Anaplasma phagocytophilum and Anaplasma marginale Elicit Different Gene Expression Responses in Cultured Tick Cells

The genus Anaplasma (Rickettsiales: Anaplasmataceae) includes obligate tick-transmitted intracellular organisms, Anaplasma phagocytophilum and Anaplasma marginale that multiply in both vertebrate and tick host cells. Recently, we showed that A. marginale affects the expression of tick genes that are involved in tick survival and pathogen infection and multiplication. However, the gene expression profile in A. phagocytophilum-infected tick cells is currently poorly characterized. The objectives of this study were to characterize tick gene expression profile in Ixodes scapularis ticks and cultured ISE6 cells in response to infection with A. phagocypthilum and to compare tick gene expression responses in A. phagocytophilum- and A. marginale-infected tick cells by microarray and real-time RT-PCR analyses. The results of these studies demonstrated modulation of tick gene expression by A. phagocytophilum and provided evidence of different gene expression responses in tick cells infected with A. phagocytophilum and A. marginale. These differences in Anaplasma-tick interactions may reflect differences in pathogen life cycle in the tick cells.


Introduction
Ticks transmit pathogens that greatly impact both human and animal health [1]. The genus Anaplasma (Rickettsiales: Anaplasmataceae) includes obligate tick-transmitted intracellular organisms found exclusively within membranebound inclusions or vacuoles in the cytoplasm of both vertebrate and tick host cells [2,3]. A. marginale infects cattle and wild ruminants and causes bovine anaplasmosis [2]. A. phagocytophilum infects humans and wild and domesticated animals [2,4,5] and is the causative agent of human, equine and canine granulocytic anaplasmosis and tick-borne fever of ruminants [5,6]. In the United States, A. phagocytophilum is transmitted by Ixodes scapularis and I. pacificus [2,4].
The ticks and the pathogens they transmit have evolved molecular interactions that affect their survival and life cycle [3]. The A. phagocytophilum outer membrane proteins that are involved in interactions with tick cells have been identified and partially characterized [7,8]. Recently, we identified and characterized tick molecules that are involved in A. marginale-tick interactions, demonstrating that A. marginale affects the expression of tick genes essential for tick survival and pathogen infection and multiplication [9]. However, tick molecules that are affected by and participate in A. phagocytophilum infection and multiplication are currently poorly characterized [10,11].
The objectives of this study were the characterization of tick gene expression profiles in I. scapularis ticks and cultured tick cells in response to infection with A. phagocytophilum and to compare tick gene expression responses in A. phagocytophilum-and A. marginale-infected cultured tick cells by microarray and real-time RT-PCR analyses. The results reported herein demonstrated modulation of tick gene expression by A. phagocytophilum and identified differentially expressed genes that are relevant for the understanding of basic biological questions of A. phagocytophilum life cycle in I. scapularis. The results also provided evidence of differences in tick gene expression in response to infection with A. phagocytophilum or A. marginale.

Uninfected and Anaplasma-Infected Ticks and Tick Cells.
The I. scapularis nymphs uninfected and infected with A. phagocytophilum (Gaillard and Dawson strains) were obtained from a laboratory colony reared at the Centers for Disease Control and Prevention, Atlanta, Ga, USA. Tick larvae were fed on uninfected or infected mice, collected after feeding, and allowed to molt to nymphs. Animals were housed with the approval and supervision of the Institutional Animal Care and Use Committee.
The tick cell line ISE6, derived from I. scapularis embryos (provided by U.G. Munderloh, University of Minnesota, USA), was cultured in L15B medium as described previously for IDE8 cells [12], but the osmotic pressure was lowered by the addition of one-fourth sterile water by volume after Munderloh et al. [13]. The ISE6 cells were first inoculated with A. phagocytophilum-(NY18 isolate) infected HL-60 cells and maintained according to Munderloh et al. [13] until infection was established and routinely passaged. Infected ISE6 cells were frozen in liquid nitrogen and served as inoculum for uninfected cells. The ISE6 cells were initially infected with A. marginale (Virginia isolate) from a frozen inoculum of infected bovine erythrocytes. Following infection and routine passage, infected ISE6 cells were frozen in liquid nitrogen and served to infect uninfected ISE6 cells. For the current study each inoculum of infected cells was thawed and centrifuged, and the pellet was resuspended in culture medium and put on the ISE6 cells. When the infection reached approximately 80% of the tick cells, the monolayer was passaged onto uninfected ISE6 monolayers and maintained in L15B medium as described above. Monolayers of infected ISE6 cells were collected at different time points as described above. Uninfected cells were cultured in the same way but adding 1 mL of culture medium instead of infected cells. Collected cells were centrifuged at 10 000 × g for 3 minutes, and cell pellets were frozen in liquid N until used for RNA extraction.
The infection of ticks and tick cells with A. phagocytophilum or A. marginale was corroborated by major surface protein 4 (msp4) PCR [14,15].

Microarray Analysis.
Infected tick ISE6 cells were sampled at 6 days postinfection (dpi) with approximately 70% infected cells (separate cell cultures grown under similar conditions had >90% cells infected at 8 dpi). Uninfected cells were sampled at the same time point as infected cells to account for culture time effects. Total RNA was extracted from three A. marginale-infected, three A. phagocytophiluminfected, and three uninfected ISE6 cell cultures using the RNeasy Mini Kit (Qiagen) including the on-column DNA digestion with the RNase-free DNase set following manufacturer's instructions. RNA quality was checked by gel electrophoresis to verify the integrity of RNA preparations. Total RNAs (5 μg) were labeled using the 3DNA Array900 kit with Alexa Fluor dyes (Genisphere, Hatfield, Pa, USA), Superscript II (Invitrogen, Carlsbad, Calif, USA), the supplied formamide-based hybridization buffer and 24 × 60 mm LifterSlips (Erie Scientific, Portmouth, NH, USA) according to the manufacturer's (Genisphere) instructions. The microarray was constructed with 768 random I. scapularis sequences enriched for genes differentially expressed after subolesin knockdown as previously described [16] (NCBI Gene Expression Omnibus (GEO) platform accession number GPL6394 and series number GSE10222). Eight pools of 12 clones each from an unsubtracted I. scapularis cDNA library and subolesin cDNA were also arrayed and used to validate normalization. Hybridization signals were measured using a ScanArray Express (PerkinElmer, Boston, Mass, USA), and the images were processed using GenePix Pro version 4.0 (Axon, Union City, Calif, USA). Ratios were calculated as Anaplasma-infected cells versus uninfected control cells. Preprocessing of data was accomplished using R-project statistical environment (http://www.r-project.org) and Bioconductor (http://www.bioconductor.org) and the LIMMA package as previously described [17]. This included (1) removal of data points where signal was less than the background plus two standard deviations in both channels, (2) removal of data points where signal was less than 200 RFU in both channels, (3) removal of poor quality spots flagged during image processing, (4) removal of spots with less than 50% valid biological and technical replicates, (5) log transformation of the background subtracted mean signal ratios, and (6) normalization using global Lowess intensitydependent normalization. Normalized ratio values obtained for each probe were averaged across 3 biological replicates, and four technical replicates and significant differences were defined as P-value ≤ .05 and displaying an expression fold change greater than 2-fold in either A. phagocytophilum or A. marginale infected cells.

Sequence Analysis and Database
Search. Partial sequences were determined for cDNA sequences identified as differentially expressed in the microarray analysis. Multiple sequence alignment was performed using the program AlignX (Vector NTI Suite V 8.0, InforMax, Invitrogen, Carisbad, Calif, USA) to exclude vector sequences and to identify redundant (not unique) sequences. Searches for sequence similarity were performed with the BLASTX program (http://www.ncbi.nlm.nih.gov/BLAST) against the Table 1: RT-PCR oligonucleotide primers and conditions for the characterization of the expression profiles of differentially expressed tick genes.
Gene ID (a) Upstream/downstream primer sequences PCR annealing temperature (a) IDs for I. scapularis genes are described herein and in de la Fuente et al. [9]. The A. phagocytophilum and A. marginale infection levels were evaluated in tick ISE6 cells at 2, 5, and 8 dpi by real-time PCR of msp4 and normalizing against tick 16S rDNA sequences using the QuantiTec SYBR Green PCR kit (Qiagen) in an iQ5 thermal cycler (Bio-Rad) as described above. Known amounts of the full length A. phagocytophilum and A. marginale msp4 PCR product were used to construct a standard for quantitation of pathogens per cell.

Nucleotide Sequence Accession Numbers.
The nucleotide sequences of the ESTs reported in this paper have been deposited in the GenBank database under accession numbers FL685631-FL685658.

A. phagocytophilum Modulates Gene Expression in
Infected I. scapularis Nymphs and Tick ISE6 Cells. The infection with A. marginale has been shown to modulate tick gene expression [9]. However, the effect of A. phagocytophilum infection on tick gene expression is unknown. Here, two experimental approaches were used to characterize gene expression profiles in tick cells infected with A. phagocytophilum. In the first approach, tick gene expression was characterized by microarray analysis of RNA from infected and uninfected tick ISE6 cells. In the second approach, genes identified as differentially expressed in tick IDE8 cells and ticks infected with A. marginale were used to characterize the effect of A. phagocytophilum on tick gene expression in infected I. scapularis nymphs and tick ISE6 cells by real-time RT-PCR.
The microarray analysis showed in A. phagocytophiluminfected tick ISE6 cells the upregulation of genes C4B10 with homology to von Willebrand factor and R1E12 with homology to ribosomal protein L32, C4A10, C3C3, C4A1, and C3D9 with unknown function and the downregulation of genes C3B2 with homology to an aspartic protease and R2A12, C3A7, R2G1, R2D6, C3C11, and R3D4 with unknown function ( Table 2). The expression of other genes with homology to troponin I (C2E6), putative secreted salivary protein (C4G3), and ML domain-containing protein (R4G5) did not change after infection of tick ISE6 cells with A. phagocytophilum ( Table 2).
The mRNA levels of selected genes differentially expressed in A. phagocytophilum-infected tick ISE6 cells were evaluated by real-time RT-PCR in infected and uninfected cells ( Figure 1). Similar to microarray hybridization results, the analysis of mRNA levels by real-time RT-PCR showed significant upregulation of C4B10 (von Willebrand factor) and R1E12 (ribosomal protein L32) and downregulation of C3B2 (aspartic protease) in tick ISE6 cells infected with A. phagocytophilum (Figure 1). The mRNA levels of genes identified previously as differentially expressed in tick IDE8 cells and ticks infected with A. marginale [9] were also evaluated by real-time RT-PCR in infected and uninfected tick ISE6 cells. The mRNA levels of genes differentially expressed in A. marginale-infected ISE6 cells were similar to those reported previously in infected IDE8 cells [9] and data not shown. The results in A. phagocytophilum-infected ISE6 cells showed that pathogen infection significantly upregulated the expression of U2A8 (signal sequence receptor delta), 1I5B9 (ixodegrin-2A RGD containing protein), and 1I4G12 (unknown function) and downregulated the expression of 2I3A7 (NADH-ubiquinoe oxidoreductase) and 1I1H6 (glutathione S-transferase (GST)) in tick ISE6 cells (Figure 1).

Discussion
We have shown previously that A. marginale modulates gene expression in infected ticks and tick cells [9]. In the experiment reported herein we hypothesized that A. marginale and A. phagocytophilum may elicit similar gene expression responses in infected cultured tick cells. To test this hypothesis, gene expression profiles were compared between A. phagocytophilum and A. marginale in infected tick ISE6 cells. The results showed that A. phagocytophilum modulates gene expression in infected I. scapularis nymphs  Comparative and Functional Genomics and cultured tick ISE6 cells but with different gene expression profiles when compared with A. marginale. These results suggested that A. marginale and A. phagocytophilum produce different differential gene expression profiles in infected tick cells. These differences in Anaplasma-tick interactions may reflect differences in pathogen developmental cycle in the tick cells. Alternatively, differences in gene expression profiles between A. phagocytophilum-and A. marginaleinfected tick ISE6 cells may be due to the fact that I. scapularis is not a natural vector of A. marginale. However, I. scapularis-cultured cells have shown to provide functionally relevant data for the study of tick-A. marginale interactions [9]. The differences in gene expression between A. marginale-and A. phagocytophilum-infected tick cells could be attributed to nonspecific responses to the presence of bacterial components that differ between the two Anaplasma species While this explanation is potentially possible, it is more likely that gene expression profiles resulted from Anaplasma intracellular infection because at least for some genes differential expression persisted until 8 dpi, when >90% cells were infected. Taken together the results reported here consistently provided differences in gene expression profiles between A. phagocytophilum-and A. marginaleinfected tick cells. Importantly, sampling time points during Anaplasma infection of tick ISE6 cells may be important to characterize the expression of particular genes. Although not addressed in this study, these differences may be also present during tick feeding and development.
The genes differentially expressed in I. scapularis nymphs and tick ISE6 cells infected with A. phagocytophilum included some genes such as GST and ferritin shown previously to affect A. marginale infection and/or multiplication in ticks and/or tick cells [9]. However, while GST and ferritin were upregulated and downregulated after A. marginale infection, respectively, they were regulated in the opposite direction in A. phagocytophilum-infected ticks and tick cells. GST, ferritin, and aspartic protease (C3B2), also found to be differentially expressed in A. phagocytophilum-infected ISE6 cells, have been reported to be regulated by tick feeding or infection with other pathogens [12][13][14][15][16][17][18]. Other genes differentially expressed after A. phagocytophilum infection such as U2A8 (signal sequence receptor delta), 1I5B9 (ixodegrin-2A RGD containing protein), 2I3A7 (NADH-ubiquinone oxidoreductase), 2IP10 (ubiquitin C variant 5-like), 2I3A3 (gamma actin-like protein), C4B10 (von Willebrand factor), C2E6 (troponin I), and R1E12 (ribosomal protein L32) constitute new findings and may be involved in infection and/or multiplication of the pathogen in ticks or are part of tick cell immune response to moderate infection levels. As shown previously for A. marginale [9], RNA interference experiments may help to characterize the function of differentially expressed genes during A. phagocytophilum infection of ticks and tick ISE6 cells.
The expression of selected genes was analyzed in I. scapularis ISE6 cells and nymphs infected with A. phagocytophilum. These experiments allowed us to compare the results of gene expression in vitro and in vivo. The nymphs were selected for analysis because this stage plays an important role during pathogen transmission to humans [5]. The ISE6 cell line was obtained from embryos of I. scapularis, one of the natural vectors of A. phagocytophilum [19]. However, this cell line is heterogeneous in the cell types represented [19], which may have different susceptibility and response to pathogen infection. Although cultured tick cells have been shown to be a good model for the study of tick-Anaplasma interactions [19][20][21], these results demonstrated that differences may exist between I. scapularis-cultured tick cells and nymphs in the mRNA levels of certain genes, at least under the experimental conditions used herein. These differences may account for differences in gene expression between infected ISE6 cells and tick whole nymph tissues and/or due to differences in the infection levels in both systems. An additional source of potential differences in gene expression between I. scapularis ISE6 cells and nymph tissues could be attributed to the fact that different cells types may be infected in both systems resulting in different responses to infection. Finally, although less likely, these differences may be related to differences between the NY18 isolate used to infect ISE6 cells and the Gaillard and Dawson strains used to infect I. scapularis nymphs.
Recent studies have characterized A. marginale and A. phagocytophilum proteins that are involved in interactions with tick cells [7,8,22]. However, tick-Anaplasma coevolution also involves genetic traits of the vector as demonstrated recently in studies on the role of tick proteins in the infection and transmission of A. marginale [9,11] and A. phagocytophilum [10,11]. Furthermore, genetic factors have been associated with intraspecific variation in vector competence for a variety of vector-borne pathogens, including A. phagocytophilum [23] and A. marginale [24,25].

Conclusions
In summary, we have characterized the gene expression profile in A. phagocytophilum-infected I. scapularis nymphs and cultured ISE6 cells. Interestingly, differential gene expression seems to differ between A. marginale and A. phagocytophilum infected cultured tick cells. Future experiments would provide detailed information on the role of these genes during A. phagocytophilum life cycle in ticks. These results provide fundamental information toward understanding tick-Anaplasma interactions and may lead to formulations of new interventions for the prevention of the transmission of tick-borne pathogens.

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The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the CDC or the United States Department of Health and Human Services.