Ehrlichia chaffeensis Co-Opts Phagocytic Hemocytes for Systemic Dissemination in the Lone Star Tick, Amblyomma americanum.

INTRODUCTION: Hematophagous arthropods can acquire and transmit several pathogens of medical importance. In ticks, the innate immune system is crucial in the outcome between vector-pathogen interaction and overall vector competence. However, the specific immune response(s) elicited by the immune cells known as hemocytes remains largely undefined in Ehrlichia chaffeensis and its competent tick vector, Amblyomma americanum. METHODS: We utilized injection of clodronate liposome to deplete tick granulocytes combined with infection with E. chaffeensis to demonstrate their essential role in microbial infection. RESULTS: Here, we show that granulocytes, professional phagocytic cells, are integral in eliciting immune responses against commensal and pathogen infection. The chemical depletion of granulocytes led to decreased phagocytic efficiency of tissue-associated hemocytes. We demonstrate that E. chaffeensis can infect circulating hemocytes, and both cell-free plasma and hemocytes from E. chaffeensis-infected ticks can establish Ehrlichia infection in recipient ticks. Lastly, we provide evidence to show that granulocytes play a dual role in E. chaffeensis infection. Depleting granulocytic hemocytes increased Ehrlichia load in the salivary gland and midgut tissues. In contrast, granulocyte depletion led to a reduced systemic load of Ehrlichia. CONCLUSION: This study has identified multiple roles for granulocytic hemocytes in the control and systemic dissemination of E. chaffeensis infection.

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

Ehrlichia chaffeensis causes human monocytic ehrlichiosis (HME), which is one of the most prevalent, life-threatening emerging infectious zoonoses. The life cycle of E. chaffeensis includes ticks and mammals, in which E. chaffeensis proteins are expressed differentially contributing to bacterial survival and infection. Among the E. chaffeensis P28-OMP outer membrane proteins, OMP-1B and P28 are predominantly expressed in tick cells and mammalian macrophages, respectively. The mechanisms regulating this differential expression have not been comprehensively studied. Here, we demonstrate that the transcriptional regulators EcxR and Tr1 regulate the differential expression of omp-1B and p28 in E. chaffeensis. Recombinant E. chaffeensis Tr1 bound to the promoters of omp-1B and p28, and transactivated omp-1B and p28 promoter-EGFP fusion constructs in Escherichia coli. The consensus sequence of Tr1 binding motifs was AC/TTATA as determined with DNase I footprint assay. Tr1 showed a higher affinity towards the p28 promoter than the omp-1B promoter as determined with surface plasmon resonance. EcxR activated the tr1 expression in response to a temperature decrease. At 37°C low level of Tr1 activated the p28 expression. At 25°C high level of Tr1 activated the omp-1B expression, while repressing the p28 expression by binding to an additional site upstream of the p28 gene. Our data provide insights into a novel mechanism mediated by Tr1 regulating E. chaffeensis differential gene expression, which may aid in the development of new therapeutics for HME.

Effective control of the motile stages of Amblyomma americanum and reduced Ehrlichia spp. prevalence in adults via permethrin treatment of white-tailed deer in coastal Connecticut, USA.

The lone star tick, Amblyomma americanum, is a common human-biting species whose range has been largely restricted to the southeastern United States, until recent detections of established populations on Long Island, New York and throughout coastal southern New England. We evaluated the effectiveness of topical treatment of 10 % permethrin delivered via 4-poster devices to white-tailed deer, Odocoileus virginianus, in the management of a newly discovered A. americanum population in Norwalk, Connecticut. Using a high-density deployment of one 4-poster device/12.7 ha, we were successful in significantly reducing densities of host-seeking adults (93 % reduction), nymphs (92 %), and larvae (96 %) from 2018 to 2020. We also documented a significant reduction (87 %) in parasitizing adults and nymphs on white-tailed deer from 2018 to 2019. The prevalence of Ehrlichia chaffeensis and Ehrlichia ewingii combined in host-seeking adults declined significantly from 47 % at the time the A. americanum population was discovered in 2017 to 7% in 2020. However, the prevalence in nymphs remained static (∼9%) throughout the study period. These data demonstrate that, when properly deployed in a density-dependent manner in terms of deer abundance, 4-poster devices can effectively manage parasitizing and host-seeking A. americanum populations and reduce the prevalence of two ehrlichial species of public health importance.

Protein and DNA Biosynthesis Demonstrated in Host Cell-Free Phagosomes Containing Anaplasma phagocytophilum or Ehrlichia chaffeensis in Axenic Media.

Rickettsiae belong to the Anaplasmataceae family, which includes mostly tick-transmitted pathogens causing human, canine, and ruminant diseases. Biochemical characterization of the pathogens remains a major challenge because of their obligate parasitism. We investigated the use of an axenic medium for growth of two important pathogens-Anaplasma phagocytophilum and Ehrlichia chaffeensis-in host cell-free phagosomes. We recently reported that the axenic medium promotes protein and DNA biosynthesis in host cell-free replicating form of E. chaffeensis, although the bacterial replication is limited. We now tested the hypothesis that growth on axenic medium can be improved if host cell-free rickettsia-containing phagosomes are used. Purification of phagosomes from A. phagocytophilum- and E. chaffeensis-infected host cells was accomplished by density gradient centrifugation combined with magnet-assisted cell sorting. Protein and DNA synthesis was observed for both organisms in cell-free phagosomes with glucose-6-phosphate and/or ATP. The levels of protein and DNA synthesis were the highest for a medium pH of 7. The data demonstrate bacterial DNA and protein synthesis for the first time in host cell-free phagosomes for two rickettsial pathogens. The host cell support-free axenic growth of obligate pathogenic rickettsiae will be critical in advancing research goals in many important tick-borne diseases impacting human and animal health.

Ehrlichia chaffeensis TRP120-mediated ubiquitination and proteasomal degradation of tumor suppressor FBW7 increases oncoprotein stability and promotes infection.

Ehrlichia chaffeensis (E. chaffeensis) exploits evolutionarily conserved Notch and Wnt host cell signaling pathways to downregulate innate immune host defenses and promote infection. The multifunctional E. chaffeensis TRP120 effector which has HECT E3 ubiquitin ligase activity, interacts with the host nuclear tumor suppressor F-BOX and WD domain repeating-containing 7 (FBW7). FBW7 is the substrate recognition subunit of the Skp1-cullin-1-FBOX E3 ubiquitin (Ub) ligase complex (SCF) known to negatively regulate a network of oncoproteins (Notch, cyclin E, c-Jun, MCL1 and cMYC). In this study, we demonstrate that TRP120 and FBW7 colocalize strongly in the nucleus by confocal immunofluorescent microscopy and interactions between TRP120 and FBW7 FBOX and WD40 domains were demonstrated by ectopic expression and co-immunoprecipitation. Although FBW7 gene expression increased during E. chaffeensis infection, FBW7 levels significantly decreased (>70%) by 72 h post infection. Moreover, an iRNA knockdown of FBW7 coincided with increased E. chaffeensis infection and levels of Notch intracellular domain (NICD), phosphorylated c-Jun, MCL-1 and cMYC, which are negatively regulated by FBW7. An increase in FBW7 K48 ubiquitination was detected during infection by co-IP, and FBW7 degradation was inhibited in infected cells treated with the proteasomal inhibitor bortezomib. Direct TRP120 ubiquitination of native and recombinant FBW7 was demonstrated in vitro and confirmed by ectopic expression of TRP120 HECT Ub ligase catalytic site mutant. This study identifies the tumor suppressor, FBW7, as a TRP120 HECT E3 Ub ligase substrate, and demonstrates that TRP120 ligase activity promotes ehrlichial infection by degrading FBW7 to maintain stability of Notch and other oncoproteins involved in cell survival and apoptosis.

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 Ehrlichia chaffeensis lacks most factors that could respond to oxidative stress (a host cell defense mechanism). We previously found that the C terminus of Ehrlichia surface invasin, entry-triggering protein of Ehrlichia (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. A mammalian transmembrane glycoprotein CD147 (basigin) binds to the EtpE-DNase X complex and is required for Ehrlichia entry and infection of host cells. Here, we found that bone marrow-derived macrophages (BMDM) from myeloid cell lineage-selective CD147-null mice had significantly reduced Ehrlichia-induced or EtpE-C-induced blockade of ROS generation in response to phorbol myristate acetate. In BMDM from CD147-null mice, nucleofection with CD147 partially restored the Ehrlichia-mediated inhibition of ROS generation. Indeed, CD147-null mice as well as their BMDM were resistant to Ehrlichia infection. Moreover, in human monocytes, anti-CD147 partially abrogated EtpE-C-induced blockade of ROS generation. Both Ehrlichia and EtpE-C could block activation of the small GTPase Rac1 (which in turn activates phagocyte NADPH oxidase) and suppress activation of Vav1, a hematopoietic-specific Rho/Rac guanine nucleotide exchange factor by phorbol myristate acetate. Vav1 suppression by Ehrlichia was CD147 dependent. E. chaffeensis is the first example of pathogens that block Rac1 activation to colonize macrophages. Furthermore, Ehrlichia uses EtpE to hijack the unique host DNase X-CD147-Vav1 signaling to block Rac1 activation.IMPORTANCEEhrlichia chaffeensis is an obligatory intracellular bacterium with the capability of causing an emerging infectious disease called human monocytic ehrlichiosis. E. chaffeensis preferentially infects monocytes and macrophages, professional phagocytes, equipped with an arsenal of antimicrobial mechanisms, including rapid reactive oxygen species (ROS) generation upon encountering bacteria. As Ehrlichia isolated from host cells are readily killed upon exposure to ROS, Ehrlichia must have evolved a unique mechanism to safely enter phagocytes. We discovered that binding of the Ehrlichia surface invasin to the host cell surface receptor not only triggers Ehrlichia entry but also blocks ROS generation by the host cells by mobilizing a novel intracellular signaling pathway. Knowledge of the mechanisms by which ROS production is inhibited may lead to the development of therapeutics for ehrlichiosis as well as other ROS-related pathologies.

Subversion of RAB5-regulated autophagy by the intracellular pathogen Ehrlichia chaffeensis.

Intracellular pathogens often exploit RAB functions to establish a safe haven in which to survive and proliferate. Ehrlichia chaffeensis, an obligatory intracellular bacterium, resides in specialized membrane-bound inclusions that have early endosome-like characteristics, e.g., resident RAB5 GTPase and RAB5 effectors, including VPS34 (the catalytic subunit of class III phosphatidylinositol 3-kinase), but the inclusions lack late endosomal or lysosomal markers. Within inclusions, Ehrlichia obtains host-derived nutrients by inducing RAB5-regulated autophagy using Ehrlichia translocated factor-1 deployed by its type IV secretion system. This manipulation of RAB5 by a bacterial molecule offers a simple strategy for Ehrlichia to avoid destruction in lysosomes and obtain nutrients, membrane components, and a homeostatic intra-host-cell environment in which to grow.

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

Ehrlichia chaffeensis, 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 Ehrlichia translocated factor-2 (Etf-2), a previously predicted effector of the Ehrlichia type IV secretion system, and recombinant Etf-2 (cloned into the Ehrlichia genome) are secreted into the host-cell cytoplasm and localize to ehrlichial inclusions. Ectopically expressed Etf-2-GFP also localized to inclusions and membranes of early endosomes marked with RAB5 and interacted with GTP-bound RAB5 but not with a GDP-bound RAB5. Etf-2, although lacking a RAB GTPase-activating protein (GAP) Tre2-Bub2-Cdc16 (TBC) domain, contains two conserved TBC domain motifs, namely an Arg finger and a Gln finger, and site-directed mutagenesis revealed that both Arg188 and Gln245 are required for Etf-2 localization to early endosomes. The yeast two-hybrid assay and microscale thermophoresis revealed that Etf-2 binds tightly to GTP-bound RAB5 but not to GDP-bound RAB5. However, Etf-2 lacks RAB5-specific GAP activity. Etf-2 localized to bead-containing phagosomes as well as endosomes containing beads coated with the C-terminal fragment of EtpE (entry-triggering protein of Ehrlichia), an Ehrlichia outer-membrane invasin, and significantly delayed RAB5 dissociation from and RAB7 localization to phagosomes/endosomes and RABGAP5 localization to endosomes. Thus, binding of Etf-2 to RAB5-GTP appears to delay RAB5 inactivation by impeding RABGAP5 localization to endosomes. This suggests a unique mechanism by which RAB5 is sequestered on ehrlichial inclusions to benefit bacterial survival and replication.

Ehrlichia chaffeensis TRP75 Interacts with Host Cell Targets Involved in Homeostasis, Cytoskeleton Organization, and Apoptosis Regulation To Promote Infection.

Ehrlichia chaffeensis is an obligately intracellular bacterium that exhibits tropism for mononuclear phagocytes. The mechanisms involved in E. chaffeensis infection of the host cell and evasion of host defenses are not fully defined, but a subset of type 1 secreted tandem repeat protein (TRP) effectors play important roles. Recently, we determined molecular interactions of TRP120, TRP47, and TRP32 with the eukaryotic host cell. In this investigation, we used yeast two-hybrid analysis to reveal that another E. chaffeensis tandem repeat protein, TRP75, interacts with a diverse group of human proteins associated with organismal and tissue homeostasis, multiple metabolic processes and regulation, response to reactive oxygen species, signal transduction, and protein modifications. Thirteen identified host target proteins associated with actin cytoskeleton reorganization or apoptosis were examined in detail and confirmed to interact with TRP75 at different levels as determined by coimmunoprecipitation assays. These protein interactions were visualized by immunofluorescence confocal microscopy during infection and colocalized with Ehrlichia morulae with different intensities. Moreover, small interfering RNAs (siRNAs) (n = 86) were used to knock down identified TRP75-interacting host proteins separately, and their influence on ehrlichial infection was investigated by real-time quantitative PCR (qPCR). Knockdown of 74/86 (86%) TRP75 target proteins had a significant negative effect on ehrlichial infection. The results of this study further support the idea of a role of Ehrlichia TRPs as effectors that interact with a complex array of host proteins to promote ehrlichial infection.IMPORTANCE Human monocytic ehrlichiosis (HME) is caused by an obligatory intracellular bacterium, E. chaffeensis, and is one of the most prevalent, life-threatening emerging infectious zoonoses in the United States. The mechanisms through which E. chaffeensis invades and establishes an intracellular niche are not well understood but are dependent on secreted ehrlichial effector proteins. The significance of this study is in addressing how intracellular pathogens, particularly those with small genomes such as Ehrlichia, exploit a limited number of secreted effector proteins such as tandem repeat proteins (TRPs) to manipulate complex eukaryotes and to regulate host cell processes through molecular pathogen-host interplay. The results of our studies highlight the broader role of ehrlichial TRPs in promoting infection and help define the mechanisms through which obligately intracellular bacteria modulate host cell function for survival.

Ehrlichia chaffeensis TRP120 Effector Targets and Recruits Host Polycomb Group Proteins for Degradation To Promote Intracellular Infection.

Ehrlichia chaffeensis has a group of well-characterized type I secreted tandem repeat protein (TRP) effectors that have moonlighting capabilities. TRPs modulate various cellular processes, reprogram host gene transcription as nucleomodulins, function as ubiquitin ligases, and directly activate conserved host cell signaling pathways to promote E. chaffeensis infection. One TRP-interacting host target is polycomb group ring finger protein 5 (PCGF5), a member of the polycomb group (PcG) protein family and a component of the polycomb repressive complex 1 (PRC1). The current study demonstrates that during early infection, PCGF5 strongly colocalizes with TRP120 in the nucleus and later dramatically redistributes to the ehrlichial vacuole along with other PCGF isoforms. Ectopic expression and immunoprecipitation of TRP120 confirmed the interaction of TRP120 with multiple different PCGF isoforms. At 48 h postinfection, a dramatic redistribution of PCGF isoforms from the nucleus to the ehrlichial vacuole was observed, which also temporally coincided with proteasomal degradation of PCGF isoforms and TRP120 expression on the vacuole. A decrease in PRC1-mediated repressive chromatin mark and an altered transcriptional activity in PRC1-associated Hox genes primarily from HOXB and HOXC clusters were observed along with the degradation of PCGF isoforms, suggesting disruption of the PRC1 in E. chaffeensis-infected cells. Notably, small interfering RNA (siRNA)-mediated knockdown of PCGF isoforms resulted in significantly increased E. chaffeensis infection. This study demonstrates a novel strategy in which E. chaffeensis manipulates PRC complexes through interactions between TRP120 and PCGF isoforms to promote infection.

Ehrlichia chaffeensis TRP120 Moonlights as a HECT E3 Ligase Involved in Self- and Host Ubiquitination To Influence Protein Interactions and Stability for Intracellular Survival.

Ehrlichia chaffeensis secretes tandem repeat protein (TRP) effectors that are involved in a diverse array of host cell interactions, some of which directly activate cell signaling pathways and reprogram host gene transcription to promote survival in the mononuclear phagocyte. However, the molecular details of these effector-host interactions and roles in pathobiology are incompletely understood. In this study, we determined that the E. chaffeensis effector TRP120 is posttranslationally modified by ubiquitin (Ub) and that ubiquitination occurs through intrinsic and host-mediated HECT ligase activity. A functional HECT E3 ligase domain with a conserved catalytic site was identified in the C-terminal region of TRP120, and TRP120 autoubiquitination occurred in vitro in the presence of host UbcH5b/c E2 enzymes. TRP120 ubiquitination sites were mapped using a high-density microfluidic peptide array and confirmed by ectopic expression of TRP120 lysine mutants in cells. Moreover, we determined that the HECT E3 ubiquitin ligase, Nedd4L, interacts with TRP120 during infection and also mediates TRP120 ubiquitination. Nedd4L knockdown resulted in the reduction of TRP120-Ub, decreased ehrlichial infection, and reduced recruitment of a known TRP120-interacting host protein, PCGF5, to ehrlichial inclusions. TRP120-mediated PCGF5 polyubiquitination was associated with a reduction in PCGF5 levels. Inhibition of ubiquitination with small molecules also significantly decreased ehrlichial infection, indicating that the Ub pathway is critical for ehrlichial intracellular replication and survival. The current study identified a novel E. chaffeensis ubiquitin ligase and revealed an important role for the ubiquitin pathway in effector-host interactions and pathogen-mediated host protein stability in order to promote intracellular survival.

Protein aggregation in Ehrlichia chaffeensis during infection of mammalian cells.

Ehrlichia chaffeensis is an obligatory intracellular pathogen transmitted through infected ticks to humans and other vertebrates. We investigated the extent of protein aggregation in E. chaffeensis during infection of canine macrophage cell line, DH82. We discovered that the size of the aggregated fraction of E. chaffeensis proteins increased during the first 48 h post infection. We also incubated the infected cells with guanidinium chloride (GuHCl), a known inhibitor of the protein-disaggregating molecular chaperone ClpB. Up to 0.5 mM GuHCl had no impact on the host cells, whereas the viability of the pathogen was reduced by ∼60% in the presence of the inhibitor. Furthermore, we found that the size of the aggregated protein fraction in E. chaffeensis increased significantly in cultures supplemented with 0.5 mM GuHCl, which also resulted in the preferential accumulation of ClpB with the aggregated proteins. Altogether, our results suggest that an exposure of E. chaffeensis to the stressful environment of a host cell results in an increased aggregation of the pathogen's proteins, which is exacerbated upon inhibition of ClpB. Our studies establish a link between protein quality control and pathogen survival during infection of a host.

Amblyomma americanum ticks infected with in vitro cultured wild-type and mutants of Ehrlichia chaffeensis are competent to produce infection in naïve deer and dogs.

Monocytic ehrlichiosis in people caused by the intracellular bacterium, Ehrlichia chaffeensis, is an emerging infectious disease transmitted by the lone star tick, Amblyomma americanum. Tick transmission disease models for ehrlichiosis require at least two hosts and two tick blood feeding episodes to recapitulate the natural transmission cycle. One blood feeding is necessary for the tick to acquire the infection from an infected host and the next feeding is needed to transmit the bacterium to a naïve host. We have developed a model for E. chaffeensis transmission that eliminates the entire tick acquisition stage while still producing high numbers of infected ticks that are also able to transmit infections to naïve hosts. Fully engorged A. americanum nymphs were ventrally needle-infected, possibly into the midgut, and following molting, the unfed adult ticks were used to infect naive deer and dogs. We have also described using the ticks infected by this method the transmission of both wild-type and transposon mutants of E. chaffeensis to its primary reservoir host, white tailed deer and to another known host, dog. The infection progression and IgG antibody responses in deer were similar to those observed with transmission feeding of ticks acquiring infection by natural blood feeding. The pathogen infections acquired by natural tick transmission and by feeding needle-infected ticks on animals were also similar to intravenous infections in causing persistent infections. Needle-infected ticks having the ability to transmit pathogens will be a valuable resource to substantially simplify the process of generating infected ticks and to study infection systems in vertebrate hosts where interference of other pathogens could be avoided.

Occurrence of Amblyomma americanum (Acari: Ixodidae) and Human Infection With Ehrlichia chaffeensis in Wisconsin, 2008-2015.

Because of the increasing incidence of human ehrlichiosis in Wisconsin, we assessed reports of human infections by Ehrlichia chaffeensis and the distribution of its vector, the lone star tick (Amblyomma americanum (L.)). From 2008 through 2015, 158 probable and confirmed human cases of E. chaffeensis infections were reported to the Wisconsin Department of Health Services. Five cases without travel history outside of Wisconsin were confirmed as E. chaffeensis by polymerase chain reaction. Surveillance for the vector occurred from 2008 through 2015 and was based on active and passive methods, including examination of white-tailed deer, collections from live-trapped small mammals, submissions of ticks removed from wild and domestic animals through the Wisconsin Surveillance of Animals for Ticks (SWAT) program, digital or physical submissions by the public to the University of Wisconsin Insect Diagnostic or Medical Entomology laboratories, and active tick dragging. More than 50 lone star ticks (46 adults, 6 nymphs, and 1 larva) were identified. Lone star ticks were more commonly found in south central Wisconsin, particularly in Dane County, where discovery of more than one life stage in a single year indicates possible establishment.

Ehrlichia chaffeensis TRP32 Nucleomodulin Function and Localization Is Regulated by NEDD4L-Mediated Ubiquitination.

Ehrlichia chaffeensis is an obligately intracellular bacterium that reprograms the mononuclear phagocyte through diverse effector-host interactions to modulate various host cell processes. In a previous study, we reported that the E. chaffeensis nucleomodulin TRP32 regulates transcription of host genes in several biologically relevant categories, including cell differentiation and proliferation. In this study, we investigate the effect of ubiquitination on TRP32 function and localization within the host cell. TRP32 is both mono- and polyubiquitinated on multiple lysine residues during infection and when ectopically expressed. Despite lacking a canonical PPxY motif, TRP32 interacted with, and was modified by the human HECT E3 ubiquitin (Ub) ligase NEDD4L. TRP32 ubiquitination was not by K48-linked polyUb chains, nor was it degraded by the proteasome; however, TRP32 was modified by K63-linked polyUb chains detected both in the cytosol and nucleus. HECT ligase inhibitor, heclin, altered the subnuclear localization of ectopically expressed TRP32 from a diffuse nuclear pattern to a lacy, punctate pattern with TRP32 distributed around the periphery of the nucleus and nucleoli. When a TRP32 lysine null (K-null) mutant was ectopically expressed, it exhibited a similar phenotype as single lysine mutants (K63R, K93R, and K123R). However, the K-null mutant showed increased amounts of cytoplasmic TRP32 compared to single lysine mutants or heclin-treated cells ectopically expressing TRP32. These alterations in localization corresponded to changes in TRP32 transcriptional repressor function with heclin-treated and single lysine mutants unable to repress transcription of a TRP32 target genes in a luciferase assay.

Panola Mountain Ehrlichia in Amblyomma maculatum From the United States and Amblyomma variegatum (Acari: Ixodidae) From the Caribbean and Africa.

Panola Mountain Ehrlichia (PME) has been suggested as an emerging pathogen of humans and dogs. Domestic goats and white-tailed deer (Odocoileus virginianus) are also susceptible and likely serve as reservoirs. Experimentally, both the lone star tick (Amblyomma americanum (L.)) and the Gulf Coast tick (Amblyomma maculatum Koch) can transmit PME among deer and goats. In the current study, we detected PME in adult wild-caught A. maculatum from the United States and Amblyomma variegatum (F.) from the Caribbean and Africa. This significantly expands the range, potential tick vectors, and risk for exposure to PME.

Note on Ehrlichia chaffeensis, Ehrlichia ewingii, and "Borrelia lonestari" infection in lone star ticks (Acari: Ixodidae), Nebraska, USA.

The lone star tick, Amblyomma americanum (L.) (Acari: Ixodidae), is established in southeastern Nebraska yet the prevalence of tick-associated microorganisms is not known. An initial PCR-based analysis for Ehrlichia chaffeensis, Ehrlichia ewingii, and Borrelia infection in host-seeking adult ticks collected in southeast Nebraska was conducted. A total of 251 adult ticks collected in six sites in southeast Nebraska were tested. E. chaffeensis, E. ewingii, and Borrelia spp. were present, and the prevalence of each was approximately 1.6%. This study demonstrates that Ehrlichia spp. are present in Nebraska lone star tick populations.

Ehrlichia chaffeensis Exploits Canonical and Noncanonical Host Wnt Signaling Pathways To Stimulate Phagocytosis and Promote Intracellular Survival.

Ehrlichia chaffeensis invades and survives in phagocytes by modulating host cell processes and evading innate defenses, but the mechanisms are not fully defined. Recently we have determined that E. chaffeensis tandem repeat proteins (TRPs) are type 1 secreted effectors involved in functionally diverse interactions with host targets, including components of the evolutionarily conserved Wnt signaling pathways. In this study, we demonstrated that induction of host canonical and noncanonical Wnt pathways by E. chaffeensis TRP effectors stimulates phagocytosis and promotes intracellular survival. After E. chaffeensis infection, canonical and noncanonical Wnt signalings were significantly stimulated during early stages of infection (1 to 3 h) which coincided with dephosphorylation and nuclear translocation of ß-catenin, a major canonical Wnt signal transducer, and NFATC1, a noncanonical Wnt transcription factor. In total, the expression of ∼44% of Wnt signaling target genes was altered during infection. Knockdown of TRP120-interacting Wnt pathway components/regulators and other critical components, such as Wnt5a ligand, Frizzled 5 receptor, ß-catenin, nuclear factor of activated T cells (NFAT), and major signaling molecules, resulted in significant reductions in the ehrlichial load. Moreover, small-molecule inhibitors specific for components of canonical and noncanonical (Ca(2+) and planar cell polarity [PCP]) Wnt pathways, including IWP-2, which blocks Wnt secretion, significantly decreased ehrlichial infection. TRPs directly activated Wnt signaling, as TRP-coated microspheres triggered phagocytosis which was blocked by Wnt pathway inhibitors, demonstrating a key role of TRP activation of Wnt pathways to induce ehrlichial phagocytosis. These novel findings reveal that E. chaffeensis exploits canonical and noncanonical Wnt pathways through TRP effectors to facilitate host cell entry and promote intracellular survival.

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. To infect cells, Ehrlichia uses the C terminus of the outer membrane invasin entry-triggering protein (EtpE) of Ehrlichia (EtpE-C), which directly binds the mammalian cell surface glycosylphosphatidyl inositol-anchored protein, DNase X. How this binding drives Ehrlichia entry is unknown. Here, using affinity pulldown of host cell lysates with recombinant EtpE-C (rEtpE-C), we identified two new human proteins that interact with EtpE-C: CD147 and heterogeneous nuclear ribonucleoprotein K (hnRNP-K). The interaction of CD147 with rEtpE-C was validated by far-Western blotting and coimmunoprecipitation of native EtpE with endogenous CD147. CD147 was ubiquitous on the cell surface and also present around foci of rEtpE-C-coated-bead entry. Functional neutralization of surface-exposed CD147 with a specific antibody inhibited Ehrlichia internalization and infection but not binding. Downregulation of CD147 by short hairpin RNA (shRNA) impaired E. chaffeensis infection. Functional ablation of cytoplasmic hnRNP-K by a nanoscale intracellular antibody markedly attenuated bacterial entry and infection but not binding. EtpE-C also interacted with neuronal Wiskott-Aldrich syndrome protein (N-WASP), which is activated by hnRNP-K. Wiskostatin, which inhibits N-WASP activation, and cytochalasin D, which inhibits actin polymerization, inhibited Ehrlichia entry. Upon incubation with host cell lysate, EtpE-C but not an EtpE N-terminal fragment stimulated in vitro actin polymerization in an N-WASP- and DNase X-dependent manner. Time-lapse video images revealed N-WASP recruitment at EtpE-C-coated bead entry foci. Thus, EtpE-C binding to DNase X drives Ehrlichia entry by engaging CD147 and hnRNP-K and activating N-WASP-dependent actin polymerization. IMPORTANCE: Ehrlichia chaffeensis, an obligate intracellular bacterium, causes a blood-borne disease called human monocytic ehrlichiosis, one of the most prevalent life-threatening emerging tick-transmitted infectious diseases in the United States. The survival of Ehrlichia bacteria, and hence, their ability to cause disease, depends on their specific mode of entry into eukaryotic host cells. Understanding the mechanism by which E. chaffeensis enters cells will create new opportunities for developing effective therapies to prevent bacterial entry and disease in humans. Our findings reveal a novel cellular signaling pathway triggered by an ehrlichial surface protein called EtpE to induce its infectious entry. The results are also important from the viewpoint of human cell physiology because three EtpE-interacting human proteins, DNase X, CD147, and hnRNP-K, are hitherto unknown partners that drive the uptake of small particles, including bacteria, into human cells.