RESUMEN
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.
Asunto(s)
Cultivo Axénico , ADN/biosíntesis , Ehrlichia chaffeensis/metabolismo , Biosíntesis de Proteínas , Carbono/farmacología , Sistema Libre de Células , Diatrizoato de Meglumina/metabolismo , Ehrlichia chaffeensis/ultraestructura , Concentración de Iones de Hidrógeno , Modelos Biológicos , ARN/biosíntesis , ARN Ribosómico 16S/genéticaRESUMEN
BACKGROUND: Ehrlichia chaffeensis is an emerging tick-borne rickettsial pathogen responsible for human monocytic ehrlichiosis. Despite the induction of an active host immune response, the pathogen has evolved to persist in its vertebrate and tick hosts. Understanding how the organism progresses in tick and vertebrate host cells is critical in identifying effective strategies to block the pathogen transmission. Our recent molecular and proteomic studies revealed differences in numerous expressed proteins of the organism during its growth in different host environments. METHODOLOGY/PRINCIPAL FINDINGS: Transmission electron microscopy analysis was performed to assess morphological changes in the bacterium within macrophages and tick cells. The stages of pathogen progression observed included the attachment of the organism to the host cells, its engulfment and replication within a morulae by binary fission and release of the organisms from infected host cells by complete host cell lysis or by exocytosis. E. chaffeensis grown in tick cells was highly pleomorphic and appears to replicate by both binary fission and filamentous type cell divisions. The presence of Ehrlichia-like inclusions was also observed within the nucleus of both macrophages and tick cells. This observation was confirmed by confocal microscopy and immunoblot analysis. CONCLUSIONS/SIGNIFICANCE: Morphological differences in the pathogen's progression, replication, and processing within macrophages and tick cells provide further evidence that E. chaffeensis employs unique host-cell specific strategies in support of adaptation to vertebrate and tick cell environments.
Asunto(s)
Ehrlichia chaffeensis/ultraestructura , Macrófagos/microbiología , Microscopía Electrónica de Transmisión/métodos , Garrapatas/microbiología , Animales , Western Blotting , Ehrlichia chaffeensis/crecimiento & desarrollo , Microscopía ConfocalRESUMEN
Ehrlichia chaffeensis, an obligatory intracellular bacterium, has two forms in mammalian cells: small dense-cored cells (DC) with dense nucleoid and larger reticulate cells (RC) with uniformly dispersed nucleoid. We have determined by electron microscopy that DC but not RC attaches to and enters into the host cells and RC but not DC multiples inside the host cells. Analysis of outer membrane protein expression by confocal microscopy showed that RC expressed the 28 kDa outer membrane protein (p28), the intermediate form, which were transforming from RC to DC, expressed both gp120 and p28, and the mature DC expressed gp120 only. The TCID50 of DC is 6 log10 higher than RC. We conclude that E. chaffeensis has a developmental cycle, in which the DC attaches to and enters into the host cells, and transforms into RC and the RC multiplies by binary fission for 48 h and then matures into DC at 72 h.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Ehrlichia chaffeensis/crecimiento & desarrollo , Macrófagos/microbiología , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Diferenciación Celular , División Celular , Línea Celular , Ehrlichia chaffeensis/metabolismo , Ehrlichia chaffeensis/patogenicidad , Ehrlichia chaffeensis/ultraestructura , Macrófagos/citología , Macrófagos/ultraestructura , Microscopía Confocal , Microscopía Electrónica de Transmisión , Modelos Biológicos , Reacción en Cadena de la PolimerasaRESUMEN
Ehrlichia chaffeensis is an obligatory intracellular bacterium that infects the monocyte-macrophage. E. chaffeensis, which is transmitted to humans by ticks primarily from infected deer, causes human monocytic ehrlichiosis, an acute febrile systemic illness. This paper reviews current knowledge of clinical and biological aspects of infections caused by E. chaffeensis.
Asunto(s)
Ehrlichia chaffeensis/patogenicidad , Ehrlichiosis/inmunología , Ehrlichiosis/microbiología , Animales , Vectores Artrópodos/microbiología , Cricetinae , Dermacentor/microbiología , Ehrlichia chaffeensis/inmunología , Ehrlichia chaffeensis/aislamiento & purificación , Ehrlichia chaffeensis/ultraestructura , Ehrlichiosis/epidemiología , Ehrlichiosis/parasitología , HumanosRESUMEN
Diagnosis of human monocytotropic ehrlichiosis (HME) generally depends on serology that detects the antibody response to immunodominant proteins of Ehrlichia chaffeensis. Protein immunoblotting was used to evaluate the reaction of the antibodies in patients' sera with the recombinant E. chaffeensis 120- and 28-kDa proteins as well as the 106- and the 37-kDa proteins. The cloning of the genes encoding the latter two proteins is described in this report. Immunoelectron microscopy demonstrated that the 106-kDa protein is located at the surfaces of ehrlichiae and on the intramorular fibrillar structures associated with E. chaffeensis. The 37-kDa protein is homologous to the iron-binding protein of gram-negative bacteria. Forty-two serum samples from patients who were suspected to have HME were tested by immunofluorescence (IFA) using E. chaffeensis antigen and by protein immunoblotting using recombinant E. chaffeensis proteins expressed in Escherichia coli. Thirty-two serum samples contained IFA antibodies at a titer of 1:64 or greater. The correlation of IFA and recombinant protein immunoblotting was 100% for the 120-kDa protein, 41% for the 28-kDa protein, 9.4% for the 106-kDa protein, and 0% for the 37-kDa protein. None of the recombinant antigens yielded false-positive results. All the sera reactive with the recombinant 28- or the 106-kDa proteins also reacted with the recombinant 120-kDa protein.
Asunto(s)
Proteínas Bacterianas , Ehrlichia chaffeensis/genética , Ehrlichiosis/diagnóstico , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/ultraestructura , Secuencia de Bases , Clonación Molecular , Ehrlichia chaffeensis/aislamiento & purificación , Ehrlichia chaffeensis/ultraestructura , Ehrlichiosis/microbiología , Humanos , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Alineación de SecuenciaRESUMEN
Ultrastructural characteristics of 15 strains and isolates of ehrlichiae belonging to three genogroups, or clades of genetically related organisms united in the genera Ehrlichia, Cowdria, Anaplasma, Neorickettsia and a strain of Wolbachia pipientis which represents a fourth genogroup in this cluster of species, were studied in continuous cell culture or in vivo: E. canis (Oklahoma strain and VHE isolate), E. muris (AS 145), E. chaffeensis (Arkansas, 91HE17 and Sapulpa), human granulocytic ehrlichiae (HGE)(BDS, 96HE27, 96HE37, #54, #55 and #72), E. equi (MRK), E. sennetsu (Miyayama), E. risticii (HRC-IL). Wolbachia pipientis was studied in the naturally infected Aedes albopictus mosquito cell line Aa23. All organisms were similar in the normal ultrastructure of individual cells and in the ability to form abnormal, pathological ehrlichial cells of the same type irrespective of the species. Normally all ehrlichiae studied in cell culture existed in two morphological forms - reticulate and dense-cored cells, both of which could divide by binary fission. Most alterations were related to their membranes, especially the cell wall. Differences in the structure of intravacuolar microcolonies (morulae) of ehrlichiae and their inter-relations with the host cells allowed differentiation of the genogroups: the E. canis-E. chaffeensis-E. muris genogroup formed large morulae, with many ehrlichiae, often suspended in a fibrillar matrix, and the host cell mitochondria and endoplasmic reticulum usually aggregated near the morulae and were in contact with the morula membrane; the E. phagocytophila-E. equi-HGE group morulae had no fibrillar matrix, no contacts with host cell mitochodria, and they did not aggregate around the morulae; E. sennetsu-E. risticii group usually developed in small individual vacuoles that did not fuse with each other and divided along with the ehrlichiae.
Asunto(s)
Ehrlichia/ultraestructura , Animales , Células Cultivadas , Ehrlichia/clasificación , Ehrlichia/genética , Ehrlichia chaffeensis/ultraestructura , Caballos , Humanos , Macrófagos/microbiología , Ratones , Ratones Endogámicos BALB C , ARN Ribosómico 16S/análisisRESUMEN
Several immunodominant major proteins ranging from 23 to 30 kDa were identified in the outer membrane fractions of Ehrlichia chaffeensis and Ehrlichia canis. The N-terminal amino acid sequence of a 28-kDa protein of E. chaffeensis (one of the major proteins) was determined. The gene (p28), almost full length, encoding the 28-kDa protein was cloned by PCR with primers designed based on the N-terminal sequence of the E. chaffeensis 28-kDa protein and the consensus sequence between the C termini of the Cowdria ruminantium MAP-1 and Anaplasma marginale MSP-4 proteins. The p28 gene was overexpressed, and antibody to the recombinant protein was raised in a rabbit. The antibody and serum from a patient infected with E. chaffeensis reacted with the recombinant protein, three proteins (29, 28, and 25 kDa) of E. chaffeensis, and a 30-kDa protein of E. canis. Immunoelectron microscopy with the rabbit antibody revealed that the antigenic epitope of the 28-kDa protein was exposed on the surface of E. chaffeensis. Southern blot analysis with a 32P-labeled p28 gene probe revealed multiple copies of genes homologous to p28 in the E. chaffeensis genome. Six copies of the p28 gene were cloned and sequenced from the genomic DNA by using the same probe. The open reading frames of these gene copies were tandemly arranged with intergenic spaces. They were nonidentical genes and contained a semivariable region and three hypervariable regions in the predicted protein molecules. One of the gene copies encoded a protein with an internal amino acid sequence identical to the chemically determined N-terminal amino acid sequence of a 23-kDa protein of E. chaffeensis. Immunization with the recombinant P28 protein protected mice from infection with E. chaffeensis. These findings suggest that the 30-kDa-range proteins of E. chaffeensis represent a family of antigenically related homologous proteins encoded by a single gene family.
Asunto(s)
Antígenos Bacterianos , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/inmunología , Ehrlichia chaffeensis/genética , Ehrlichia chaffeensis/inmunología , Ehrlichia/genética , Ehrlichia/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antibacterianos/biosíntesis , Anticuerpos Antibacterianos/inmunología , Proteínas Bacterianas/genética , Clonación Molecular , ADN Bacteriano/análisis , ADN Bacteriano/genética , Ehrlichia chaffeensis/ultraestructura , Expresión Génica , Genes Bacterianos , Genoma Bacteriano , Humanos , Epítopos Inmunodominantes , Masculino , Proteínas de la Membrana/genética , Ratones , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Pruebas de Neutralización , Sistemas de Lectura Abierta , Filogenia , Reacción en Cadena de la Polimerasa , ARN Ribosómico 16S/genética , Conejos , Proteínas Recombinantes/inmunología , Mapeo Restrictivo , Alineación de Secuencia , Análisis de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
Two new isolates of Ehrlichia chaffeensis (designated Jax and St. Vincent) were obtained from patients with fatal ehrlichial infections. Patients developed characteristic manifestations of severe disease due to E. chaffeensis, including marked thrombocytopenia, pulmonary insufficiency, and encephalopathy. Primary isolation was achieved in DH82 cells; the Jax and St. Vincent isolates were detected within 19 and 8 days postinoculation, respectively. The isolates were characterized by molecular evaluation of the 16S rRNA gene, the groESL heat shock operon, a 120-kDa immunodominant protein gene, and an incompletely characterized repetitive-motif sequence (variable-length PCR target [VLPT]). The sequences were compared with those of the corresponding molecular regions in the type isolate (Arkansas). St. Vincent contained one fewer repeat unit in both the 120-kDa protein gene and the VLPT compared with corresponding sequences of the Jax and Arkansas isolates. 16S rRNA gene sequences from the two new isolates had 100% identity to the corresponding sequences of the 91HE17 and Sapulpa isolates of E. chaffeensis, and to the corrected 16S rRNA gene sequence of the Arkansas isolate. The Jax isolate grew more slowly than the St. Vincent isolate in DH82 cells, and both of the new isolates grew more slowly than the extensively passaged Arkansas isolate. Although specific associations between ehrlichial pathogenicity and genotype were not identified from these comparisons, recovery of this organism from a spectrum of clinical presentations remains an integral step in understanding mechanisms of disease caused by E. chaffeensis.
Asunto(s)
Ehrlichia chaffeensis/aislamiento & purificación , Ehrlichiosis/microbiología , Análisis de Varianza , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Chaperonina 10/genética , Chaperonina 60/genética , Ehrlichia chaffeensis/genética , Ehrlichia chaffeensis/ultraestructura , Ehrlichiosis/sangre , Ehrlichiosis/etiología , Ehrlichiosis/mortalidad , Femenino , Humanos , Epítopos Inmunodominantes/genética , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
Ehrlichia chaffeensis is an obligatory intracellular bacterium which infects macrophages and monocytes. Double immunofluorescence labeling was used to characterize the nature of E. chaffeensis inclusion in the human promyelocytic leukemia cell line THP-1. E. chaffeensis was labeled with dog anti-E. chaffeensis serum and fluorescein isothiocyanate-conjugated anti-dog immunoglobulin G (IgG). Lissamine rhodamine-conjugated anti-mouse IgG was used to label various mouse monoclonal antibodies. Ehrlichial inclusions did not fuse with lysosomes, since they were not labeled with anti-CD63 or anti-LAMP-1. The ehrlichial inclusions were slightly acidic, since they weakly accumulated 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine and stained weakly positive for vacuolar type H+ ATPase. Some ehrlichial inclusions were labeled positive with antibodies against HLA-DR, HLA-ABC, and beta2 microglobulin, while other inclusions in the same cell were labeled negative. The inclusions were labeled strongly positive for transferrin receptors (TfRs) and negative for the clathrin heavy chain. Time course labeling for TfRs showed that up to 3 h postinfection, most of the ehrlichial inclusions were negative for TfRs. After 6 h postinfection, 100% of the ehrlichial inclusions became TfR positive and the intensity of labeling was increased during the subsequent 3 days. Reverse transcription-PCR showed a gradual increase in the level of TfR mRNA postinfection, which reached a peak at 24 h postinfection. These results suggest that ehrlichial inclusions are early endosomes which selectively accumulate TfRs and that the ehrlichiae up-regulate TfR mRNA expression.
Asunto(s)
Ehrlichia chaffeensis/ultraestructura , Endosomas/metabolismo , Receptores de Transferrina/metabolismo , Animales , Adhesión Bacteriana , Transporte Biológico , Línea Celular , Perros , Ehrlichia chaffeensis/metabolismo , Humanos , RatonesRESUMEN
A new Ehrlichia strain, designated as Ehrlichia chaffeensis, Sapulpa strain, was isolated from a patient from Oklahoma with severe ehrlichiosis. Isolation of the ehrlichial pathogen was achieved by inoculating patient blood onto HEL cells and DH82 cells. Antigenic properties of the new isolate were characterized with monoclonal antibodies, homologous patient serum, and polyclonal rabbit serum by Western immunoblotting. The results showed antigenic differences and protein size variation of Sapulpa strain compared with the other 2 strains of E. chaffeensis. Sequencing of the 16S rRNA gene showed 100% identity to that of E. chaffeensis, strain 91HE17. Polymerase chain reaction and sequencing of DNA homologous to the 120-kDa protein gene of E. chaffeensis, Arkansas strain, showed that this gene of Sapulpa strain was smaller than that of Arkansas strain and contained a repeat region with three tandem repeat units.
Asunto(s)
Ehrlichia chaffeensis/genética , Animales , Antígenos Bacterianos/sangre , Secuencia de Bases , ADN Ribosómico/química , Ehrlichia chaffeensis/inmunología , Ehrlichia chaffeensis/ultraestructura , Variación Genética , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Ribosómico 16S/genética , ConejosRESUMEN
Ehrlichia chaffeensis, an obligately intracellular bacterium with tropism for monocytes, is the etiologic agent of human monocytic ehrlichiosis. To determine the nature and ultrastructural location of E. chaffeensis antigens, monoclonal antibodies (MAbs) to E. chaffeensis were developed. The MAbs were used for immunofluorescence and Western immunoblotting analysis of the antigens of density gradient-purified ehrlichiae. Monoclonal antibody 6A1 recognized an epitope of a 30-kD protein. This antibody reacted with a strain-specific epitope of E. chaffeensis, Arkansas strain, and did not cross-react with any other ehrlichia tested. Monoclonal antibodies 3C7 and 7C1-B recognized Arkansas strain proteins of 30 and 29 kD and reacted with E. chaffeensis (strain 91HE17) proteins of 31 and 29 kD and an E. canis protein of 30 kD. Lack of reactivity of these two MAbs with E. sennetsu and E. risticii suggests that the epitope is group-specific. Monoclonal antibody 5D11 recognized a 58-kD protein of both strains of E. chaffeensis as well as E. canis, apparently a group-specific, conformation-independent epitope. Monoclonal antibody 7C1-C reacted with 58- and 88-kD proteins of both the Arkansas and 91HE17 strains. Trypsin treatment destroyed the reactivity of E. chaffeensis antigens with all the MAbs when tested by Western immunoblotting, indicating that these antigens are proteins with trypsin-sensitive epitopes. Immunoelectron microscopy of negatively stained intact E. chaffeensis organisms showed that the 30- and 29-kD proteins are present on the surface of the ehrlichial cell wall along with the previously localized 28-kD protein.
Asunto(s)
Anticuerpos Monoclonales , Proteínas Bacterianas/análisis , Ehrlichia chaffeensis/química , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Antígenos Bacterianos/análisis , Antígenos Bacterianos/química , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/química , Proteínas Bacterianas/inmunología , Western Blotting , Línea Celular , Chlorocebus aethiops , Perros , Ehrlichia chaffeensis/inmunología , Ehrlichia chaffeensis/ultraestructura , Epítopos/análisis , Epítopos/inmunología , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Hibridomas , Sueros Inmunes/inmunología , Ratones , Ratones Endogámicos BALB C , Microscopía Inmunoelectrónica , Peso Molecular , Conejos , Células VeroRESUMEN
The ultrastructure of Ehrlichia chaffeensis (Arkansas strain) was studied in non-irradiated and irradiated monolayers of mouse embryo, Vero, BGM and L929 cells, and in non-irradiated DH82 cells. Within the intracellular parasitophorous vacuoles (morulae), two types of ehrlichial cells were found regularly--those with uniformly dispersed nucleoid filaments and ribosomes (dense-cored cells), which represent the normal life cycle of ehrlichiae. In addition, large reticulate cells were observed, forming long projections of the cell wall, protrusions of cytoplasmic membrane into the periplasmic space, or budding of protoplast fragments (minute forms) into the periplasmic space. Ehrlichiae with abnormalities of protoplast fission were found, apparently leading to formation of giant, multilobular or elongated rod-like ehrlichiae. Morulae were usually surrounded by cisterns of granular endoplasmic reticulum and mitochondria and often contained vesicles, long tubules 25nm in diameter, probably originating from the ehrlichial cell wall, and fibrillar ehrlichial antigen apparently shed from the surface of the cell wall. Some cells contained, in addition to normal morulae, a whole morula that had become dense and contained degenerating ehrlichiae. These results indicate that as well as normal growth and reproduction, ehrlichiae exhibit pathological events: they can be remarkably damaged inside the host cell vacuoles, presumably phagolysosomes, or enter a process morphologically similar to bacterial L-transformation.