Multiple p44 Genes Encoding Major Outer Membrane Proteins Are Expressed in the Human Granulocytic Ehrlichiosis Agent (original) (raw)
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Infection and Immunity, 2002
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°C, but their expression levels decreased in the organisms cultivated at 24°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.
Host Cell–Specific Expression of a p44 Epitope by the Human Granulocytic Ehrlichiosis Agent
The Journal of Infectious Diseases, 2001
The human granulocytic ehrlichiosis agent (HGEa) survives extreme differences between ticks and humans, possibly by use of differential expression of specific antigens for survival in different hosts. The role of the immunodominant p44 antigens is unknown. In this study, HGEa cultured in human or tick cells was probed with human, mouse, and hamster serum and with monoclonal antibodies (MAbs). p44 antigens were strongly expressed in human HL-60 cells but were strikingly reduced in tick cells. In HGEa alternately grown in HL-60 or tick cells, a p44 epitope recognized by MAb R5E4 was expressed in human but not tick cells. This was not a temperature effect, because incubation of infected tick cells at 37 C did not induce expression of the p44 epitope. The p44 antigen predominates in human but not tick cells and may be involved in regulatory changes that mediate survival of the HGEa by immune modulation after tick transmission.
Journal of Clinical Microbiology
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 ...
1998
The major outer membrane proteins (OMPs) of the human granulocytic ehrlichiosis (HGE) agent, with molecular sizes of 44 to 47 kDa, are immunodominant antigens in human infection. Monoclonal antibodies (MAbs) to the OMPs were made by immunizing BALB/c mice with the purified HGE agent and then by fusing spleen cells with myeloma cells. The immunologic specificities of three MAbs (3E65, 5C11, and 5D13) were examined with five human HGE agent isolates and one tick isolate. By Western blot analysis, all three MAbs recognized the HGE agent but not Ehrlichia chaffeensis, Ehrlichia sennetsu, Ehrlichia canis, or their host cells. MAb 3E65 reacted with a 44-kDa protein in the homologous human isolate but not in the remaining five isolates. The two remaining MAbs recognized proteins with molecular sizes of 44 to 47 kDa in all six isolates. Western blot results with the OMP fraction of the six isolates were consistent with results with the whole HGE agent. Immunofluorescent-antibody staining and immunogold labeling with these MAbs showed that these antigens were primarily present on the membrane of the HGE agent. MAbs 5C11 and 5D13 recognized the recombinant 44-kDa protein by Western immunoblot analysis, but MAb 3E65 did not. Passive immunization with MAb 3E65 was more effective in protecting mice from HGE agent infection than with MAbs 5C11 and 5D13. These MAbs would be useful for analyzing the role of the major OMP antigens in HGE agent infection and for serodiagnosis.
Journal of clinical microbiology, 1997
The etiologic agent of human granulocytic ehrlichiosis (HGE) is an obligate intracellular bacterium. In 1996, blood specimens from 53 patients suspected of having HGE were examined by indirect fluorescent antibody (IFA) testing with the HGE agent no. 13 isolate as the antigen, by nested PCR, and by culture. All patients resided in Westchester County, N.Y. Twelve patient specimens were positive for IFA (titer > or = 1:40). Seven of these were also positive by PCR. Of the seven specimens positive by both IFA testing and PCR, the HGE agent was isolated from four (no. 2, 3, 6, and 11) and continuously cultured in HL-60 cells. These were confirmed as the HGE agent by sequencing of 16S rDNA. Both purified whole-cell organisms and the outer membrane fractions of the new isolates were compared with no. 13 isolate and a tick (USG) isolate by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblot analysis. No. 11 and 13 isolates had identical SDS-PAGE p...
1998
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.
Clinical and Vaccine Immunology, 2000
Enzyme-linked immunosorbent assay (ELISA) for human granulocytic ehrlichiosis (HGE) using two different recombinant P44 proteins (rP44 and rP44-2hv) of the HGE agent as antigens was evaluated. Sera from a total of 72 healthy humans both from regions where HGE is nonendemic and regions where HGE is endemic were used as negative controls to determine the cutoff value for ELISA. Sera from a total of 14 patients (nine from whom the HGE agent was isolated and five who were HGE-PCR positive) were used as positive controls. One hundred nine sera from 72 patients in an area where HGE is endemic who were suspected of having HGE were examined by ELISA and indirect immunofluorescence assay (IFA). All IFA-negative sera were negative by both ELISAs. Of 39 sera that were IFA positive, 35 and 27 were positive by ELISA using rP44 and rP44-2hv, respectively, indicating that the use of rP44 is more sensitive. Western blot analysis of the four rP44-ELISA-negative IFA-positive sera using whole HGE agen...
Journal of Infectious Diseases, 1996
Human granulocytic ehrlichiosis (HGE) is a tickborne illness caused by an agent closely related to Ehrlichia equi and Ehrlichia phagocytophila. The clinical presentation is nonspecific, and diagnosis is made infrequently. Sixty-six sera were obtained from 54 patients in Minnesota and Wisconsin with undifferentiated fever during the summer and fall of 1993. Serologic reactivity with E. equi was observed in 6 (11 %), including 4 seroconversions, 1 stable titer, and 1 seroreversion. Of the seropositive patients, HGE agent DNA was detected by polymerase chain reaction in the first serum from 4 of 5 patients and was present in the serum of 1 of 2 untreated patients after 21 days, even when E. equi antibodies also were present. HGE is a significant and potentially frequent cause of undifferentiated fever in Wisconsin and Minnesota during seasons with tick activity. The agent may persist in untreated patients for at least 1 month or may be cleared earlier, even if not treated with doxycycline or tetracycline.
The American Journal of Tropical Medicine and Hygiene, 1999
Immunodominant proteins in the range of 42-45 kD are important for the serodiagnosis of human granulocytic ehrlichiosis (HGE). Antigens from human isolates of the etiologic agent of HGE cultivated in HL-60 cells were used to immunize BALB/c mice and generate a panel of hybridomas secreting monoclonal antibodies. Using an enzyme immunoassay, an immunofluorescent assay (IFA), and Western blotting, we showed that culture supernatants and ascites of these hybridomas were reactive with human isolates of the etiologic agent of HGE, Ehrlichia equi and E. phagocytophila. Following screening and subcloning, we selected three stable hybridomas, R1B10, R5E4, and R5A9, which were determined to be of the isotypes IgG 3 , IgG 1 , and IgG 2a , respectively. These results suggest that the epitopes of the 42-45-kD protein recognized by these three monoclonal antibodies are conserved among E. equi, E. phagocytophila, and the etiologic agent of HGE. Western blot analysis showed reactivity with the 44-kD protein of human isolates of the HGE agent. None of the monoclonal antibodies were reactive with HL-60 cells that were not infected with the HGE agent. No cross-reactivity with related intracellular pathogens could be detected when undiluted supernatants from hybridoma cultures were allowed to react by IFA with antigens from E. chaffeensis, E. risticii, E. platys, Rickettsia rickettsii, R. prowazekii, or Coxiella burnetii. The additivity index of two antibodies, R5E4 and R1B10 was near zero, suggesting that these two antibodies may compete for the same epitope of the 44-kD protein, while monoclonal antibody R5A9 appears to interact with a different epitope. The antibodies secreted by these hybridomas may be useful as immunologic agents in serodiagnostic, immunohistochemical, and other studies of the etiologic agent of HGE.
Journal of Clinical Microbiology, 2001
Laboratory diagnosis of human ehrlichioses is routinely made by an indirect immunofluorescence assay (IFA) using cultured ehrlichia-infected whole cells as antigen. Concern has been raised that incorrect diagnoses of human monocytic ehrlichiosis (HME) or human granulocytic ehrlichiosis (HGE) may be made on the basis of serologic cross-reactivity between Ehrlichia chaffeensis and the agent of HGE. The present study examined whether two recombinant major outer membrane proteins, rP30 and rP44, that were previously shown to be sensitive and specific serodiagnostic antigens for HME and HGE, respectively, could be used to discriminate IFA dually reacting sera. Thirteen dually IFA-reactive sera, three sera that were IFA positive only with E. chaffeensis, and three sera that were IFA positive only with the HGE agent were examined by Western immunoblot analysis using purified whole organisms and recombinant proteins as antigens. All 16 E. chaffeensis IFA-positive sera reacted with rP30. However, none of these sera reacted with rP44, regardless of IFA reactivity with the HGE agent. The three HGE-agent-only IFA-positive sera reacted only with rP44, not with rP30. Western immunoblotting using purified E. chaffeensis and the HGE agent as antigens suggested that heat shock and other proteins, but not major outer membrane proteins, cross-react between the two organisms. Therefore, Western immunoblot analysis using rP44 and rP30 may be useful in discriminating dually HME and HGE IFA-reactive sera.