Molecular Cloning and Sequencing of Three Granulocytic<i>Ehrlichia</i>Genes Encoding High-Molecular-Weight Immunoreactive Proteins

Storey, James R.; Doros-Richert, Linda A.; Gingrich-Baker, Cindy; Munroe, Kenneth J.; Mather, Thomas N.; Coughlin, Richard T.; Beltz, Gerald A.; Murphy, Cheryl I.

doi:10.1128/iai.66.4.1356-1363.1998

Cited by 44 publications

(29 citation statements)

References 43 publications

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“…1E). Protein identification by mass spectrometry from the immunoprecipitated 160 kDa band matched to the 160 kDa protein of the A. phagocytophilum USG3 strain (Storey et al ., 1998; GenBank Accession number: ) with 25% sequence coverage (Supplementary Fig. S1), rather than to a host cell protein.…”

Section: Resultsmentioning

confidence: 99%

Anaplasma phagocytophilum AnkA secreted by type IV secretion system is tyrosine phosphorylated by Abl-1 to facilitate infection

et al. 2007

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SummaryAnaplasma phagocytophilum, the agent of human granulocytic anaplasmosis, is an obligate intracellular bacterium of granulocytes. A. phagocytophilum specifically induces tyrosine phosphorylation of a 160 kDa protein (P160) in host cells. However, identity of P160, kinases involved, and effects of tyrosine phosphorylation on bacterial infection remain largely unknown. Here, we demonstrated through proteomic analysis that P160, an abundant and rapidly tyrosinephosphorylated protein throughout infection, was AnkA of bacterial origin. Differential centrifugation and confocal microscopy revealed that AnkA was rarely retained within A. phagocytophilum or its inclusion, but localized mainly in the cytoplasm of infected cells. Using Cre recombinase reporter assay of Agrobacterium tumefaciens, we proved that AnkA could be secreted by VirB/D4-dependent type IV secretion (T4S) system. Yeast two-hybrid and coimmunoprecipitation analyses demonstrated that AnkA could bind to Abl-interactor 1 (Abi-1), an adaptor protein that interacts with Abl-1 tyrosine kinase, thus mediating AnkA phosphorylation. AnkA and Abl-1 were critical for bacterial infection, as infection was inhibited upon host cytoplasmic delivery of anti-AnkA antibody, Abl-1 knockdown with targeted siRNA, or treatment with a specific pharmacological inhibitor of Abl-1. These data establish AnkA as the first proven T4S substrate in members of obligate intracellular a-proteobacteria; furthermore, it demonstrated that AnkA plays an important role in facilitating intracellular infection by activating Abl-1 signalling pathway, and suggest a novel approach to treatment of human granulocytic anaplasmosis through inhibition of host cell signalling pathways.

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Section: Resultsmentioning

confidence: 99%

Anaplasma phagocytophilum AnkA secreted by type IV secretion system is tyrosine phosphorylated by Abl-1 to facilitate infection

et al. 2007

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“…The molecular basis of antigenic variation in ehrlichiae is under investigation (2,24). Antigens that have been identified and cloned from the HGE agent indicate that a complex array of proteins may contribute to IFA reactivity (12,14,21,23). A major outer membrane protein antigen that is approximately 44 kDa in molecular size and is encoded by a gene that is part of a multigene family has been identified in protein immunoblots and cloned (2,5,13,16,23).…”

Section: Discussionmentioning

confidence: 99%

Inter- and Intralaboratory Comparison of Ehrlichia equi and Human Granulocytic Ehrlichiosis (HGE) Agent Strains for Serodiagnosis of HGE by the Immunofluorescent-Antibody Test

Walls

Agüero-Rosenfeld

Bakken³

et al. 1999

J Clin Microbiol

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Human granulocytic ehrlichiosis (HGE) is usually diagnosed by immunofluorescent antibody (IFA) serology with Ehrlichia equi-infected neutrophils or HGE agent-infected cultured HL60 cells. The HGE agent and E. equi are antigenically diverse, and interpretation of serologic results is also often variable. Thus, we investigated the sensitivity and specificity of various HGE agent and E. equi antigens used for IFA diagnosis by three different laboratories. Serum samples from 28 patients with well-characterized HGE and 9 patients with suspected HGE who were investigated by PCR, blood smear examinations, and serology were used, along with 9 serum samples from patients with other rickettsial and ehrlichial infections. Each serum sample was tested with up to 10 different antigen preparations. Overall, qualitative IFA results agreed in 70% of the samples. Titers among antigens were similar (r = 0.89 to 0.96), but titers of individual samples varied by fourfold or more in 5 of 81 (6%) of the serum samples. Sensitivity ranged from 100% to 82%, and specificity varied from 100% to 67%, but these differences were not significant, even among those tested in the same laboratory or between two different laboratories. Antibodies were detected in 14 to 44% of acute-phase sera from confirmed HGE patients. Most false-positive reactions resulted with Ehrlichia chaffeensis; when these sera were excluded, the specificity of most antigens was 91 to 100%. These data indicate that IFA results often agree and that IFA is useful for diagnosis of HGE in convalescence. However, without further standardization, variability among serologic tests using E. equi and HGE agent isolates for diagnosis of HGE will occasionally provide discrepant results and confound diagnosis.

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“…1A), among several bacterial proteins recognized by serum from individuals infected with A. phagocytophilum (IJdo et al, 1997). Another bacterial protein recognized by immune sera is AnkA (Storey et al, 1998;Caturegli et al, 2000). Based on the hypothesis that intracellular organisms may interfere with phosphorylation events in the host cell, we examined the phosphorylation of bacterial proteins during infection of HL-60 with A. phagocytophilum.…”

Section: Anaplasma Phagocytophilum Infection Results In Phosphorylatimentioning

confidence: 99%

“…5B). For example, our AnkA sequence (NCH-1 strain) contains one additional EPIYA-A as compared with the one in the USG3 and BDS strains (Storey et al, 1998;Caturegli et al, 2000). Based on the AnkA sequence analysis we investigated if tyrosine phosphorylation occurred at the C-terminus.…”

Section: Anka Contains Epiya Motifs That Are Target Sequences For Tyrmentioning

confidence: 99%

Anaplasma phagocytophilum AnkA is tyrosine-phosphorylated at EPIYA motifs and recruits SHP-1 during early infection

IJdo¹,

2007

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Molecular Cloning and Sequencing of Three GranulocyticEhrlichiaGenes Encoding High-Molecular-Weight Immunoreactive Proteins

Cited by 44 publications

References 43 publications

Anaplasma phagocytophilum AnkA secreted by type IV secretion system is tyrosine phosphorylated by Abl-1 to facilitate infection

Anaplasma phagocytophilum AnkA secreted by type IV secretion system is tyrosine phosphorylated by Abl-1 to facilitate infection

Inter- and Intralaboratory Comparison of Ehrlichia equi and Human Granulocytic Ehrlichiosis (HGE) Agent Strains for Serodiagnosis of HGE by the Immunofluorescent-Antibody Test

Anaplasma phagocytophilum AnkA is tyrosine-phosphorylated at EPIYA motifs and recruits SHP-1 during early infection

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