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PubMed2004

Glycosylation of Anaplasma marginale major surface protein 1a and its putative role in adhesion to tick cells.

Anaplasma marginale, the causative agent of bovine anaplasmosis, is a tick-borne rickettsial pathogen of cattle that multiplies in erythrocytes and tick cells. Major surface protein 1a (MSP1a) and MSP1b form the MSP1 complex of A. marginale, which is involved in adhesion of the pathogen to host cells. In this study we tested the hypothesis that MSP1a and MSP1b were glycosylated, because the observed molecular weights of both proteins were greater than the deduced molecular masses. We further hypothesized that the glycosylation of MSP1a plays a role in adhesion of A. marginale to tick cells. Native and Escherichia coli-derived recombinant MSP1a and MSP1b proteins were shown by gas chromatography to be glycosylated and to contain neutral sugars. Glycosylation of MSP1a appeared to be mainly O-linked to Ser/Thr residues in the N-terminal repeated peptides. Glycosylation may play a role in adhesion of A. marginale to tick cells because chemical deglycosylation of MSP1a significantly reduced its adhesive properties. Although the MSP1a polypeptide backbone alone was adherent to tick cell extract, the glycans in the N-terminal repeats appeared to enhance binding and may cooperatively interact with one or more surface molecules on host cells. These results demonstrated that MSP1a and MSP1b are glycosylated and suggest that the glycosylation of MSP1a plays a role in the adhesion of A. marginale to tick cells.

Amino Acid Sequence↗
PubMed2004

Anaplasma phagocytophilum infection induces protracted neutrophil degranulation.

Anaplasma phagocytophilum-infected neutrophil degranulation could exacerbate inflammation. Thus, the degranulation of infected neutrophils was assayed. Infected neutrophils expressed CD11b and CD66b, and supernatants of infected neutrophils showed more proMMP-9 and MMP-9 activity than controls and continued to do so for > or =18 h. Degranulation-related inflammatory tissue injury may account for some clinical manifestations in human granulocytic anaplasmosis.

Anaplasma phagocytophilum↗
PubMed2004

CD4+ T lymphocytes from Anaplasma marginale major surface protein 2 (MSP2) vaccinees recognize naturally processed epitopes conserved in MSP3.

Major surface protein 2 (MSP2) and MSP3 of the persistent bovine ehrlichial pathogen Anaplasma marginale are immunodominant proteins that undergo antigenic variation. The recently completed sequence of MSP3 revealed blocks of amino acids in the N and C termini that are conserved with MSP2. This study tested the hypothesis that CD4+ T cells specific for MSP2 recognize naturally processed epitopes conserved in MSP3. At least one epitope in the N terminus and two in the C terminus of MSP2 were also processed from MSP3 and presented to CD4+ T lymphocytes from MSP2-immunized cattle. This T-lymphocyte response to conserved and partially conserved epitopes may contribute to the immunodominance of MSP2 and MSP3.

Amino Acid Sequence↗
PubMed2004

Reinfection with Anaplasma phagocytophilum in BALB/c mice and cross-protection between two sympatric isolates.

Infection with Anaplasma phagocytophilum in white-footed mice results in partial protection against reinfection with the same agent. However, humans and domestic animals may be sequentially exposed to different isolates of the agent circulating in the same or adjacent foci. We investigated whether immune response to a tick-borne infection with A. phagocytophilum provides protection against homologous and heterologous challenges. BALB/c mice were infected with one of the two sympatric isolates of A. phagocytophilum via tick bite and challenged 16 weeks later by Ixodes scapularis nymphs infected with either the same or the alternative isolate. As controls, groups of infected mice were challenged by uninfected ticks to confirm an absence of reactivation of the original infection or groups of naive mice were fed upon by ticks from cohorts used for an infectious challenge. Xenodiagnostic I. scapularis larvae were fed upon each mouse at 14 and 21 days postchallenge (PCH) and tested for the presence of A. phagocytophilum as freshly molted nymphs. Blood samples for quantitative PCR were collected at 7, 14, 21, and 70 days PCH. Serum samples were collected weekly to monitor development of immune response. The proportion of infected animals, levels of bacteremia, and the prevalence of infection in xenodiagnostic ticks were higher in groups of control mice exposed to A. phagocytophilum for the first time than in mice reinfected with either homologous or heterologous isolates. The presence of antibodies against A. phagocytophilum did not protect mice from a challenge with either homologous or heterologous isolates, however the ensuing reinfection was significantly milder and of a shorter duration than the first infection with either isolate.

Anaplasma phagocytophilum↗
PubMed2004

Neutrophil NADPH oxidase is reduced at the Anaplasma phagocytophilum phagosome.

The intracellular organism Anaplasma phagocytophilum causes human granulocytic ehrlichiosis and specifically infects and multiplies in neutrophilic granulocytes. Previous reports have suggested that, for its survival, this bacterium suppresses the neutrophil respiratory burst. To investigate the mechanism of survival, we first assessed the kinetics of A. phagocytophilum entry into neutrophils by using double-labeling confocal microscopy. At 30, 60, 120, and 240 min of incubation, 25, 50, 55, and 70% of neutrophils contained bacteria, respectively. The neutrophil respiratory burst in the presence of A. phagocytophilum was assessed by a kinetic cytochrome c assay and by measurement of oxygen consumption. Neutrophils in the presence of A. phagocytophilum did not produce a significant respiratory burst, but A. phagocytophilum did not inhibit the neutrophil respiratory burst when phorbol myristate acetate was added. Immunoelectron microscopy of neutrophils infected with A. phagocytophilum or Escherichia coli revealed that NADPH oxidase subunits gp91(phox) and p22(phox) were significantly reduced at the A. phagocytophilum phagosome after 1 and 4 h of incubation. In neutrophils incubated simultaneously with A. phagocytophilum and E. coli for 30, 60, and 90 min, gp91(phox) was present on 20, 14, and 10% of the A. phagocytophilum phagosomes, whereas p22(phox) was present in 11, 5, and 4% of the phagosomes, respectively. Similarly, on E. coli phagosomes, gp91(phox) was present in 62, 64, and 65%, whereas p22(phox) was detected in 54, 48, and 48%. We conclude that A. phagocytophilum does not suppress a global respiratory burst and that, under identical conditions in the same cells, A. phagocytophilum, but not E. coli, significantly reduces gp91(phox) and p22(phox) from its phagosome membrane.

Anaplasma phagocytophilum↗
PubMed2005

Anaplasma phagocytophilum modulates gp91phox gene expression through altered interferon regulatory factor 1 and PU.1 levels and binding of CCAAT displacement protein.

Infection of neutrophil precursors with Anaplasma phagocytophilum, the causative agent of human granulocytic ehrlichiosis, results in downregulation of the gp91(phox) gene, a key component of NADPH oxidase. We now show that repression of gp91(phox) gene transcription is associated with reduced expression of interferon regulatory factor 1 (IRF-1) and PU.1 in nuclear extracts of A. phagocytophilum-infected cells. Loss of PU.1 and IRF-1 correlated with increased binding of the repressor, CCAAT displacement protein (CDP), to the promoter of the gp91(phox) gene. Reduced protein expression of IRF-1 was observed with or without gamma interferon (IFN-gamma) stimulation, and the defect in IFN-gamma signaling was associated with diminished binding of phosphorylated Stat1 to the Stat1 binding element of the IRF-1 promoter. The diminished levels of activator proteins and enhanced binding of CDP account for the transcriptional inhibition of the gp91(phox) gene during A. phagocytophilum infection, providing evidence of the first molecular mechanism that a pathogen uses to alter the regulation of genes that contribute to an effective respiratory burst.

Anaplasma phagocytophilum↗
PubMed2005

Effects of Anaplasma phagocytophilum on host cell ferritin mRNA and protein levels.

Ferritin is a major intracellular iron storage protein and also functions as a cytoprotectant by sequestering iron to minimize the formation of reactive oxygen species. Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis, is an obligate intracellular bacterium that colonizes neutrophils. We have previously reported that human promyelocytic HL-60 cells infected with A. phagocytophilum demonstrate increased transcription of ferritin heavy chain and also that the bacterium stimulates neutrophil NADPH oxidase assembly and degranulation during the initial hours of infection (J. A. Carlyon, W. T. Chan, J. Galan, D. Roos, and E. Fikrig, J. Immunol. 169:7009-7018, 2002, and J. A. Carlyon, D. Abdel-Latif, M. Pypaert, P. Lacy, and E. Fikrig, Infect. Immun. 72:4772-4783, 2004). In this study, we assessed ferritin mRNA and protein levels during A. phagocytophilum infection in vitro using HL-60 cells and neutrophils and in vivo using neutrophils from infected mice. The addition of A. phagocytophilum, as well as Escherichia coli and serum-opsonized zymosan, to neutrophils results in a pronounced increase in ferritin light-chain transcription and a concomitant rise in ferritin protein levels. Neutrophils from A. phagocytophilum-infected mice demonstrate elevated ferritin heavy-chain mRNA expression, a phenomenon consistent with infections by intracellular pathogens. Notably, ferritin protein levels of infected HL-60 cells were markedly diminished in a dose- and time-dependent manner. These studies provide insight into the effects A. phagocytophilum has on the ferritin levels of its host cell.

Anaplasma phagocytophilum↗
PubMed2005

Early transcriptional response of human neutrophils to Anaplasma phagocytophilum infection.

Anaplasma phagocytophilum, an unusual obligate intracellular pathogen that persists within neutrophils, causes human anaplasmosis (previously known as human granulocytic ehrlichiosis). To study the effects of this pathogen on the transcriptional profile of its host cell, we performed a comprehensive DNA microarray analysis of the early (4-h) transcriptional response of human neutrophils to A. phagocytophilum infection. A. phagocytophilum infection resulted in the up- and down-regulation of 177 and 67 neutrophil genes, respectively. These data were verified by quantitative reverse transcription-PCR of selected genes. Notably, the up-regulation of many antiapoptotic genes, including the BCL2A1, BIRC3, and CFLAR genes, and the down-regulation of the proapoptotic TNFSF10 gene were observed. Genes involved in inflammation, innate immunity, cytoskeletal remodeling, and vesicular transport also exhibited differential expression. Vascular endothelial growth factor was also induced. These data suggest that A. phagocytophilum may alter selected host pathways in order to facilitate its survival within human neutrophils. To gain further insight into the bacterium's influence on host cell gene expression, this report presents a detailed comparative analysis of our data and other gene expression profiling studies of A. phagocytophilum-infected neutrophils and promyelocytic cell lines.

Anaplasma phagocytophilum↗
PubMed2005

Identification of novel antigenic proteins in a complex Anaplasma marginale outer membrane immunogen by mass spectrometry and genomic mapping.

Immunization with purified Anaplasma marginale outer membranes induces complete protection against infection that is associated with CD4+ T-lymphocyte-mediated gamma interferon secretion and immunoglobulin G2 (IgG2) antibody titers. However, knowledge of the composition of the outer membrane immunogen is limited. Recent sequencing and annotation of the A. marginale genome predicts at least 62 outer membrane proteins (OMP), enabling a proteomic and genomic approach for identification of novel OMP by use of IgG serum antibody from outer membrane vaccinates. Outer membrane proteins were separated by two-dimensional electrophoresis, and proteins recognized by total IgG and IgG2 in immune sera of outer membrane-vaccinated cattle were detected by immunoblotting. Immunoreactive protein spots were excised and subjected to liquid chromatography-tandem mass spectrometry. A database search of the A. marginale genome identified 24 antigenic proteins that were predicted to be outer membrane, inner membrane, or membrane-associated proteins. These included the previously characterized surface-exposed outer membrane proteins MSP2, operon associated gene 2 (OpAG2), MSP3, and MSP5 as well as recently identified appendage-associated proteins. Among the 21 newly described antigenic proteins, 14 are annotated in the A. marginale genome and include type IV secretion system proteins, elongation factor Tu, and members of the MSP2 superfamily. The identification of these novel antigenic proteins markedly expands current understanding of the composition of the protective immunogen and provides new candidates for vaccine development.

Anaplasma marginale↗
PubMed2005

Anaplasma phagocytophilum delay of neutrophil apoptosis through the p38 mitogen-activated protein kinase signal pathway.

Human granulocytic anaplasmosis is caused by the obligate intracellular bacterium Anaplasma phagocytophilum. The bacterium avoids host innate defenses in part by infecting, surviving in, and propagating in neutrophils, as well as by inhibiting neutrophil apoptosis. However, the mechanisms of A. phagocytophilum survival in neutrophils and the inhibition of spontaneous apoptosis are not well understood. In this study, we demonstrated that antiapoptotic Mcl-1 protein (Bcl-2 family) expression is maintained and that inhibition of procaspase-3 processing occurs in A. phagocytophilum-infected human neutrophils. An evaluation of p38 mitogen-activated protein kinase (MAPK) showed evidence of increased phosphorylation with infection. Moreover, antagonism of p38 MAPK by the inhibitor SB203580 reversed apoptosis inhibition in live or heat-killed A. phagocytophilum-infected neutrophils. A role for the autocrine or paracrine production of antiapoptotic interleukin 8 (IL-8) expressed with A. phagocytophilum infection was excluded by the use of IL-8-, IL-8R1 (CXCR1)-, and IL-8R2 (CXCR2)-blocking antibodies. As previously demonstrated, the antiapoptotic effect was initially mediated by exposure to A. phagocytophilum components in heat-killed bacteria. However, an important role for active infection is demonstrated by the additional delay in apoptosis with intracellular growth and the refractory abrogation of this response by the p38 MAPK inhibitor 3 to 6 h after neutrophil infection. These results suggest that the initial activation of the p38 MAPK pathway leading to A. phagocytophilum-delayed neutrophil apoptosis is bypassed with active intracellular infection. Moreover, active intracellular infection contributes more to the overall delay in apoptosis than do components of heat-killed A. phagocytophilum alone.

Anaplasma phagocytophilum↗
PubMed2005

Defective phagocytosis in Anaplasma phagocytophilum-infected neutrophils.

Anaplasma phagocytophilum infection induces functional neutrophil changes. Using both Candida albicans and fluorescent-aggregate phagocytosis assays, we examined whether neutrophil and dimethyl sulfoxide-differentiated HL-60 cell infection impairs internalization. A. phagocytophilum infection significantly decreased phagocytosis compared to that of controls (P < 0.05). This further impairment of neutrophil function may promote opportunistic infections and exacerbate disease.

Anaplasma phagocytophilum↗
PubMed2005

Establishment of cloned Anaplasma phagocytophilum and analysis of p44 gene conversion within an infected horse and infected SCID mice.

Diverse p44 alleles at the p44 expression locus (p44Es) encoding surface-exposed major membrane proteins, P44s, of Anaplasma phagocytophilum were hypothesized to be garnered by recombination to enact antigenic variation. However, this hypothesis has not been proven so far, due to inability to clone this obligate intragranulocytic rickettsia. To define the p44E recombination, we developed a novel method to clone A. phagocytophilum. This isogenic cloned population containing a defined p44E was used to infect a naive horse and severe combined immunodeficiency (SCID) mice. During a 58-day infection period in the blood of the horse, p44E conversion was evident in a total of 11 new p44Es, 48% (115/242) of the sequenced p44E population. During a 50-day infection period in the blood of SCID mice, p44E conversion was manifested in a total of 13 new p44Es, 42% (192/460) of the p44E population. Thus, similar levels of p44E convertants were detected in either the presence or absence of an acquired immune system, suggesting that T- and B-cell immune pressure was not essential for recombination and/or selection of the p44E variants. Analysis of sequentially changed p44Es revealed that the entire central hypervariable region of donor p44 pseudogenes or of donor full-length p44s replaced the same region of the resident p44E as a cassette. Putative recombination points were detected within p44 conserved regions flanking the central hypervariable region by the TOPALi analysis. Our results unambiguously demonstrated p44E recombination. The cloning method developed would facilitate precise analysis of the recombination process and the extent of diversity which the recombination creates in the antigenic repertoire.

Anaplasma phagocytophilum↗
PubMed2006

Reservoir competency of goats for the Ap-variant 1 strain of Anaplasma phagocytophilum.

Field-collected ticks were used to infect goats with either Ap-ha, a strain associated with human disease, or a variant strain, Ap-Variant 1, of Anaplasma phagocytophilum. Goats were shown to be competent as a reservoir for Ap-Variant 1, and challenge and immunosuppression studies were used to further examine infection in the goat model.

Anaplasma phagocytophilum↗
PubMed2006

Two monoclonal antibodies with defined epitopes of P44 major surface proteins neutralize Anaplasma phagocytophilum by distinct mechanisms.

Anaplasma phagocytophilum is an obligatory intracellular bacterium that causes human granulocytic anaplasmosis. The polymorphic 44-kDa major outer membrane proteins of A. phagocytophilum are dominant antigens recognized by patients and infected animals. However, the ability of anti-P44 antibody to neutralize the infection has been unclear due to a mixture of P44 proteins with diverse hypervariable region amino acid sequences expressed by a given bacterial population and lack of epitope-defined antibodies. Monoclonal antibodies (MAbs) 5C11 and 3E65 are directed to different domains of P44 proteins, the N-terminal conserved region and P44-18 central hypervariable region, respectively. Passive immunization with either MAb 5C11 or 3E65 partially protects mice from infection with A. phagocytophilum. In the present study, we demonstrated that the two monoclonal antibodies recognize bacterial surface-exposed epitopes of naturally folded P44 proteins and mapped these epitopes to specific peptide sequences. The two MAbs almost completely blocked the infection of the A. phagocytophilum population that predominantly expressed P44-18 in HL-60 cells by distinct mechanisms: MAb 5C11 blocked the binding, but MAb 3E65 did not block binding or internalization. Instead, MAb 3E65 inhibited internalized A. phagocytophilum to develop into microcolonies called morulae. Some plasma from experimentally infected horses and mice reacted with these two epitopes. Taken together, these data indicate the presence of at least two distinct bacterial surface-exposed neutralization epitopes in P44 proteins. The results indicate that antibodies directed to certain epitopes of P44 proteins have a critical role in inhibiting A. phagocytophilum infection of host cells.

Anaplasma phagocytophilum↗
PubMed2006

Analysis of involvement of the RecF pathway in p44 recombination in Anaplasma phagocytophilum and in Escherichia coli by using a plasmid carrying the p44 expression and p44 donor loci.

Anaplasma phagocytophilum, the etiologic agent of human granulocytic anaplasmosis, has a large paralog cluster (approximate 90 members) that encodes the 44-kDa major outer membrane proteins (P44s). Gene conversion at a single p44 expression locus leads to P44 antigenic variation. Homologs of genes for the RecA-dependent RecF pathway, but not the RecBCD or RecE pathways, of recombination were detected in the A. phagocytophilum genome. In the present study, we examined whether the RecF pathway is involved in p44 gene conversion. The recombination intermediate structure between a donor p44 and the p44 expression locus of A. phagocytophilum was detected in an HL-60 cell culture by Southern blot analysis followed by sequencing the band and in blood samples from infected SCID mice by PCR, followed by sequencing. The sequences were consistent with the RecF pathway recombination: a half-crossover structure, consisting of the donor p44 locus connected to the 3' conserved region of the recipient p44 in the p44 expression locus in direct orientation. To determine whether the p44 recombination intermediate structure can be generated in a RecF-active Escherichia coli strain, we constructed a double-origin plasmid carrying the p44 expression locus and a donor p44 locus and introduced the plasmid into various E. coli strains. The recombination intermediate was recovered in an E. coli strain with active RecF recombination pathway but not in strains with deficient RecF pathway. Our results support the view that the p44 gene conversion in A. phagocytophilum occurs through the RecF pathway.

Anaplasma phagocytophilum↗
PubMed1972

Deoxyribonucleic acid of Anaplasma marginale.

Deoxyribonucleic acid from isolated marginal bodies and calf erythrocytes infected with Anaplasma marginale is found to be double stranded and to contain 51 moles per cent guanine plus cytosine.

Anaplasma↗
PubMed1967

Evidence of lactate dehydrogenase in Anaplasma marginale.

Extracts from preparations of partially purified Anaplasma marginale revealed low levels of lactate dehydrogenase (LDH). Enzyme inhibition by immune sera further indicated that A. marginale possesses a protein moiety the same as that of the normal red blood cell (RBC), although data suggested an alteration of LDH(1) from that observed in normal RBC. Bimodal isozyme distribution was detected after electrophoresis of the extracts. One isozyme approached the cathode and the other the anode, and both appeared to be nicotinamide adenine dinucleotide-dependent. Heterogeneity of parasite and host cell isozymes was established on the basis of zone electrophoresis on cellulose acetate strips.

Anaplasma↗