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J Bacteriol. Dec 2007; 189(23): 8651–8659.
Published online Sep 28, 2007. doi:  10.1128/JB.00881-07
PMCID: PMC2168941

Anaplasma phagocytophilum p44 mRNA Expression Is Differentially Regulated in Mammalian and Tick Host Cells: Involvement of the DNA Binding Protein ApxR[down-pointing small open triangle]

Abstract

The natural life cycle of Anaplasma phagocytophilum, an obligatory intracellular bacterium that causes human granulocytic anaplasmosis, consists of alternate infection of two distinct hosts, ticks and mammals, in which bacterial surface proteins are expected to have a critical role. The present study investigated regulation of A. phagocytophilum p44 genes, which encode the P44 major surface proteins. Quantitative real-time reverse transcription-PCR analysis revealed that the amount of p44 mRNA obtained from spleens of A. phagocytophilum-infected SCID mice was approximately 10-fold greater than the amount obtained from salivary glands of A. phagocytophilum-infected Ixodes scapularis nymphs. Similarly, the amount of p44 mRNA obtained from A. phagocytophilum-infected HL-60 cells per bacterium was significantly greater than the amount obtained from infected ISE6 tick cells. The relative amount of p44 mRNA was approximately threefold higher in A. phagocytophilum-infected HL-60 cells cultured at 37°C than in A. phagocytophilum-infected HL-60 cells cultured at 28°C. Although there are more than 100 p44 paralogs, we observed expression mainly from the p44 expression locus (p44E) in various host environments. Interestingly, transcription of the A. phagocytophilum gene encoding the DNA binding protein ApxR was also significantly greater in A. phagocytophilum-infected HL-60 cells than in infected ISE6 tick cells. Gel mobility shift and DNase I protection assays revealed recombinant ApxR binding to the promoter regions of p44E and apxR. ApxR also transactivated the p44E and apxR promoter regions in a lacZ reporter assay. These results indicate that p44 genes and apxR are specifically up-regulated in the mammalian host environment and suggest that ApxR not only is positively autoregulated but also acts as a transcriptional regulator of p44E.

The obligatory intracellular bacterium Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis (5, 11), is well adapted to a natural cycle of alternately infecting vertebrates and blood-sucking ticks. A. phagocytophilum surface proteins at the host-pathogen interface are expected to be differentially regulated in the two divergent host environments so that the bacterium can infect and acquire nutrients from the hosts and evade killing by the host immune systems.

The polymorphic 44-kDa major outer membrane proteins (P44s, Msp2s) of A. phagocytophilum are the primary bacterial surface-exposed antigens recognized by immune systems of human granulocytic anaplasmosis patients, experimentally infected mice, and horses (8, 14, 20, 30, 33-35). P44 has been demonstrated to function as a porin, facilitating transport of some sugars and amino acids through the bacterial membrane (13). Notably, 113 p44 genes are present in the A. phagocytophilum genome, including 22 full-length p44 genes, 64 shorter p44 genes (without a start codon), 21 fragmented p44 genes (containing only the 5′ or 3′ conserved region), and 6 truncated p44 genes (containing only the hypervariable region) (12). One special polymorphic p44 gene in the p44 (msp2) expression locus is designated p44E (p44ES) (3, 18). Expression of either a full-length or shorter p44 occurs after the hypervariable region of a p44 replaces the p44E currently occupying the p44 expression locus by nonreciprocal recombination in a RecF-dependent manner (3, 16, 18, 19). Each expressed p44 can be identified by its signature central hypervariable sequence. While many studies have shown expression of different p44 species in different host environments (3, 15, 16, 18, 30, 34), little is known about the regulation of p44 transcription. Unlike the well-studied bovine erythrocytic agent Anaplasma marginale, which has a single full-length msp2 gene and thus a single msp2 expression locus (4), there are many full-length p44 genes in the A. phagocytophilum genome which need to be examined. Thus, the aim of the present study was to characterize the influences of mammalian and tick host environments on total p44 and p44E expression levels. Because transcription of the A. phagocytophilum gene encoding the DNA binding protein ApxR that regulates the tr1 promoter in A. phagocytophilum (28) was also significantly changed in tick and mammalian environments, we also examined the possible involvement of ApxR in p44E transcriptional regulation.

MATERIALS AND METHODS

A. phagocytophilum and cell culture.

The A. phagocytophilum HZ strain (23) was propagated in HL-60 cells in RPMI 1640 medium supplemented with 5% heat-inactivated fetal bovine serum (US Bio-Technologies, Parkerford, PA) and 2 mM l-glutamine in a humidified 5% CO2 atmosphere at 37°C. HL-60 cells are a promyelocytic human leukemia cell line that can be induced to differentiate to neutrophil-like cells in response to some chemical stimuli (7). A. phagocytophilum strain HGE2 (11) was propagated in ISE6 Ixodes scapularis tick cells in L15B medium (with 80 mM glucose) supplemented with 10% heat-inactivated fetal bovine serum and 10% tryptose phosphate broth at 34°C (Sigma, St. Louis, MO) as described previously (22). No antibiotic was used in the culture. The degree of bacterial infection in host cells was assessed by Diff-Quik staining (Baxter Scientific Products, Obetz, OH) of cytocentrifuged preparations.

Tick salivary gland and mouse spleen samples.

Salivary glands from transmission-fed I. scapularis nymphs infected with A. phagocytophilum strain NTN-1 (25) preserved in RNAlater (QIAGEN, Valencia, CA) (9) were homogenized with a Kontes pellet pestle motor (Kimble-Kontes, Vineland, NJ) for 3 min, followed by passage through a 26-gauge 0.375-in needle 30 times. Four-week-old ICR SCID male mice (Taconic Farm Inc., Germantown, NY) were inoculated intraperitoneally with 106 HL-60 cells infected with A. phagocytophilum strain HZ (80% infected cells). Plasma and spleen specimens were collected from three mice on day 26 postinoculation.

Quantitative real-time RT-PCR.

Total DNA was extracted with a QIAamp blood kit (Qiagen), and total RNA was extracted with an RNeasy Protect mini kit (Qiagen). For cDNA synthesis, 2 μg of total RNA from A. phagocytophilum HZ-infected HL-60 cells or HGE2-infected ISE6 cells, 5 μg of total RNA from one tick salivary gland, or ~30 mm3 of mouse spleen tissue was first treated with DNase I at a final concentration of 0.1 U/μl (Invitrogen, Carlsbad, CA) at 25°C for 15 min. To stop the reaction, EDTA was added to a final concentration of 2.5 mM and the DNase I was heat inactivated at 65°C for 10 min. Reverse transcription (RT) reactions were performed by adding 200 ng of random hexamers and deoxynucleoside triphosphates (final concentration, 0.5 mM each). The mixtures were then heated at 70°C for 5 min and chilled on ice to denature the RNA, and then they were reverse transcribed using a final volume of 20 μl (for cell culture samples) or 40 μl (for tick and spleen samples). Reaction mixtures containing 200 U of Superscript III reverse transcriptase (Invitrogen), 2 μl of 0.1 μM dithiothreitol, 2 U of RNaseOUT RNase inhibitor (Invitrogen), and an appropriate amount of the manufacturer's reaction buffer were incubated at 50°C for 50 min. To control for DNA contamination, an identical reaction mixture was prepared without reverse transcriptase. Quantitative PCR was performed with a real-time instrument (MX3000P; Stratagene, Austin, TX) using a Brilliant SYBR green QPCR core reagent kit (Stratagene) as previously described (6) and primers shown in Table S1 in the supplemental material.

To compare transcription levels for different cDNA fragments, the corresponding target DNA fragments were amplified by PCR from the A. phagocytophilum genomic DNA and cloned into the pCR II vector from a TA cloning kit (Invitrogen) to serve as control DNA standards. The plasmid copy number was calculated based on the concentration of the DNA and the plasmid size. Tenfold serial dilutions (from 102 to 108 copies) of the control plasmids were used in the real-time PCR to construct the standard curve. Assays with a linear regression R value of >0.99 were acceptable. Tenfold serial dilutions of each cDNA specimen were also tested using the real-time PCR. The cDNA copy number for each specimen was calculated based on the standard curve.

5′ RACE.

The 5′ rapid amplification of cDNA ends (5′ RACE) assay was performed using a 5′ RACE kit (Invitrogen). The cDNAs were prepared as described above, tails were added by adding cytosine residues at the 3′ end using terminal transferase (Invitrogen), and the cDNAs were amplified by using two sets of hemi-nested PCR primers, with each set containing an oligo(dG)-linked primer (Invitrogen) and a locus-specific primer (Fig. (Fig.1;1; see Table S1 in the supplemental material). Thirty-five cycles of amplification were performed as follows: 1 min at 94°C, 1 min at 55°C, and 1 min at 72°C. The PCR product bands were purified by agarose gel electrophoresis, excised from the gel, recovered using a QIAEX II gel extraction kit (Qiagen), and then cloned into the pCR2.1 TOPO vector with a TA cloning kit (Invitrogen). Inserts were sequenced with an Applied Biosystems 3730 DNA analyzer using a BigDye terminator cycle sequencing reaction kit (Applied Biosystems, Inc., Foster City, CA). Sequence assembly, alignment, and analysis were performed using SeqMan and MegAlign programs (DNAStar, Inc. Madison, WI).

FIG. 1.
Schematic representation of the promoter region of p44E in the p44 expression locus. The 213-bp intergenic region between omp-1N and p44E (p44EI) is indicated by a horizontal line. The bent arrow at −157 represents the transcriptional start site ...

Electrophoretic mobility shift assay (EMSA).

The 213-bp intergenic region between omp-1N and p44E (p44EI) and a promoter fragment (350 bp) upstream of the apxR gene were amplified by PCR with the primer pairs shown in Table S1 in the supplemental material and were purified as described above. Recombinant ApxR (rApxR) purified as described by Wang et al. (28) or bovine serum albumin (BSA) as a negative control was incubated with amplified DNA fragments (4 pmol each) for 30 min at 4°C in 20 μl of binding buffer (10 mM Tris-HCl [pH 7.5], 50 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol, 2.5% glycerol, 0.1% [wt/vol] NP-40). Samples were electrophoresed as previously described (28). The probes were visualized by staining with ethidium bromide (0.5 μg/ml) in 0.5× Tris-borate-EDTA buffer (0.044 M Tris base, 0.044 M boric acid, 0.001 M EDTA; pH 8.0) for 20 min.

DNase I footprint analysis.

A 338-bp DNA fragment upstream of p44E and a 350-bp DNA fragment upstream of apxR were amplified by PCR with the primer sets shown in Table S1 in the supplemental material, except that the 5′ end primers were 5′ labeled with 6-carboxyfluorescein (FAM) by Applied Biosystems, Inc. (Foster City, CA). A FAM-labeled probe (300 ng) was incubated with 5 or 1.25 μM rApxR or BSA as a control under the conditions described previously (36). Based on the results of DNase I (Worthington Biochemicals, Lakewood, NJ) optimization experiments, 0.02 U of DNase I was added to each reaction mixture and incubated for 5 min at 25°C. The reaction was terminated by heating the mixture at 75°C for 10 min. The digested DNA fragments were immediately purified using a QIAquick PCR purification kit (Qiagen) and eluted in 40 μl Tris buffer (2 mM Tris, pH 8.5). Nondigested DNA was used for sequencing reactions with the 5′ FAM-labeled forward primers (see Table S1 in the supplemental material) and a Thermo Sequenase dye primer manual cycle sequencing kit (USB, Inc., Cleveland, OH). The digested DNA and sequencing reaction products were analyzed with a 3730 DNA analyzer (Applied Biosystems, Inc.) (36). The sequences were then analyzed with GeneMapper software (Applied Biosystems, Inc.) to convert the DNase I digestion map into sequencing data to identify the exact sequences that were protected.

Construction of lacZ reporter fusion plasmids for β-galactosidase assays.

The full-length lacZ gene was amplified from plasmid pQF50 using Pfu DNA polymerase (Stratagene, La Jolla, CA). The 213-bp p44EI region and a 350-bp DNA fragment upstream of apxR were amplified by PCR using primers shown in Table S1 in the supplemental material. The lacZ transcriptional fusion plasmid was constructed by cloning the amplified p44EI or apxR promoter fragments upstream of the promoterless lacZ gene in pACYC184 (New England Biolabs, Beverly, MA). Escherichia coli BL21(DE3) cells already containing either the empty pET29a(+) vector (Novagen Inc., Madison, WI) or pApxR [a pET29a(+)-based vector expressing full-length ApxR-His6 under control of the T7 promoter] (28) were transformed with the pACYC184 construct containing either the p44EI or apxR promoter fragments fused to lacZ. After overnight culture, transformants were subcultured in LB medium supplemented with 50 μg/ml kanamycin and 25 μg/ml chloramphenicol at 37°C for 2 h, which was followed by isopropyl-β-d-thiogalactopyranoside (IPTG) induction for 1 h. β-Galactosidase activity was measured as described previously (21).

Western blot analysis.

After IPTG induction, a sample of each E. coli suspension was dissolved in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis gel loading buffer (50 mM Tris-HCl [pH 6.8], 2% SDS, 0.1% bromophenol blue, 10% glycerol, 5% β-mercaptoethanol) and incubated at 95°C for 10 min. Approximately 20 μg of total protein was loaded in each well of a 15% SDS-polyacrylamide gel, electrophoresed, and transferred to a nitrocellulose membrane. The membrane was incubated with peroxidase-conjugated antipolyhistidine antibody (A7058; Sigma) at a 1:500 dilution, and reacting bands were visualized by enhanced chemiluminescence (GE Healthcare, Piscataway, NJ).

Statistical analyses.

Statistical analyses were performed by using analysis of variance and Tukey's honestly significant difference (HSD) test or Student's t test, and a P value of <0.01 was considered significant.

RESULTS

Comparison of p44 expression in mice and ticks.

Previous studies showed that p44E can be cotranscribed from upstream genes or transcribed from its own promoter (18) and that full-length p44-1 can be expressed from its own genomic locus (32). Therefore, in order to compare p44 expression, first we compared p44 expression from different genomic regions by designing three PCR target regions of approximately 100 bp each, as shown in Fig. Fig.1.1. Region R1 (119 bp) transcription is expected to occur from promoters upstream of omp-1N or genes further upstream (1, 3, 18). Region R2 (124 bp) transcription represents p44E transcription, because the 27-bp region upstream of the ATG start codon is unique to p44E based on a BLAST search of the entire A. phagocytophilum genome. Region N1 (146 bp) represents the total p44 expression because all p44 transcripts examined so far have a conserved sequence in this region (16, 18). A comparison of transcription levels for the different genomic locations was performed by estimating the resulting cDNA copy numbers. To control for primer-based differences in PCR efficiency, cDNA copy numbers were estimated based on the standard curve generated by using serial dilutions of the target DNA fragments cloned into pCR II. The results were then normalized to A. phagocytophilum 16S rRNA levels (Table (Table1)1) or 16S rRNA gene levels.

TABLE 1.
Quantitative real-time RT-PCR analysis of two regions of the p44 expression locus (R1 and R2) and the total p44 genes (N1) normalized to bacterial 16S rRNA from the spleens of three individual A. phagocytophilum-infected SCID micea

The R2 and N1 transcription levels from A. phagocytophilum in spleens from three individual SCID mice were similar and approximately 5- to 10-fold greater than the R1 transcription levels (Table (Table1).1). This indicates that in A. phagocytophilum residing in the mouse spleen, most p44 transcription occurs in a monocistronic fashion at p44E and from the promoter upstream of p44E (p44EI).

To confirm this result, 5′ RACE was performed with RNA specimens from infected mouse spleens using primers specific to the p44 5′ conserved region (Fig. (Fig.1;1; see Table Table11 in the supplemental material). The assay revealed an obvious approximately 600-bp band (Fig. (Fig.2A).2A). There was no DNA contamination, as no PCR product was detected in the samples that lacked reverse transcriptase (Fig. (Fig.2A).2A). Five PCR clones were sequenced, and for each clone the 5′ end of the cDNA was 157 bp upstream of p44E (Fig. (Fig.2B).2B). This monocistronic start site matched the 5′ end previously found by 5′ RACE in four strains of A. phagocytophilum, including the HZ strain cultured in HL-60 cells at 37°C, but was reported to be 199 bp upstream of p44E, because the downstream (second) ATG was chosen as the putative translational start site (18).

FIG. 2.
Analysis of the A. phagocytophilum p44E transcriptional start sites from infected mouse spleen tissue specimens. (A) Agarose gel electrophoresis of 5′ RACE products generated using the p44-specific primer shown in Table S1 in the supplemental ...

The salivary glands of I. scapularis nymphal ticks that had been infected as larvae were examined for p44 expression immediately after the ticks fed on naïve mice (9). In each salivary gland dissected from three individual ticks, the R1 and N1 transcription levels were similar, suggesting that a substantial portion of the transcription was polycistronic and, therefore, started upstream of the −157 transcriptional start site in tick salivary glands (Table (Table2).2). Relative to A. phagocytophilum 16S rRNA levels, the R1 transcript copy number in tick salivary glands was approximately 10-fold lower than that in mouse spleen tissue; likewise, the N1 transcript copy number was approximately 100-fold lower. Although the R2 transcript could not be directly measured due to the limited amount of RNA that could be recovered from individual tick salivary glands, the results suggest that there was negligible monocistronic transcription from the −157 start site in the tick salivary glands. These results also imply that most p44 transcription in ticks and mice takes place at the p44 expression locus.

TABLE 2.
Quantitative real-time RT-PCR analysis of the total p44 genes (N1) and the R1 region of the p44 expression locus normalized to bacterial 16S rRNA from the salivary glands of three individual A. phagocytophilum-infected transmission-fed ticks

p44E expression is down-regulated at a lower temperature and in tick cells.

In A. phagocytophilum-infected HL-60 cells cultured at 37 or 28°C, the transcript levels of R2 were similar to the total p44 (N1) transcript levels, indicating that the p44E transcript represents the primary p44 transcript. At both 28 and 37°C, the transcript levels of R1 were approximately one-third the transcript levels of R2, suggesting that only approximately one-third of the p44E transcription in HL-60 cells was polycistronic, initiating upstream of the −157 start site (Fig. (Fig.3).3). The R1, R2, and N1 transcript levels in A. phagocytophilum cultured at 28°C were approximately one-half to one-third lower than those in A. phagocytophilum cultured at 37°C (Fig. (Fig.3).3). A. phagocytophilum 16S rRNA levels were used to normalize the input RNA for pairs of specimens obtained from two different culture temperatures. The ratios of the 16S rRNA to the 16S rRNA gene were similar (approximately 600:1) at 24 h after the culture temperature was shifted from 37 to 28°C or after the culture temperature was maintained at 37°C. Therefore, the p44 expression per unit of 16S rRNA or per unit of 16S rRNA gene (per bacterium) was significantly down-regulated in A. phagocytophilum in HL-60 cells cultured at 28°C compared to cells cultured at 37°C.

FIG. 3.
Transcription of p44 is down-regulated at 28°C. Quantitative real-time RT-PCR was performed with primers targeting the R1, R2, and N1 regions of p44E (see Table S1 in the supplemental material) to compare mRNA copy numbers at 37 and 28°C ...

Because p44 expression by A. phagocytophilum was significantly down-regulated in the tick salivary gland and in an HL-60 28°C culture, we examined whether p44E expression was also down-regulated in tick cells in culture. In infected ISE6 tick cells cultured at 34°C, the relative amount of p44E transcript per bacterium was approximately 5,000-fold lower than that in infected HL-60 cells cultured at 37°C (0.153 ± 0.011 transcript per bacterium versus 764 ± 138 transcript per bacterium; n = 3).

ApxR binds to the promoter region of p44E and transactivates p44E in a lacZ reporter assay.

A. phagocytophilum ApxR is a 119-amino-acid (12.5-kDa) protein, which was found to bind and transactivate tr1 in a lacZ reporter assay (28). Therefore, we tested the binding of ApxR to a p44EI probe in an EMSA and found that increasing the concentration of the rApxR protein shifted the probe in a dose-dependent manner (Fig. (Fig.4A).4A). Dose-dependent multiple bands suggested that there was more than one binding site upstream of tr1 (four binding sites) (28). This also suggested that there was coordinate binding of rApxR or rApxR was aggregated at a higher concentration.

FIG. 4.
rApxR binds to and transactivates the p44E upstream region (p44EI). (A) Dose response of rApxR (5, 1, and 0.2 μg) binding to the 213-bp p44EI fragment (4 pmol per lane) in an EMSA. DNA was visualized by ethidium bromide staining, and the free ...

DNase I protection assays revealed the DNA sequences to which rApxR bound. The protected regions of the sense strand were determined by comparing the sequence of a DNA sample protected by 5 or 1.25 μM rApxR to the sequence of an unprotected DNA sample incubated with BSA (Fig. (Fig.4B).4B). There were two tandem regions that were protected by rApxR: from base −206 to base −183 (region I) and from base −124 to base −101 (region II) (Fig. (Fig.4B4B and and4C4C).

A transcriptional fusion was then constructed by cloning the 213-bp p44E promoter region (p44EI) upstream of the promoterless lacZ gene in pACYC184. The lacZ reporter fusion was transformed into E. coli that already contained either an IPTG-inducible pApxR vector or the corresponding empty control pET29a(+) vector. The β-galactosidase activity significantly increased in the p44EI-lacZ construct when rApxR expression was induced and was significantly greater than the activity of the negative control (E. coli transformed with the control vector) (Fig. (Fig.4D).4D). The background β-galactosidase activity with the empty pET vector in the presence of IPTG was likely due to T7 polymerase in E. coli strain BL21(DE3) which was also induced by IPTG, as even with the empty pET vector transcription could be activated if T7 polymerase bound to the putative promoter sequence.

Positive autoregulation of apxR.

The level of relative ApxR mRNA expression by each bacterium was approximately 1,000-fold lower in infected ISE6 tick cells than in infected HL-60 cells (0.424 × 10−2 ± 0.034 × 10−2 transcript per bacterium versus 441 × 10−2 ± 6.23 × 10−2 transcript per bacterium; n = 3). We examined possible positive autoregulation of apxR. Using an EMSA, we found that increasing the concentration of the rApxR protein shifted the upstream promoter region of apxR (Fig. (Fig.5A).5A). Using DNase I protection assays, the protected regions of the sense strand were determined by comparing the sequences of a DNA sample protected by 5 or 1.25 μM rApxR to the sequence of an unprotected DNA sample treated with BSA (Fig. (Fig.5B).5B). There were two tandem regions that were protected by rApxR: from base −244 to base −222 (region I) and from base −96 to base −75 (region II) (Fig. (Fig.5B5B and and5C).5C). Likewise, rApxR up-regulated lacZ expression driven by the apxR promoter in a lacZ reporter assay (Fig. (Fig.5D5D).

FIG. 5.
Autoregulation of ApxR. (A) Dose response of rApxR (5, 1, and 0.2 μg) binding to the 350-bp apxR promoter fragment in an EMSA. DNA was visualized by ethidium bromide staining, and the free probe is indicated by the arrow and is shown in the leftmost ...

DISCUSSION

The present study showed that A. phagocytophilum p44 transcription is not constitutive but is highly regulated and responsive to temperature and tick and mammalian host environments. This result is in agreement with a previous study that showed that there was a significant reduction in the amount of total P44 protein in infected HL-60 cells cultured at 24°C compared to cells cultured at 37°C (34). Because P44 is the major outer membrane protein, has porin activity (13), and is capable of eliciting neutralizing antibodies (29), changes in p44 transcript amounts and consequently P44 protein amounts would alter the nutrient acquisition, physiology, and immune avoidance of A. phagocytophilum in ticks and mammals.

p44E is the second gene identified in A. phagocytophilum that is regulated by ApxR. Based on sequences found at ApxR binding sites upstream of tr1 and the paucity of predicted DNA binding proteins in the A. phagocytophilum genome, we predicted that ApxR may be a global regulator of gene expression in this bacterium (28). The up-regulation of apxR mRNA levels in mammalian HL-60 cells suggests that ApxR may specifically regulate transcription during the mammalian stage of the A. phagocytophilum life cycle rather than during the tick stage of the bacterial life cycle. ApxR may, therefore, also coordinate expression of many additional A. phagocytophilum genes that are required during mammalian infection. The present data revealed that ApxR can be positively autoregulated. Autoregulation is an important means of regulating a variety of biological processes, including cell cycle control, biorhythmic oscillations, and pattern formation in development (10). While negative feedback is used to maintain homeostasis, recently the PhoP/PhoQ two-component system in Salmonella was shown to be regulated by a positive feedback mechanism in order to allow jump-start transcription of PhoP-regulated genes involved in mouse pathogenesis (24). Similarly, the positive feedback regulation of apxR may allow amplification of the mammalian environmental signals to ensure rapid establishment of a new phenotype of A. phagocytophilum regulated by ApxR to face the challenging new environment following tick transmission.

p44E transcription appears to be monocistronic only in infected HL-60 cell cultures and spleens of infected mice. Barbet et al. reported a lack of a monocistronic promoter in the p44-msp2 intergenic region based on analysis of the “G region” in the A. phagocytophilum genome (1). The G region (242 bp) spans coordinates 1290042 to 1290283 of the A. phagocytophilum HZ genome sequence (GenBank accession no. NC 007797), whereas the intergenic space between omp-1N and p44E spans coordinates 1290170 to 1290383. As observed in our lacZ reporter assay, without ApxR the activity of the monocistronic promoter is weak, and the promoter activity could not be detected, perhaps due to the absence of ApxR in the promoter assay system in the previous study (1). We also observed cotranscription of p44E with upstream genes that appeared to be constitutive, as this gene was expressed under all environmental conditions examined. Polycistronic expression of p44E from A. phagocytophilum was observed in mammalian cell cultures at both 28 and 37°C, in tick salivary glands, and in mouse spleens at concentrations of 2.3 × 102 to 5.0 × 103 mol mRNA/106 mol 16S rRNA, which are similar to the levels of polycistronic expression of msp2 from A. marginale in bovine erythrocytes (1). In A. marginale, tr1, omp-1, and opag 2 upstream of msp2 were expressed at levels of 2.1 × 10 to 2.8 × 102 mol/106 mol 16S rRNA in bovine erythrocytes and in IDE8 tick cells cultured at 26, 34, and 37°C (1). Details of coexpression of p44 with upstream genes in the p44 expression locus remain to be investigated, since this may provide steady-state basal expression to maintain the phenotype in tick cells.

Here, ticks were infected with the A. phagocytophilum NTN-1 strain from Massachusetts, mice and HL-60 cell cultures were infected with the HZ strain, and ISE6 tick cell cultures were infected with the HGE2 strain from Minnesota; however, the sequences of the p44 expression locus, as well as the conserved 5′ and 3′ regions of p44 sequences flanking the central hypervariable region, in strains of A. phagocytophilum in the United States are quite conserved (2, 9, 12, 17). We sequenced apxR of A. phagocytophilum strain HGE2, including 15-bp upstream and 13-bp downstream regions which were identical to those of strain HZ (not including primer sequences). The GenBank accession number of HGE2 apxR is EF626971. This suggests that the differences that we observed in p44E expression between ticks and mammals are not strain specific.

This work showed that p44E is the primary expression locus for p44 genes under the environmental conditions examined; however, it is not the sole expression locus because several full-length p44 transcripts have been shown to be expressed at significantly lower levels (31, 33). The biological function of this small population of full-length p44 transcripts remains to be analyzed. The expression levels of the p30 paralogs from Ehrlichia canis (26) and of the omp-1 paralogs from Ehrlichia chaffeensis (27), both of which belong to the OMP-1/Msp2/P44 superfamily (12), have also been shown to vary in response to environmental conditions (26, 27). In addition, apxR is conserved among members of the family Anaplasmataceae (28). These facts indicate that there has been evolutionary pressure among members of the family Anaplasmataceae to conserve differential gene expression of the major outer membrane proteins, presumably allowing the presence of distinct bacterial surface properties in ticks and mammals.

Supplementary Material

[Supplemental material]

Acknowledgments

We thank Ulrike G. Munderloh for generously providing uninfected and HGE2-infected ISE6 cells and for helpful instructions. We thank Michael Zianni at the Plant-Microbe Genomics Facility, The Ohio State University, for help with the DNase footprint analysis. We thank S. Felek for providing tick specimens.

This research was partially supported by grant R01 AI47407, and the A. phagocytophilum genome sequence project was supported by grant R01 AI 47885 from the National Institutes of Health.

Footnotes

[down-pointing small open triangle]Published ahead of print on 28 September 2007.

Supplemental material for this article may be found at http://jb.asm.org/.

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