Molecular cloning of a gene encoding the immunogenic 21 kDa protein of Cowdria ruminantium

Mahan, Suman M.; McGuire, Travis C.; Semu, S. M.; Bowie, Michael V.; Jongejan, Frans; Rurangirwa, F. R.; Barbet, A. F.

doi:10.1099/13500872-140-8-2135

Cited by 41 publications

(39 citation statements)

References 7 publications

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“…Another major immunoreactive protein (MAP2) related to Anaplasma marginale MSP5 has been identified and molecularly characterized in E. canis, E. chaffeensis, and E. ruminantium with a molecular mass (ϳ21 kDa) similar to that of the gp19 protein identified in this study (1,2,17). However, there is no amino acid homology between MAP2 and gp19, and thus, these proteins are molecularly and immunologically distinct.…”

Section: Vol 75 2007mentioning

confidence: 85%

Identification of a Glycosylated Ehrlichia canis 19-Kilodalton Major Immunoreactive Protein with a Species-Specific Serine-Rich Glycopeptide Epitope

McBride

Doyle²,

Zhang³

et al. 2007

Infect Immun

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Ehrlichia canis has a small subset of major immunoreactive proteins that includes a 19-kDa protein that elicits an early Ehrlichia-specific antibody response in infected dogs. We report herein the identification and molecular characterization of this highly conserved 19-kDa major immunoreactive glycoprotein (gp19) ortholog of the Ehrlichia chaffeensis variable-length PCR target (VLPT) protein. E. canis gp19 has substantial carboxyl-terminal amino acid homology (59%) with E. chaffeensis VLPT and the same chromosomal location; however, the E. chaffeensis VLPT gene (594 bp) has tandem repeats that are not present in the E. canis gp19 gene (414 bp). Consistent with other ehrlichial glycoproteins, the gp19 protein exhibited a larger-thanpredicted mass (ϳ3 kDa), O-linked glycosylation sites were predicted in an amino-terminal serine/threonine/ glutamate (STE)-rich patch (26 amino acids), carbohydrate was detected on the recombinant gp19 protein, and the neutral sugars glucose and galactose were detected on the recombinant amino-terminal polypeptide. E. canis gp19 composition consists of five predominant amino acids, cysteine, glutamate, tyrosine, serine, and threonine, concentrated in the STE-rich patch and a carboxyl-terminal domain predominated by cysteine and tyrosine (55%). The amino-terminal STE-rich patch contained a major species-specific antibody epitope strongly recognized by serum from an E. canis-infected dog. The recombinant glycopeptide epitope was substantially more reactive with antibody than the synthetic (nonglycosylated) peptide, and periodate treatment of the recombinant glycopeptide epitope reduced its immunoreactivity, demonstrating the importance of a carbohydrate immunodeterminant(s). The gp19 protein was present on reticulate and dense-cored cells, and it was found extracellularly in the fibrillar matrix and associated with the morula membrane, the host cell cytoplasm, and the nucleus.Ehrlichia canis is a tick-transmitted obligately intracellular bacterium that causes moderate to severe and sometimes fatal disease in wild and domestic canids. The genomes of E. canis and other organisms in the genus, including Ehrlichia chaffeensis and Ehrlichia ruminantium, exhibit a high degree of genomic synteny, paralogous protein families, a large proportion of proteins with transmembrane helices and/or signal sequences, tandem repeats and ankyrin domains in proteins associated with host-pathogen interactions, and a unique serine-threonine bias associated with a potential for O glycosylation and phosphorylation (6,10,11,18). A small subset of the approximately 1,000 proteins (including hypothetical proteins) encoded by each of these genomes is recognized by antibody (8,20,25,32). Several of the major immunoreactive proteins identified and molecularly characterized are serine-rich glycoproteins that are secreted. Many of these glycoproteins have tandem repeats; however, one has numerous eukaryote-like ankyrin domains (7,20,25,27,31,35).Numerous proteins that contain tandem repeats have been identified in E. ...

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Section: Vol 75 2007mentioning

confidence: 85%

Identification of a Glycosylated Ehrlichia canis 19-Kilodalton Major Immunoreactive Protein with a Species-Specific Serine-Rich Glycopeptide Epitope

McBride

Doyle²,

Zhang³

et al. 2007

Infect Immun

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“…Genes encoding both the MAP2 protein of C. ruminantium and the MSP5 protein of A. marginale were found to be single-copy genes that were highly conserved between various isolates of organisms within their respective species. Recombinant proteins developed from these genes have been used as diagnostic antigens to identify infected animals (4,24,26,37).…”

Section: Discussionmentioning

confidence: 99%

“…Cells were harvested when 90 to 100% of them were infected, and ehrlichiae were purified as described previously (10). Genomic DNA of E. canis was isolated by treatment of purified organisms with 5 mg of lysozyme per ml, 100 g of proteinase K per ml, and 2% (wt/vol) sodium dodecyl sulfate (SDS), followed by phenol-chloroform extraction and ethanol precipitation (26).…”

Section: Methodsmentioning

confidence: 99%

“…Both map2 and msp5 genes have been shown to be conserved between various isolates of their respective organisms (4,37). The recombinant products of these genes are presently being used in ELISAs to diagnose infections with these agents (24,26). In this study, we report the cloning and expression of map2 from E. canis and examine the potential value of the recombinant MAP2 protein (rMAP2) for the rapid serodiagnosis of canine monocytic ehrlichiosis.…”

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confidence: 99%

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Recombinant Major Antigenic Protein 2 ofEhrlichia canis: a Potential Diagnostic Tool

Alleman¹,

McSherry²,

Barbet

et al. 2001

J Clin Microbiol

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The major antigenic protein 2 (MAP2) of Ehrlichia canis was cloned and expressed. The recombinant protein was characterized and tested in an enzyme-linked immunosorbent assay (ELISA) format for potential application in the serodiagnosis of canine monocytic ehrlichiosis. The recombinant protein, which contained a C-terminal polyhistidine tag, had a molecular mass of approximately 26 kDa. The antigen was clearly identified by Western immunoblotting using antihistidine antibody and immune serum from an experimentally infected dog. The recombinant MAP2 (rMAP2) was tested in an ELISA format using 141 serum samples from E. canis immunofluorescent antibody (IFA)-positive and IFA-negative dogs. Fifty-five of the serum samples were from dogs experimentally or naturally infected with E. canis and were previously demonstrated to contain antibodies reactive with E. canis by indirect immunofluorescence assays. The remaining 86 samples, 33 of which were from dogs infected with microorganisms other than E. canis, were seronegative. All of the samples from experimentally infected animals and 36 of the 37 samples from naturally infected animals were found to contain antibodies against rMAP2 of E. canis in the ELISA. Only 3 of 53 IFA-negative samples tested positive on the rMAP2 ELISA. There was 100% agreement among IFA-positive samples from experimentally infected animals, 97.3% agreement among IFA-positive samples from naturally infected animals, and 94.3% agreement among IFA-negative samples, resulting in a 97.2% overall agreement between the two assays. These data suggest that rMAP2 of E. canis could be used as a recombinant test antigen for the serodiagnosis of canine monocytic ehrlichiosis.Ehrlichia canis is an obligate intracellular, gram-negative bacterium and the causative agent of canine monocytic ehrlichiosis. Canine monocytic ehrlichiosis is a tick-borne rickettsial disease which is transmitted by the brown dog tick, Rhipicephalus sanguineus (19). Interest in E. canis infection has heightened over the last decade, fueled by the recent discovery of a very closely related organism, Ehrlichia chaffeensis, the causative agent of human monocytic ehrlichiosis (15). More recently, it has been shown that dogs are susceptible to experimental infection with E. chaffeensis (11), and natural infections with E. chaffeensis have also been identified in healthy and clinically ill dogs by PCR analysis (5,11,25,29). This suggests that dogs may serve as an important reservoir for this human pathogen.Sequence analysis of the 16S rRNA genes of E. canis and E. chaffeensis revealed that E. chaffeensis is more closely related to E. canis than to any other species (3). These organisms, along with Cowdria ruminantium, a rickettsial disease agent of cattle, are phylogenetically related and are all placed within the E. canis genogroup (13,36, 48). There is considerable antigenic cross-reactivity between the Ehrlichia spp. and other closely related organisms, such as C. ruminantium (8,9,23,30,32). Serological assays able to distinguish between i...

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“…Although a few genes from C. ruminantium have been identified and cloned (Mahan et al, 1994 ;Van Vliet et al, 1994 ;Lally et al, 1995 ;Brayton et al, 1997), nothing was previously known about the structure and organization of the genome. Recent advances in the purification of C. ruminantium (De Villiers et al, 1998), using a combination of Percoll density-gradient centrifugation (Tamura et al, 1982) and high-gradient magnetic cell separation (MACS) (Miltenyi et al, 1990), have allowed us to study some of Downloaded from www.microbiologyresearch.org by IP: 13.66.222.141…”

Section: Introductionmentioning

confidence: 99%

Genome size and genetic map of Cowdria ruminantium

et al. 2000

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Cowdria ruminantium is the cause of a serious tick-borne disease of domestic ruminants, known as heartwater or cowdriosis. The organism belongs to the tribe Ehrlichieae, which contains obligate intracellular pathogens, causing several important animal and human diseases. Although a few C. ruminantium genes have been cloned and sequenced, very little is known about the size, gross structure and organization of the genome. This paper presents a complete physical map and a preliminary genetic map for C. ruminantium. Chromosomal C. ruminantium DNA was examined by PFGE and Southern hybridization. PFGE analysis revealed that C. ruminantium has a circular chromosome approximately 1576 kb in size. A physical map was derived by combining the results of PFGE analysis of DNA fragments resulting from digestion of the whole genome with KspI, RsrII and SmaI and Southern hybridization analysis with a series of gene probes and isolated macrorestriction fragments. A genetic map for C. ruminantium with a mean resolution of 290 kb was established, the first for a member of the Ehrlichieae. A total of nine genes or cloned C. ruminantium DNA fragments were mapped to specific KspI, RsrII and SmaI fragments, including the major antigenic protein gene, map-1.

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Molecular cloning of a gene encoding the immunogenic 21 kDa protein of Cowdria ruminantium

Cited by 41 publications

References 7 publications

Identification of a Glycosylated Ehrlichia canis 19-Kilodalton Major Immunoreactive Protein with a Species-Specific Serine-Rich Glycopeptide Epitope

Identification of a Glycosylated Ehrlichia canis 19-Kilodalton Major Immunoreactive Protein with a Species-Specific Serine-Rich Glycopeptide Epitope

Recombinant Major Antigenic Protein 2 ofEhrlichia canis: a Potential Diagnostic Tool

Genome size and genetic map of Cowdria ruminantium

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