Browse
Submit Data
Databases
API
Help

Dataset Information

27 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Cloning of the gene encoding the 44-kilodalton antigen of the agent of human granulocytic ehrlichiosis and characterization of the humoral response.

ABSTRACT: Antibodies in the sera of patients with human granulocytic ehrlichiosis (HGE) commonly recognize a 44-kDa antigen. We cloned the gene encoding the 44-kDa protein of the agent of HGE (aoHGE) by probing an aoHGE lambda ZAP II genomic DNA expression library with sera from aoHGE-infected mice. The gene, hge-44, is part of a multigene family, with sequence similarity to the Anaplasma marginale msp-2 genes. RNA-PCR studies confirmed that hge-44 is expressed by aoHGE cultured in HL-60 cells and by aoHGE during murine infection. Recombinant HGE-44, expressed and purified as a glutathione transferase fusion protein, was used as the substrate in immunoblots to help diagnose HGE. Antibodies in eight sera from eight patients with HGE and in two sera from two aoHGE-infected mice bound recombinant HGE-44. Antibodies in the sera of healthy individuals or patients with Ehrlichia chaffeensis or Borrelia burgdorferi infection did not recognize HGE-44. We conclude that hge-44 is a member of a multigene family and that hge-44 is expressed and elicits specific antibodies during infection.

SUBMITTER: Ijdo JW

PROVIDER: S-EPMC108341 | biostudies-literature | 1998 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Cloning and expression of the 44-kilodalton major outer membrane protein gene of the human granulocytic ehrlichiosis agent and application of the recombinant protein to serodiagnosis.

Project description:A 44-kDa major outer membrane protein of the human granulocytic ehrlichiosis (HGE) agent is an immunodominant antigen in human infection. A gene encoding this protein was cloned and sequenced. Southern blot results revealed the existence of multigenes homologous to the P44 gene in the genome of the HGE agent. The recombinant 44-kDa protein (rP44) was expressed by using expression vector pET30a. The reactivity of the affinity-purified rP44 was evaluated by Western immunoblot analysis and dot blot immunoassay. Western immunoblot analysis showed that mouse anti-rP44 serum reacted with 44- to 42-kDa proteins in six different HGE agent strains tested except strain 2, in which three proteins of 42, 40, and 38 kDa were recognized. Eleven HGE patient serum samples, a horse anti-HGE serum, and a horse anti-Ehrlichia equi serum recognized the rP44 protein. This suggests that rP44 is an HGE-E. equi group-specific antigen. Neither human anti-Ehrlichia chaffeensis serum nor rabbit anti-Borrelia burgdorferi serum reacted with rP44. Sera from two patients coinfected with the HGE agent and B. burgdorferi reacted positively with rP44 and the HGE agent. Sera from 20 HGE patients with indirect fluorescent-antibody (IFA) titers ranging from 1:20 to 1:2,560 gave distinct positive reactions in a dot immunoblot assay. There was a positive correlation between the color densities of the dot reactions and the IFA titers when greater than 50 ng of recombinant antigen per dot was used. The use of the affinity-purified rP44 protein as antigen would provide a more specific, consistent, and simpler serodiagnosis for HGE than the use of whole infected cells or purified HGE agents.

| S-EPMC104897 | biostudies-literature

Characterization of an immunoreactive protein from the agent of human granulocytic ehrlichiosis.

Project description:A gene that is homologous to the Ehrlichia chaffeensis groEL operon was recovered and characterized by broad-range PCR amplification of whole blood from patients with human granulocytic ehrlichiosis (HGE) and from infected HL60 cell cultures. Sequence analysis of an 820-bp DNA fragment recovered directly from human blood showed 76.5 and 76.3% identity with cognate sequences from E. chaffeensis and Cowdria ruminantium, respectively. Analysis of a 1.6-kb DNA fragment derived from an HGE agent-infected HL60 cell culture indicated a near-complete open reading frame that contained 75.6 and 75.2% sequence identity with the E. chaffeensis and C. ruminantium groEL sequences, respectively. Phylogenetic analysis of this fragment showed that the HGE agent-derived sequence was related to, but distinct from, the sequences of E. chaffeensis and C. ruminantium. Polyvalent antibody responses to a recombinant fusion protein based on the HGE agent groEL homolog were detected in three of three BALB/c mice that were infected by syringe inoculation with a Wisconsin strain of the HGE agent (WI-1) and nine of nine mice infected by Ixodes scapularis (Ixodes dammini) tick inoculation of an isolate from Nantucket Island, Mass. (NCH-1). No response was detected in mice infected with Borrelia burgdorferi or in control BALB/c mice. Further characterization of the sensitivity and specificity of immune responses to this protein will be facilitated by the use of recombinant fusion proteins or peptides based on the HGE agent-specific groEL homolog.

| S-EPMC232724 | biostudies-other

Evidence of the human granulocytic ehrlichiosis agent in Ixodes ricinus ticks in Switzerland.

Project description:A total of 1,667 Ixodes ricinus ticks were collected from five regions in Switzerland where there have been sporadic occurrences of granulocytic ehrlichiosis in dogs and horses. The ticks were examined for rickettsiae of the Ehrlichia phagocytophila group via nested PCR. Twenty-one ticks (1.3%) were positive; 3 (0.5%) were nymphs, 6 (1.3%) were adult males, and 12 (1.9%) were adult females. The number of positive ticks varied with the stage of development and with the geographical origin. Nucleotide sequencing of the isolated PCR products identified these products as part of the 16S rRNA gene of Ehrlichia. In addition, these products had 100% homology with the agent of human granulocytic ehrlichiosis. The occurrence of this agent in I. ricinus in Switzerland presents a potential danger of transmission of granulocytic ehrlichiosis to dogs, horses, and humans.

| S-EPMC84767 | biostudies-literature

Differential expression of the p44 gene family in the agent of human granulocytic ehrlichiosis.

Project description:Using reverse transcription-PCR targeting of the p44 genes of the agent of human granulocytic ehrlichiosis (HGE) with primers flanking the hypervariable region, we show differential expression in a murine model of HGE infection and during tick transmission. The p44 genes were differentially expressed in salivary glands of infected nymphal ticks removed during transmission feeding but not in nonfeeding infected ticks. Similarly, the p44 genes were differentially expressed in infected C3H mice, in SCID mice, and in cultured HGE bacteria. Thus, differential p44 expression exists in vivo and in vitro and could provide a basis for antigenic variation.

| S-EPMC128253 | biostudies-literature

Invasion and intracellular development of the human granulocytic ehrlichiosis agent in tick cell culture.

Project description:Human granulocytotropic ehrlichias are tick-borne bacterial pathogens that cause an acute, life-threatening illness, human granulocytic ehrlichiosis (HGE). Ehrlichias within neutrophil granulocytes that invade tick bite sites are likely ingested by the vector, to be transmitted to another mammalian host during the tick's next blood meal. Thus, the cycle of replication and development in the vector is prerequisite to mammalian infection, and yet these events have not been described. We report tick cell culture isolation of two strains of the HGE agent directly from an infected horse and a dog and have also established a human isolate from HL60 culture in tick cells, proving that the blood stages of the HGE agent are infectious for tick cells, as are those replicating in the human cell line HL60. This required changes to the culture system, including a new tick cell line. In tick cell layers, the HGE agent induced foci of infection that caused necrotic plaques and eventual destruction of the culture. Using the human isolate and electron microscopy, we monitored adhesion, internalization, and replication in vector tick cells. Both electron-lucent and -dense forms adhered to and entered cells by a mechanism reminiscent of phagocytosis. Ehrlichial cell division was initiated soon after, resulting in endosomes filled with numerous ehrlichias. During early development, pale ehrlichias with a tight cell wall dominated, but by day 2, individual bacteria condensed into dark forms with a rippled membrane. These may become compacted into clumps where individual organisms are barely discernible. Whether these are part of an ehrlichia life cycle or are degenerating is unknown.

| S-EPMC85271 | biostudies-literature

Molecular cloning and characterization of the 120-kilodalton protein gene of Ehrlichia canis and application of the recombinant 120-kilodalton protein for serodiagnosis of canine ehrlichiosis.

Project description:The 120-kDa outer membrane protein (p120) is a potential adhesin of Ehrlichia chaffeensis, and recombinant p120 is very useful for serodiagnosis of human monocytotropic ehrlichiosis. The analogous gene of p120 in Ehrlichia canis was cloned, sequenced, and expressed. Like the E. chaffeensis p120, the E. canis p120 contains tandem repeat units. However, neither the repeat number nor the amino acid sequences in the repeats are identical in the two Ehrlichia species. The repeat units are hydrophilic and by probability analysis are predicted to be surface exposed in both species. The repeat regions of the p120s of the two species have common amino acid sequences that are predicted to be surface exposed. The overall amino acid sequence of the E. canis p120 is 30% homologous to that of E. chaffeensis p120. Protein immunoblotting demonstrated that the recombinant E. canis p120 reacted with convalescent sera from dogs with canine ehrlichiosis. These results indicate that the recombinant p120 is a potential antigen for the serodiagnosis of canine ehrlichiosis.

| S-EPMC88726 | biostudies-literature

Cloning and sequence analysis of a highly polymorphic Cryptosporidium parvum gene encoding a 60-kilodalton glycoprotein and characterization of its 15- and 45-kilodalton zoite surface antigen products.

Project description:The apicomplexan parasite Cryptosporidium parvum is a major cause of serious diarrheal disease in both humans and animals. No efficacious chemo- or immunotherapies have been identified for cryptosporidiosis, but certain antibodies directed against zoite surface antigens and/or proteins shed by gliding zoites have been shown to neutralize infectivity in vitro and/or to passively protect against, or ameliorate, disease in vivo. We previously used monoclonal antibody 11A5 to identify a 15-kDa surface glycoprotein that was shed behind motile sporozoites and was recognized by several lectins that neutralized parasite infectivity for cultured epithelial cells. Here we report the cloning and sequence analysis of the gene encoding this 11A5 antigen. Surprisingly, the gene encoded a 330-amino-acid, mucin-like glycoprotein that was predicted to contain an N-terminal signal peptide, a homopolymeric tract of serine residues, 36 sites of O-linked glycosylation, and a hydrophobic C-terminal peptide specifying attachment of a glycosylphosphatidylinositol anchor. The single-copy gene lacked introns and was expressed during merogony to produce a 60-kDa precursor which was proteolytically cleaved to 15- and 45-kDa glycoprotein products that both localized to the surface of sporozoites and merozoites. The gp15/45/60 gene displayed a very high degree of sequence diversity among C. parvum isolates, and the numerous single-nucleotide and single-amino-acid polymorphisms defined five to six allelic classes, each characterized by additional intra-allelic sequence variation. The gp15/45/60 single-nucleotide polymorphisms will prove useful for haplotyping and fingerprinting isolates and for establishing meaningful relationships between C. parvum genotype and phenotype.

| S-EPMC101708 | biostudies-literature

Cloning, characterization, and expression of a 200-kilodalton diagnostic antigen of Babesia bigemina.

Project description:Current serological tests for Babesia bigemina use semipurified merozoite antigens derived from infected erythrocytes. One of the major drawbacks of these tests is that antigen quality can vary from batch to batch. Since the quality of the antigen contributes to the sensitivity and specificity of serological tests, the use of standardized recombinant antigens should ensure consistency in assay quality. Previously, a 200-kDa merozoite antigen (p200) was identified as a candidate diagnostic antigen for use in a serological assay for the detection of B. bigemina antibodies in infected cattle. In this study, we have cloned, characterized, and expressed p200. A 3.5-kbp cDNA clone encoding p200 was isolated and shown to be almost full length, lacking approximately 300 bp at the 5' end. The predicted amino acid sequence shows that p200 consists of a long, highly charged central repeat region of an uninterrupted alpha helix, indicative of a fibrous protein. Immunoelectron microscopy localized p200 to the merozoite cytoplasm, suggesting that the antigen may be a structural protein involved in forming filament structures within the cytoskeleton. The 3.5-kbp cDNA was expressed in bacteria as a fusion protein with glutathione S-transferase (GST), but the yield was poor. To improve the yield, cDNA fragments encoding antigenic domains of p200 were expressed as fusions with GST. One of these fusion proteins, C1A-GST, is composed of a 7-kDa fragment of the p200 repeat region and contains epitopes that react strongly with sera from cattle experimentally infected with B. bigemina. Recombinant C1A-GST should permit the development of an improved enzyme-linked immunosorbent assay for the detection of antibodies against B. bigemina.

| S-EPMC86771 | biostudies-literature

Transcript heterogeneity of the p44 multigene family in a human granulocytic ehrlichiosis agent transmitted by ticks.

Project description:Human granulocytic ehrlichiosis (HGE) is an emerging tick-borne zoonosis caused by a strain of Anaplasma phagocytophila called the HGE agent, an obligatory intracellular bacterium. The agent expresses immunodominant 44-kDa outer membrane proteins (P44s) encoded by a multigene family. The present study established an experimental process for transmission of the HGE agent from infected mice (a reservoir model) to nymphal Ixodes scapularis ticks (a biological vector) and subsequently to horses (a patient model) by the adult infected ticks. Overall, a total of 20 different p44 transcripts were detected in the mammals, ticks, and cell cultures. Among them, a transcript from a p44-18 gene was major at acute stage in mice and horses but minor in ticks. Both mRNA and protein produced from the p44-18 gene were detected in the HGE agent cultivated in HL-60 cells at 37 degrees C, but their expression levels decreased in the organisms cultivated at 24 degrees C, suggesting that temperature is one of the factors that influence the expression of members of the p44 multigene family. Several additional p44 transcripts that were not detected in the mammals at the acute stage of infection were detected in ticks. Phylogenetic analysis of the 20 different p44 transcripts revealed that the major transcripts found in mammals and ticks were distinct, suggesting a difference in surface properties between populations of the HGE agent in different host environments. The present study provides new information for understanding the role of the p44 multigene family in transmission of the HGE agent between mammals and ticks.

| S-EPMC127761 | biostudies-literature

Isolation of the etiologic agent of human granulocytic ehrlichiosis from the white-footed mouse (Peromyscus leucopus).

Project description:We examined white-footed mice (Peromyscus leucopus) from Minnesota for infection with the etiologic agent of human granulocytic ehrlichiosis (HGE). From April to September 1997, we collected P. leucopus from Washington County, Minnesota, an area enzootic for HGE. Blood was cultivated in HL60 cells for isolation of the HGE agent. Of 59 mice examined, only a single mouse was culture positive for the HGE agent. The 16S ribosomal DNA sequence of the isolate was determined to be identical to that of the HGE agent. The isolate was reactive with monoclonal antibodies to the 44-kDa antigen of the HGE agent and was infectious for laboratory mice.

| S-EPMC87725 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data