Analysis and Ultrastructural Localization of Ehrlichia chaffeensis Proteins with Monoclonal Antibodies

Chen, Sheng-Min; Popov, Vsevolod L.; Feng, Huimin; Walker, David H.

doi:10.4269/ajtmh.1996.54.405

Cited by 33 publications

(29 citation statements)

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“…Interestingly, R4, the most divergent repeat, was not reactive by Western immunoblotting but was reactive with antibody in ELISA, suggesting that a distinct conformational epitope was present in R4. Conformational epitopes have been described for Ehrlichia and Anaplasma species (6,23). Thus, R4 contributes to the immunoreactivity of VLPT independent of R3.…”

Section: Discussionmentioning

confidence: 99%

A Variable-Length PCR Target Protein of Ehrlichia chaffeensis Contains Major Species-Specific Antibody Epitopes in Acidic Serine-Rich Tandem Repeats

Luo¹,

Zhang²,

Wakeel³

et al. 2008

Infect Immun

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109

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Ehrlichia chaffeensis and E. canis have a small subset of tandem repeat (TR)-containing proteins that elicit strong host immune responses and are associated with host-pathogen interactions. In a previous study, we molecularly characterized a highly conserved 19-kDa major immunoreactive protein (gp19) of E. canis and identified the corresponding TR-containing ortholog variable-length PCR target (VLPT) protein in E. chaffeensis. In this study, the native 32-kDa VLPT protein was identified and the immunodeterminants defined in order to further understand the molecular basis of the host immune response to E. chaffeensis. Synthetic and/or recombinant polypeptides corresponding to various regions of VLPT were used to localize major antibody epitopes to the TR-containing region. Major antibody epitopes were identified in three nonidentical repeats (R2, R3, and R4), which reacted strongly with antibodies in sera from an E. chaffeensis-infected dog and human monocytotropic ehrlichiosis patients. VLPT-R3 and VLPT-R2 reacted most strongly with antibody, and the epitope was further localized to a nearly identical proximal 17-amino-acid region common between these repeats that was species specific. The epitope in R4 was distinct from that of R2 and R3 and was found to have conformational dependence. VLPT was detected in supernatants from infected cells, indicating that the protein was secreted. VLPT was localized on both reticulate and dense-core cells, and it was found extracellularly in the morula fibrillar matrix and associated with the morula membrane.Ehrlichia chaffeensis is a tick-transmitted, obligately intracellular bacterium which causes human monocytotropic ehrlichiosis (HME), an emerging life-threatening disease in humans, and also causes mild to severe disease in canines (28). Recently, a number of studies demonstrated that humoral immunity plays an essential role in host defenses against ehrlichial pathogens (10,33,34,36). Furthermore, a small subset of E. chaffeensis proteins, many of which contain tandem repeats (TRs), appear to be the primary targets of the humoral immune response and are considered to be the major immunoreactive proteins (4,5,15,30). However, the characteristics of the immunodeterminants that shape the humoral immune response to Ehrlichia species are not fully defined, nor has their role in protective immunity been determined.Major immunoreactive proteins of E. chaffeensis include 30). Some of these proteins (200-, 120-, 47-, and 28-kDa proteins) have been identified and molecularly characterized, including the corresponding orthologs in Ehrlichia canis respectively) (7,16,20,25,27,(38)(39)(40). Most recently, a strongly acidic 19-kDa major immunoreactive protein (gp19) of E. canis was identified. The gp19 gene has the same relative chromosomal location as and substantial amino acid homology in a C-terminal cysteine-tyrosine-rich domain to the previously reported variable-length PCR target (VLPT) protein identified in E. chaffeensis (19). The VLPT gene has 90-bp TRs that vary in number (2 t...

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Section: Discussionmentioning

confidence: 99%

A Variable-Length PCR Target Protein of Ehrlichia chaffeensis Contains Major Species-Specific Antibody Epitopes in Acidic Serine-Rich Tandem Repeats

Luo¹,

Zhang²,

Wakeel³

et al. 2008

Infect Immun

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109

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“…The E. chaffeensis Arkansas isolate was continuously cultivated in the canine macrophage cell line DH82 at 37 or 34°C (6). E. chaffeensis in DH82 cells was also cultivated at 25°C.…”

Section: Methodsmentioning

confidence: 99%

Ehrlichia chaffeensis Expresses Macrophage- and Tick Cell-Specific 28-Kilodalton Outer Membrane Proteins

et al. 2005

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Ehrlichia chaffeensis, a tick-transmitted rickettsial agent, causes human monocyte/macrophage-tropic ehrlichiosis. In this study, proteomic approaches were used to demonstrate host cell-specific antigenic expression by E. chaffeensis. The differentially expressed antigens include those from the 28-kDa outer membrane protein (p28-Omp) multigene locus. The proteins expressed in infected macrophages are the products of p28-Omp19 and p28-Omp20 genes, whereas in tick cells, the protein expressed is the p28-Omp14 gene product. The differentially expressed proteins are posttranslationally modified by phosphorylation and glycosylation to generate multiple expressed forms. Host cell-specific protein expression is not influenced by growth temperatures and is reversible. Host cell-specific protein expression coupled with posttranslational modifications may be a hallmark for the pathogen's adaptation to a dual-host life cycle and its persistence.

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“…Ten E. chaffeensis isolates obtained from whole blood or bone marrow of acutely ill patients with HME (Table 1) were cultivated in the canine macrophage cell line DH82, as described previously (3). All isolates were obtained from moderately to seriously ill patients, including two patients who died from the infection.…”

Section: Methodsmentioning

confidence: 99%

Molecular Heterogeneity of Ehrlichia chaffeensis Isolates Determined by Sequence Analysis of the 28-Kilodalton Outer Membrane Protein Genes and Other Regions of the Genome

2003

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Ehrlichia chaffeensis, a tick-transmitted rickettsial agent, is responsible for human monocytic ehrlichiosis (HME). In this study, we genetically mapped 10 isolates obtained from HME patients. Sequence analysis of the 28-kDa outer membrane protein (OMP) multigene locus spanning 6 of the 22 tandemly arranged genes identified three distinct genetic groups with shared homology among isolates within each group. Isolates in Groups I and III contained six genes each, while Group II isolates had a gene deletion. There were two regions on the locus where novel gene deletion or insertion mutations occurred, resulting in the net loss of one gene in Group II isolates. Numerous nucleotide differences among genes in isolates of each group also were detected. The shared homology among isolates in each group for the 28-kDa OMP locus suggests the derivation of clonal lineages. Transcription and translation analysis of the locus revealed differences in the expressed genes of different group isolates. Analysis of the 120-kDa OMP gene and variable-length PCR target gene showed size variations resulting from loss or gain of long, direct repeats within the protein coding sequences. To our knowledge this is the first study that looked at several regions of the genome simultaneously, and we provide the first evidence of heterogeneity resulting from gene deletion and insertion mutations in the E. chaffeensis genome. Diversity in different genomic regions could be the result of a selection process or of independently evolved genes.

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Analysis and Ultrastructural Localization of Ehrlichia chaffeensis Proteins with Monoclonal Antibodies

Cited by 33 publications

References 0 publications

A Variable-Length PCR Target Protein of Ehrlichia chaffeensis Contains Major Species-Specific Antibody Epitopes in Acidic Serine-Rich Tandem Repeats

A Variable-Length PCR Target Protein of Ehrlichia chaffeensis Contains Major Species-Specific Antibody Epitopes in Acidic Serine-Rich Tandem Repeats

Ehrlichia chaffeensis Expresses Macrophage- and Tick Cell-Specific 28-Kilodalton Outer Membrane Proteins

Molecular Heterogeneity of Ehrlichia chaffeensis Isolates Determined by Sequence Analysis of the 28-Kilodalton Outer Membrane Protein Genes and Other Regions of the Genome

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