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Ehrlichiosis is a potentially life-threatening disease caused by infection with the obligatory intracellular bacteria Ehrlichia species. Ehrlichia japonica infection of mice provides an animal model of ehrlichiosis as it recapitulates full-spectrum and lethal ehrlichiosis in humans. The E. japonica transposon mutant of EHF0962, which encodes a previously uncharacterized hypothetical protein, is attenuated in both infection and virulence in mice. EHF0962 was hence named here as resistance-inducing protein of Ehrlichia (RipE). Using this ΔripE mutant, we studied how RipE protein contributes to Ehrlichia pathogenesis. Ehrlichia species have an intracellular developmental cycle and a brief extracellular stage to initiate a new cycle of infection. Majority of RipE proteins were expressed on the surface of the smaller infectious dense-core stage of bacteria. Extracellular ΔripE E. japonica contained significantly less adenosine triphosphate (ATP) and lost infectivity more rapidly in culture compared with wild-type (WT) E. japonica. Genetic complementation in the ΔripE mutant or overexpression of ripE in WT E. japonica significantly increased bacterial ATP levels, and RipE-overexpressing E. japonica was more virulent in mice than WT E. japonica. RipE is conserved among Ehrlichia species. Immunization of mice with recombinant RipE induced an in vitro infection-neutralizing antibody, significantly prolonged survival time after a lethal dose of E. japonica challenge, and cross-protected mice from infection by Ehrlichia chaffeensis, the agent of human monocytic ehrlichiosis. Our findings shed light on the extracellular stage of Ehrlichia, highlighting the importance of RipE and ATP levels in Ehrlichia for extracellular resistance and the next cycle of infection. Thus, RipE is a critical Ehrlichia protein for infection as such can be a potential vaccine target for ehrlichiosis.

BackgroundMicroRNAs (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. Therefore, changes in an arthropod host’s miRNA profile in response to pathogen invasion could be critical in understanding host-pathogen dynamics. Additionally, this area of study could provide insights into discovering new targets for disease control and prevention. The main objective of the present study is to investigate the functional role of differentially expressed miRNAs upon Ehrlichia chaffeensis, a tick-borne pathogen, infection in tick vector, Amblyomma americanum.MethodsSmall RNA libraries from uninfected and E. chaffeensis-infected Am. americanum midgut and salivary gland tissues were prepared using the Illumina Truseq kit. Small RNA sequencing data was analyzed using miRDeep2 and sRNAtoolbox to identify novel and known miRNAs. The differentially expressed miRNAs were validated using a quantitative PCR assay. Furthermore, a miRNA inhibitor approach was used to determine the functional role of selected miRNA candidates.ResultsThe sequencing of small RNA libraries generated >147 million raw reads in all four libraries and identified a total of >250 miRNAs across the four libraries. We identified 23 and 14 differentially expressed miRNAs in salivary glands, and midgut tissues infected with E. chaffeensis, respectively. Three differentially expressed miRNAs (miR-87, miR-750, and miR-275) were further characterized to determine their roles in pathogen infection. Inhibition of target miRNAs significantly decreased the E. chaffeensis load in tick tissues, which warrants more in-depth mechanistic studies.ConclusionsThe current study identified known and novel miRNAs and suggests that interfering with these miRNAs may impact the vectorial capacity of ticks to harbor Ehrlichia. This study identified several new miRNAs for future analysis of their functions in tick biology and tick-pathogen interaction studies.

ABSTRACTEhrlichia species are obligatory intracellular bacteria that cause a potentially fatal disease, human ehrlichiosis. The biomolecular mechanisms of tick acquisition of Ehrlichia and transmission between ticks and mammals are poorly understood. Ehrlichia japonica infection of mice recapitulates the full spectrum of human ehrlichiosis. We compared the pathogenicity and host acquisition of wild-type E. japonica with an isogenic transposon mutant of E. japonica that lacks tandem repeat protein 120 (TRP120) (ΔTRP120). Both wild-type and ΔTRP120 E. japonica proliferated similarly in cultures of mammalian and tick cells. Upon inoculation into mice, both wild-type and ΔTRP120 E. japonica multiplied to high levels in various tissues, with similar clinical chemistry and hematologic changes, proinflammatory cytokine induction, and fatal disease. However, the blood levels of ΔTRP120 E. japonica were almost undetectable within 24 h, whereas the levels of the wild type increased exponentially. Greater than 90% of TRP120 was released from infected cells into the culture medium. Mouse blood monocytes exposed to native TRP120 from culture supernatants showed significantly reduced cell surface expression of the transmigration-related markers Ly6C and CD11b. Larval ticks attached to mice infected with either wild-type or ΔTRP120 E. japonica imbibed similar amounts of blood and subsequently molted to nymphs at similar rates. However, unlike wild-type E. japonica, the ΔTRP120 mutant was minimally acquired by larval ticks and subsequent molted nymphs and, thus, failed to transmit to naïve mice. Thus, TRP120 is required for bacteremia but not disease. These findings suggest a novel mechanism whereby an obligatory intracellular bacterium manipulates infected blood monocytes to sustain the tick–mammal transmission cycle.IMPORTANCEEffective prevention of tick-borne diseases such as human ehrlichiosis requires an understanding of how disease-causing organisms are acquired. Ehrlichia species are intracellular bacteria that require infection of both mammals and ticks, involving cycles of transmission between them. Mouse models of ehrlichiosis and tick–mouse transmission can advance our fundamental understanding of the pathogenesis and prevention of ehrlichiosis. Herein, a mutant of Ehrlichia japonica was used to investigate the role of a single Ehrlichia factor, named tandem repeat protein 120 (TRP120), in infection of mammalian and tick cells in culture, infection and disease progression in mice, and tick acquisition of E. japonica from infected mice. Our results suggest that TRP120 is necessary only for Ehrlichia proliferation in circulating mouse blood and ongoing bacteremia to permit Ehrlichia acquisition by ticks. This study provides new insights into the importance of bacterial factors in regulating bacteremia, which may facilitate tick acquisition of pathogens.

ABSTRACT The tick-borne obligatory intracellular bacterium Anaplasma phagocytophilum infects granulocytes of humans as well as domesticated and wild animals, causing a febrile disease collectively called granulocytic anaplasmosis. The host species specificity and zoonotic potential of A. phagocytophilum strains remain unclear. In the USA, human granulocytic anaplasmosis (HGA) is primarily reported in Wisconsin and Minnesota, the northeastern coastal states, and northern California. In horses, A. phagocytophilum causes equine granulocytic anaplasmosis (EGA). Although EGA has been reported in the same US regions as HGA, horses with a definitive diagnosis of EGA have not been reported in Ohio, where the incidence of HGA is low. In this study, samples from archived blood smears or whole blood from three horses from three different counties in Ohio that presented in 2018, 2020, and 2021 with clinical signs and laboratory findings consistent with EGA were evaluated by PCR and sequencing. Sequences of ankA (encoding ankyrin A) of the three A. phagocytophilum strains were identical and related to strains isolated from humans in the northeastern USA yet distinct from those in California or Europe based on phylogenetic analysis. Each Ohio strain contains unique A. phagocytophilum p44 genes encoding the immunodominant major outer membrane proteins, underscoring the antigenic diversity of A. phagocytophilum strains that cause EGA in Ohio. From 2018 to 2023, 14 additional cases of EGA were identified by serodiagnosis in Ohio and nearby locations in Pennsylvania, pointing to a heretofore unrecognized prevalence of EGA and the risk of transmission of anaplasmosis to humans and animals in this region. IMPORTANCE The tick-borne obligatory intracellular bacterium Anaplasma phagocytophilum 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 A. phagocytophilum strains remain unclear. In this study, ankA (encoding ankyrin A) and p44 gene sequences of A. phagocytophilum were determined in clinical specimens from horses in Ohio and compared with those found in A. phagocytophilum strains from various hosts and geographic regions. With increasing numbers of seropositive horses, the study points out the unrecognized prevalence and uncharacterized strains of A. phagocytophilum infection in horses and the importance of A. phagocytophilum molecular testing for the prevention of equine and human granulocytic anaplasmosis.

ABSTRACT 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 E. chaffeensis porin P28s/OMP-1s, OMP-1B is predominantly expressed by E. chaffeensis in A. americanum ticks, whereas VirB2-4 is a pilus protein of the type IV secretion system essential for E. chaffeensis infection of host cells. Immunization with recombinant OMP-1B (rOMP-1B) or recombinant VirB2-4 (rVirB2-4) protected mice from E. chaffeensis infection as effectively as Entry-triggering protein of Ehrlichia immunization. Dogs vaccinated with a nanoparticle vaccine composed of rOMP-1B or rVirB2-4 and an immunostimulating complex developed high antibody titers against the respective antigen. Upon challenge with E. chaffeensis-infected A. americanum ticks, E. chaffeensis was undetectable in the blood of rOMP-1B or rVirB2-4 immunized dogs on day 3 or 6 post-tick attachment and for the duration of the experiment, whereas dogs sham-vaccinated with the complex alone were persistently infected for the duration of the experiment. E. chaffeensis exponentially replicates in blood-feeding ticks to facilitate transmission. Previously infected ticks removed from OMP-1B-immunized dogs showed significantly lower bacterial load relative to ticks removed from sham-immunized dogs, suggesting in-tick neutralization. Peripheral blood leukocytes from rVirB2-4-vaccinated dogs secreted significantly elevated amounts of interferon-γ soon after tick attachment by ELISpot assay and reverse transcription-quantitative PCR, suggesting interferon-γ-mediated Ehrlichia inhibition. Thus, Ehrlichia surface-exposed proteins OMP-1B and VirB2-4 represent new potential vaccine candidates for blocking tick-borne ehrlichial transmission. IMPORTANCE Ehrlichia are tick-borne pathogens that cause a potentially fatal illness—ehrlichiosis—in animals and humans worldwide. Currently, no vaccine is available for ehrlichiosis, and treatment options are limited. Ticks are biological vectors of Ehrlichia, i.e., Ehrlichia exponentially replicates in blood-sucking ticks before infecting animals. Ticks also inoculate immunomodulatory substances into animals. Thus, it is important to study effects of candidate vaccines on Ehrlichia infection in both animals and ticks and the immune responses of animals shortly after infected tick challenge. Here, we investigated the efficacy of vaccination with functionality-defined two surface-exposed outer membrane proteins of Ehrlichia chaffeensis, OMP-1B and VirB2-4, in a mouse infection model and then in a dog-tick transmission model. Our results begin to fill gaps in our understanding of Ehrlichia-derived protective antigens against tick-transmission and immune correlates and mechanisms that could help future development of vaccines for immunization of humans and animals to counter tick-transmitted ehrlichiosis.

Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes human monocytic ehrlichiosis, an emerging, potentially fatal tick-borne infectious disease. The bacterium enters human cells via 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 E. chaffeensis 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. For example, the Ehrlichia translocated factor (Etf)-1 enters mitochondria and inhibits mitochondria-mediated apoptosis of host cells. Etf-1 also induces autophagy mediated by the small GTPase RAB5, the result being the liberation of catabolites for proliferation inside host cells. Moreover, Etf-2 competes with the RAB5 GTPase-activating protein, for binding to RAB5-GTP on the surface of E. chaffeensis inclusions, which blocks GTP hydrolysis and consequently prevents the fusion of inclusions with host-cell lysosomes. Etf-3 binds ferritin light chain to induce ferritinophagy to obtain intracellular iron. To enable E. chaffeensis to rapidly adapt to the host environment and proliferate, the bacterium must acquire host membrane cholesterol and glycerophospholipids for the purpose of producing large amounts of its own membrane. Future studies on the arsenal of unique Ehrlichia molecules and their interplay with host-cell components will undoubtedly advance our understanding of the molecular mechanisms of obligatory intracellular infection and may identify hitherto unrecognized signaling pathways of human hosts. Such data could be exploited for development of treatment and control measures for ehrlichiosis as well as other ailments that potentially could involve the same host-cell signaling pathways that are appropriated by E. chaffeensis.

Cholesterol influences membrane fluidity and forms membrane microdomains called lipid rafts that serve as organizing centers for the assembly of signaling molecules. Flotillin (FLOT) is a cholesterol-binding lipid-raft protein.

The obligatory intracellular bacterium Anaplasma phagocytophilum causes human granulocytic anaplasmosis, an emerging zoonosis. Anaplasma 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, ER-Golgi exit site protein of Anaplasma (EgeA). In cells infected by Anaplasma, secreted native EgeA, EgeA-GFP, and the C-terminal half of EgeA (EgeA-C)-GFP localized to Anaplasma-containing inclusions. In uninfected cells, EgeA-C-GFP localized to cis-Golgi, whereas the N-terminal half of EgeA-GFP localized to the ER. Pull-down assays identified EgeA-GFP binding to a transmembrane protein in the ER, Transport and Golgi organization protein 1 (TANGO1). By yeast two-hybrid analysis, EgeA-C directly bound Sec1 family domain-containing protein 1 (SCFD1), a host protein of the cis-Golgi network that binds TANGO1 at ER-Golgi exit sites (ERES). Both TANGO1 and SCFD1 localized to the Anaplasma inclusion surface. Furthermore, knockdown of Anaplasma EgeA or either host TANGO1 or SCFD1 significantly reduced Anaplasma infection. TANGO1 and SCFD1 prevent ER congestion and stress by facilitating transport of bulky or unfolded proteins at ERES. A bulky cargo collagen and the ER-resident chaperon BiP were transported into Anaplasma inclusions, and several ER stress marker genes were not up-regulated in Anaplasma-infected cells. Furthermore, EgeA transfection reduced collagen overexpression-induced BiP upregulation. These results suggest that by binding to the two ERES proteins, EgeA redirects the cargo-adapted ERES to pathogen-occupied inclusions and reduces ERES congestion, which facilitates Anaplasma nutrient acquisition and reduces ER stress for Anaplasma survival and proliferation. [ABSTRACT FROM AUTHOR]

Background: MicroRNAs (miRNAs) represent a subset of small noncoding RNAs and carry tremendous potential for regulating gene expression at the posttranscriptional 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. Therefore, changes in an arthropod host's miRNA profile in response to pathogen invasion could be critical in understanding hostpathogen dynamics. Additionally, this area of study could provide insights into discovering new targets for disease control and prevention. The main objective of the present study is to investigate the functional role of differentially expressed miRNAs upon Ehrlichia chaffeensis, a tick-borne pathogen, infection in tick vector, Amblyomma americanum. Methods: Small RNA libraries from uninfected and E. chaffeensis-infected Am. americanum midgut and salivary gland tissues were prepared using the Illumina Truseq kit. Small RNA sequencing data was analyzed using miRDeep2 and sRNAtoolbox to identify novel and known miRNAs. The differentially expressed miRNAs were validated using a quantitative PCR assay. Furthermore, a miRNA inhibitor approach was used to determine the functional role of selected miRNA candidates. Results: The sequencing of small RNA libraries generated >147 million raw reads in all four libraries and identified a total of >250 miRNAs across the four libraries. We identified 23 and 14 differentially expressed miRNAs in salivary glands, and midgut tissues infected with E. chaffeensis, respectively. Three differentially expressed miRNAs (miR-87, miR-750, and miR-275) were further characterized to determine their roles in pathogen infection. Inhibition of target miRNAs significantly decreased the E. chaffeensis load in tick tissues, which warrants more in-depth mechanistic studies. Conclusions: The current study identified known and novel miRNAs and suggests that interfering with these miRNAs may impact the vectorial capacity of ticks to harbor Ehrlichia. This study identified several new miRNAs for future analysis of their functions in tick biology and tick-pathogen interaction studies. [ABSTRACT FROM AUTHOR]