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An emerging theme among vacuole-adapted bacterial pathogens is the ability to hijack ubiquitin machinery to modulate host cellular processes and secure pathogen survival. Mono- and polyubiquitination differentially dictate the subcellular localization, activity, and fate of protein substrates. Monoubiquitination directs membrane traffic from the plasma membrane to the endosome and has been shown to promote autophagy. Anaplasma phagocytophilum is an obligate intracellular bacterium that replicates within a host cell-derived vacuole that co-opts membrane traffic and numerous other host cell processes. Here, we show that monoubiquitinated proteins decorate the A. phagocytophilum-occupied vacuolar membrane (AVM) during infection of promyelocytic HL-60 cell, endothelial RF/6A cells, and to a lesser extent, embryonic tick ISE6 cells. Monoubiquitinated proteins are present on the AVM upon its formation and continue to accumulate throughout infection. Tetracycline-mediated inhibition of de novo bacterial protein synthesis promotes the loss of ubiquitinated proteins from the AVM. This effect is reversible, as removal of tetracycline restores AVM ubiquitination to pretreatment levels. These results demonstrate a novel mechanism by which A. phagocytophilum remodels the composition of its host cell-derived vacuolar membrane and present the first example of a Rickettsiales pathogen co-opting ubiquitin during intracellular residence.

Anaplasma phagocytophilum is an obligate intracellular bacterium that invades neutrophils to cause the emerging infectious disease human granulocytic anaplasmosis. A. phagocytophilum undergoes a biphasic developmental cycle, transitioning between an infectious dense-cored cell (DC) and a noninfectious reticulate cell (RC). To gain insights into the organism's biology and pathogenesis during human myeloid cell infection, we conducted proteomic analyses on A. phagocytophilum organisms purified from HL-60 cells. A total of 324 proteins were unambiguously identified, thereby verifying 23.7% of the predicted A. phagocytophilum proteome. Fifty-three identified proteins had been previously annotated as hypothetical or conserved hypothetical. The second most abundant gene product, after the well-studied major surface protein 2 (P44), was the hitherto hypothetical protein APH_1235. APH_1235 homologs are found in other Anaplasma and Ehrlichia species but not in other bacteria. The aph_1235 RNA level is increased 70-fold in the DC form relative to that in the RC form. Transcriptional upregulation of and our ability to detect APH_1235 correlate with RC to DC transition, DC exit from host cells, and subsequent DC binding and entry during the next round of infection. Immunoelectron microscopy pronouncedly detects APH_1235 on DC organisms, while detection on RC bacteria minimally, at best, exceeds background. This work represents an extensive study of the A. phagocytophilum proteome, discerns the complement of proteins that is generated during survival within human myeloid cells, and identifies APH_1235 as the first known protein that is pronouncedly upregulated on the infectious DC form.

Mast cells are sentinels for infection. Upon exposure to pathogens, they release their stores of proinflammatory cytokines, chemokines, and histamine. Mast cells are also important for the control of certain tick-borne infections. Anaplasma phagocytophilum is an obligate intracellular tick-transmitted bacterium that infects neutrophils to cause the emerging disease granulocytic anaplasmosis. A. phagocytophilum adhesion to and infection of neutrophils depend on sialylated and α1,3-fucosylated glycans. We investigated the hypotheses that A. phagocytophilum invades mast cells and inhibits mast cell activation. We demonstrate that A. phagocytophilum binds and/or infects murine bone marrow-derived mast cells (BMMCs), murine peritoneal mast cells, and human skin-derived mast cells. A. phagocytophilum infection of BMMCs depends on α1,3-fucosylated, but not sialylated, glycans. A. phagocytophilum binding to and invasion of BMMCs do not elicit proinflammatory cytokine secretion. Moreover, A. phagocytophilum-infected cells are inhibited in the release of tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), IL-13, and histamine following stimulation with IgE or antigen. Thus, A. phagocytophilum mitigates mast cell activation. These findings potentially represent a novel means by which A. phagocytophilum usurps host defense mechanisms and shed light on the interplay between mast cells and vector-borne bacterial pathogens.

Anaplasma phagocytophilum infects neutrophils and myeloid, endothelial, and tick cell lines to reside within a host cell-derived vacuole that is indispensible for its survival. Here, we identify APH_0032 as an Anaplasma-derived protein that associates with the A. phagocytophilum-occupied vacuolar membrane (AVM). APH_0032 is a 66.1 kDa acidic protein that electrophoretically migrates with an apparent molecular weight of 130 kDa. It contains a predicted transmembrane domain and tandemly arranged direct repeats that comprise 46% of the protein. APH_0032 is undetectable on Anaplasma organisms bound to the surfaces of HL-60 cells, but is detected on the AVM and surfaces of intravacuolar bacteria beginning 24 h post-infection. APH_0032 localizes to the AVM in HL-60, THP-1, HMEC-1, and ISE6 cells. APH_0032, along with APH_1387, which encodes a confirmed AVM protein, is transcribed during A. phagocytophilum infection of tick salivary glands and murine neutrophils. APH_0032 localizes to the AVM in neutrophils recovered from infected mice. The Legionella pneumophila Dot/IcM type IV secretion system (T4SS) can heterologously secrete a CyaA-tagged version of the A. phagocytophilum VirB/D T4SS effector, AnkA, but fails to secrete CyaA-tagged APH_0032 or APH_1387. These data confirm APH_0032 as an Anaplasma-derived AVM protein and hint that neither it nor APH_1387 are T4SS effectors., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Anaplasma phagocytophilum is an obligate intracellular bacterium that infects neutrophils to reside within a host cell-derived vacuole. The A. phagocytophilum-occupied vacuole (ApV) fails to mature along the endocytic pathway and is non-fusogenic with lysosomes. Rab GTPases regulate membrane traffic. To better understand how the bacterium modulates the ApV's selective fusogencity, we examined the intracellular localization of 20 green fluorescent protein (GFP) or red fluorescent protein (RFP)-tagged Rab GTPases in A. phagocytophilum-infected HL-60 cells. GFP-Rab4A, GFP-Rab10, GFP-Rab11A, GFP-Rab14, RFP-Rab22A and GFP-Rab35, which regulate endocytic recycling, and GFP-Rab1, which mediates endoplasmic reticulum to Golgi apparatus trafficking, localize to the ApV. Fluorescently tagged Rabs are recruited to the ApV upon its formation and remain associated throughout infection. Endogenous Rab14 localizes to the ApV. Tetracycline treatment concomitantly promotes loss of recycling endosome-associated GFP-Rabs and acquisition of GFP-Rab5, GFP-Rab7, and the lysosomal marker, LAMP-1. Wild-type and GTPase- deficient versions, but not GDP-restricted versions of GFP-Rab1, GFP-Rab4A and GFP-Rab11A, localize to the ApV. Strikingly, GFP-Rab10 recruitment to the ApV is guanine nucleotide-independent. These data establish that A. phagocytophilum selectively recruits Rab GTPases that are primarily associated with recycling endosomes to facilitate its intracellular survival and implicate bacterial proteins in regulating Rab10 membrane cycling on the ApV.

Anaplasma phagocytophilum is an obligate intracellular bacterium that has evolved mechanisms to hijack polymorphonuclear neutrophil (PMN) receptors and signaling pathways to bind, infect, and multiply within the host cell. E-selectin is upregulated during inflammation and is a requisite endothelial receptor that supports PMN capture, rolling, and activation of integrin-mediated arrest. Ligands expressed by PMN that mediate binding to endothelium via E-selectin include sialyl Lewis x (sLe(x))-expressing ligands such as P-selectin glycoprotein ligand-1 (PSGL-1) and other glycolipids and glycoproteins. As A. phagocytophilum is capable of binding to sLe(x)-expressing ligands expressed on PMN, we hypothesized that acute bacterial adhesion to PMN would subsequently attenuate PMN recruitment during inflammation. We assessed the dynamics of PMN recruitment and migration under shear flow in the presence of a wild-type strain of A. phagocytophilum and compared it with a strain of bacteria that binds to PMN independent of PSGL-1. Acute bacterial engagement with PMN resulted in transient PMN arrest and minimal PMN polarization. Although the wild-type pathogen also signaled activation of beta2 integrins and elicited a mild intracellular calcium flux, downstream signals including PMN transmigration and phosphorylation of p38 mitogen-activated protein kinase (MAPK) were inhibited. The mutant strain bound less well to PMN and failed to activate beta2 integrins and induce a calcium flux but did result in decreased PMN arrest and polarization that may have been partially mediated by a suppression of p38 MAPK activation. This model suggests that A. phagocytophilum binding to PMN under shear flow during recruitment to inflamed endothelium interferes with normal tethering via E-selectin and navigational signaling of transendothelial migration.

Obligate vacuolar pathogens produce proteins that localize to the host cell-derived membranes of the vacuoles in which they reside, yielding unique organelles that are optimally suited for pathogen survival. Anaplasma phagocytophilum is an obligate vacuolar bacterium that infects neutrophils and causes the emerging and potentially fatal disease human granulocytic anaplasmosis. Here we identified APH_1387 as the first A. phagocytophilum-derived protein that associates with the A. phagocytophilum-occupied vacuolar membrane (AVM). APH_1387, also referred to as P100, is a 61.4-kDa acidic protein that migrates with an apparent molecular weight of 115 kDa on SDS-PAGE gels. It carries 3 tandem direct repeats that comprise 58% of the protein. Each APH_1387 repeat carries a bilobed hydrophobic alpha-helix domain, which is a structural characteristic that is consistent with the structure of chlamydia-derived proteins that traverse inclusion membranes. APH_1387 is not detectable on the surfaces of A. phagocytophilum dense core organisms bound at the HL-60 cell surface, but abundant APH_1387 is detected on the surfaces of intravacuolar reticulate cell and dense core organisms. APH_1387 accumulates on the AVM throughout infection. It associates with the AVM in human HL-60, THP-1, and HMEC-1 cells and tick ISE6 cells. APH_1387 is expressed and localizes to the AVM in neutrophils recovered from A. phagocytophilum-infected mice. This paper presents the first direct evidence that A. phagocytophilum actively modifies its host cell-derived vacuole.

Anaplasma phagocytophilum is an obligate intracellular bacterium that infects granulocytes to cause human granulocytic anaplasmosis. The susceptibilities of human neutrophils and promyelocytic HL-60 cells to A. phagocytophilum are linked to bacterial usage of P-selectin glycoprotein ligand 1 (PSGL-1) as a receptor for adhesion and entry. A. phagocytophilum undergoes a biphasic developmental cycle, transitioning between a smaller electron dense-cored cell (DC), which has a dense nucleoid, and a larger, pleomorphic electron lucent reticulate cell (RC), which has a dispersed nucleoid. The pathobiological roles of each form have not been elucidated. To ascertain the role of each form, we used electron microscopy to monitor bacterial binding, entry, and intracellular development within HL-60 cells. Only DCs were observed binding to and inducing uptake by HL-60 cells. By 12 h, internalized DCs had transitioned to RCs, which had initiated replication. By 24 h, large RC numbers were observed within individual inclusions. Reinfection had occurred by 36 h, as individual, vacuole-enclosed DCs and RCs were again observed. The abilities of DC- and RC-enriched A. phagocytophilum populations to bind and/or infect HL-60 cells or Chinese hamster ovary cells transfected to express PSGL-1 (PSGL-1 CHO) were compared. Only DCs bound PSGL-1 CHO cells and did so in a PSGL-1-blocking antibody-inhibitable manner. These results demonstrate that the respective roles of A. phagocytophilum DCs and RCs are consistent with analogous forms of other obligate intracellular pathogens that undergo biphasic development and hint that the PSGL-1-targeting adhesin(s) may be upregulated or optimally posttranslationally modified on DCs.

Anaplasma phagocytophilum, Ehrlichia chaffeensis and Ehrlichia ewingii are emerging tick-borne pathogens and are the causative agents of human granulocytic anaplasmosis, human monocytic ehrlichiosis and E. ewingii ehrlichiosis, respectively. Collectively, these are referred to as human ehrlichioses. These obligate intracellular bacterial pathogens of the family Anaplasmataceae are transmitted by Ixodes spp. or Amblyomma americanum ticks and infect peripherally circulating leukocytes to cause infections that range in clinical spectra from asymptomatic seroconversion to mild, severe or, in rare instances, fatal disease. This review describes: the ecology of each pathogen; the epidemiology, clinical signs and symptoms of the human diseases that each causes; the choice methods for diagnosing and treating human ehrlichioses; recommendations for patient management; and is concluded with suggestions for potential future research.

Many microbial pathogens alter expression and/or posttranslational modifications of their surface proteins in response to dynamics within their host microenvironments to retain optimal interactions with their host cells and/or to evade the humoral immune response. Anaplasma phagocytophilum is an intragranulocytic bacterium that utilizes sialyl Lewis x (sLe(x))-modified P-selectin glycoprotein ligand 1 as a receptor for infecting myeloid cells. Bacterial populations that do not rely on this receptor can be obtained through cultivation in sLe(x)-defective cell lines. A. phagocytophilum major surface protein 2 [Msp2(P44)] is encoded by members of a paralogous gene family and is speculated to play roles in host adaptation. We assessed the complement of Msp2(P44) paralogs expressed by A. phagocytophilum during infection of sLe(x)-competent HL-60 cells and two HL-60 cell lines defective for sLe(x) expression. Multiple Msp2(P44) and N-terminally truncated 25- to 27-kDa isoforms having various isoelectric points and electrophoretic mobilities were expressed in each cell line. The complement of expressed msp2(p44) paralogs and the glycosyl residues modifying Msp2(P44) varied considerably among bacterial populations recovered from sLe(x)-competent and -deficient host cells. Thus, loss of host cell sLe(x) expression coincided with both differential expression and glycosylation of A. phagocytophilum Msp2(P44). This reinforces the hypothesis that this bacterium is able to generate a large variety of surface-exposed molecules that could provide great antigenic diversity and result in multiple binding properties.

Anaplasma phagocytophilum is an obligate intracellular bacterium that infects neutrophils to cause granulocytic anaplasmosis in humans and mammals. P-selectin glycoprotein ligand-1 (PSGL-1) and the tetrasaccharide sialyl Lewis x (sLe(x)), which caps the PSGL-1 N-terminus, are confirmed A. phagocytophilum receptors. A. phagocytophilum is capable of sLe(x)-modified PSGL-1-dependent and -independent infection. PSGL-1 N-terminus-mediated entry is dependent on spleen tyrosine kinase (Syk). Here, we determined that PSGL-1-independent entry does not alter bacterial replication and investigated whether it involves Syk using NCH-1A2, an enriched subpopulation of A. phagocytophilum NCH-1 obtained through cultivation in a sLe(x)-deficient HL-60 cell line, HL-60 A2. Pharmacological inhibition of Syk nearly abolishes NCH-1 infection, but does not alter NCH-1A2 invasion and only marginally reduces NCH-1A2 propagation. This phenomenon was confirmed by a competitive infection assay using PSGL-1-dependent and -independent A. phagocytophilum organisms transformed to express mCherry or green fluorescent protein respectively. We also assayed for delivery and tyrosine phosphorylation of the A. phagocytophilum effector, AnkA, following NCH-1or NCH-1A2 incubation with HL-60 or HL-60 A2 cells in the presence of PSGL-1 blocking antibody. PSGL-1 N-terminus recognition promotes optimal AnkA delivery while binding to sLe(x) or the unknown receptor is comparably less important for this process.

Anaplasma phagocytophilum is the etiologic agent of human granulocytic anaplasmosis. MSP2(P44), the bacterium's major surface protein, is encoded by a paralogous gene family and has been implicated in a variety of pathobiological processes, including antigenic variation, host adaptation, adhesion, porin activity, and structural integrity. The consensus among several studies performed at the DNA and RNA levels is that a heterogeneous mix of a limited number of msp2(p44) transcripts is expressed by A. phagocytophilum during in vitro cultivation. Such analyses have yet to be extended to the protein level. In this study, we used proteomic and molecular approaches to determine that MSP2(P44)-18 is the predominant if not the only paralog expressed and is modified into multiple 42- to 44-kDa isoforms by A. phagocytophilum strain HGE1 during infection of HL-60 cells. The msp2(p44) expression profile was homogeneous for msp2(p44)-18. Thus, MSP2(P44)-18 may have a fitness advantage in HL-60 cell culture in the absence of selective immune pressure. Several novel 22- to 27-kDa MSP2 isoforms lacking most of the N-terminal conserved region were also identified. A. phagocytophilum MSP2(P44) orthologs expressed by other pathogens in the family Anaplasmataceae are glycosylated. Gas chromatography revealed that recombinant MSP2(P44)-18 is modified by glucose, galactose, xylose, mannose, and trace amounts of other glycosyl residues. These data are the first to confirm differential modification of any A. phagocytophilum MSP2(P44) paralog and the first to provide evidence for expression of truncated versions of such proteins.

Anaplasma phagocytophilum is an obligate intracellular bacterial pathogen that principally inhabits neutrophils. However, infection with A. phagocytophilum results in a moderate to marked thrombocytopenia. In host neutrophils, A. phagocytophilum uses sialylated ligands, primarily P-selectin glycoprotein ligand-1 (PSGL-1), to enter its host cell. PSGL-1 is expressed on a wide array of haematopoietic cells, including megakaryocytes. In this study, it was hypothesized that (i) cells of the megakaryocytic lineage (MEG-01 cells) would be susceptible to A. phagocytophilum infection and (ii) infection may induce alterations in platelet production contributing to infection-induced thrombocytopenia. It was found that MEG-01 cells are susceptible to infection. MEG-01 cells expressing abundant sialylated ligands were the most susceptible to infection, and the absence of sialylation, or blocking of PSGL-1, limited infection susceptibility. However, infected MEG-01 cells produced proplatelets and platelet-like particles comparable to uninfected cells. These results highlight a novel target of pathogen infection and suggest that the pathogen may utilize similar strategies to gain access to megakaryocytes. Direct pathogen modification of platelet production may not play a role in infection-induced thrombocytopenia.

Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis, is an obligate intracellular bacterium that infects neutrophils and neutrophil precursors. Bacterial recognition of P-selectin glycoprotein ligand-1 (PSGL-1) and the alpha2,3-sialylated- and alpha1,3-fucosylated-moiety sialyl-Lewis x (sLe(x)), which modifies the PSGL-1 N terminus, is important for adhesion to and invasion of myeloid cells. We have previously demonstrated that A. phagocytophilum organisms of the NCH-1 strain that utilize an sLe(x)-modified PSGL-1-independent means of entry can be enriched for by cultivation in undersialylated HL-60 cells that are unable to construct sLe(x). Because it was unknown whether other A. phagocytophilum isolates share this ability, we extended our studies to the geographically diverse strains HZ and HGE1. HL-60 A2 is a clonal cell line that is defective for sialylation and alpha1,3-fucosyltransferase. HL-60 A2 cell surfaces, therefore, not only lack sLe(x) but also are virtually devoid of any other sialic acid- and/or alpha1,3-fucose-modified glycan. By cultivating HZ and HGE1 in HL-60 A2 cells, we enriched for bacterial subpopulations (termed HZA2 and HGE1A2) that bind and/or infect myeloid cells in the absence of sialic acid and alpha1,3-fucose and in the presence of antibody that blocks the N terminus of PSGL-1. Thus, multiple A. phagocytophilum isolates share the ability to use sLe(x)-modified PSGL-1-dependent and -independent routes of entry into myeloid cells. HZA2 and HGE1A2 represent enriched bacterial populations that will aid dissection of the complexities of the interactions between A. phagocytophilum and host myeloid cells.

Anaplasma phagocytophilum, the aetiologic agent of human granulocytic anaplasmosis, is an obligate intracellular bacterium that colonizes neutrophils and neutrophil precursors. The granulocytotropic bacterium uses multiple adhesins that cooperatively bind to the N-terminal region of P-selectin glycoprotein ligand-1 (PSGL-1) and to sialyl Lewis x (sLe(x)) expressed on myeloid cell surfaces. Recognition of sLe(x) occurs through interactions with alpha2,3-sialic acid and alpha1,3-fucose. It is unknown whether other bacteria-host cell interactions are involved. In this study, we have enriched for A. phagocytophilum organisms that do not rely on sialic acid for cellular adhesion and entry by maintaining strain NCH-1 in HL-60 cells that are severely undersialylated. The selected bacteria, termed NCH-1A, also exhibit lessened dependencies on PSGL-1 and alpha1,3-fucose. Optimal adhesion and invasion by NCH-1A require interactions with the known determinants (sialic acid, PSGL-1 and alpha1,3-fucose), but none of them is absolutely necessary. NCH-1A binding to sLe(x)-modified PSGL-1 requires recognition of the known determinants in the same manners as other A. phagocytophilum strains. These data suggest that A. phagocytophilum expresses a separate adhesin from those targeting sialic acid, alpha1,3-fucose and the N-terminal region of PSGL-1. We propose that NCH-1A upregulates expression of this adhesin.

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.

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 is a gram-negative obligate intracellular bacterium that persists within neutrophils. We assessed the impact of A. phagocytophilum infection in NB4 promyelocytic leukemic cells using high-density oligoarray, two-dimensional differential gel electrophoresis and liquid chromatography-mass spectrometry. Our Affymetrix data revealed that A. phagocytophilum altered the expression of transcription factors, cell adhesion molecules, signal transduction genes, and proinflammatory cytokines. However, the expression of Toll-like receptors, MYD88, RNF36, IRF3, and TBK1 and inhibitors of the NF-kappaB gene was not altered. A. phagocytophilum infection also altered the apoptotic program of NB4 cells and resulted in increased transcription of antiapoptotic genes (MCL1 and BFL1). The transcription and translation of iron-metabolism genes (light polypeptide ferritin chain, transferrin, and the transferrin receptor) were significantly altered, suggesting a possible link between A. phagocytophilum infection and iron metabolism. Our study clearly demonstrates multifactorial effects of A. phagocytophilum infection on NB4 promyelocytic leukemic cell machinery.

Anaplasma phagocytophilum is the etiologic agent of human granulocytic anaplasmosis (formerly human granulocytic ehrlichiosis), an emerging and potentially deadly disease in the United States, Europe, and Asia. A. phagocytophilum is an obligate intracellular bacterium that displays a unique tropism for neutrophils. Studying this fascinating organism not only provides insight into microbial invasion and intracellular survival strategies, but also offers a unique approach to understanding neutrophil biology and host defense mechanisms. This unit describes the inoculation and maintenance of A. phagocytophilum from an infected blood sample into eukaryotic cell culture or laboratory mice. Cytological staining and immunofluorescent methods for assessing A. phagocytophilum infection are also presented. In addition, this unit describes isolation of viable, host cell-free bacterial preparations from infected cells, as well as the cryopreservation of infected cultures. Lastly, fluorescent labeling of live A. phagocytophilum for the purpose of tracking infection is provided.

Anaplasma phagocytophilum, the etiologic agent of human anaplasmosis, is a bacterial pathogen that specifically colonizes neutrophils. Neutrophils utilize the NADPH oxidase complex to generate superoxide (O(2)(-)) and initiate oxidative killing of microorganisms. A. phagocytophilum's unique tropism for neutrophils, however, indicates that it subverts and/or avoids oxidative killing. We therefore examined the effects of A. phagocytophilum infection on neutrophil NADPH oxidase assembly and reactive oxygen species (ROS) production. Following neutrophil binding, Anaplasma invasion requires at least 240 min. During its prolonged association with the neutrophil plasma membrane, A. phagocytophilum stimulates NADPH oxidase assembly, as indicated by increased cytochrome b(558) mobilization to the membrane, as well as colocalization of Rac and p22(phox). This initial stimulation taxes the host neutrophil's finite oxidase reserves, as demonstrated by time- and bacterial-dose-dependent decreases in secondary activation by N-formyl-methionyl-leucyl-phenylalanine (FMLP) or phorbol myristate acetate (PMA). This stimulation is modest, however, and does not diminish oxidase stores to nearly the extent that Escherichia coli, serum-opsonized zymosan, FMLP, or PMA do. Despite the apparent activation of NADPH oxidase, no change in ROS-dependent chemiluminescence is observed upon the addition of A. phagocytophilum to neutrophils, indicating that the bacterium may scavenge exogenous O(2)(-). Indeed, A. phagocytophilum rapidly detoxifies O(2)(-) in a cell-free system. Once internalized, the bacterium resides within a protective vacuole that excludes p22(phox) and gp91(phox). Thus, A. phagocytophilum employs at least two strategies to protect itself from neutrophil NADPH oxidase-mediated killing.

Anaplasma phagocytophilum is an aetiological agent of human granulocytic ehrlichiosis, an emerging tick-borne zoonosis in the United States and Europe. This obligate intracellular bacterium is unique in that it colonizes polymorphonuclear leucocytes (neutrophils). Neutrophils are key players in innate immunity. These short-lived phagocytes ingest invading microorganisms and destroy them by various means, which include fusing the bacteria-containing phagosome with acidic lysosomes as well as directing toxic oxidative and proteolytic compounds into the phagosomal lumen. Its tropism for neutrophils indicates that A. phagocytophilum uses strategies for evading and/or neutralizing these microbicidal activities. This review focuses on some of the mechanisms that A. phagocytophilum uses for neutrophil adhesion, surviving within the hostile intracellular environment of its host neutrophil and for effectively disseminating to naïve host cells.

Anaplasma phagocytophilum causes human granulocytic ehrlichiosis, the second most common tick-borne disease in the United States. Mice are natural reservoirs for this bacterium and man is an inadvertent host. A phagocytophilum's tropism for human neutrophils is linked to neutrophil expression of P-selectin glycoprotein ligand-1 (PSGL-1), as well as sialylated and alpha1,3-fucosylated glycans. To determine whether A phagocytophilum uses similar molecular features to infect murine neutrophils, we assessed in vitro bacterial binding to neutrophils from and infection burden in wild-type mice; mice lacking alpha 1,3-fucosyltransferases Fuc-TIV and Fuc-TVII; or mice lacking PSGL-1. Binding to Fuc-TIV-/-/Fuc-TVII-/- neutrophils and infection of Fuc-TIV-/-/Fuc-TVII-/- mice were significantly reduced relative to wild-type mice. A phagocytophilum binding to PSGL-1-/- neutrophils was modestly reduced, whereas sialidase treatment significantly decreased binding to both wild-type and PSGL-1-/- neutrophils. A phagocytophilum similarly infected PSGL-1-/- and wild-type mice in vivo. A phagocytophilum induced comparable levels of chemokines from wild-type and PSGL-1-/- neutrophils in vitro, while those induced from Fuc-TIV-/-/Fuc-TVII-/- neutrophils were appreciably reduced. Therefore, A phagocytophilum infection in mice, as in humans, requires sialylation and alpha1,3-fucosylation of neutrophils. However, murine infection does not require neutrophil PSGL-1 expression, which has important implications for understanding how A phagocytophilum binds and infects neutrophils.

Colonization of neutrophils by the bacterium Anaplasma phagocytophilum causes the disease human granulocytic ehrlichiosis. The pathogen also infects mice, its natural host. Like binding of P-selectin, binding of A. phagocytophilum to human neutrophils requires expression of P-selectin glycoprotein ligand-1 (PSGL-1) and alpha1-3-fucosyltransferases that construct the glycan determinant sialyl Lewis x (sLex). Binding of A. phagocytophilum to murine neutrophils, however, requires expression of alpha1-3-fucosyltransferases but not PSGL-1. To further characterize the molecular features that A. phagocytophilum recognizes, we measured bacterial binding to microspheres bearing specific glycoconjugates or to cells expressing human PSGL-1 and particular glycosyltransferases. Like P-selectin, A. phagocytophilum bound to purified human PSGL-1 and to glycopeptides modeled after the N terminus of human PSGL-1 that presented sLex on an O-glycan. Unlike P-selectin, A. phagocytophilum bound to glycopeptides that contained sLex but lacked tyrosine sulfation or a specific core-2 orientation of sLex on the O-glycan. A. phagocytophilum bound only to glycopeptides that contained a short amino acid sequence found in the N-terminal region of human but not murine PSGL-1. Unlike P-selectin, A. phagocytophilum bound to cells expressing PSGL-1 in cooperation with sLex on both N-and O-glycans. Moreover, bacteria bound to microspheres coupled independently with glycopeptide lacking sLex and with sLex lacking peptide. These results demonstrate that, unlike P-selectin, A. phagocytophilum binds cooperatively to a nonsulfated N-terminal peptide in human PSGL-1 and to sLex expressed on PSGL-1 or other glycoproteins. Distinct bacterial adhesins may mediate these cooperative interactions.

Anaplasma phagocytophila, the etiologic agent of human granulocytic ehrlichiosis, is an emerging bacterial pathogen that invades neutrophils and can be cultivated in HL-60 cells. Infected neutrophils and HL-60 cells fail to produce superoxide anion (O(2)(-)), which is partially attributable to the fact that A. phagocytophila inhibits transcription of gp91(phox), an integral component of NADPH oxidase. cDNA microarray and RT-PCR analyses demonstrated that transcription of the gene encoding Rac2, a key component in NADPH oxidase activation, was down-regulated in infected HL-60 cells. Quantitative RT-PCR demonstrated that rac2 mRNA expression was reduced 7-fold in retinoic acid-differentiated HL-60 cells and 50-fold in neutrophils following A. phagocytophila infection. Rac2 protein expression was absent in infected HL-60 cells. Rac1 and Rac2 are interchangeable in their abilities to activate NADPH oxidase. HL-60 cells transfected to express myc-tagged rac1 and gp91(phox) from the CMV immediate early promoter maintained the ability to generate O(2)(-) 120 h postinfection. A. phagocytophila proliferation was severely inhibited in these cells. These results directly attribute the inhibition of rac2 and gp91(phox) transcription to the loss of NADPH oxidase activity in A. phagocytophila-infected cells and demonstrate its importance to bacterial intracellular survival.

Here, we describe the molecular and immunological characterization of the bdr gene family of Borrelia turicatae, a relapsing-fever spirochete. Nine bdr alleles belonging to two different subfamilies were sequenced and localized to linear plasmids. Anti-Bdr antiserum was generated and used to analyze Bdr expression in pre- and postinfection isogenic populations. The analyses presented here provide a detailed characterization of the Bdr proteins in a relapsing-fever spirochete species, enhancing our understanding of these proteins at the genus-wide level.

Species of the genus Borrelia cause human and animal infections, including Lyme disease, relapsing fever, and epizootic bovine abortion. The borrelial genome is unique among bacterial genomes in that it is composed of a linear chromosome and a series of linear and circular plasmids. The plasmids exhibit significant genetic redundancy and carry 175 paralogous gene families, most of unknown function. Homologous alleles on different plasmids could influence the organization and evolution of the Borrelia genome by serving as foci for interplasmid homologous recombination. The plasmid-carried Borrelia direct repeat (bdr) gene family encodes polymorphic, acidic proteins with putative phosphorylation sites and transmembrane domains. These proteins may play regulatory roles in Borrelia. We describe recent progress in the characterization of the Borrelia bdr genes and discuss the possible influence of this gene family on the biology, pathogenesis, and evolution of the Borrelia genome.

The ospE gene family of the Lyme disease spirochetes encodes a polymorphic group of immunogenic lipoproteins. The ospE genes are one of several gene families that are flanked by a highly conserved upstream sequence called the upstream homology box, or UHB, element. Earlier analyses in our lab demonstrated that ospE-related genes are characterized by defined hypervariable domains (domains 1 and 2) that are predicted to be hydrophilic, surface exposed, and antigenic. The flanking of hypervariable domain 1 by DNA repeats may indicate that recombination contributes to ospE diversity and thus ultimately to antigenic variation. Using an isogeneic clone of Borrelia burgdorferi B31G (designated B31Gc1), we demonstrate that the ospE-related genes undergo mutation and rearrangement during infection in mice. The mutations that develop during infection resulted in the generation of OspE proteins with altered antigenic characteristics. The data support the hypothesized role of OspE-related proteins in immune system evasion.

B. turicatae, a causative agent of relapsing fever, carries a polymorphic gene family that is homologous to the bdr gene family of the Lyme disease spirochetes (previously referred to as the rep+ or ORF-E gene family). Here we demonstrate that bdr related genes are widely distributed among pathogenic Borrelia species and exist as large, polymorphic, plasmid carried, gene families. Twenty distinct bdr alleles were identified in isolates of the relapsing fever spirochete, B. hermsii, and were localized to linear plasmids. Cloning and sequence analyses demonstrate that the putative Bdr functional domains (i.e. the phosphorylation motifs and the transmembrane C-terminal domain) are conserved across the genus while other regions of these proteins exhibit variability. An assessment of the evolutionary relationships among all known Bdr protein sequences obtained from five pathogenic Borrelia species revealed that there are distinct Bdr sub-families. The recognition of distinct phyletic clusters serves as the basis of a revised and simplified nomenclature for the bdr proteins that can be applied genus wide. At the biological level the delineation of multiple bdr sub-families within isogeneic populations raises the possibility that there may be functional partitioning among alleles. In summary, the distribution and conservation of the Bdr proteins suggests that they are important in the biology/pathogenesis of the Borrelia at the genus wide level., (Copyright 2000 Academic Press.)

A series of related genes that are flanked at their 5' ends by a conserved upstream sequence element called the upstream homology box (UHB) have been identified in Borrelia burgdorferi. These genes have been referred to as the UHB or erp gene family. We previously demonstrated that among a limited number of B. burgdorferi isolates, the UHB gene family is variable in composition and organization. Prior to this report the UHB gene family in other species of the B. burgdorferi sensu lato complex had not been studied, and if this family is important in the pathogenesis or biology of the Lyme disease spirochetes, then a wide distribution among species and isolates of the B. burgdorferi sensu lato complex would be expected. To assess this, we screened for the UHB element by Southern hybridization and determined its restriction fragment length polymorphism (RFLP) patterns. The UHB element was found to be carried by all B. burgdorferi sensu lato complex species tested (B. burgdorferi, B. garinii, B. afzelii, B. japonica, B. valaisiana sp. nov., and B. andersonii), but the RFLP patterns varied widely at both the inter- and intraspecies levels. Variation in both the number and size of the hybridizing restriction fragments was evident. PCR analyses also revealed the presence of polymorphic, ospE-related alleles in many isolates. Sequence analyses identified the molecular basis of the polymorphisms as being primarily insertions and deletions. Sequence variation and the insertions and deletions were found to be clustered in two distinct domains (variable domains 1 and 2). In many isolates variable domain 1 is flanked by direct repeat elements, some as long as 38 bp. Computer analyses of the deduced amino acid sequences encoded within variable domain 1 predict them to be hydrophilic, surface exposed, and antigenic. The analyses conducted here suggest that the UHB gene family, as evidenced by the variable UHB RFLP patterns, is not evolutionarily stable and that the polymorphic ospE alleles are derived from a common ancestral gene which has been modified through mutation or recombination events. The characterization of ospE-related genes of the UHB gene family among B. burgdorferi sensu lato species will prove important in attempts to construct a model for UHB gene family organization and in deciphering the role of the UHB gene family in the biology and pathogenesis of the Lyme disease spirochetes.

Borrelia turicatae is one of several spirochete species that can cause relapsing fever. Here, we describe the identification and characterization of a gene from B. turicatae and other relapsing-fever spirochetes that exhibits homology with the rep+ and ORF-E gene families of the Lyme disease spirochetes. This gene, which we have designated repA, encodes a putative protein of 30.2 kDa with an isoelectric point of 4.69. The central region of RepA harbors a series of amino acid repeat motifs which exhibit homology with casein kinase 2 phosphorylation sites. Through Southern hybridization analyses, we demonstrate that repA (or a closely related sequence) is multicopy in the relapsing-fever spirochetes and is carried on variably sized linear plasmids in both Borrelia parkeri and B. turicatae. Transcriptional analyses demonstrate that repA is expressed, albeit at low levels, during in vitro cultivation of B. turicatae. Transcriptional start site analysis revealed that repA is preceded by a consensus ribosomal binding site and an appropriately spaced promoter element. The sequence conservation, unique features, and multicopy status of repA and its homologs suggest that RepA may play an important genus-wide role in the biology of the Borrelia.

Plasmid cp8.3 of Borrelia afzelii IP21 carries several open reading frames (ORFs) and a 184-bp inverted repeat (IR) element. It has been speculated that this plasmid may encode factors involved in virulence or infectivity. In this report, we have characterized the distribution, molecular variability, and organization of ORFs 1, 2, and 4 and the IR elements among isolates of the Borrelia burgdorferi sensu lato complex. ORFs 1 and 2 are contained within a segment of cp8.3 that is bordered by the IR elements, while ORF 4 resides just outside of the IR-bordered region. By PCR, ORF 4 was amplified from most isolates while ORFs 1 and 2 were amplified from only some B. afzelii isolates. However, Southern hybridization analyses with ORF 1, 2, and 4 probes detected related sequences even in some isolates that were PCR negative. The ORF restriction fragment length polymorphism patterns varied widely even among isolates of the same species. Two-dimensional contour-clamped homogeneous electric field-pulsed-field gel electrophoresis and Southern hybridization detected ORF 1-, 2-, and 4-related sequences on linear and circular plasmids. In addition, an ORF 4-related sequence was detected on a previously uncharacterized, circular plasmid that is greater than 70 kb in size. The IR elements originally identified on plasmid cp8.3 of B. afzelii IP21 were also analyzed by Southern hybridization. Related sequences were detected in some but not all B. burgdorferi sensu lato isolates. These sequences are carried on plasmids in addition to cp8.3 in some isolates. Single-primer PCR analyses demonstrated that in some isolates these sequences exist with IR orientation. The data presented here demonstrate that the IR elements and the ORF 1-, 2-, and 4-related sequences are multicopy and are variable in organization and in genomic location among isolates of the B. burgdorferi sensu lato complex. These analyses provide additional evidence for the highly variable organization of the plasmid component of the B. burgdorferi sensu lato genome.

In this study we report on the molecular characterization of a series of genes that constitute a gene family related to ospE and ospF. Some members of this family appear to represent recombined or variant forms of ospE and ospF. Variant ospE and ospF genes were found in several Borrelia burgdorferi isolates, demonstrating that their occurrence is not a phenomenon relevant to only a single isolate. Hybridization analyses revealed that the upstream sequence originally identified 5' of the full-length ospEF operon exists in multiple copies ranging in number from two to six depending on the isolate. This repeated sequence, which we refer to as the upstream homology box (UHB), carries a putative promoter element. In some isolates, UHB elements were found to flank copies of ospE and ospF that exist independently of each other. We refer to this group of UHB-flanked genes collectively as the UHB gene family. The evolutionary relationships among UHB gene family members were assessed through DNA sequence analysis and gene tree construction. These analyses suggest that some UHB-flanked genes might actually represent divergent forms of other previously described genes. Analysis of the restriction fragment length polymorphism patterns of the UHB-flanked genes among B. burgdorferi isolates demonstrated that these patterns are highly variable among isolates, suggesting that these genes are not phylogenetically conserved. The variable restriction fragment length polymorphism patterns could indicate recombinational activity in these sequences. The presence of numerous copies of the UHB elements and the high degree of homology among UHB-flanked genes could provide the necessary elements to allow for homologous recombination, leading to the generation of recombination variants of UHB gene family members.