Agent Of Human Monocytic Ehrlichiosis Research Articles

CtrA activates the expression of glutathione S-transferase conferring oxidative stress resistance to Ehrlichia chaffeensis.

Ehrlichia chaffeensis, the causative agent of human monocytic ehrlichiosis (HME), is a Gram-negative obligatory intracellular bacterium, which infects and multiplies in human monocytes and macrophages. Host immune cells produce reactive oxygen species (ROS) to eliminate E. chaffeensis upon infection. E. chaffeensis global transcriptional regulator CtrA activates the expression of GshA and GshB to synthesize glutathione (GSH), the most potent natural antioxidant, upon oxidative stress to combat ROS damage. However, the mechanisms exploited by E. chaffeensis to utilize GSH are still unknown. Here, we found that in E. chaffeensis CtrA activated the expression of glutathione S-transferase (GST) upon oxidative stress, and E. chaffeensis GST utilizes GSH to eliminate ROS and confers the oxidative stress resistance to E. chaffeensis. We found that CtrA bound to the promoter regions of 211 genes, including gst, in E. chaffeensis using chromatin immunoprecipitation coupled to deep sequencing (ChIP-seq). Recombinant E. chaffeensis CtrA directly bound to the gst promoter region determined with electrophoretic mobility shift assay (EMSA), and activated the gst expression determined with reporter assay. Recombinant GST showed GSH conjugation activity towards its typical substrate 2,4-dinitrochlorobenzene (CDNB) in vitro and peptide nucleic acid (PNA) transfection of E. chaffeensis, which can knock down the gst transcription level, reduced bacterial survival upon oxidative stress. Our results demonstrate that E. chaffeensis CtrA regulates GSH utilization, which plays a critical role in resistance to oxidative stress, and aid in the development of new therapeutics for HME.

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.

<p class="Body"><strong>Detection of a <em>Rickettsia </em>sp. and an <em>Ehrlichia chaffeensis</em>-like organism in ticks parasitizing the endangered Florida Grasshopper Sparrow (<em>Ammodramus savannarum floridanus</em>)</strong></p>

Between 2013 and 2015, 163 resident endangered Florida Grasshopper Sparrows (Ammodramus savannarum floridanus) and four migratory Eastern Grasshopper Sparrows (A. savannarum pratensis) were examined for the presence of ticks in peninsular Florida. Thirteen Amblyomma maculatum and seven Haemaphysalis chordeilis ticks were removed from 13 Florida Grasshopper Sparrows. Two A. maculatum were discovered on two Eastern Grasshopper Sparrows. Polymerase chain reaction (PCR) and sequencing of resultant amplicons of some of the tick specimens were performed to determine if ticks were infected with pathogenic bacteria. Salivary gland and midgut contents of five of six (83%) of the H. chordeilis tested positive for a novel Rickettsia closely related to, but distinct from, Rickettsia aeschlimannii (causative agent of Mediterranean spotted fever-like illness), an infectious zoonotic bacterium that has not been previously reported in the United States. Four of 14 (29%) of the A. maculatum tested positive for an agent most closely related to an uncultured Ehrlichia previously isolated from Oriental house rats (Rattus tanezumi; 97.5% identity to GenBank KM817187), which is genetically similar to Ehrlichia chaffeensis (causative agent of human monocytic ehrlichiosis), another infectious zoonotic bacterium. Blood from 16 Florida Grasshopper Sparrows and one Eastern Grasshopper Sparrow tested negative for spotted fever group rickettsiae, Anaplasma spp. and Ehrlichia spp. We recommend that additional collections and screening of ticks and blood from Florida Grasshopper Sparrows be undertaken to determine the rates of infection with rickettsiae and ehrlichiae in these imperiled songbirds.

THE IDENTIFICATION OF «NEW» PATHOGENS FOR FOCAL INFECTIONS IN IXODES TICKS ON THE TERRITORY OF TULA REGION

The most of ixodes ticks in Tula region belongs to the group of pasture mites. It is generally accepted to estimate the tick’s contamination by the tick-borne encephalitis virus and raoueti inducing Lyme Borellia. The aim of the present work was to educe the aetiologic agents of the set of potentially-enable infections out of ticks Dermacentor reticulatus, Ixodes ricinus and Ixodes сrenulatus collected at the different terrains of Tula Region by PCR method. The results: a considerable number of pathogenic rickettsiae R. raoultii was educes from the ticks D. reticulatus, which including them as the component of mixed infection together with the human monocytic ehrlichiosis agent. R. raoultii was determined in more than a half of the cases in ticks I. ricinus including the mixed infection together with ticks’ borreliosis virus and Kemerovo fever agent. Conclusion. The reasons, induced the quantity changes of the ticks’ distribution at Tula Region terrains, apparently promote the rise in frequency of the ticks contamination with the agents of herd tick-transmissive infection. It demand an infectiologist’s attention rise and dictate the necessity of the above mentioned diseases monitoring as well as Fr. tularensis, the tick-borne encephalitis virus and Lyme disease.

THE IDENTIFICATION OF «NEW» PATHOGENS FOR FOCAL INFECTIONS IN IXODES TICKS ON THE TERRITORY OF TULA REGION

The most of ixodes ticks in Tula region belongs to the group of pasture mites. It is generally accepted to estimate the tick’s contamination by the tick-borne encephalitis virus and raoueti inducing Lyme Borellia. The aim of the present work was to educe the aetiologic agents of the set of potentially-enable infections out of ticks Dermacentor reticulatus, Ixodes ricinus and Ixodes сrenulatus collected at the different terrains of Tula Region by PCR method. The results: a considerable number of pathogenic rickettsiae R. raoultii was educes from the ticks D. reticulatus, which including them as the component of mixed infection together with the human monocytic ehrlichiosis agent. R. raoultii was determined in more than a half of the cases in ticks I. ricinus including the mixed infection together with ticks’ borreliosis virus and Kemerovo fever agent. Conclusion. The reasons, induced the quantity changes of the ticks’ distribution at Tula Region terrains, apparently promote the rise in frequency of the ticks contamination with the agents of herd tick-transmissive infection. It demand an infectiologist’s attention rise and dictate the necessity of the above mentioned diseases monitoring as well as Fr. tularensis, the tick-borne encephalitis virus and Lyme disease.

Protein and DNA synthesis demonstrated in cell-free Ehrlichia chaffeensis organisms in axenic medium

Ehrlichia chaffeensis, a tick-transmitted rickettsial bacterium, is the causative agent of human monocytic ehrlichiosis. Biochemical characterization of this and other related Rickettsiales remains a major challenge, as they require a host cell for their replication. We investigated the use of an axenic medium for E. chaffeensis growth, assessed by protein and DNA synthesis, in the absence of a host cell. E. chaffeensis organisms harvested from in vitro cultures grown in a vertebrate cell line were fractionated into infectious dense-core cells (DC) and the non-infectious replicating form, known as reticulate cells (RC) by renografin density gradient centrifugation and incubated in the axenic medium containing amino acids, nucleotides, and different energy sources. Bacterial protein and DNA synthesis were observed in RCs in response to glucose-6-phosphate, although adenosine triphosphate, alpha-ketoglutarate or sodium acetate supported protein synthesis. The biosynthetic activity could not be detected in DCs in the axenic medium. While the data demonstrate de novo protein and DNA synthesis under axenic conditions for E. chaffeensis RCs, additional modifications are required in order to establish conditions that support bacterial replication, and transition to DCs.

The transcriptome of the lone star tick, Amblyomma americanum, reveals molecular changes in response to infection with the pathogen, Ehrlichia chaffeensis.

The lone star tick, Amblyomma americanum, is an obligatory ectoparasite of many vertebrates and the primary vector of Ehrlichia chaffeensis, the causative agent of human monocytic ehrlichiosis. This study aimed to investigate the comparative transcriptomes of A. americanum underlying the processes of pathogen acquisition and of immunity towards the pathogen. Differential expression of the whole body transcripts in six different treatments were compared: females and males that were E. chaffeensis non-exposed, E. chaffeensis-exposed/uninfected, and E. chaffeensis-exposed/infected. The Trinity assembly pipeline produced 140,574 transcripts from trimmed and filtered total raw sequence reads (approximately 117M reads). The gold transcript set of the transcriptome data was established to minimize noise by retaining only transcripts homologous to official peptide sets of Ixodes scapularis and A. americanum ESTs and transcripts covered with high enough frequency from the raw data. Comparison of the gene ontology term enrichment analyses for the six groups tested here revealed an up-regulation of genes for defense responses against the pathogen and for the supply of intracellular Ca++ for pathogen proliferation in the pathogen-exposed ticks. Analyses of differential expression, focused on functional subcategories including immune, sialome, neuropeptides, and G protein-coupled receptor, revealed that E. chaffeensis-exposed ticks exhibited an upregulation of transcripts involved in the immune deficiency (IMD) pathway, antimicrobial peptides, Kunitz, an insulin-like peptide, and bursicon receptor over unexposed ones, while transcripts for metalloprotease were down-regulated in general. This study found that ticks exhibit enhanced expression of genes responsible for defense against E. chaffeensis.

Vaccination with an Attenuated Mutant of Ehrlichia chaffeensis Induces Pathogen-Specific CD4+ T Cell Immunity and Protection from Tick-Transmitted Wild-Type Challenge in the Canine Host.

Ehrlichia chaffeensis is a tick-borne rickettsial pathogen and the causative agent of human monocytic ehrlichiosis. Transmitted by the Amblyomma americanum tick, E. chaffeensis also causes disease in several other vertebrate species including white-tailed deer and dogs. We have recently described the generation of an attenuated mutant strain of E. chaffeensis, with a mutation in the Ech_0660 gene, which is able to confer protection from secondary, intravenous-administered, wild-type E. chaffeensis infection in dogs. Here, we extend our previous results, demonstrating that vaccination with the Ech_0660 mutant protects dogs from physiologic, tick-transmitted, secondary challenge with wild-type E. chaffeensis; and describing, for the first time, the cellular and humoral immune responses induced by Ech_0660 mutant vaccination and wild-type E. chaffeensis infection in the canine host. Both vaccination and infection induced a rise in E. chaffeensis-specific antibody titers and a significant Th1 response in peripheral blood as measured by E. chaffeensis antigen-dependent CD4+ T cell proliferation and IFNγ production. Further, we describe for the first time significant IL-17 production by peripheral blood leukocytes from both Ech_0660 mutant vaccinated animals and control animals infected with wild-type E. chaffeensis, suggesting a previously unrecognized role for IL-17 and Th17 cells in the immune response to rickettsial pathogens. Our results are a critical first step towards defining the role of the immune system in vaccine-induced protection from E. chaffeensis infection in an incidental host; and confirm the potential of the attenuated mutant clone, Ech_0660, to be used as a vaccine candidate for protection against tick-transmitted E. chaffeensis infection.

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

ABSTRACTObligate 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.

Experimental infection of Rhipicephalus sanguineus with Ehrlichia chaffeensis

Ehrlichia chaffeensis, the etiologic agent of human monocytic ehrlichiosis, is a tick-borne rickettsial pathogen that is infective to a wide range of mammals, including dogs and people. Amblyomma americanum, the lone star tick, is considered the primary vector of E. chaffeensis, but this pathogen has been detected in other tick species, including the brown dog tick, Rhipicephalus sanguineus. We hypothesized that the Arkansas strain of E. chaffeensis is infective to R. sanguineus, and used a novel PCR assay to test for acquisition of this pathogen by R. sanguineus and A. americanum ticks that were simultaneously fed on experimentally infected dogs. Although E. chaffeensis was not frequently detected in peripheral blood of these dogs, the pathogen was detected in both tick species and in canine lung, kidney, lymph node, bone marrow and frontal lobe samples. One dog (AFL) was maintained for several years, and ticks again acquired E. chaffeensis from this dog 566 days after intradermal inoculation with E. chaffeensis, but the pathogen was not detected in ticks fed on the same dog at 764 or 1086 days after the intradermal inoculation.

Detection of Vector-Borne Agents in Lone Star Ticks, <I>Amblyomma americanum</I> (Acari: Ixodidae), From Mississippi

In this study, we evaluated Amblyomma americanum (lone star tick) in Mississippi for the presence of Ehrlichia chaffeensis, causative agent of human monocytic ehrlichiosis; Ehrlichia ewingii, causative agent of human and canine granulocytic ehrlichiosis; Borrelia lonestari, putative agent of southern tick-associated rash illness; Francisella tularensis, the agent of tularemia; and Rickettsia spp., particularly R. amblyommii, a suspected pathogen. We collected adult A. americanum from four regions of Mississippi: Northeast, Northwest, Southeast, and East. Of the ticks collected, 192 were dissected and DNA was extracted for nested polymerase chain reaction (PCR) assays to detect the above bacteria. In all, 3% of tick extracts had evidence of Borrelia sp., 4% for E. chaffeensis, 6% for E. ewingii, and 44% for a Rickettsia species. As determined by sequencing, most Rickettsia spp. were R. amblyommii. In addition, extracts from 42 pools (total of 950) of larval A. americanum collected in Southwest Mississippi were tested for the presence of E. chaffeensis and Rickettsia species. Of these extracts from pools, nine of 37 (24%) were PCR positive for a Rickettsia sp., most often, R. amblyommii; none had evidence of E. chaffeensis, supporting the ability of lone star ticks to transovarially transmit R. amblyommii, but not E. chaffeensis. This study demonstrates E. chaffeensis, E. ewingii, "B. lonestari", and R. amblyommii in A. americanum by PCR for the first time in Mississippi. Understanding the prevalence and epidemiology of these agents in Mississippi should increase awareness of tick-borne disease in the medical community.

Detection of Anaplasmataceae in ticks collected in Morocco

Bacteria within the Anaplasmataceae include Gram-negative intracellular bacteria that have been known for a long time as the agents of veterinary diseases. However, in the recent years, five members of Anaplasmataceae have been reported to infect humans, including Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis, Ehrlichia chaffeensis, the agent of human monocytic ehrlichiosis, and E. ewingii, E. canis and Neorickettsia sennetsu [1]. The abundance of ticks in North Africa represents potential risks for animal and human public health. However, agents within the Anaplasmataceae family have been poorly investigated in North Africa, including in Morocco. In this work, we analysed ticks collected in this country for evidence of infection by Anaplasmataceae.

Liver Transcriptome Profiles Associated with Strain-SpecificEhrlichia chaffeensis-Induced Hepatitis in SCID Mice

Infection of humans with Ehrlichia chaffeensis, the etiologic agent of human monocytic ehrlichiosis, can cause hepatitis of various levels of severity. When the three human isolates of E. chaffeensis, each belonging to a different genogroup, are inoculated into severe combined immunodeficiency mice, the order of severity of clinical signs and bacterial burden detected in the liver is as follows (from greatest to least severity and highest to lowest burden): strain Wakulla, followed by strain Liberty, followed by strain Arkansas. In this article, we used microarray analysis to define transcriptional profiles characteristic of the histopathological features in the mouse liver. Cytokine and chemokine profiles and their receptor profiles were strikingly different among the three strains of E. chaffeensis: gamma interferon, CCL5, CXCL1, CXCL2, CXCL7, CXCL9, interleukin 2 receptor gamma (IL2Rgamma), IL21R, CCR2, and CXCR6 were highly upregulated with strain Arkansas; and tumor necrosis factor (TNF), CCL2, CCL3, CCL5, CCL6, CCL12, CCL20, CXCL2, CXCL7, CXCL9, CXCL13, TNF receptor superfamily 9 (TNFRSF9), TNFRSF13beta, IL1R2, IL2Rgamma, IL20Rbeta, IL21R, CCR1, CCR2, and CXCR4 were highly upregulated with strain Wakulla. With strain Liberty, only CXCL13 was highly upregulated, and IL13Ralpha2 was downregulated. In livers infected with the Arkansas strain, monocytes/macrophages and NK cells were enriched in the granulomas and an increase in NK cell marker mRNAs was detected. Livers infected with the Wakulla strain displayed infiltration of significantly more neutrophils and an increase in neutrophil marker mRNAs. Genes commonly upregulated in liver tissue infected with the three strains are other host innate immune and inflammatory response genes, including those encoding several acute-phase proteins. Genes downregulated commonly are related to host physiologic functions. The results suggest that marked modulation of host cytokine and chemokine profiles by E. chaffeensis strains underlies the distinct host liver disease.

Four VirB6 Paralogs and VirB9 Are Expressed and Interact inEhrlichia chaffeensis-Containing Vacuoles

The type IV secretion system is an important virulence factor in several host cell-associated pathogens, as it delivers various bacterial macromolecules to target eukaryotic cells. Genes homologous to several virB genes and virD4 of Agrobacterium tumefaciens are found in an intravacuolar pathogen Ehrlichia chaffeensis, the tick-borne causative agent of human monocytic ehrlichiosis. In particular, despite its small genome size, E. chaffeensis has four tandem virB6 paralogs (virB6-1, -2, -3, and -4) that are 3- to 10-fold larger than A. tumefaciens virB6. The present study for the first time illustrates the relevance of the larger quadruple VirB6 paralogs by demonstrating the protein expression and interaction in E. chaffeensis. All four virB6 paralogs were cotranscribed in THP-1 human leukemia and ISE6 tick cell cultures. The four VirB6 proteins and VirB9 were expressed by E. chaffeensis in THP-1 cells, and amounts of these five proteins were similar in isolated E. chaffeensis-containing vacuoles and vacuole-free E. chaffeensis. In addition, an 80-kDa fragment of VirB6-2 was detected, which was strikingly more prevalent in E. chaffeensis-containing vacuoles than in vacuole-free E. chaffeensis. Coimmunoprecipitation analysis revealed VirB9 interaction with VirB6-1 and VirB6-2; VirB6-4 interaction with VirB6-1, VirB6-2, and VirB6-3; and VirB6-2 80-kDa fragment interaction with VirB6-3 and VirB6-4. The interaction of VirB9 and VirB6-2 was confirmed by far-Western blotting. The results suggest that E. chaffeensis VirB9, the quadruple VirB6 proteins, and the VirB6-2 80-kDa fragment form a unique molecular subassembly to cooperate in type IV secretion.

Defining the immune response to Ehrlichia species using murine models

Pathogenic bacteria belonging to the family Anaplasmataceae include species of the genera Ehrlichia and Anaplasma. Ehrlichia chaffeensis, first known as the causative agent of human monocytic ehrlichiosis, also infects several vertebrate hosts including white-tailed deer, dogs, coyotes and goats. E. chaffeensis is transmitted from the bite of an infected hard tick, such as Amblyomma americanum. E. chaffeensis and other tick-transmitted pathogens have adapted to both the tick and vertebrate host cell environments. Although E. chaffeensis persists in both vertebrate and tick hosts for long periods of time, little is known about that process. Immunological studies will be valuable in assessing how the pathogen persists in nature in both vertebrate and invertebrate hosts. Understanding the host immune response to the pathogen originating from dual host backgrounds is also important to develop effective methods of diagnosis, control and treatment. In this paper, we provide our perspective of the current understanding of the immune response against E. chaffeensis in relation to other related Anaplasmataceae pathogens.

Total, Membrane, and Immunogenic Proteomes of Macrophage- and Tick Cell-Derived Ehrlichia chaffeensis Evaluated by Liquid Chromatography-Tandem Mass Spectrometry and MALDI-TOF Methods

Ehrlichia chaffeensis, a tick-transmitted rickettsial, is the causative agent of human monocytic ehrlichiosis. To examine protein expression patterns, we analyzed total, membrane, and immunogenic proteomes of E. chaffeensis originating from macrophage and tick cell cultures. Total proteins resolved by one-dimensional gel electrophoresis and subjected to liquid chromatography-electrospray ionization ion trap mass spectrometry allowed identification of 134 and 116 proteins from macrophage- and tick cell-derived E. chaffeensis, respectively. Because a majority of immunogenic proteins remained in the membrane fraction, individually picked total and immunogenic membrane proteins were also surveyed by liquid chromatography-tandem mass spectrometry and matrix-assisted laser desorption ionization-time of flight methods. The analysis aided the identification of 48 additional proteins. In all, 278 genes of the E. chaffeensis genome were verified as functional genes. They included genes for DNA and protein metabolism, energy metabolism and transport, membrane proteins, hypothetical proteins, and many novel proteins of unknown function. The data reported in this study suggest that the membrane of E. chaffeensis is very complex, having many expressed proteins. This study represents the first and the most comprehensive analysis of E. chaffeensis-expressed proteins. This also is the first study confirming the expression of nearly one-fourth of all predicted genes of the E. chaffeensis genome, validating that they are functionally active genes, and demonstrating that classic shotgun proteomic approaches are feasible for tick-transmitted intraphagosomal bacteria. The identity of novel expressed proteins reported in this study, including the large selection of membrane and immunogenic proteins, will be valuable in elucidating pathogenic mechanisms and developing effective prevention and control methods.

Proteomic Analysis of and Immune Responses toEhrlichia chaffeensisLipoproteins

Ehrlichia chaffeensis is an obligately intracellular gram-negative bacterium and is the etiologic agent of human monocytic ehrlichiosis (HME). Although E. chaffeensis induces the generation of several cytokines and chemokines by leukocytes, E. chaffeensis lacks lipopolysaccharide and peptidoglycan. Bioinfomatic analysis of the E. chaffeensis genome, however, predicted genes encoding 15 lipoproteins and 3 posttranslational lipoprotein-processing enzymes. The present study showed that by use of multidimensional liquid chromatography followed by tandem mass spectrometry, all predicted lipoproteins as well as lipoprotein-processing enzymes were expressed by E. chaffeensis cultured in the human promyelocytic leukemia cell line HL-60. Consistent with this observation, a signal peptidase II inhibitor, globomycin, was found to inhibit E. chaffeensis infection and lipoprotein processing in HL-60 cell culture. To study in vivo E. chaffeensis lipoprotein expression and host immune responses to E. chaffeensis lipoproteins, 13 E. chaffeensis lipoprotein genes were cloned into a mammalian expression vector. When the DNA constructs were inoculated into naïve dogs, or when dogs were infected with E. chaffeensis, the animals developed delayed-type hypersensitivity reactions at cutaneous sites of the DNA construct deposition and serum antibodies to these lipoproteins. This is the first demonstration of lipoprotein expression and elicitation of immune responses by a member of the order Rickettsiales. Multiple lipoproteins expressed by E. chaffeensis in vitro and in vivo may play key roles in pathogenesis and immune responses in HME.

Use of Drosophila S2 Cells as a Model for Studying Ehrlichia chaffeensis Infections

Ehrlichia chaffeensis is an obligate intracellular bacterium and the causative agent of human monocytic ehrlichiosis. Although this pathogen grows in several mammalian cell lines, no general model for eukaryotic cellular requirements for bacteria replication has yet been proposed. We found that Drosophila S2 cells are permissive for the growth of E. chaffeensis. We saw morulae (aggregates of bacteria) by microscopy, detected the E. chaffeensis 16S rRNA gene by reverse transcriptase PCR, and used immunocytochemistry to detect E. chaffeensis in S2 and mammalian cells. Bacteria grown in S2 cells reinfected mammalian macrophages. S2 cells were made nonpermissive for E. chaffeensis through incubation with lipopolysaccharide. Our results demonstrate that S2 cells are an appropriate system for studying the pathogenesis of E. chaffeensis. The use of a Drosophila system has the potential to serve as a model system for studying Ehrlichia due to its completed genome, ease of genetic manipulation, and the availability of mutants.

Biochemical Activities of Three Pairs of Ehrlichia chaffeensis Two-Component Regulatory System Proteins Involved in Inhibition of Lysosomal Fusion

Ehrlichia chaffeensis, the etiologic agent of human monocytic ehrlichiosis, replicates in early endosomes by avoiding lysosomal fusion in monocytes and macrophages. In E. chaffeensis we predicted three pairs of putative two-component regulatory systems (TCSs) designated PleC-PleD, NtrY-NtrX, and CckA-CtrA based on amino acid sequence homology. In the present study to determine biochemical pairs and specificities of the TCSs, the recombinant proteins of the three putative histidine kinase (HK) kinase domains (rPleCHKD, rNtrYHKD, and MBP-rCckAHKD) and the full-length forms of three putative response regulators (RRs) (rPleD, rNtrX, and rCtrA) as well as the respective mutant recombinant proteins (rPleCHKDH244A, rNtrYHKDH498A, MBP-rCckAHKDH449A, rPleDD53A, rNtrXD59A, and rCtrAD53A) were expressed and purified as soluble proteins. The in vitro HK activity, the specific His residue-dependent autophosphorylation of the kinase domain, was demonstrated in the three HKs. The specific Asp residue-dependent in vitro phosphotransfer from the kinase domain to the putative cognate RR was demonstrated in each of the three RRs. Western blot analysis of E. chaffeensis membrane and soluble fractions using antibodies specific for each recombinant protein detected PleC and CckA in the membrane fraction, whereas it detected NtrY, NtrX, and PleD in the soluble fraction. CtrA was found in the two fractions at similar levels. E. chaffeensis was sensitive to closantel, an HK inhibitor. Closantel treatment induced lysosomal fusion of the E. chaffeensis inclusion in a human monocytic leukemia cell line, THP-1 cells, implying that functional TCSs are essential in preventing lysosomal fusion of the E. chaffeensis inclusion compartment.

Primary and Secondary Infection withEhrlichia chaffeensisin White-Tailed Deer (Odocoileus virginianus)

White-tailed deer (Odocoileus virginianus) are the principal reservoir host for Ehrlichia chaffeensis, causative agent of human monocytic ehrlichiosis (HME). Because white-tailed deer maintain a long-term infection with E. chaffeensis and because deer can be naturally exposed to multiple strains of E. chaffeensis, we evaluated the response to secondary infection of E. chaffeensis in deer. For primary infection, six white-tailed deer were injected with 5.4 x 10(6) DH82 cells infected with the Arkansas strain of E. chaffeensis (Ark) and two control deer were injected with noninfected DH82 cells. On post-infection day 54, three E. chaffeensis (Ark) infected deer and one naive deer were injected with 4.2 x 10(6) cells infected with strain WTD-6045B E. chaffeensis, which differs from the Arkansas strain by number of nucleotide repeats in the variable length PCR target (VLPT) gene; three other Arkansas strain infected deer were injected with noninfected DH82 cells. All animals were monitored for 31 additional days. All deer in the primary infection became positive by PCR amplification of the 16S rRNA or VLPT genes and/or cell culture by DPI-8. PCR amplification of the VLPT gene on whole blood, cell culture, and tissues detected primary and/or secondary strains in all deer exposed to both primary and secondary strains; in one deer, the primary strain was cultured from the lymph node. Our culture results demonstrated that both strains were present; however, PCR detection suggests that the secondary strain may have been circulating in blood at higher levels. In conclusion, this study provides evidence that primary infection of deer with E. chaffeensis does not protect against subsequent exposure and confirms that deer can be simultaneously coinfected with at least two different strains of E. chaffeensis.