INFECTION AND IMMUNITY, May 2010, p. 1864–1873 Vol. 78, No. 50019-9567/10/$12.00 doi:10.1128/IAI.01418-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Anaplasma phagocytophilum APH_1387 Is Expressed throughoutBacterial Intracellular Development and Localizes to the

Pathogen-Occupied Vacuolar Membrane�

Bernice Huang,1 Matthew J. Troese,1 Shaojing Ye,2† Jonathan T. Sims,2‡ Nathan L. Galloway,1Dori L. Borjesson,3 and Jason A. Carlyon1*

Department of Microbiology and Immunology, Virginia Commonwealth University School of Medicine, Richmond, Virginia 232981;Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky College of Medicine, Lexington,

Kentucky 405042; and Department of Pathology, Microbiology, and Immunology, University of California School ofVeterinary Medicine, Davis, California 956163

Received 18 December 2009/Returned for modification 18 January 2010/Accepted 15 February 2010

Obligate vacuolar pathogens produce proteins that localize to the host cell-derived membranes of thevacuoles 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 emergingand 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 apparentmolecular weight of 115 kDa on SDS-PAGE gels. It carries 3 tandem direct repeats that comprise 58% of theprotein. Each APH_1387 repeat carries a bilobed hydrophobic alpha-helix domain, which is a structuralcharacteristic that is consistent with the structure of chlamydia-derived proteins that traverse inclusionmembranes. APH_1387 is not detectable on the surfaces of A. phagocytophilum dense core organisms bound atthe HL-60 cell surface, but abundant APH_1387 is detected on the surfaces of intravacuolar reticulate cell anddense core organisms. APH_1387 accumulates on the AVM throughout infection. It associates with the AVMin human HL-60, THP-1, and HMEC-1 cells and tick ISE6 cells. APH_1387 is expressed and localizes to theAVM in neutrophils recovered from A. phagocytophilum-infected mice. This paper presents the first directevidence that A. phagocytophilum actively modifies its host cell-derived vacuole.

Obligate vacuolar pathogens remodel the host cell-derivedcompartments in which they reside into unique organellescalled pathogen-occupied vacuoles (PVs). PVs are develop-mentally arrested and sequestered outside the normal endo-cytic continuum and are optimal niches for intracellular sur-vival (29). The PV membrane (PVM) provides a crucialinterface between the host and the pathogen. Pathogen-en-coded proteins that localize to the PVM play critical pathobio-logical roles, which include providing structural integrity to thePVM, hijacking vesicular traffic, and intercepting host signaltransduction pathways. Therefore, identification and study ofpathogen-derived PVM proteins are crucial for understandingthe survival strategies of intravacuolar pathogens.

Anaplasma phagocytophilum is an Ixodes sp. tick-transmittedobligate vacuolar bacterium that infects granulocytes andcauses the emerging and potentially fatal disease human gran-

ulocytic anaplasmosis (HGA) (14, 47). A. phagocytophilum is amember of the family Anaplasmataceae, which contains othertick-transmissible pathogens that infect peripheral white andred blood cells. Other Anaplasmataceae pathogens includeEhrlichia chaffeensis, the causative agent of human monocyticehrlichiosis, and Anaplasma marginale, which infects bovineerythrocytes. HGA is an important cause of morbidity and isthe second most common tick-transmitted disease in theUnited States. The clinical manifestations of HGA range fromsubclinical infection to severe disease, including death. Thenonspecific symptoms include fever, chills, headache, malaise,and myalgia, and the severe complications include prolongedfever, shock, leucopenia, thrombocytopenia, high levels of C-reactive protein and hepatic transaminases, pneumonitis, acuterenal failure, and hemorrhages. Fatal opportunistic infectionshave resulted when antibiotic therapy was not provided in atimely fashion.

Neutrophils are the primary effector cells of microbial killingand use oxidative and proteolytic mechanisms to destroy en-gulfed pathogens. Yet A. phagocytophilum establishes a vacu-olar safe haven within neutrophils, as well as mammalian my-eloid and endothelial cell lines and tick embryonic cell lines(21, 34, 47, 53). An A. phagocytophilum-occupied vacuole(ApV) lacks early endosomal markers and most late endoso-mal markers, does not acidify, is not sensitive to brefeldin Atreatment, and avoids fusion with lysosomes and NADPHoxidase-carrying secretory vesicles and specific granules (10,28, 33, 51). Tetracycline treatment promotes lysosomal fusion

* Corresponding author. Mailing address: Department of Microbi-ology and Immunology, Virginia Commonwealth University School ofMedicine, Molecular Medicine Research Building, 1220 East BroadStreet, Room 4052, P.O. Box 980678, Richmond, VA 23298-0678.Phone: (804) 628-3382. Fax: (804) 828-9946. E-mail: jacarlyon@vcu.edu.

† Present address: Department of Molecular and Cellular Biochem-istry, University of Kentucky College of Medicine, Lexington, KY40504.

‡ Present address: Department of Molecular and Biomedical Phar-macology, University of Kentucky College of Medicine, Lexington, KY40504.

� Published ahead of print on 8 March 2010.

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of the ApV (23), which confirms that bacterial protein synthe-sis is required to prevent ApV maturation and suggests thatbacterial proteins incorporated into the ApV membrane(AVM) are likely to be important mediators of its alteredfusogenic properties.

Because PVM alteration by pathogen-derived proteins is aparadigm of intracellular pathogenesis and there have been noprevious reports of A. phagocytophilum-encoded proteins thatlocalize to the AVM, we sought to identify and characterizesuch proteins. Since A. phagocytophilum is an obligate intra-cellular bacterium, we hypothesized that bacterial proteins in-duced in host cells represent AVM candidate proteins. Ananalogous strategy was used to identify the first three chlamyd-ial inclusion membrane proteins (Inc proteins) (4, 42, 43).Storey et al. identified 3 such A. phagocytophilum proteins,P100, P130, and P160, which had apparent molecular masses of100, 130, and 160 kDa, by screening an A. phagocytophilumgenomic expression library with convalescent dog serum (45).Recombinant forms of these proteins were recognized byHGA patient antisera. P160, which has since been renamedAnkA because it carries a series of ankyrin repeats (13), is thefirst A. phagocytophilum type IV effector that was identifiedand does not localize to the AVM (27, 31, 40). P100 and P130correspond to APH_1387 and APH_0032, respectively, in theannotated A. phagocytophilum proteome (25). Both of theseproteins are acidic, carry tandem direct repeats, and containsegments that putatively traverse the AVM. Because it is in-duced upon infection and because it shares secondary structurecharacteristics with chlamydial Inc proteins, we performed de-tailed studies of APH_1387 and identified it as the first A.phagocytophilum-encoded protein that localizes to the AVM.This paper presents the first direct evidence that A. phagocy-tophilum actively modifies the host cell-derived organelle inwhich it resides and is an important step in deciphering thispathogen’s intracellular survival strategies.

MATERIALS AND METHODS

Cell lines and in vitro cultivation of A. phagocytophilum. The human promy-elocytic cell lines HL-60 (ATCC CCL-240; American Type Culture Collection[ATCC], Manassas, VA) and THP-1 (ATCC TIB-202) and A. phagocytophilumstrain NCH-1-infected HL-60 and THP-1 cells were cultivated in Iscove’s mod-ified Dulbecco’s medium supplemented with 10% fetal bovine serum (FBS)(IMDM-10) (Invitrogen, Carlsbad, CA) at 37°C in a humidified incubator in thepresence of 5% CO2. In some experiments, A. phagocytophilum strain HZ, whichwas a gift from Ralph Horwitz of New York Medical College (Valhalla, NY) andYasuko Rikihisa of Ohio State University (Columbus, OH), was used. Thehuman microvascular endothelial cell line HMEC-1 (1) was obtained from theCenters for Disease Control and Prevention (Atlanta, GA). Uninfected and A.phagocytophilum-infected HMEC-1 cells were propagated in MCDM 131(Mediatech, Herndon, VA) supplemented with 10 ng/ml epidermal growth factor(Becton Dickinson, Franklin Lakes, NJ), 1.0 �g/ml hydrocortisone (Sigma, St.Louis, MO), and 10% FBS in the presence of 5% CO2 in a humidified incubator.

In silico sequence analyses. The entire APH_1387 sequence was analyzed usingseveral algorithms in order to obtain clues that would help predict its secondarystructure and its ability to associate with the AVM. TMPred (www.ch.embnet.org/software/TMPRED_form.html) was used to determine if APH_1387 has a trans-membrane domain. Protean, which is part of the Lasergene software package (ver-sion 8.02; DNASTAR, Madison, WI), was used to assess whether APH_1387 hasregions of alpha helices and beta strands, alpha amphipathic sequences, and hydro-phobicity, using the Garnier-Osguthorpe-Robson, Eisenberg, and Kyte-Doolittlealgorithms, respectively (19, 22, 30). BLASTP (blast.ncbi.nlm.nih.gov/Blast.cgi) wasused to identify protein sequences to which APH_1387 exhibits homology. TheSWISS-MODEL (swissmodel.expasy.org), 3Djigsaw (bmm.cancerresearchuk.org/�3djigsaw), ESyPred3D (www.fundp.ac.be/sciences/biologie/urbm/bioinfo/esypred),

and Geno3d (geno3d-pbil.ibcp.fr/cgi-bin/geno3d_automat.pl?page�/GENO3D/geno3d_home.html) algorithms were used in attempts to predict the tertiary struc-ture of APH_1387.

Generation of maltose-binding protein (MBP)-APH_1387. The pMal-c2x vec-tor (New England Biolabs, Ipswich, MA) was made into a Gateway destinationvector using the Gateway vector conversion system (Invitrogen, Carlsbad, CA) byfollowing the manufacturer’s protocol; this yielded pMal-c2x/DEST, which wastransformed into One Shot ccdB Survival competent Escherichia coli cells (In-vitrogen). pMal-c2x/DEST was isolated from overnight cultures of transformantsusing a Qiaprep Spin miniprep kit (QIAGEN, Valencia, CA). The APH_1387gene was amplified using primers 5�-CACCATGTATGGTATAGATATAGAGCTAAG-3� and 5�-CTAATAACTTAGAACATCTTCATCG-3� and PlatinumPfx DNA polymerase (Invitrogen); the underlined nucleotides correspond tonucleotides in a Gateway-compatible sequence. After we confirmed that theamplicon was the expected size by agarose gel electrophoresis, the amplicon waspurified using a QIAquick PCR purification column (QIAGEN) and clonedwithout ligation into pENTR/D-Topo (Invitrogen) by following the manufactur-er’s instructions, which yielded the entry plasmid pENTR-APH_1387. The entryplasmid was transformed into chemically competent E. coli One Shot cells (In-vitrogen) and subsequently isolated from an overnight culture. The insert se-quence and cloning junctions were verified.

One hundred fifty nanograms of pENTR-APH_1387 was incubated with 150ng of pMal-c2x/DEST and LR Clonase II (Invitrogen) at 25°C for 1 h to facilitaterecombination of the APH_1387 insert downstream of the gene encoding mal-tose-binding protein (MBP) of pMal-c2x/DEST, which yielded pMal-c2x-APH_1387. Proteinase K was added to a final concentration of 0.18 �g/�l, andthen the preparation was incubated at 37°C for 10 min, after which the LRrecombination reaction mixtures were transformed into E. coli DH5� cells (No-vagen, Madison, WI). Cultures of the transformants were grown in Luria-Bertanimedium containing 100 �g ml�1 ampicillin at 37°C with shaking at 250 rpm.When the cultures were in the mid-logarithmic phase of growth (optical densityat 600 nm [OD600], 0.4), expression of MBP-APH_1387 was induced at 37°C byadding isopropyl-�-D-thiogalactopyranoside to a final concentration of 0.5 mM,and the cultures were grown for another 3 h. Portions (80 ml) of the inducedbacterial suspensions were harvested by centrifugation at 4,000 � g for 10 min at4°C. Each pellet was resuspended in 5 ml column buffer (20 mM Tris-HCl [pH7.4], 200 mM NaCl, 1 mM EDTA) and frozen overnight at �20°C. Frozen pelletswere thawed in cold water and placed in an ice-water bath, in which they weresonicated. Soluble crude extracts were recovered by centrifugation at 9,000 � gfor 20 min at 4°C. The crude extracts were loaded onto columns containingamylose resin (New England Biolabs) at a flow rate of 1 ml per min. The columnswere washed with 12 column volumes of column buffer. MBP-APH_1387 waseluted in column buffer containing 10 mM maltose, and 3-ml MBP-APH_1387fractions were collected and were visualized by Coomassie brilliant blue stainingfollowing resolution by SDS-PAGE. Desired fractions were pooled and concen-trated by centrifugation through an Amicon Ultra-4 centrifugal filter with a50-kDa cutoff (Millipore, Bedford, MA) to minimize the content of undesiredbreakdown products. Concentrated protein preparations were quantified usingthe Bradford assay (7).

Generation of anti-APH_1387. MBP-APH_1387 was submitted to AnimalPharm Services (Healdsburg, CA) for production of rabbit polyclonal antiserum.Immunoreactivity against MBP-APH_1387 and native A. phagocytophilumAPH_1387 was confirmed by Western blotting, as was the lack of recognition ofAPH_1387 by preimmune serum. Anti-MBP-APH_1387 is referred to below asanti-APH_1387.

Preparation of host cell-free A. phagocytophilum populations containing DCand RC organisms or only DC organisms. To isolate host cell-free A. phagocy-tophilum dense core (DC) and reticulate cell (RC) organisms, infected (�90%)HL-60 cells were mechanically disrupted by repeated passage through a 27-gaugeneedle. To isolate DC organisms, infected HL-60 cells were subjected to eight 8-sbursts on ice interspersed with 8-s rest periods using a Misonix S4000 ultrasonicprocessor (Farmingdale, NY) with an amplitude setting of 30, which destroyedhost cells and fragile RC organisms but not hardy DC organisms, as confirmedby transmission electron microscopy (data not shown). Bacteria were separatedfrom unbroken host cells and debris by differential centrifugation as describedpreviously (48).

Western blotting. Whole-cell lysates were fractionated by SDS-PAGE, trans-ferred to nitrocellulose, and screened using anti-APH_1387 followed by horse-radish peroxidase-conjugated goat anti-rabbit IgG, as described previously (11).Actin and A. phagocytophilum Msp2 (P44) were detected using anti-human actinmonoclonal antibody (MAb) (Sigma, St. Louis, MO) and anti-Msp2 (P44) MAb20B4 (a gift from J. Stephen Dumler of Johns Hopkins University, Baltimore,MD) (37, 44), respectively. To quantitatively assess the increase in APH_1387

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band signal intensity over the course of infection of HL-60 cells, densitometrywas performed using ImageJ (National Institutes of Health, United States)(rsb.info.nih.gov/ij), and the ratios of the densitometric values for APH_1387 andactin at different time points were plotted.

Infection time courses. Host cell-free A. phagocytophilum bacteria were iso-lated and synchronous infections were established as described previously (48).At the appropriate time points after addition of bacteria, aliquots were removedand processed for Western blot analysis as described above or for immunofluo-rescence or immunoelectron microscopy as described below.

Laser scanning confocal microscopy (LSCM). C3H/HeN scid mice were in-fected with NCH-1 exactly as previously described (6). On day 8 postinfection,whole blood was collected by cardiocentesis in EDTA and centrifuged, and theleukocyte-rich buffy coat was removed and cytospun (Cytospin 4; Thermo Shan-don, Pittsburgh, PA) onto glass slides at 113 � g for 5 min. The slides were fixedin ice-cold methanol and stored at �20°C until they were used. Ulrike Munder-loh (University of Minnesota, Minneapolis, MN) kindly provided slides of para-formaldehyde-fixed A. phagocytophilum-infected ISE6 cells. A. phagocytophilum-infected HL-60, THP-1, or HMEC-1 cells were cytospun onto glass slides at 70 �g for 2 min, which was followed by fixation in 4% (vol/vol) paraformaldehyde inphosphate-buffered saline (PBS) for 1 h. All slides were placed in ice-coldmethanol for 30 s to facilitate permeabilization and stored at �20°C until theywere needed. Slides were screened using rabbit anti-APH_1387 and mouseanti-Msp2 (P44) MAb 20B4, both at a dilution of 1:500, followed by Alexa Fluor594-conjugated goat anti-rabbit IgG and Alexa Fluor 488-conjugated goat anti-mouse IgG (Invitrogen), respectively, at dilutions of 1:1,000, as described previ-ously (9). Slides were mounted with the Prolong gold antifade reagent (Invitro-gen) and examined to determine the presence of ApVs and surface-boundbacteria with a TCS-SP2 AOBS confocal laser scanning microscope (LeicaMicrosystems, Bannockburn, IL) at the Virginia Commonwealth University De-partment of Neurobiology and Anatomy Microscopy Core Facility.

Immunoelectron microscopy. HL-60 cells were incubated with host cell-free A.phagocytophilum for 40 min at 37°C. The cells were washed three times with PBSand centrifuged at 300 � g for 5 min to remove unbound bacteria. At appropriatetime points 9 � 106 cells were washed with cacodylate buffer and fixed in 1 ml of3% paraformaldehyde-0.05% glutaraldehyde in 0.1 M cacodylate buffer for 45min at 4°C. The cells were washed four times by resuspending them in 1 mlcacodylate buffer and incubating them at room temperature for 15 min. Next, theVirginia Commonwealth University Department of Neurobiology and AnatomyMicroscopy Core Facility transferred washed cells onto 200-mesh Formvar-coated nickel grids. The grids were washed twice with PBS, blocked with 0.1%bovine serum albumin (BSA)-PBS for 1 h, and then stained sequentially for 1 heach with anti-APH_1387 (1:200) and goat anti-rabbit IgG (1:10) conjugated to6-nm gold particles (Electron Microscopy Sciences, Hatfield, PA). Grids wereviewed and images were recorded using a JEM-1230 transmission electron mi-croscope (JOEL, Tokyo, Japan) equipped with a Gatan UltraScan 4000SP 4K �4K charge-coupled device camera.

Statistical analyses. The Student’s t test (paired), performed using the Prism4.0 software package (GraphPad, San Diego, CA), was used to assess statisticalsignificance. A P value of 0.05 was considered statistically significant.

RESULTS

APH_1387 has limited homology to any previously de-scribed protein but does have predicted secondary structuralcharacteristics that are consistent with those of chlamydia-derived PVM proteins. APH_1387 is an acidic protein (pI 3.67)that contains 578 amino acids and has a predicted molecularmass of 61.4 kDa. It has 3 tandemly arranged direct repeatsconsisting of 93 amino acids (amino acids 180 to 272), 122amino acids (amino acids 304 to 425), and 130 amino acids(amino acids 428 to 557) that together comprise 58% of theprotein (Fig. 1A). BLASTP searches using the entireAPH_1387 sequence, the tandem repeat region (amino acids180 to 557), or individual repeats identified no proteins thatexhibited homology with APH_1387. A BLASTP search usingthe amino-terminal nonrepeat region (amino acids 1 to 179)identified a 93-amino-acid stretch exhibiting 32.3% identity toAM_924 and AMF_1226 of the A. marginale St. Maries andFlorida strains, respectively (8, 16). APH_1387 is predicted to

consist largely of amphipathic alpha helices (Fig. 1B). Chla-mydial Inc proteins possess transmembrane and/or hydropho-bic domains that facilitate insertion into the PVM (3, 4, 41).Likewise, many PVM proteins possess hydrophilic domainsthat extend into the host cytoplasm to interact with host pro-teins (41). Although APH_1387 lacks a typical transmembranedomain, a 24-amino-acid sequence (AQVPVVAEAELPGVEAAEAIVPSL) that is part of each of the 3 direct repeatsconstitutes a bilobed hydrophobic domain (amino acids 200 to223, 324 to 347, and 448 to 471) (Fig. 1B). Bilobed hydrophobicdomains are highly conserved among all chlamydial Inc pro-teins and are hypothesized to facilitate insertion of these pro-teins into the chlamydial inclusion membrane (41). Betweenthe 3 hydrophobic domains are alpha-helical stretches that arelargely hydrophilic. This periodicity of hydrophobic and hydro-philic domains could conceivably enable APH_1387 to traversethe AVM multiple times. Attempts to predict a tertiary struc-ture for APH_1387 using SWISS-MODEL, ESyPred3D,3Djigsaw, and GENO3D were unsuccessful because none ofthese programs was able to identify similar sequences withknown structures on which to model the APH_1387 structure.Sequencing of the PCR products obtained for the entireAPH_1387 coding sequence of A. phagocytophilum strainsNCH-1, HZ, and HGE-1 revealed that this gene’s sequence isthe same in these 3 strains and the USG3 strain, the strain inwhich it was originally sequenced (45) (data not shown).

APH_1387 is more abundant in A. phagocytophilum RC or-ganisms than in DC organisms. We initiated our characteriza-tion of APH_1387 by expressing it as an MBP fusion protein inE. coli and using the recombinant protein to raise rabbit poly-clonal antiserum. To test the efficacy of anti-APH_1387 and todetermine if APH_1387 is expressed during A. phagocytophi-

FIG. 1. Schematic diagrams of the A. phagocytophilum APH_1387,sequence, and secondary structure. (A) The amino (N)-terminal region(amino acids 1 to 179) precedes the repeat region (amino acids 180 to549), which precedes a short carboxy (C)-terminal region (amino acids550 to 578). The repeat region consists of 3 tandem direct repeats (indi-cated by open arrows) consisting of 93, 122, and 130 amino acids. Under-lining indicates the locations of the bilobed hydrophobic domains. aa,amino acids. (B) Diagrams of the sequence and secondary structure ofAPH_1387. The scale indicates 50-amino-acid intervals. The open andfilled boxes indicate alpha helical (Alpha regions) and beta strand (Betaregions) regions, respectively, as determined using the Garnier-Osguthorpe-Robson algorithm. In the Alpha amphipathic diagram,striped boxes indicate regions that are predicted to form alpha helices andare comprised of amphipathic amino acids, as determined by the Eisen-berg algorithm. For the Hydrophobicity diagram, the Kyte-Doolittle al-gorithm was used to determine hydrophobic (histogram above the x axis)and hydrophilic (histogram below the axis) regions. The 24-amino-acidsequence constituting a bilobed hydrophobic domain occurs at aminoacids 200 to 223, 324 to 347, and 448 to 471. All of the analyses wereperformed using Protean, which is part of the Lasergene software pack-age, as described in Materials and Methods.

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lum infection of promyelocytic HL-60 cells, we performedWestern blot analyses. Anti-APH_1387 recognized APH_1387as a single band at �115 kDa in an A. phagocytophilum-in-fected cell lysate but not in an uninfected HL-60 cell lysate(Fig. 2). A. phagocytophilum has a biphasic developmental cy-cle in which an infectious DC organism binds, enters, andchanges into a replicative RC organism that subsequently di-vides by binary fission (48). The numerous RC organisms,which are noninfectious, revert to DC organisms before theyare released to infect naïve host cells. We confirmed that a hostcell-free population of A. phagocytophilum DC and RC organ-isms can be recovered following syringe lysis, which does notdamage the fragile RC organisms, while a pure DC organismpopulation can be recovered following sonication, which de-stroys the RC organisms (data not shown). To assess whetherRC or DC organisms express more APH_1387, we screenedWestern-blotted lysates of host cell-free A. phagocytophilumorganisms recovered following syringe lysis (DC and RC or-ganisms) or sonication (only DC organisms) that were normal-ized using the levels of Msp2 (P44), which is a constitutivelyexpressed outer membrane protein (24) (Fig. 2). The intensityof the APH_1387 band was considerably higher for the lysatederived from DC and RC organisms than for the lysate gen-erated from DC organisms. After longer exposures, additionalanti-APH_1387-reactive bands at �61 and 90 kDa were de-tected in lysates derived from the DC and RC organisms, aswell as infected HL-60 cells (data not shown).

APH_1387 is induced following bacterial entry into hostcells and localizes to the AVM. We next screened A. phagocy-tophilum-infected HL-60 cells with anti-APH_1387 in conjunctionwith a MAb against Msp2 (P44) (37, 44) at 0.7, 8.5, and 24 hpostinfection and visualized the cells by LSCM. At 0.7 h postin-fection, very little or no APH_1387 was detected on Msp2 (P44)-positive organisms that were bound to the HL-60 cell surface (Fig.3A to D). By 8.5 h, ApVs containing individual A. phagocytophi-lum bacteria were detected. At this time point, the AVM waspositive for APH_1387 (Fig. 3E to H). At 24 h, the AVM wasstrikingly distinguished from enclosed bacteria by exclusive stain-

ing for APH_1387 (Fig. 3I to L). A. phagocytophilum bacteriawithin ApVs that were positive for both Msp2 (P44) andAPH_1387 were green [corresponding to Msp2 (P44) staining]spheroid organisms, each of which was surrounded by a red ring(corresponding to APH_1387 staining) or a yellow ring [corre-sponding to APH_1387 and Msp2 (P44) staining] (Fig. 3J to L).At 24 h, 94% 3.5% of the ApVs were positive for APH_1387.The AVM was negative for Msp2 (P44) at all time points.

APH_1387 localizes to the AVM during A. phagocytophiluminfection of human myeloid and endothelial cell lines and tickembryonic cell lines. In addition to HL-60 cells, A. phagocyto-philum infects and resides in ApVs in the human monocyticcell line THP-1, the human microvascular endothelial cell lineHMEC-1, and the Ixodes scapularis embryonic cell line ISE6(21, 34, 47, 53). To determine if APH_1387 is expressed andlocalizes to the AVM in each of these cell lines, A. phagocyto-philum-infected THP-1, HMEC-1, and ISE6 cells were exam-ined by LSCM at 24 h postinfection. As observed for HL-60cells, virtually all ApVs in each cell line were positive forAPH_1387 (Fig. 3M to X). Indeed, at 24 h postinfection 93%of ApVs were positive for APH_1387 in THP-1 cells (data notshown). Notably, the plasma membranes of heavily infectedTHP-1 cells (Fig. 3O and P), ISE6 cells (Fig. 3W and X), andHL-60 cells (data not shown) and occasionally uninfected cellsthat were adjacent to the heavily infected cells were also pos-itive for APH_1387.

APH_1387 is expressed and localizes to the AVM through-out A. phagocytophilum intracellular development in HL-60cells. We next assessed the temporal expression and AVM local-ization patterns of APH_1387 over the course of a synchronous A.phagocytophilum infection of HL-60 cells. Host cell-free bacteriawere added to HL-60 cells and allowed to bind for 40 min, whichwas followed by removal of unbound organisms. We and otherworkers have determined that it takes 4 h for bound A. phagocy-tophilum DC organisms to internalize in nascent vacuoles (5, 10,28). Beginning at 8 h after addition of bacteria, which corre-sponded to �4 h after entry, APH_1387 was weakly detected asa 115-kDa band by immunoblot analysis (Fig. 4A and B). Theintensity of this band increased continually during the timecourse. Beginning at 18 h, several additional bands were alsodetected, and the primary bands were at �61 and 90 kDa. As wehave shown previously (12), the intensity of the Msp2 (P44) band,which is used as an infection control, increases throughout thecourse of an infection. APH_1387 was detected on �80% of theApVs at 12 h and on �90% of the ApVs at all later time pointsby LSCM (Fig. 4C).

To more closely examine when APH_1387 is expressed andassociates with the AVM during the course of infection, wescreened synchronously infected HL-60 cells over a 48-h periodby using immunoelectron microscopy. APH_1387-negative spher-oid DC organisms were bound to the HL-60 cell surface at 0.7 h(Fig. 5A), and some organisms had internalized into nascentvacuoles by 4 h (Fig. 5B). By 8 h, which corresponded to �4 hafter entry, the internalized bacteria had changed into elongated,pleomorphic RC organisms and had begun to replicate. The sur-faces of the replicating RC organisms were positive forAPH_1387, as were portions of the AVM (Fig. 5C). MoreAPH_1387 was detected on the AVM and on intravacuolar A.phagocytophilum bacteria as the infection progressed (Fig. 5D toG). At 24 h, individual HL-60 cells contained numerous ApVs,

FIG. 2. Screening of A. phagocytophilum-infected HL-60 cells andhost cell-free bacterial populations with anti-APH_1387. Western-blottedlysates of uninfected (lane U) and A. phagocytophilum-infected HL-60cells (lane I), as well as host cell-free A. phagocytophilum populationsconsisting of RC and DC organisms (lane RC� DC) or only DC organ-isms (lane DC), were screened with anti-APH_1387. MBP-APH_1387was used as a positive control. The blot was stripped and screened withanti-Msp2 (P44) to confirm that equivalent amounts of lysates derivedfrom RC and DC organisms and from only DC organisms were used. Theresults are representative of the results of two separate experiments.

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each of which harbored several DC organisms that had roughouter membranes or contained multiple mature RC organismsthat were changing into DC organisms (Fig. 5F), which is consis-tent with our previous report on the replication kinetics of A.phagocytophilum in HL-60 cells (48). At this time point,APH_1387 heavily decorated the AVM, as well as the surfaces ofintravacuolar DC and RC organisms. APH_1387 labeling of the

AVM was most pronounced for mature ApVs that containedseveral A. phagocytophilum bacteria at 48 h (Fig. 5G and H).

APH_1387 is expressed and localizes to the AVM in vivo.Next, we investigated APH_1387 expression in vivo. MBP-APH_1387, but not MBP alone, was recognized by antiserumfrom an HGA patient (Fig. 6A), which confirmed a previousreport by Storey and colleagues (45). Likewise, Msp2 (P44)-

FIG. 3. A. phagocytophilum APH_1387 is detectable on the surfaces of intravacuolar bacteria and localizes to the AVM in infected host cells.(A to L) HL-60 cells were synchronously infected with A. phagocytophilum. At 0.7 h (A to D), 8.5 h (E to H), and 24 h (I to L) postinfection, thecells were fixed and viewed by LSCM to determine immunoreactivity with antibodies against Msp2 (P44) (major bacterial surface protein; usedto identify bacteria in panels B, F, and J) and APH_1387 (C, G, and K). Differential interfering contrast images are shown in panels A, E, and I.Merged fluorescent images are shown in panels D, H, and L. (M to X) A. phagocytophilum-infected THP-1 (M to P), HMEC-1 (Q to T), and ISE6(U to X) cells were fixed and viewed by LSCM to determine immunoreactivity with antibodies against Msp2 (P44) (N, R, and V) and APH_1387(O, S, and W). Differential interfering contrast images are shown in panels M, Q, and U. Merged fluorescent images are shown in panels P, T, andX. In panel H the arrowheads indicate individual Msp2 (P44)-positive A. phagocytophilum organisms enclosed in APH_1387-positive vacuoles. Inpanels L, P, T, and X the arrows indicate representative AVMs or portions of AVMs that exhibit exclusive staining for APH_1387. Intravacuolarbacteria are positive for Msp2 (P44) or both Msp2 (P44) and APH_1387. The results are representative of the results of three separate experiments.

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and APH_1387-positive ApVs were detected by LSCM in mu-rine buffy coats isolated from A. phagocytophilum-infectedmice at 8 days postinfection (Fig. 6B to E).

DISCUSSION

APH_1387 is the first identified A. phagocytophilum-derivedprotein that localizes to the AVM. It is expressed very earlyfollowing A. phagocytophilum entry into a nascent host cell-de-rived vacuole, is expressed continually throughout the intracellu-lar part of the bacterium’s life cycle, and heavily decorates theAVM. APH_1387 potentially traverses the AVM 3 or fewer timesby means of the 3 bilobed hydrophobic alpha-helical domains thatare predicted to be in each direct repeat. These 24-amino-acidhydrophobic domains could easily traverse the AVM, as theirlength exceeds the minimum length for transmembrane helices(20 amino acids) (38). The bilobed hydrophobicity motif ofAPH_1387 is a feature that is shared with all chlamydial Incproteins (41). In contrast to APH_1387, however, each chlamyd-ial Inc protein has a single bilobed hydrophobic motif that is 40 to60 amino acids long. Portions of pathogen-derived PVM proteinsare typically exposed on the PVM’s cytoplasmic face, where theyproject into the cytoplasm to interact with host cell proteins (41).While anti-APH_1387-mediated immunogold labeling demon-strated that APH_1387 localizes to the AVM, we cannot accu-rately determine whether one or more portions of APH_1387 arepresented on the AVM’s cytoplasmic face.

APH_1387 is expressed and modifies the AVM in vivo inneutrophils during murine infection and in vitro during intra-cellular residence in human HL-60, THP-1, HMEC-1, and tickISE6 cells, and it is expressed throughout the course of intra-cellular development. The examination of A. phagocytophilumwhole-genome transcription profile data that was performedby Nelson and colleagues for infected HL-60, HMEC-1, andISE6 cells revealed that APH_1387 is transcribed in each cellline (35), which corroborates the findings of our protein ex-pression analyses. Thus, APH_1387 is conceivably importantfor A. phagocytophilum survival in all eukaryotic host cells thatthis bacterium infects. This hypothesis is further supported bythe high degree of sequence conservation in at least 4 geo-graphically diverse A. phagocytophilum strains. Notably, anti-APH_1387 stains the plasma membranes of heavily infectedhost cells and uninfected host cells that are adjacent to heavilyinfected cells, which suggests that APH_1387 may associatewith the host cell plasma membrane.

Tandem repeat proteins of pathogenic bacteria have beenimplicated in hijacking host signaling pathways, adhesion, im-mune evasion, and other host-pathogen interactions (15, 20,26, 27, 31, 50, 52, 56). The genomes of A. phagocytophilum andE. chaffeensis each encode multiple acidic tandem repeat-car-rying proteins. Some examples that have been studied or iden-tified are P47, P120, and a variable-length PCR target proteinof E. chaffeensis and A. phagocytophilum APH_1387 andAPH_0032 (P130) (18, 32, 45, 54). Like APH_1387, P47 andP120 have been observed to be associated with the PVM (18,39). Unlike APH_1387, which is found on intravacuolar RCand DC organisms, P47 and P120 are preferentially expressedby DC ehrlichiae (18, 39). A protein alignment of APH_1387and P120 (encoded by ECH_0039 in the annotated E. chaffeen-sis genome) (55) that was performed by Storey and colleagues

FIG. 4. Kinetics of APH_1387 expression and AVM localization inA. phagocytophilum-infected HL-60 cells. HL-60 cells were synchro-nously infected with A. phagocytophilum. At the times postinfectionindicated, aliquots were processed and analyzed using Western blot-ting and densitometric analysis (A and B) or immunofluorescencemicroscopy (C). (A) Western blots of A. phagocytophilum-infectedHL-60 whole-cell lysates screened with antibodies against APH_1387,Msp2 (P44) (infection control), and actin (loading control). (B) Den-sitometry to quantify the intensities of APH_1387 and actin bands. Theratio of the APH_1387 densitometric value to the actin densitometricvalue is shown for each time point. (C) Percentages of ApVs [based onthe presence of Msp2 (P44)-positive A. phagocytophilum bacteria] thatare positive for APH_1387 staining of intravacuolar bacteria and ex-clusive staining of the AVM. The data are the means and standarddeviations for 3 separate experiments. At least 948 Msp2 (P44)-posi-tive ApVs were scored for APH_1387 for each time point.

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revealed that two short repeat segments that help compriseeach APH_1387 tandem repeat region share sequence similar-ity with portions of the tandem repeat region of P120 (45). E.chaffeensis P47 is an acidic (pI 4.2) tandem repeat-carryingprotein that interacts with host molecules involved in cell sig-naling, transcriptional regulation, and vesicle trafficking (50).The target host molecules with which APH_1387 presumablyinteracts and/or the pathobiological role of APH_1387 islikely unique to A. phagocytophilum intracellular survival, asAPH_1387 exhibits very little or no homology with any previ-ously described protein. Alternatively, APH_1387 may be im-portant for adding structural integrity to the AVM and mayhave no target ligand. The only other known Anaplasmataceaepathogen-derived inclusion membrane protein that has been

identified is Ehrlichia canis AF21920, which is a slightly basic(pI 8.49) 24.0-kDa protein with an unknown function that lackstandem repeats but does carry 5 nonlobed hydrophobic do-mains that are predicted to traverse the PVM (46).

Upon electrophoresis APH_1387 migrates primarily as a115-kDa band and also produces less prominent bands; theprimary less prominent bands are a 90-kDa band and a band ata predicted molecular mass of 61.4 kDa. While other workershave attributed this aberrant migration to glycosylation (17),our assessments of both native and recombinant APH_1387indicate that neither protein is glycosylated (unpublisheddata). All Anaplasmataceae acidic tandem repeat proteins an-alyzed to date migrate at molecular weights considerablyhigher than their predicted molecular weights. Alternatively,

FIG. 5. Assessment of A. phagocytophilum APH_1387 expression and localization to the AVM by immunoelectron microscopy. HL-60 cellswere synchronously infected with A. phagocytophilum. At 0.7 h (A), 4 h (B), 8 h (C), 12 h (D), 18 h (E), 24 h (F), and 48 h (G and H) after additionof bacteria, samples were fixed and screened with anti-APH_1387 followed by goat anti-rabbit IgG conjugated to 6-nm gold particles and examinedby electron microscopy. Representative results of two separate experiments are shown. (A and B) Asterisks indicate bound or newly internalizedA. phagocytophilum DC organisms. (C to F) Arrows indicate representative portions of the AVM that are labeled with gold particles. (H) Mag-nification of the region in panel G indicated by a box. Scale bars, 0.5 �m.

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the acidic nature of APH_1387 may prevent it from amplybinding SDS, which would retard proper SDS-PAGE resolu-tion, as has been observed for other acidic proteins and wasrecently reported for E. chaffeensis P47 by Wakeel and col-leagues (A. Wakeel, X. Zhang, and J. W. McBride, presentedat the Twenty-Third Meeting of the American Society forRickettsiology).

Large amounts of APH_1387 associated with the surfaces ofintravacuolar A. phagocytophilum RC and DC organisms aredetected by indirect immunofluorescence and immunoelectronmicroscopy. Yet APH_1387 was not detected on the surfacesof glutaraldehyde-fixed host cell-free A. phagocytophilum bac-teria that were recovered following mechanical disruption ofinfected HL-60 cells, washed with PBS, and added to naïveHL-60 cells, and it was only weakly detected on methanol-permeabilized bacteria by indirect immunofluorescence anal-ysis. Accordingly, we hypothesize that APH_1387 is not inte-grated into the A. phagocytophilum outer membrane butinstead is loosely associated with it and is easily washed awayduring the isolation procedure. We further hypothesize thatthis is because APH_1387 is transiently associated with thesurfaces of intravacuolar bacteria while it is being secreted andthat it subsequently integrates into the AVM. It is not knownhow APH_1387 is secreted. A. phagocytophilum encodes a typeIV secretion system that is homologous to the system ofAgrobacterium tumefaciens (36), and the A. phagocytophilumeffector, AnkA, can be heterologously secreted by A. tumefa-ciens (31). The C termini of type IV substrates have a netpositive charge compared to the rest of the protein sequence(49). This is not the case for the APH_1387 C terminus, whichhas a net negative charge (pI 3.34). Moreover, type IV secre-tion system-mediated delivery of effectors is contact dependent(2, 49). Thus, APH_1387 is probably not delivered to the AVMvia type IV secretion. Also, A. phagocytophilum lacks genesencoding type II and type III secretion systems (25).

Multiple pathogen-encoded PVM proteins have been foundfor a diverse array of obligate vacuolar pathogens. Indeed, �45and �65 Inc proteins have been confirmed and/or are pre-dicted for Chlamydia pneumoniae and Chlamydia trachomatis,respectively (41). Thus, it is likely that APH_1387 is just one ofa multitude of AVM proteins that are waiting to be identifiedand functionally characterized. Dissecting the roles of A.phagocytophilum-derived AVM proteins, confirming theirroute of delivery, and identifying their eukaryotic host cellligands are areas that are ripe for investigation and are essen-

tial for fully comprehending the intracellular survival strategiesof this unusual bacterial pathogen that effectively resides in theprimary effector cell for killing microbes.

ACKNOWLEDGMENTS

We thank J. Stephen Dumler of Johns Hopkins University for pro-viding MAb 20B4; Erol Fikrig of Yale University for providing HGApatient antiserum; Ulrike Munderloh for providing A. phagocytophi-lum-infected ISE6 cells; Yasuko Rikihisa and Ralph Horwitz for pro-viding A. phagocytophilum strain HZ; Naomi Walker and DexterReneer for technical assistance; and Jere W. McBride and Richard T.Marconi for invaluable discussions.

This work was supported by NIH grants DK065039 and AI072683and by funds from the National Research Fund for Tick-Borne Dis-eases. The Virginia Commonwealth University Department of Neuro-biology and Anatomy Microscopy Facility is supported in part by fundsfrom NIH-NINDS Center core grant 5P30NS047463.

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