First detection of Ehrlichia chaffeensis, Ehrlichia canis, and Anaplasma ovis in Rhipicephalus bursa ticks collected from sheep, Turkey.

Anaplasmosis and ehrlichiosis are important tick-borne rickettsial diseases of medical and veterinary importance that cause economic losses in livestock. In this study, the prevalence of Anaplasma ovis, Ehrlichia canis and Ehrlichia chaffeensis was investigated in ticks collected from sheep in various farms in Van province, which is located in the Eastern Anatolian Region of Turkey. The ticks used in this study were collected by random sampling in 26 family farm business in 13 districts of Van province. A total of 688 ticks were collected from 88 sheep and 88 tick pools were created. All ticks identified morphologically as Rhipicephalus bursa. Phylogenetic analysis of Chaperonin and 16S rRNA gene sequences confirmed A. ovis, E. canis and E. chaffeensis in this study. Of the 88 tick pools tested, 28.41% (25/88) were positive for at least one pathogen. Anaplasma DNA was detected in five of the 88 pools (5.68%), E. canis DNA was detected in 19 of the 88 pools (21.59%), and E. chaffeensis DNA was detected in one of the 88 pools (1.14%) of R. bursa ticks. To our knowledge, this is the first report describing the presence of A. ovis, E. canis, and E. chaffeensis in R. bursa ticks collected from sheep in Turkey. Further studies are needed to investigate other co-infections in sheep in Turkey.

Ehrlichia Wnt SLiM ligand mimic deactivates the Hippo pathway to engage the anti-apoptotic Yap-GLUT1-BCL-xL axis.

Ehrlichia chaffeensis TRP120 effector has evolved short linear motif (SLiM) ligand mimicry to repurpose multiple evolutionarily conserved cellular signaling pathways, including Wnt, Notch, and Hedgehog. In this investigation, we demonstrate that E. chaffeensis and recombinant TRP120 deactivate Hippo signaling, resulting in the activation of Hippo transcription coactivator Yes-associated protein (Yap). Moreover, a homologous 6 amino acid (QDVASH) SLiM shared by TRP120 and Wnt3a/5a ligands phenocopied Yap and ß-catenin activation induced by E. chaffeensis, rTRP120, and Wnt5a. Similar Hippo gene expression profiles were also stimulated by E. chaffeensis, rTRP120, SLiM, and Wnt5a. Single siRNA knockdown of Hippo transcription co-activator/factors, Yap, and transcriptional enhanced associate domain (TEAD) significantly decreased E. chaffeensis infection. Yap activation was abolished in THP-1 Wnt Frizzled-5 (Fzd5) receptor knockout cells (KO), demonstrating Fzd5 receptor dependence. In addition, the TRP120-Wnt-SLiM antibody blocked Hippo deactivation (Yap activation). Expression of anti-apoptotic Hippo target gene SLC2A1 (encodes glucose transporter 1; GLUT1) was upregulated by E. chaffeensis and corresponded to increased levels of GLUT1. Conversely, siRNA knockdown of SLC2A1 significantly inhibited infection. Higher GLUT1 levels correlated with increased B cell lymphoma-extra large (BCL-xL) and decreased BCL2-associated X, apoptosis regulator (Bax) levels. Moreover, blocking Yap activation with the inhibitor Verteporfin induced apoptosis that corresponded to significant reductions in GLUT1 and BCL-xL levels and activation of Bax and Caspase-3 and -9. This study identifies a novel shared Wnt/Hippo SLiM ligand mimic and demonstrates that E. chaffeensis deactivates the Hippo pathway to engage the anti-apoptotic Yap-GLUT1-BCL-xL axis.

Ehrlichia Notch signaling induction promotes XIAP stability and inhibits apoptosis.

Ehrlichia chaffeensis has evolved multiple strategies to evade innate defenses of the mononuclear phagocyte. Recently, we reported the E. chaffeensis tandem repeat protein (TRP)120 effector functions as a Notch ligand mimetic and a ubiquitin ligase that degrades the nuclear tumor suppressor, F-box and WD repeat domain-containing 7, a negative regulator of Notch. The Notch intracellular domain (NICD) is known to inhibit apoptosis primarily by interacting with X-linked inhibitor of apoptosis protein (XIAP) to prevent degradation. In this study, we determined that E. chaffeensis activation of Notch signaling increases XIAP levels, thereby inhibiting apoptosis through both the intrinsic and executioner pathways. Increased NICD and XIAP levels were detected during E. chaffeensis infection and after TRP120 Notch ligand mimetic peptide treatment. Conversely, XIAP levels were reduced in the presence of Notch inhibitor DAPT. Cytoplasmic and nuclear colocalization of NICD and XIAP was observed during infection and a direct interaction was confirmed by co-immunoprecipitation. Procaspase levels increased temporally during infection, consistent with increased XIAP levels; however, knockdown (KD) of XIAP during infection significantly increased apoptosis and Caspase-3, -7, and -9 levels. Furthermore, treatment with SM-164, a second mitochondrial activator of caspases (Smac/DIABLO) antagonist, resulted in decreased procaspase levels and increased caspase activation, induced apoptosis, and significantly decreased infection. In addition, RNAi KD of XIAP also decreased infection and significantly increased apoptosis. Moreover, ectopic expression of TRP120 HECT Ub ligase catalytically defective mutant in HeLa cells decreased NICD and XIAP levels and increased caspase activation compared to HeLa cells with functional HECT Ub ligase catalytic activity (TRP120-WT). This investigation reveals a mechanism whereby E. chaffeensis modulates Notch signaling to stabilize XIAP and inhibit apoptosis.

Molecular detection and genetic characterization of Ehrlichia canis and Ehrlichia sp. in neotropical primates from Brazil.

The Anaplasmataceae family includes obligate, arthropod-transmitted intracellular bacteria that can be zoonotic and potentially fatal. Studies focusing on the interaction between neotropical primates and the agents of this family are scarce. The present study aimed to identify agents of the Anaplasmataceae family in the whole blood of free-living and captive neotropical primates in the State of Mato Grosso, Central-West Brazil. Thirty-eight samples of six nonhuman primate (NHP) species were collected in seven municipalities and analysed through polymerase chain reaction (PCR), nucleotide sequencing, and phylogenetic analysis of the dsb, groEL, 16S rRNA, and gltA genes. DNA fragments similar to those of Ehrlichia canis were detected in Sapajus apella and Ehrlichia chaffeensis from Mico melanurus. The sequences generated in this study and homologous sequences retrieved from GenBank® were used for phylogenetic analyses to characterize the Ehrlichial agents detected in NHPs. The agents were then grouped into clades corresponding to different isolates from the NHP species. In addition, an Anaplasma sp. closely related to Anaplasma marginale was identified in two S. apella individuals. These findings shed light on the susceptibility of neotropical NHPs to Anaplasmataceae agents. These bacteria are known to be transmitted by ticks, which can also serve as possible sources of infection for other animals, including humans.

Genome-Wide Subtraction Analysis and Reverse Vaccinology to Detect Novel Drug Targets and Potential Vaccine Candidates Against Ehrlichia chaffeensis.

Human monocytotropic ehrlichiosis is an emerging tick-borne infection caused by the obligate intracellular pathogen, Ehrlichia chaffeensis. The non-specific symptoms can range from a self-limiting fever to a fatal septic-like syndrome and may be misdiagnosed. The limited treatment choices including doxycycline are effective only in the initiation phase of the infection. It seems that novel therapeutic targets and new vaccine strategies could be effective to control this pathogen. This study is comprised of two major phases. First, the common proteins retrieved through subtractive analysis and potential drug targets were evaluated by subcellular localization, homology prediction, metabolic pathways, druggability, essentiality, protein-protein interaction networks, and protein data bank availability. In the second phase, surface-exposed proteins were assessed based on antigenicity, allergenicity, physiochemical properties, B cell and T cell epitopes, conserved domains, and protein-protein interaction networks. A multi-epitope vaccine was designed and characterized using molecular dockings and immune simulation analysis. Six proteins including WP_011452818.1, WP_011452723.1, WP_006010413.1, WP_006010278.1, WP_011452938.1, and WP_006010644.1 were detected. They belong to unique metabolic pathways of E. chaffeensis that are considered as new essential drug targets. Based on the reverse vaccinology, WP_011452702.1, WP_044193405.1, WP_044170604.1, and WP_006010191.1 proteins were potential vaccine candidates. Finally, four B cell epitopes, including SINNQDRNC, FESVSSYNI, SGKKEISVQSN, and QSSAKRKST, were used to generate the multi-epitope vaccine based on LCL platform. The vaccine showed strong interactions with toll-like receptors and acceptable immune-reactivity by immune simulation analysis. The findings of this study may represent a turning point in developing an effective drug and vaccine against E. chaffeensis. However, further experimental analyses have remained.

Tick innate immune responses to hematophagy and Ehrlichia infection at single-cell resolution.

Introduction: Ticks rely on robust cellular and humoral responses to control microbial infection. However, several aspects of the tick's innate immune system remain uncharacterized, most notably that of the immune cells (called hemocytes), which are known to play a significant role in cellular and humoral responses. Despite the importance of hemocytes in regulating microbial infection, our understanding of their basic biology and molecular mechanisms remains limited. Therefore, we believe that a more detailed understanding of the role of hemocytes in the interactions between ticks and tick-borne microbes is crucial to illuminating their function in vector competence and to help identify novel targets for developing new strategies to block tick-borne pathogen transmission. Methods: This study examined hemocytes from the lone star tick (Amblyomma americanum) at the transcriptomic level using the 10X genomics single-cell RNA sequencing platform to analyze hemocyte populations from unfed, partially blood-fed, and Ehrlichia chaffeensis-infected ticks. The functional role of differentially expressed hemocyte markers in hemocyte proliferation and Ehrlichia dissemination was determined using an RNA interference approach. Results and discussion: Our data exhibit the identification of fourteen distinct hemocyte populations. Our results uncover seven distinct lineages present in uninfected and Ehrlichia-infected hemocyte clusters. The functional characterization of hemocytin, cystatin, fibronectin, and lipocalin demonstrate their role in hemocyte population changes, proliferation, and Ehrlichia dissemination. Conclusion: Our results uncover the tick immune responses to Ehrlichia infection and hematophagy at a single-cell resolution. This work opens a new field of tick innate immunobiology to understand the role of hemocytes, particularly in response to prolonged blood-feeding (hematophagy), and tick-microbial interactions.

Evaluating EcxR for Its Possible Role in Ehrlichia chaffeensis Gene Regulation.

Ehrlichia chaffeensis, a tick-transmitted intraphagosomal bacterium, is the causative agent of human monocytic ehrlichiosis. The pathogen also infects several other vertebrate hosts. E. chaffeensis has a biphasic developmental cycle during its growth in vertebrate monocytes/macrophages and invertebrate tick cells. Host- and vector-specific differences in the gene expression from many genes of E. chaffeensis are well documented. It is unclear how the organism regulates gene expression during its developmental cycle and for its adaptation to vertebrate and tick host cell environments. We previously mapped promoters of several E. chaffeensis genes which are recognized by its only two sigma factors: σ32 and σ70. In the current study, we investigated in assessing five predicted E. chaffeensis transcription regulators; EcxR, CtrA, MerR, HU and Tr1 for their possible roles in regulating the pathogen gene expression. Promoter segments of three genes each transcribed with the RNA polymerase containing σ70 (HU, P28-Omp14 and P28-Omp19) and σ32 (ClpB, DnaK and GroES/L) were evaluated by employing multiple independent molecular methods. We report that EcxR binds to all six promoters tested. Promoter-specific binding of EcxR to several gene promoters results in varying levels of gene expression enhancement. This is the first detailed molecular characterization of transcription regulators where we identified EcxR as a gene regulator having multiple promoter-specific interactions.

Genetic characterization of a novel Ehrlichia chaffeensis genotype from an Amblyomma tenellum tick from South Texas, USA.

Ehrlichia chaffeensis is the causative agent of human monocytotropic ehrlichiosis (HME), a disease that ranges in severity from mild to fatal infection. Ehrlichia chaffeensis is maintained in a zoonotic cycle involving white-tailed deer (Odocoileus virginianus) as the main vertebrate reservoir and lone star ticks (Amblyomma americanum) as its principal vector. Through complete genomic analysis from human ehrlichial isolates and DNA sequences obtained from deer and tick specimens, nine strains of E. chaffeensis have been characterized. Few studies have examined the genetic diversity of E. chaffeensis in ticks, and some of these investigations have identified that the genetic sequences coincide with the circulating strains reported so far. Here, we report the first evidence of E. chaffeensis DNA from an unfed Amblyomma tenellum (formerly Amblyomma imitator) collected in South Texas. We characterized the genetic variation of this E. chaffeensis genotype using conserved gene markers such as rRNA, dsb, and groEL. We also used gene targets useful to distinguish genotypes, such as the variable length PCR target gene (VLPT) and 120-kDa gene, encoding the tandem-repeat proteins TRP32 and TRP120, respectively. Our results suggest a novel E. chaffeensis genotype that exhibited greater variability than other genotypes of E. chaffeensis and highlights the role for A. tenellum as a potential vector of E. chaffeensis.

Ehrlichia SLiM ligand mimetic activates Hedgehog signaling to engage a BCL-2 anti-apoptotic cellular program.

Ehrlichia chaffeensis (E. chaffeensis) has evolved eukaryotic ligand mimicry to repurpose multiple cellular signaling pathways for immune evasion. In this investigation, we demonstrate that TRP120 has a novel repetitive short linear motif (SLiM) that activates the evolutionarily conserved Hedgehog (Hh) signaling pathway to inhibit apoptosis. In silico analysis revealed that TRP120 has sequence and functional similarity with Hh ligands and a candidate Hh ligand SLiM was identified. siRNA knockdown of Hh signaling and transcriptional components significantly reduced infection. Co-immunoprecipitation and surface plasmon resonance demonstrated that rTRP120-TR interacted directly with Hh receptor Patched-2 (PTCH2). E. chaffeensis infection resulted in early upregulation of Hh transcription factor GLI-1 and regulation of Hh target genes. Moreover, soluble recombinant TRP120 (rTRP120) activated Hh and induced gene expression consistent with the eukaryotic Hh ligand. The TRP120-Hh-SLiM (NPEVLIKD) induced nuclear translocation of GLI-1 in THP-1 cells and primary human monocytes and induced a rapid and expansive activation of Hh pathway target genes. Furthermore, Hh activation was blocked by an α-TRP120-Hh-SLiM antibody. TRP120-Hh-SLiM significantly increased levels of Hh target, anti-apoptotic protein B-cell lymphoma 2 (BCL-2), and siRNA knockdown of BCL-2 dramatically inhibited infection. Blocking Hh signaling with the inhibitor Vismodegib, induced a pro-apoptotic cellular program defined by decreased mitochondria membrane potential, significant reductions in BCL-2, activation of caspase 3 and 9, and increased apoptotic cells. This study reveals a novel E. chaffeensis SLiM ligand mimetic that activates Hh signaling to maintain E. chaffeensis infection by engaging a BCL-2 anti-apoptotic cellular program.

Ehrlichia SLiM Ligand Mimetic Activates Notch Signaling in Human Monocytes.

Ehrlichia chaffeensis evades innate host defenses by reprogramming the mononuclear phagocyte through mechanisms that involve the exploitation of multiple evolutionarily conserved cellular signaling pathways, including Notch. This immune evasion strategy is directed in part by tandem repeat protein (TRP) effectors. Specifically, the TRP120 effector activates and regulates Notch signaling through interactions with the Notch receptor and the negative regulator, F-Box and WD repeat domain-containing 7 (FBW7). However, the specific molecular interactions and motifs required for E. chaffeensis TRP120-Notch receptor interaction and activation have not been defined. To investigate the molecular basis of TRP120 Notch activation, we compared TRP120 with endogenous canonical/noncanonical Notch ligands and identified a short region of sequence homology within the tandem repeat (TR) domain. TRP120 was predicted to share biological function with Notch ligands, and a function-associated sequence in the TR domain was identified. To investigate TRP120-Notch receptor interactions, colocalization between TRP120 and endogenous Notch-1 was observed. Moreover, direct interactions between full-length TRP120, the TRP120 TR domain containing the putative Notch ligand sequence, and the Notch receptor LBR were demonstrated. To molecularly define the TRP120 Notch activation motif, peptide mapping was used to identify an 11-amino acid short linear motif (SLiM) located within the TRP120 TR that activated Notch signaling and downstream gene expression. Peptide mutants of the Notch SLiM or anti-Notch SLiM antibody reduced or eliminated Notch activation and NICD nuclear translocation. This investigation reveals a novel molecularly defined pathogen encoded Notch SLiM mimetic that activates Notch signaling consistent with endogenous ligands. IMPORTANCE E. chaffeensis infects and replicates in mononuclear phagocytes, but how it evades innate immune defenses of this indispensable primary innate immune cell is not well understood. This investigation revealed the molecular details of a ligand mimicry cellular reprogramming strategy that involved a short linear motif (SLiM), which enabled E. chaffeensis to exploit host cell signaling to establish and maintain infection. E. chaffeensis TRP120 is a moonlighting effector that has been associated with cellular activation and other functions, including ubiquitin ligase activity. Herein, we identified and demonstrated that a SLiM present within each tandem repeat of TRP120 activated Notch signaling. Notch is an evolutionarily conserved signaling pathway responsible for many cell functions, including cell fate, development, and innate immunity. This study is significant because it revealed the first molecularly defined pathogen encoded SLiM that appears to have evolved de novo to mimic endogenous Notch ligands. Understanding Notch activation during E. chaffeensis infection provides a model to study pathogen exploitation of signaling pathways and will be useful in developing molecularly targeted countermeasures for inhibiting infection by a multitude of disease-causing pathogens that exploit cell signaling through molecular mimicry.

Functional Characterization of Multiple Ehrlichia chaffeensis Sodium (Cation)/Proton Antiporter Genes Involved in the Bacterial pH Homeostasis.

Ehrlichia chaffeensis causes human monocytic ehrlichiosis. Little is known about how this and other related tick-borne rickettsia pathogens maintain pH homeostasis in acidified phagosomes and the extracellular milieu. The membrane-bound sodium (cation)/proton antiporters are found in a wide range of organisms aiding pH homeostasis. We recently reported a mutation in an antiporter gene of E. chaffeensis (ECH_0379) which causes bacterial in vivo attenuation. The E. chaffeensis genome contains 10 protein coding sequences encoding for predicted antiporters. We report here that nine of these genes are transcribed during the bacterial growth in macrophages and tick cells. All E. chaffeensis antiporter genes functionally complemented antiporter deficient Escherichia coli. Antiporter activity for all predicted E. chaffeensis genes was observed at pH 5.5, while gene products of ECH_0179 and ECH_0379 were also active at pH 8.0, and ECH_0179 protein was complemented at pH 7.0. The antiporter activity was independently verified for the ECH_0379 protein by proteoliposome diffusion analysis. This is the first description of antiporters in E. chaffeensis and demonstrates that the pathogen contains multiple antiporters with varying biological functions, which are likely important for the pH homeostasis of the pathogen's replicating and infectious forms.

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

Iron is essential for survival and proliferation of Ehrlichia chaffeensis, an obligatory intracellular bacterium that causes an emerging zoonosis, human monocytic ehrlichiosis. However, how Ehrlichia acquires iron in the host cells is poorly understood. Here, we found that native and recombinant (cloned into the Ehrlichia genome) Ehrlichia translocated factor-3 (Etf-3), a previously predicted effector of the Ehrlichia type IV secretion system (T4SS), is secreted into the host cell cytoplasm. Secreted Etf-3 directly bound ferritin light chain with high affinity and induced ferritinophagy by recruiting NCOA4, a cargo receptor that mediates ferritinophagy, a selective form of autophagy, and LC3, an autophagosome biogenesis protein. Etf-3-induced ferritinophagy caused ferritin degradation and significantly increased the labile cellular iron pool, which feeds Ehrlichia Indeed, an increase in cellular ferritin by ferric ammonium citrate or overexpression of Etf-3 or NCOA4 enhanced Ehrlichia proliferation, whereas knockdown of Etf-3 in Ehrlichia via transfection with a plasmid encoding an Etf-3 antisense peptide nucleic acid inhibited Ehrlichia proliferation. Excessive ferritinophagy induces the generation of toxic reactive oxygen species (ROS), which could presumably kill both Ehrlichia and host cells. However, during Ehrlichia proliferation, we observed concomitant up-regulation of Ehrlichia Fe-superoxide dismutase, which is an integral component of Ehrlichia T4SS operon, and increased mitochondrial Mn-superoxide dismutase by cosecreted T4SS effector Etf-1. Consequently, despite enhanced ferritinophagy, cellular ROS levels were reduced in Ehrlichia-infected cells compared with uninfected cells. Thus, Ehrlichia safely robs host cell iron sequestered in ferritin. Etf-3 is a unique example of a bacterial protein that induces ferritinophagy to facilitate pathogen iron capture.

Ehrlichia TRP effectors: moonlighting, mimicry and infection.

Intracellular bacteria have evolved various strategies to evade host defense mechanisms. Remarkably, the obligately intracellular bacterium, Ehrlichia chaffeensis, hijacks host cell processes of the mononuclear phagocyte to evade host defenses through mechanisms executed in part by tandem repeat protein (TRP) effectors secreted by the type 1 secretion system. In the past decade, TRP120 has emerged as a model moonlighting effector, acting as a ligand mimetic, nucleomodulin and ubiquitin ligase. These defined functions illuminate the diverse roles TRP120 plays in exploiting and manipulating host cell processes, including cytoskeletal organization, vesicle trafficking, cell signaling, transcriptional regulation, post-translational modifications, autophagy and apoptosis. This review will focus on TRP effectors and their expanding roles in infection and provide perspective on Ehrlichia chaffeensis as an invaluable model organism for understanding infection strategies of obligately intracellular bacteria.

Ehrlichia chaffeensis TRP120 Is a Wnt Ligand Mimetic That Interacts with Wnt Receptors and Contains a Novel Repetitive Short Linear Motif That Activates Wnt Signaling.

Ehrlichia chaffeensis expresses the TRP120 multifunctional effector, which is known to play a role in phagocytic entry, on the surface of infectious dense-cored ehrlichiae, but a cognate host receptor has not been identified. We recently reported that E. chaffeensis activates canonical Wnt signaling in monocytes to promote bacterial uptake and intracellular survival and that TRP120 was involved in this activation event. To identify the specific mechanism of pathway activation, we hypothesized that TRP120 is a Wnt signaling ligand mimetic that initiates Wnt pathway activity through direct interaction with the Wnt pathway Frizzled family of receptors. In this study, we used confocal immunofluorescence microscopy to demonstrate very strong colocalization between E. chaffeensis and Fzd2, 4, 5, 7, and 9 as well as coreceptor LRP5 at 1 to 3 h postinfection. Direct binding between TRP120 and multiple Fzd receptors was further confirmed by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). Interfering RNA knockdown of Wnt receptors, coreceptors, and signaling pathway components significantly reduced E. chaffeensis infection, demonstrating that complex and redundant interactions are involved in Wnt pathway exploitation. We utilized in silico approaches to identify a repetitive short linear motif (SLiM) in TRP120 that is homologous to Wnt ligands and used mutant SLiM peptides and an α-TRP120-Wnt-SLiM antibody to demonstrate that the TRP120 Wnt SLiM activates the canonical Wnt pathway and promotes E. chaffeensis infection. This study reports the first example of bacterial mimicry of Wnt pathway ligands and highlights a pathogenic mechanism with potential for targeting by antimicrobial therapeutics.IMPORTANCE Upon infecting mammalian hosts, Ehrlichia chaffeensis establishes a replicative niche in microbe-eating immune system cells where it expertly orchestrates infection and spread. One of the ways Ehrlichia survives within these phagocytes is by activating evolutionarily conserved signaling pathways including the Wnt pathway; however, the molecular details of pathway hijacking have not been defined. This study is significant because it identifies an ehrlichial protein that directly interacts with components of the Wnt receptor complex, influencing pathway activity and promoting infection. Consequentially, Ehrlichia serves as a unique tool to investigate the intricacies of how pathogens repurpose human immune cell signaling and provides an opportunity to better understand many cellular processes in health and disease. Furthermore, understanding how this bacterium utilizes its small genome to survive within cells that evolved to destroy pathogens will facilitate the development of antibacterial therapeutics that could target Ehrlichia as well as other intracellular agents of human disease.

Development and validation of real-time PCR assays for the detection of Ehrlichia species and E. chaffeensis in clinical specimens.

Ehrlichiosis, caused by Gram-negative bacteria of the genus Ehrlichia, is considered an emerging infectious disease due to the increasing number of reported cases. Symptoms are non-specific and occur within 1 to 2 weeks following the bite of an infected tick. Confirmatory laboratory diagnostic methods vary in sensitivity and specimen requirements, which can lead to delayed diagnosis. PCR testing serves as an efficient approach to Ehrlichia confirmation in the acute stage of illness. Published assays have been effectively used to detect human ehrlichiosis at limit of detections ranging from 10 to 50 genomic copies (GC) of Ehrlichia DNA. With the discovery of new species capable of human infection, we wanted to develop assays that are sensitive and encompass a wide range of Ehrlichia. Here we developed and validated two sensitive and specific real-time PCR assays (PanE1 and PanE2) for the detection of Ehrlichia species, as well as two real-time PCR assays (ECh2 and ECh4) for the detection of Ehrlichia chaffeensis, specifically. The limit of detection was determined to be 10 GC per reaction with 100% confidence, and as little as 1 GC with lower efficiencies. Accuracy was assessed at 100% correlation. Specificity from exclusivity testing demonstrated that neither the Ehrlichia species assays (n = 60), nor the E. chaffeensis specific assays (n = 64) had cross reactivity with near neighbors or environmental bacteria. A positive predictive value of 100% and a negative predictive value of ≥93% was determined by evaluating banked clinical specimens from 62 patients with the assays. These real-time PCR assays are effective tools to detect human Ehrlichia species during the acute stage of illness. Early detection of Ehrlichia infection by these real-time PCR assays can facilitate diagnosis and treatment.

Mutations in Ehrlichia chaffeensis Genes ECH_0660 and ECH_0665 Cause Transcriptional Changes in Response to Zinc or Iron Limitation.

Ehrlichia chaffeensis causes human monocytic ehrlichiosis by replicating within phagosomes of monocytes/macrophages. A function disruption mutation within the pathogen's ECH_0660 gene, which encodes a phage head-to-tail connector protein, resulted in the rapid clearance of the pathogen in vivo, while aiding in induction of sufficient immunity in a host to protect against wild-type infection challenge. In this study, we describe the characterization of a cluster of seven genes spanning from ECH_0659 to ECH_0665, which contained four genes encoding bacterial phage proteins, including the ECH_0660 gene. Assessment of the promoter region upstream of the first gene of the seven genes (ECH_0659) in Escherichia coli demonstrated transcriptional enhancement under zinc and iron starvation conditions. Furthermore, transcription of the seven genes was significantly higher under zinc and iron starvation conditions for E. chaffeensis carrying a mutation in the ECH_0660 gene compared to the wild-type pathogen. In contrast, for the ECH_0665 gene mutant with the function disruption, transcription from the genes was mostly similar to that of the wild type or was moderately downregulated. Recently, we reported that this mutation caused a minimal impact on the pathogen's in vivo growth, as it persisted similarly to the wild type. The current study is the first to describe how zinc and iron contribute to E. chaffeensis biology. Specifically, we demonstrated that the functional disruption in the gene encoding the phage head-to-tail connector protein in E. chaffeensis results in the enhanced transcription of seven genes, including those encoding phage proteins, under zinc and iron limitation. IMPORTANCE Ehrlichia chaffeensis, a tick-transmitted bacterium, causes human monocytic ehrlichiosis by replicating within phagosomes of monocytes/macrophages. A function disruption mutation within the pathogen's gene encoding a phage head-to-tail connector protein resulted in the rapid clearance of the pathogen in vivo, while aiding in induction of sufficient immunity in a host to protect against wild-type infection challenge. In the current study, we investigated if the functional disruption in the phage head-to-tail connector protein gene caused transcriptional changes resulting from metal ion limitations. This is the first study describing how zinc and iron may contribute to E. chaffeensis replication.

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.

Detection of Rickettsia amblyommatis and Ehrlichia chaffeensis in Amblyomma americanum Inhabiting Two Urban Parks in Oklahoma.

For the past 30 years, the number of people infected with causative agents of ehrlichiosis, Rocky Mountain spotted fever, and spotted fever group rickettiosis (SFGR) has increased in Oklahoma. However, there is a lack of data on pathogen prevalence within urban environments. To assess the prevalence of tick-borne pathogens in different environments, 434 Amblyomma americanum (lone star) ticks were collected from the environment in two parks in Edmond, Oklahoma. The presence of Ehrlichia spp. and spotted fever group (SFG) Rickettsia spp. was determined using quantitative real-time polymerase chain reaction (qPCR). 33.6% (146/434) of the A. americanum ticks were positive for Rickettsia amblyommatis and 15.2% (66/434) were positive for Ehrlichia chaffeensis. No ticks were positive for other SFG Rickettsiae (R. rickettsii, R. parkeri) or other Ehrlichiae (E. ewingii, and Panola Mountain Ehrlichia). These studies provide increased understanding of the potential risk for encountering tick-borne pathogens in urban environments.

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.

Multi-Locus Sequence Typing of Ehrlichia chaffeensis Reveals Extensive Genotypic Variation across the United States.

Ehrlichia chaffeensis causes human monocytic ehrlichiosis, and its principal vector is the Amblyomma americanum tick. The most frequently identified cases of ehrlichiosis come from the southeastern and south central states of the United States. In this study, a molecular typing system was developed that allows for the genetic differentiation of E. chaffeensis isolates. This multi-locus typing system included sequencing and analyzing intergenic regions ECH0033-ECH0035 and ECH0217-ECH0218, plus, variable genes variable length PCR target, 28-kDa, 120-kDa, and hemE. We examined a total of 31 unique isolates from humans and white-tailed deer, and eight DNA samples extracted from infected A. americanum collected from multiple states. This is the largest evaluation of E. chaffeensis isolates and their genotypes. Our findings show that when sequences of all six loci were concatenated and compared, the 39 samples could be separated into 23 genotypes and further grouped into six phylogenetic clades. The data in this study show no clear pattern between the geographic alignment with the genetic differentiation between the strains. As a result, this poses a challenge to understanding the spread of E. chaffeensis in the United States. Interestingly, our findings indicate that multiple strains from distant geographic origins share the same mutations, which suggests that the strains are being moved from one site to another by their hosts or vectors. In addition, we are seeing a northward shift in the lone star tick distribution in the United States. Last, some data also suggest minimal genetic mutations have occurred over time among strains that are within geographical proximity.