Development of Etf-3-specific nanobodies to prevent Ehrlichia infection and LNP-mRNA delivery in cellular and murine models.

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).

RipE expression correlates with high ATP levels in , which confers resistance during the extracellular stage to facilitate a new cycle of infection.

Ehrlichiosis is a potentially life-threatening disease caused by infection with the obligatory intracellular bacteria species. infection of mice provides an animal model of ehrlichiosis as it recapitulates full-spectrum and lethal ehrlichiosis in humans. The transposon mutant of , which encodes a previously uncharacterized hypothetical protein, is attenuated in both infection and virulence in mice.

effector EgeA facilitates infection by hijacking TANGO1 and SCFD1 from ER-Golgi exit sites to pathogen-occupied inclusions.

The obligatory intracellular bacterium causes human granulocytic anaplasmosis, an emerging zoonosis. has limited biosynthetic and metabolic capacities, yet it effectively replicates inside of inclusions/vacuoles of eukaryotic host cells. Here, we describe a unique Type IV secretion system (T4SS) effector, R-olgi xit site protein of (EgeA).

Analysis of microRNAs in response to eensis infection and their potential role in vectorial capacity.

Background: MicroRNAs (miRNAs) represent a subset of small noncoding RNAs and carry tremendous potential for regulating gene expression at the post-transcriptional level. They play pivotal roles in distinct cellular mechanisms including inhibition of bacterial, parasitic, and viral infections via immune response pathways. Intriguingly, pathogens have developed strategies to manipulate the host's miRNA profile, fostering environments conducive to successful infection.

Analysis of microRNAs in response to eensis infection and their potential role in vectorial capacity.

Background: MicroRNAs (miRNAs) represent a subset of small noncoding RNAs and carry tremendous potential for regulating gene expression at the post-transcriptional level. They play pivotal roles in distinct cellular mechanisms including inhibition of bacterial, parasitic, and viral infections via immune response pathways. Intriguingly, pathogens have developed strategies to manipulate the host's miRNA profile, fostering environments conducive to successful infection.

effector TRP120 manipulates bacteremia to facilitate tick acquisition.

Unlabelled: species are obligatory intracellular bacteria that cause a potentially fatal disease, human ehrlichiosis. The biomolecular mechanisms of tick acquisition of and transmission between ticks and mammals are poorly understood. infection of mice recapitulates the full spectrum of human ehrlichiosis.

Rab27a via its effector JFC1 localizes to Anaplasma inclusions and promotes Anaplasma proliferation in leukocytes.

Anaplasma phagocytophilum is an obligatory intracellular bacterium that causes tick-borne zoonosis called human granulocytic anaplasmosis. Mechanisms by which Anaplasma replicates inside of the membrane-bound compartment called "inclusion" in neutrophils are incompletely understood. A small GTPase Rab27a is found in the secretory granules and multivesicular endosomes.

Diagnosis of Potomac horse fever (syn. equine neorickettsiosis) in 2 foals in southwestern Ontario.

Potomac horse fever (PHF) is characterized by fever, depression, anorexia, ileus, diarrhea, and occasionally, laminitis. The disease is caused by infection with and/or . Equids of all ages may be affected; however, the condition has not been well-characterized in foals.

Strains of from horses in Ohio are related to isolates from humans in the northeastern USA.

The tick-borne obligatory intracellular bacterium infects humans as well as domesticated and wild animals, causing a febrile disease collectively called granulocytic anaplasmosis. The epidemiology and the host species specificity and zoonotic potential of strains remain unclear. In this study, (encoding ankyrin A) and gene sequences of were determined in clinical specimens from horses in Ohio and compared with those found in strains from various hosts and geographic regions.

Inhibition of infection by cell-permeable macrocyclic peptides that bind type IV secretion effector Etf-1.

is an obligatory intracellular bacterium that infects monocytes and macrophages, and causes human monocytic ehrlichiosis, an emerging life-threatening infectious disease. translocated factor-1 (Etf-1), a type IV secretion system effector, is essential for infection of host cells. Etf-1 translocates to mitochondria to block host apoptosis; furthermore, it can bind Beclin 1 (ATG6) to induce cellular autophagy and localize to -inclusion membrane to obtain host-cell cytoplasmic nutrients.

Efficacy and Immune Correlates of OMP-1B and VirB2-4 Vaccines for Protection of Dogs from Tick Transmission of Ehrlichia chaffeensis.

Ehrlichia chaffeensis, an obligatory intracellular bacterium, causes human monocytic ehrlichiosis, an emerging disease transmitted by the Lone Star tick, Amblyomma americanum. Here, we investigated the vaccine potential of OMP-1B and VirB2-4. Among the highly expressed and immunodominant porin P28s/OMP-1s, OMP-1B is predominantly expressed by in ticks, whereas VirB2-4 is a pilus protein of the type IV secretion system essential for infection of host cells.

Real-Time PCR Differential Detection of and in Cases of Potomac Horse Fever.

Potomac horse fever (PHF) is an acute and potentially fatal enterotyphlocolitis of horses with clinical signs that include anorexia, fever, diarrhea, and laminitis. Its incidence is increasing despite a commercially available vaccine. PHF is caused by Neorickettsia risticii, and the recently rediscovered and classified .

The "Biological Weapons" of : Novel Molecules and Mechanisms to Subjugate Host Cells.

is an obligatory intracellular bacterium that causes human monocytic ehrlichiosis, an emerging, potentially fatal tick-borne infectious disease. The bacterium enters human cells the binding of its unique outer-membrane invasin EtpE to the cognate receptor DNase X on the host-cell plasma membrane; this triggers actin polymerization and filopodia formation at the site of binding, and blocks activation of phagocyte NADPH oxidase that catalyzes the generation of microbicidal reactive oxygen species. Subsequently, the bacterium replicates by hijacking/dysregulating host-cell functions using Type IV secretion effectors.

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

The intracellular cholesterol transport protein Niemann-Pick type C1 (NPC1) and lipid-raft protein flotillin (FLOT) are required for cholesterol uptake by the obligatory intracellular bacterium Anaplasma phagocytophilum and for infection, and each protein localizes to membrane-bound inclusions containing replicating bacteria. Here, we found striking localization of FLOT2 in NPC1-lined vesicles and a physical interaction between FLOT2 and NPC1. This interaction was cholesterol dependent, as a CRAC (cholesterol recognition/interaction amino acid cholesterol-binding) domain mutant of FLOT2 did not interact with NPC1, and the cholesterol-sequestering agent methyl-β-cyclodextrin reduced the interaction.

Potomac horse fever in Ontario: Clinical, geographic, and diagnostic aspects.

Clinical findings, geographic locations, laboratory diagnoses, and culture isolation of spp. in Potomac horse fever (PHF) cases diagnosed in Ontario between 2015 and 2019 are described. Forty-six confirmed PHF cases occurred from late June to early September.

Iron robbery by intracellular pathogen via bacterial effector-induced ferritinophagy.

Iron is essential for survival and proliferation of an obligatory intracellular bacterium that causes an emerging zoonosis, human monocytic ehrlichiosis. However, how acquires iron in the host cells is poorly understood. Here, we found that native and recombinant (cloned into the genome) translocated factor-3 (Etf-3), a previously predicted effector of the type IV secretion system (T4SS), is secreted into the host cell cytoplasm.

An intracellular nanobody targeting T4SS effector inhibits infection.

Infection with obligatory intracellular bacteria is difficult to treat, as intracellular targets and delivery methods of therapeutics are not well known. translocated factor-1 (Etf-1), a type IV secretion system (T4SS) effector, is a primary virulence factor for an obligatory intracellular bacterium, In this study, we developed Etf-1-specific nanobodies (Nbs) by immunizing a llama to determine if intracellular Nbs block Etf-1 functions and infection. Of 24 distinct anti-Etf-1 Nbs, NbD7 blocked mitochondrial localization of Etf-1-GFP in cotransfected cells.

Insights into the mechanism regulating the differential expression of the P28-OMP outer membrane proteins in obligatory intracellular pathogen .

causes human monocytic ehrlichiosis (HME), which is one of the most prevalent, life-threatening emerging infectious zoonoses. The life cycle of includes ticks and mammals, in which proteins are expressed differentially contributing to bacterial survival and infection. Among the P28-OMP outer membrane proteins, OMP-1B and P28 are predominantly expressed in tick cells and mammalian macrophages, respectively.

Comparative Analysis of Genome of Ehrlichia sp. HF, a Model Bacterium to Study Fatal Human Ehrlichiosis.

Background: The genus Ehrlichia consists of tick-borne obligatory intracellular bacteria that can cause deadly diseases of medical and agricultural importance. Ehrlichia sp. HF, isolated from Ixodes ovatus ticks in Japan [also referred to as I.

An Entry-Triggering Protein of Is a New Vaccine Candidate against Tick-Borne Human Monocytic Ehrlichiosis.

is an obligatory intracellular bacterium that causes human monocytic ehrlichiosis, an emerging disease transmitted by the Lone Star tick, outer membrane protein entry triggering protein of (EtpE) is necessary for bacterial entry into human cells. We investigated the role of EtpE in transmission of the bacteria from tick to human cells and whether or not vaccination with EtpE can prevent transmission of ehrlichiae from ticks to mammals. An antiserum against the recombinant C terminus of EtpE (rEtpE-C), which binds a mammalian cell-surface receptor and triggers bacterial entry, significantly inhibited transmission from infected tick cells to human monocytes in culture.

Ehrlichia chaffeensis Uses an Invasin To Suppress Reactive Oxygen Species Generation by Macrophages via CD147-Dependent Inhibition of Vav1 To Block Rac1 Activation.

The obligatory intracellular pathogen lacks most factors that could respond to oxidative stress (a host cell defense mechanism). We previously found that the C terminus of surface invasin, ntry-riggering rotein of (EtpE; EtpE-C) directly binds mammalian DNase X, a glycosylphosphatidylinositol-anchored cell surface receptor and that binding is required to induce bacterial entry and simultaneously to block the generation of reactive oxygen species (ROS) by host monocytes and macrophages. However, how the EtpE-C-DNase X complex mediates the ROS blockade was unknown.

Host membrane lipids are trafficked to membranes of intravacuolar bacterium .

, a cholesterol-rich and cholesterol-dependent obligate intracellular bacterium, partially lacks genes for glycerophospholipid biosynthesis. We found here that is dependent on host glycerolipid biosynthesis, as an inhibitor of host long-chain acyl CoA synthetases, key enzymes for glycerolipid biosynthesis, significantly reduced bacterial proliferation. cannot synthesize phosphatidylcholine or cholesterol but encodes enzymes for phosphatidylethanolamine (PE) biosynthesis; however, exogenous NBD-phosphatidylcholine, Bodipy-PE, and TopFluor-cholesterol were rapidly trafficked to ehrlichiae in infected cells.

Discovery of Virulence Genes of Obligatory Intracellular Bacteria by Random Mutagenesis.

spp. are emerging tick-borne obligatory intracellular bacteria that cause febrile and sometimes fatal diseases with abnormal blood cell counts and signs of hepatitis. HF strain provides an excellent mouse disease model of fatal human ehrlichiosis.