Proteomic analysis of and immune responses to Ehrlichia chaffeensis lipoproteins

Ehrlichia chaffeensis is an obligately intracellular gram-negative bacterium and is the etiologic agent of human monocytic ehrlichiosis (HME). Although E. chaffeensis induces the generation of several cytokines and chemokines by leukocytes, E. chaffeensis lacks lipopolysaccharide and peptidoglycan. Bioinfomatic analysis of the E. chaffeensis genome, however, predicted genes encoding 15 lipoproteins and 3 posttranslational lipoprotein-processing enzymes. The present study showed that by use of multidimensional liquid chromatography followed by tandem mass spectrometry, all predicted lipoproteins as well as lipoprotein-processing enzymes were expressed by E. chaffeensis cultured in the human promyelocytic leukemia cell line HL-60. Consistent with this observation, a signal peptidase II inhibitor, globomycin, was found to inhibit E. chaffeensis infection and lipoprotein processing in HL-60 cell culture. To study in vivo E. chaffeensis lipoprotein expression and host immune responses to E. chaffeensis lipoproteins, 13 E. chaffeensis lipoprotein genes were cloned into a mammalian expression vector. When the DNA constructs were inoculated into naïve dogs, or when dogs were infected with E. chaffeensis, the animals developed delayed-type hypersensitivity reactions at cutaneous sites of the DNA construct deposition and serum antibodies to these lipoproteins. This is the first demonstration of lipoprotein expression and elicitation of immune responses by a member of the order Rickettsiales. Multiple lipoproteins expressed by E. chaffeensis in vitro and in vivo may play key roles in pathogenesis and immune responses in HME.     

Delayed clearance of Ehrlichia chaffeensis infection in CD4+ T-cell knockout mice

Human monocytic ehrlichiosis is an emerging tick-borne disease caused by the rickettsia Ehrlichia chaffeensis. To examine the role of helper T cells in host resistance to this macrophage-tropic bacterium, we assessed E. chaffeensis infections in three mouse strains with differing functional levels of helper T cells. Wild-type, C57BL/6J mice resolved infections in approximately 2 weeks. Major histocompatibility complex class II (MHCII) knockout, B6.129-Abb(tm1) mice lacking helper T cells developed persistent infections that were not resolved even after several months. CD4+ T-cell-deficient, B6.129S6-Cd4(tm1Knw) mice cleared the infection, but the clearance took 2 weeks longer than it did for wild-type mice. C57BL/6J mice resolved infection more rapidly following a second experimental challenge, but B6.129S6-Cd4(tm1Knw) mice did not. The B6.129S6-Cd4(tm1Knw) mice also developed active E. chaffeensis-specific immunoglobulin G responses that were slightly lower in concentration and slower to develop than that observed in C57BL/6J mice. E. chaffeensis-specific cytotoxic T cells were not detected following a single bacterial challenge in any mouse strain, including wild-type C57BL/6J mice. However, the cytotoxic T-cell activity developed in all three mouse strains, including the MHCII and CD4+ T-cell knockouts, when challenged with a second E. chaffeensis infection. The data reported here suggest that the cell-mediated immunity, orchestrated by CD4+ T cells is critical for conferring rapid clearance of E. chaffeensis.     

Ehrlichia chaffeensis expresses macrophage- and tick cell-specific 28-kilodalton outer membrane proteins

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.     

Total, membrane, and immunogenic proteomes of macrophage- and tick cell-derived Ehrlichia chaffeensis evaluated by liquid chromatography-tandem mass spectrometry and MALDI-TOF methods

Ehrlichia chaffeensis, a tick-transmitted rickettsial, is the causative agent of human monocytic ehrlichiosis. To examine protein expression patterns, we analyzed total, membrane, and immunogenic proteomes of E. chaffeensis originating from macrophage and tick cell cultures. Total proteins resolved by one-dimensional gel electrophoresis and subjected to liquid chromatography-electrospray ionization ion trap mass spectrometry allowed identification of 134 and 116 proteins from macrophage- and tick cell-derived E. chaffeensis, respectively. Because a majority of immunogenic proteins remained in the membrane fraction, individually picked total and immunogenic membrane proteins were also surveyed by liquid chromatography-tandem mass spectrometry and matrix-assisted laser desorption ionization-time of flight methods. The analysis aided the identification of 48 additional proteins. In all, 278 genes of the E. chaffeensis genome were verified as functional genes. They included genes for DNA and protein metabolism, energy metabolism and transport, membrane proteins, hypothetical proteins, and many novel proteins of unknown function. The data reported in this study suggest that the membrane of E. chaffeensis is very complex, having many expressed proteins. This study represents the first and the most comprehensive analysis of E. chaffeensis-expressed proteins. This also is the first study confirming the expression of nearly one-fourth of all predicted genes of the E. chaffeensis genome, validating that they are functionally active genes, and demonstrating that classic shotgun proteomic approaches are feasible for tick-transmitted intraphagosomal bacteria. The identity of novel expressed proteins reported in this study, including the large selection of membrane and immunogenic proteins, will be valuable in elucidating pathogenic mechanisms and developing effective prevention and control methods.     

Serological responses to Ehrlichia equi, Ehrlichia chaffeensis, and Borrelia burgdorferi in patients from New York State.

Serological testing at the New York State Department of Health for human granulocytic ehrlichiosis in the residents of Westchester County, N.Y., was performed with specimens from 176 patients by the indirect fluorescent-antibody (IFA) technique with Ehrlichia equi MRK-infected neutrophils. To understand whether human monocytotropic ehrlichiosis also occurs in this northeastern geographic region, specimens were also tested for antibodies to Ehrlichia chaffeensis Arkansas. Screening tests and immunoblots for Lyme disease (Borrelia burgdorferi infection) were also performed. Thirty-two patients had antibodies only to E. equi and 21 patients had antibodies to both E. equi and E. chaffeensis, whereas 12 patients had only E. chaffeensis antibodies by the IFA technique. The remaining patients did not have antibodies to either ehrlichia. Eighteen serum samples from 13 of these patients were coded and sent to the Ehrlichia Research Laboratory (Baltimore, Md.) for repeat analysis by the IFA test and for E. equi and E. chaffeensis immunoblots. Immunoblot analysis for E. equi in samples with positive IFA test results confirmed the results for eight of the nine specimens. Immunoblot analyses for E. chaffeensis were negative for all 18 serum samples. Borrelia-reactive antibodies were found in sera both from patients with granulocytic ehrlichiosis and from patients with monocytotropic ehrlichiosis from New York State. Our results suggest that E. equi antigen is an appropriate substrate for identifying human granulocytic ehrlichiosis. E. chaffeensis antigen lacks appropriate sensitivity to serve as a surrogate substrate for the detection of human granulocytic ehrlichiosis and should be used solely for the diagnosis of human monocytotropic ehrlichiosis. Heat shock proteins may, in some cases, cause cross-reactivity between B. burgdorferi and ehrlichiae.     

New Ehrlichia species closely related to Ehrlichia chaffeensis isolated from Ixodes ovatus ticks in Japan

Seven Ehrlichia strains (six HF strains and one Anan strain) that were obtained from laboratory mice by intraperitoneally inoculating homogenates of adult Ixodes ovatus collected in Japan were characterized. 16S rRNA sequences of all six HF strains were identical, and the sequences were 99.7, 98.2, and 97.7% identical to those of Anan strain, Ehrlichia chaffeensis (human monocytic ehrlichiosis agent), and E. muris, respectively. Partial GroEL amino acid sequencing also revealed that the six HF strains had identical sequences, which were 99.0, 98.5, and 97.3% identical to those of E. chaffeensis, the Anan strain, and E. canis, respectively. All HF strains were lethal to mice at higher dosages and intraperitoneal inoculation, whereas the Anan or E. muris strain induced only mild clinical signs. Light and electron microscopy of moribund mice inoculated with one of the HF strains revealed severe liver necrosis and the presence of numerous ehrlichial inclusions (morulae) in various organs. The study revealed that members of E. canis genogroup are naturally present in Ixodes ticks. HF strains that can cause severe illness in immunocompetent laboratory mice would be valuable in studying the pathogenesis and the roles of both cellular and humoral immune responses in ehrlichiosis caused by E. canis genogroup.     

The omp-1 major outer membrane multigene family of Ehrlichia chaffeensis is differentially expressed in canine and tick hosts

Sixteen of 22 omp-1 paralogs encoding 28-kDa-range immunodominant outer membrane proteins of Ehrlichia chaffeensis were transcribed in blood monocytes of dogs throughout a 56-day infection period. Only one paralog was transcribed by E. chaffeensis in three developmental stages of Amblyomma americanum ticks before or after E. chaffeensis transmission to na?ve dogs.     

Ehrlichia chaffeensis and Rochalimaea antibodies in Kawasaki disease.

Sera from 38 patients with Kawasaki disease were tested for immunofluorescent antibodies to Ehrlichia chaffeensis, Rochalimaea henselae, and R. quintana Oklahoma. Only 2.5% of the patients tested positive for E. chaffeensis, and 5% were positive for R. henselae and R. quintana Oklahoma. Our data suggest that Ehrlichia and Rochalimaea spp. do not play a unique role in the etiology of Kawasaki disease.     

Ehrlichial proliferation and acute hepatocellular necrosis in immunocompetent mice experimentally infected with the HF strain of Ehrlichia, closely related to Ehrlichia chaffeensis

Ehrlichia chaffeensis, the agent of human monocytic ehrlichiosis, is closely related to the HF strain of Ehrlichia isolated from ticks in Japan. In this study, BALB/c mice inoculated intraperitoneally with the HF strain developed severe illness and died at about day 9 post-inoculation. At necropsy, diffuse liver necrosis was evident. Ehrlichial microcolonies were observed in endothelial cells, monocytes and macrophages of the liver, bone marrow, spleen, thymus, and large and small intestine. Immunocompetent mice infected with the HF strain would provide a useful model for studying pathogenesis and immunity in acute and severe ehrlichiosis caused by E. chaffeensis and related Ehrlichia spp. Copyright Harcourt Publishers Ltd.     

Western immunoblotting analysis of the antibody responses of patients with human monocytotropic ehrlichiosis to different strains of Ehrlichia chaffeensis and Ehrlichia canis.

In order to evaluate the relative sensitivity of the detection of antibodies against various antigenic proteins of Ehrlichia chaffeensis for the diagnosis of the emerging infectious disease human monocytotropic ehrlichiosis, Western immunoblotting was performed with 27 serum samples from convalescent patients with antibodies, as demonstrated by indirect immunofluorescence assay. Among 22 patients with antibodies reactive with the 120-kDa protein, 15 showed reactivity with the 29/28-kDa protein(s) and the proteins in the 44- to 88-kDa range. Two of the serum samples with this pattern reacted with the 29/28-kDa protein(s) of only the 91HE17 strain, and one sample reacted with only that of the Arkansas strain, indicating that the antibodies were stimulated by strain-specific epitopes. Overall, antibodies to the 29/28-kDa protein(s) were detected in only 16 patients' sera, suggesting that this protein is less sensitive than the 120-kDa protein. Two of 12 serum samples from healthy blood donors had antibodies reactive with the 120-kDa protein; one of these samples reacted also with the 29/28-kDa protein(s) of Ehrlichia canis, suggesting that unrecognized ehrlichial infection might have occurred, including human infection with E. canis. A high correlation between reactivity with the 120-kDa protein by Western immunoblotting and the recombinant 120-kDa protein by dot blot supports the potential usefulness of this recombinant antigen in diagnostic serology.     

The in situ immune response in popliteal lymph nodes of mice after macrophage depletion. Differential effects of macrophages on thymus-dependent and thymus-independent immune responses

Mice were subcutaneously (SC) injected in the left hind footpad with dichloromethylene diphosphonate (Cl2MDP)-containing liposomes to eliminate macrophages lining the subcapsular sinus (SCS) and those in the medulla of draining popliteal lymph nodes (PLN). In order to study the effect of depletion of these macrophages on the in situ immune response in the PLN, liposome-treated mice were SC injected in the same footpad with thymus-independent (TI) type 1 antigen trinitrophenylated lipopolysaccharide (TNP-LPS), TI-type 2 antigen TNP-Ficoll or thymus-dependent (TD) antigen TNP-keyhole limpet haemocyanin (TNP-KLH). No major differences were observed in antibody-serum titers of liposome-treated and control animals. After primary as well as secondary immunization with the TD-antigen TNP-KLH, an increase in the number of antibody-forming cells (AFC) was found and the peak of response was delayed in the PLN of liposome-treated animals. Such differences were not observed with the TI-antigens. These results indicate that macrophages lining the SCS and those in the medulla of the PLN are not essential for the induction of an immune response. The positive effect of macrophage-depletion on the number of AFC may be explained by competition for the antigen by macrophages and other antigen-presenting cells.     

Adaptation of Ehrlichia sennetsu to canine blood monocytes: preliminary structural and serological studies with cell culture-derived Ehrlichia sennetsu.

Ehrlichia sennetsu, the causative agent of human sennetsu rickettsiosis, was successfully propagated in primary canine blood monocyte cultures. The growth cycle of this organism appears to be similar to that of Ehrlichia canis. The antigen derived from our E. sennetsu cultures was used to develop an indirect fluorescent antibody test for detection and titration of serum antibodies to the organism. Using this test system, we found that five human serum samples obtained from patients clinically diagnosed as having sennetsu rickettsiosis were positive for anti-E. sennetsu antibodies. In addition, 29% of the serum samples obtained from 200 patients having a fever of unknown origin and residing in various regions of Malaysia were also serologically positive. All sera from apparently healthy individuals were negative in the test. Dogs inoculated with cell culture-adapted E. sennetsu developed a significant specific antibody titer to E. sennetsu, and the organism was subsequently isolated from their blood. These animals showed no clinical evidence of disease. The possibility of a higher prevalence of human sennetsu rickettsiosis in Southeast Asia and the potential usefulness of the canine model for studies of human sennetsu rickettsiosis are discussed.     

A role of macrophage complement receptor CRIg in immune clearance and inflammation

Complement receptor of the immunoglobulin superfamily (CRIg), also referred to as Z39Ig and V-set and Ig domain-containing 4 (VSIG4), has recently been implicated in the clearance of systemic pathogens and autologous cells. CRIg is exclusively expressed on tissue resident macrophages and binds to multimers of C3b and iC3b that are covalently attached to particle surfaces. Next to functioning as an important clearance receptor, CRIg's extracellular domain inhibits complement activation through the alternative, but not the classical, pathway, providing a novel tool to selectively block this pathway in vivo. Here, we review a role for CRIg in immune clearance, T-cell responses and complement regulation, and discuss the implications for disease manifestation.     

A role of macrophage complement receptor CRIg in immune clearance and inflammation

Complement receptor of the immunoglobulin superfamily (CRIg), also referred to as Z39Ig and V-set and Ig domain-containing 4 (VSIG4), has recently been implicated in the clearance of systemic pathogens and autologous cells. CRIg is exclusively expressed on tissue resident macrophages and binds to multimers of C3b and iC3b that are covalently attached to particle surfaces. Next to functioning as an important clearance receptor, CRIg's extracellular domain inhibits complement activation through the alternative, but not the classical, pathway, providing a novel tool to selectively block this pathway in vivo. Here, we review a role for CRIg in immune clearance, T-cell responses and complement regulation, and discuss the implications for disease manifestation.     

Tyrosine-phosphorylated Ehrlichia chaffeensis and Ehrlichia canis tandem repeat orthologs contain a major continuous cross-reactive antibody epitope in lysine-rich repeats

A small subset of major immunoreactive proteins have been identified in Ehrlichia chaffeensis and Ehrlichia canis, including three molecularly and immunologically characterized pairs of immunoreactive tandem repeat protein (TRP) orthologs with major continuous species-specific epitopes within acidic tandem repeats (TR) that stimulate strong antibody responses during infection. In this study, we identified a fourth major immunoreactive TR-containing ortholog pair and defined a major cross-reactive epitope in homologous nonidentical 24-amino-acid lysine-rich TRs. Antibodies from patients and dogs with ehrlichiosis reacted strongly with recombinant TR regions, and epitopes were mapped to the N-terminal TR region (18 amino acids) in E. chaffeensis and the complete TR (24 amino acids) in E. canis. Two less-dominant epitopes were mapped to adjacent glutamate/aspartate-rich and aspartate/tyrosine-rich regions in the acidic C terminus of E. canis TRP95 but not in E. chaffeensis TRP75. Major immunoreactive proteins in E. chaffeensis (75-kDa) and E. canis (95-kD) whole-cell lysates and supernatants were identified with TR-specific antibodies. Consistent with other ehrlichial TRPs, the TRPs identified in ehrlichial whole-cell lysates and the recombinant proteins migrated abnormally slow electrophoretically a characteristic that was demonstrated with the positively charged TR and negatively charged C-terminal domains. E. chaffeensis TRP75 and E. canis TRP95 were immunoprecipitated with anti-pTyr antibody, demonstrating that they are tyrosine phosphorylated during infection of the host cell.     

Cellular immune responses to the murine nematode parasite Trichuris muris. II. Differential induction of TH-cell subsets in resistant versus susceptible mice.

We have analysed the production of a wide variety of cytokines by in vitro concanavalin A (Con A) stimulated mesenteric lymph node cells (MLNC) from strains of mice experiencing chronic (B10.BR, AKR) versus acute (BALB/K) infection with the nematode parasite Trichuris muris. MLNC from infected BALB/K mice produced elevated levels of the Th2-specific cytokines interleukin-5 (IL-5) and IL-9. IL-3 and IL-4 remained at or just above normal. Interferon-gamma (IFN-gamma) (a Th1-type cytokine) was secreted only in small amounts. MLNC from infected susceptible B10.BR and AKR mice produced large amounts of IFN-gamma in the relative absence of IL-4 and IL-9. IL-5 levels failed to rise significantly above normal in B10.BR mice whilst in AKR mice high levels of IL-5 were detected early post-infection (p.i.) but levels decreased dramatically as the infection proceeded to reach normal levels by Day 34. IL-3 levels were depressed below normal. Our results are consistent with the polarization of the Th-cell response during T. muris infection to give predominantly IFN-gamma-secreting Th1 cells in strains of mice unable to expel the parasite and mainly IL-4, IL-5 and IL-9 producing Th2-type cells in resistant strains.     

Differential immune responses to primary measles-mumps-rubella vaccination in Israeli children

Measles remains an important cause of morbidity and mortality worldwide, primarily due to problems associated with delivery of the live attenuated vaccine to susceptible populations. In some developed countries, there is concern about the effects of immunization on the immune system. In this study, we analyzed the responses of 12-month-old Bedouin and Jewish children living in Israel to routine measles-mumps-rubella (MMR) vaccination. Seroconversion to measles was 99% in Bedouin and 79% in Jewish children (P < 0.01), and that to mumps and rubella was 92 to 100% in both groups. Measles neutralizing antibody titers were higher in Bedouin (333 +/- 39 mIU/ml) than Jewish (122 +/- 60 mIU/ml) children (P < 0.002). Immunoglobulin G levels were higher in Bedouin than Jewish children (P = 0.007) and increased after vaccination (P = 0.0009). Leukocyte (P < 0.02) and lymphocyte (P = 0.04) counts were higher and CD4 lymphocyte percentages were lower (P < 0.001) in Bedouin than Jewish children before and after vaccination. Leukocyte counts and natural killer cell numbers did not change after vaccination, but lytic activity increased in Bedouin children (P < 0.005). Spontaneous proliferation of cultured peripheral blood mononuclear cells increased with vaccination, but there were no changes in the proliferative responses to phytohemagglutinin or tetanus toxoid. In summary, no adverse effects of MMR vaccination on immune function were detected. However, there were differences in underlying immunologic parameters and in response to the measles component of the vaccine between Bedouin and Jewish children. It is not known whether genetic differences or environmental exposure accounts for these differences.