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).

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.

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 .

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.

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.

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.

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.

Isolation and Molecular Analysis of a Novel Species That Causes Potomac Horse Fever.

Potomac horse fever (PHF), a severe and frequently fatal febrile diarrheal disease, has been known to be caused only by , an endosymbiont of digenean trematodes. Here, we report the cell culture isolation of a new species found in two locations in eastern Ontario, Canada, in 2016 and 2017 (in addition to 10 variable strains of ) from PCR-negative horses with clinical signs of PHF. Gene sequences of 16S rRNA and the major surface antigen P51 of this new species were distinct from those of all previously characterized strains and species, except for those from an uncharacterized species culture isolate from a horse with PHF in northern Ohio in 1991.

Infection by Anaplasma phagocytophilum Requires Recruitment of Low-Density Lipoprotein Cholesterol by Flotillins.

is an obligatory intracellular bacterium that proliferates in membrane-bound inclusions. is dependent on cholesterol and acquire cholesterol from low-density lipoprotein (LDL) endocytosed by mammalian host cells. The mechanism of cholesterol transport to inclusions, however, is not fully understood.

type IV secretion system effector Etf-2 binds to active RAB5 and delays endosome maturation.

, an obligatory intracellular bacterium, infects monocytes/macrophages by sequestering a regulator of endosomal traffic, the small GTPase RAB5, on its membrane-bound inclusions to avoid routing to host-cell phagolysosomes. How RAB5 is sequestered on ehrlichial inclusions is poorly understood, however. We found that native translocated factor-2 (Etf-2), a previously predicted effector of the type IV secretion system, and recombinant Etf-2 (cloned into the genome) are secreted into the host-cell cytoplasm and localize to ehrlichial inclusions.

and Its Invasin EtpE Block Reactive Oxygen Species Generation by Macrophages in a DNase X-Dependent Manner.

The obligatory intracellular pathogen lacks most genes that confer resistance to oxidative stress but can block reactive oxygen species (ROS) generation by host monocytes-macrophages. Bacterial and host molecules responsible for this inhibition have not been identified. To infect host cells, uses the C terminus of its surface invasin, entry-triggering protein of (EtpE; EtpE-C), which directly binds the mammalian cell surface receptor glycosylphosphatidylinositol-anchored protein DNase X.

Analysis of complete genome sequence and major surface antigens of Neorickettsia helminthoeca, causative agent of salmon poisoning disease.

Neorickettsia helminthoeca, a type species of the genus Neorickettsia, is an endosymbiont of digenetic trematodes of veterinary importance. Upon ingestion of salmonid fish parasitized with infected trematodes, canids develop salmon poisoning disease (SPD), an acute febrile illness that is particularly severe and often fatal in dogs without adequate treatment. We determined and analysed the complete genome sequence of N.

Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase.

Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes a potentially fatal emerging zoonosis, human monocytic ehrlichiosis. E. chaffeensis has a limited capacity for biosynthesis and metabolism and thus depends mostly on host-synthesized nutrients for growth.

Efficient Enrichment of Bacterial mRNA from Host-Bacteria Total RNA Samples.

Despite numerous advances in genomics and bioinformatics, technological hurdles remain to examine host-microbe transcriptomics. Sometimes the transcriptome of either or both can be ascertained merely by generating more sequencing reads. However, many cases exist where bacterial mRNA needs to be enriched further to enable cost-effective sequencing of the pathogen or endosymbiont.

EtpE Binding to DNase X Induces Ehrlichial Entry via CD147 and hnRNP-K Recruitment, Followed by Mobilization of N-WASP and Actin.

Unlabelled: Obligate intracellular bacteria, such as Ehrlichia chaffeensis, perish unless they can enter eukaryotic cells. E. chaffeensis is the etiological agent of human monocytic ehrlichiosis, an emerging infectious disease.