffeensis replicates within human monocytes-macrophages and causes severe flu-like symptoms accompanied by hematologic abnormalities and hepatitis. Currently, the only HME therapy is the broad-spectrum antibiotic doxycycline, which is effective only if initiated early because delayed initiation (e.g., because of misdiagnosis can lead to severe complications or death). In addition, doxycycline is contraindicated for pregnant women and children or those with drug allergies. The presence of underlying illness or injury, immunosuppression, and coinfection with other tick-borne pathogens can similarly lead to severe complications or death (3). No vaccine exists for HME. Tick-borne diseases have risen dramatically in the past 20 y and continue to rise, underscoring the importance of developing new therapeutic approaches and preventive measures (4).The type IV secretion system (T4SS) is conserved among all rickettsial organisms. The recent elucidation of critical roles of T4SS for E. chaffeensis and Anaplasma phagocytophilum infection (5) may provide potential targets for new approaches against rickettsial diseases. For example, the T4SS effectors Ehrlichial translocated factors 1 and 2 (Etf-1 and Etf-2) are critical E. chaffeensis proteins secreted via T4SS into the host cell cytoplasm, as knockdown of Etf-1 or Etf-2 by transfection of E. chaffeensis with specific antisense peptide nucleic acids significantly inhibits E. chaffeensis infection (6, 7). Secreted Etf-1 localizes to mitochondria and blocks mitochondria-mediated host cell apoptosis to keep the infected host cell alive for bacterial intracellular replication (8). A subpopulation of Etf-1 molecules that are not localized to mitochondria interacts with Beclin 1 (ATG6) and active Rab5 (Rab5-GTP), and induces Rab5-regulated autophagy for E. chaffeensis to acquire catabolites as nutrients (9). Etf-2 directly binds Rab5-GTP on Ehrlichia-containing inclusion membranes and blocks Rab5 GTPase activating protein (RabGAP-5) engagement with Rab5-GTP to prevent Ehrlichia-containing inclusions from maturing into late endosomes and fusing with lysosomes (7).Camelidae produce two types of antibodies: conventional antibodies and heavy-chain–only antibodies (10). The variable domain of the heavy chain of heavy-chain–only antibodies (VHHs) of camelids is the smallest (11 to 15 kDa) antigen-binding fragment relative to conventional antibodies. VHHs are soluble and display long surface loops, which are often larger than those of conventional murine and human antibodies (11, 12). The VHHs can be cloned into bacterial or mammalian expression plasmids (13) to produce a nanobody (Nb), a monomeric variable antibody. VHHs cloned into mammalian expression vectors can produce intracellular Nbs within mammalian cells that are superior to conventional antibodies for modulating intracellular functions because they can operate in the reducing intracellular environment, are proteolytically stable, can target subcellular sites, can penetrate cavities in target antigens, and can bind efficiently to antigens, such as enzyme catalytic sites (13–16). Although the therapeutic potential of Nbs has been investigated for several infectious diseases (14, 17–19), the use of Nbs as a therapeutic agent against intracellular bacteria such as E. chaffeensis has not been reported. In the present study, we developed an intracellular Nb approach to block T4SS effectors within mammalian cells, thereby inhibiting intracellular pathogen infection.Progress in developing effective therapy and investigative approach for obligatory intracellular pathogens has been hindered by many factors, not the least of which is the lack of safe and efficient intracellular delivery methods of macromolecules. Although cyclic peptides are generally unable to cross the cell membrane, some naturally occurring cyclic peptides (e.g., cyclosporine A) possess the unusual ability of crossing the cell membrane by passive diffusion and are orally bioavailable (20). Cyclized Arg-rich cell-permeable peptides (CPPs)—such as cyclo(FΦRRRRQ) or cFΦR4, where Φ is l-2-naphthylalanine—or newer and more effective CPPs, such as CPP9 and CPP12, that include d-arginine or d-phenylalanine, provide rapid and efficient cytosolic delivery of their linked cargo proteins into >95% of cells (21–23). They are not cytotoxic at effective concentrations and have oral and intravenous bioavailability based on preliminary pharmacokinetics in mice (22). The cyclic CPPs (and the CPP-cargo conjugates) bind directly to plasma membrane phospholipids and enter cells by endocytosis (22). They then efficiently escape from the early endosome into the cytosol unlike Tat, which escapes only from late endosomes (22–24).In the present study, we have developed anti–Etf-1 Nbs. We obtained a Nb that blocks Etf-1 functions and demonstrated its effectiveness in combination with a cyclic CPP for inhibition of E. chaffeensis infection in cell culture and in a mouse model. These findings represent a significant advance in developing therapeutic and investigative strategy of obligatory intracellular pathogens.