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. Author manuscript; available in PMC: 2012 Jan 4.
Published in final edited form as: Cell Microbiol. 2010 Mar 19;12(9):1222–1234. doi: 10.1111/j.1462-5822.2010.01464.x

Fucosylation enhances colonization of ticks by Anaplasma phagocytophilum

Joao H F Pedra 1,2,*,†, Sukanya Narasimhan 1,†, Dubravko Rendić 3, Kathleen DePonte 4, Lesley Bell-Sakyi 5, Iain B H Wilson 3, Erol Fikrig 1,6
PMCID: PMC3250644  NIHMSID: NIHMS345766  PMID: 20331643

Abstract

Fucosylated structures participate in a wide range of pathological processes in eukaryotes and prokaryotes. The impact of fucose on microbial pathogenesis, however, has been less appreciated in arthropods of medical relevance. Thus, we used the tick-borne bacterium Anaplasma phagocytophilum – the agent of human granulocytic anaplasmosis to understand these processes. Here we show that A. phagocytophilum uses α1,3-fucose to colonize ticks. We demonstrate that A. phagocytophilum modulates the expression of α1,3-fucosyltransferases and gene silencing significantly reduces colonization of tick cells. Acquisition but not transmission of A. phagocytophilum was affected when α1,3-fucosyltransferases were silenced during tick feeding. Our results uncover a novel mechanism of pathogen colonization in arthropods. Decoding mechanisms of pathogen invasion in ticks might expedite the development of new strategies to interfere with the life cycle of A. phagocytophilum.

Introduction

Glycan structures are present in cells and are added to lipids and protein structures through glycosylation (Staudacher et al., 1999; Ma et al., 2006; van Kooyk and Rabinovich, 2008). Glycosylation plays a role in enzymatic activity and stability, protein solubility and the immune response. Many of the glycans relevant to these processes contain fucose residues; indeed, the addition of this deoxyhexose appears to be an important step for a number of biological and pathological phenomena (Staudacher et al., 1999; Altmann et al., 2001; Ma et al., 2006). Fucosylation is involved in organogenesis, inflammation, cancer, cell adhesion and microbial pathogenesis; α1,2-fucose forms part of the ABO blood group antigens, whereas α1,3-fucosyl modifications include the mammalian Lewis-type epitopes. In microbial pathogenesis, α1,2-fucose epitopes serve as ligands tethering Salmonella and Campylobacter to the intestinal mucosa (Ruiz-Palacios et al., 2003; Chessa et al., 2009). On the other hand, Lewis type mimicry plays a role in gastric colonization by Helicobacter pylori (Moran, 2008) and the same function has been suggested for the sialyl Lewisx expressed on the cell surface of an oral bacterium associated with endocarditis (Hirota et al., 1995). Sialic acid binding is required for infectious cell surface receptor recognition by parvovirus (Nam et al., 2006), and Norwalk virus – a norovirus that causes non-bacterial gastroenteritis – binds to Lewis type antigens (Hutson et al., 2003).

Structural analysis of N-glycans from arthropods reveals that these molecules can be fucosylated in three different ways: fucosylation in α1,6 linkage to the innermost N-acetylglucosamine (GlcNAc); fucosylation in α1,3 linkage to the proximal GlcNAc residue; and fucosylation in α1,3 linkage to the precursor glycan LacdiNAc (N-acetylgalactosamine linked to β1,4- to N-acetylglucosamine) to yield a Lewis-like structure (GalNAcβ-1,4{Fucα-1,3}GlcNAcβ-R), found, for instance, on honeybee phospholipase (Staudacher et al., 1999; Paschinger et al., 2005). The poorly understood role of fucose in arthropods provided further impetus to investigate the importance of this carbohydrate during microbial colonization of ticks. We used the tick-borne pathogen Anaplasma phagocytophilum as a model organism to understand fucosylated-mediated processes in arthropods. A. phagocytophilum is transmitted by ticks of the genus Ixodes, infects neutrophils and causes human granulocytic anaplasmosis – the second most common tick-borne disease in the USA. We hypothesized that A. phagocytophilum would serve as a good model of infection because A. phagocytophilum resides in Ixodes scapularis salivary glands; and requires both sialylation and fucosylation in mammalian cells for cellular invasion (Goodman et al., 1999; Herron et al., 2000; Yago et al., 2003; Reneer et al., 2006; 2008; Sarkar et al., 2007). Our findings show that A. phagocytophilum modulates the expression of three α1,3-fucosyltransferases and uses α1,3-fucosylation to colonize ticks.

Results

Core α1,3-fucosylated structures are present in ticks

To gain a better understanding of the importance of fucose for microbial pathogenesis in arthropods, we studied the colonization of ticks by A. phagocytophilum. We first determined whether the tick I. scapularis had fucosylated N-glycans on the surface of proteins. Mass spectrometry analysis showed the presence of fucose in engorged nymphal I. scapularis salivary glands (Fig. 1A and B). The N-glycan profile revealed oligosaccharides of lower complexity, most notably a series of oligo- and paucimannosidic structures. Only minor amounts of hybrid, bi- and tri-antennary complex glycans were observed in the N-glycan spectra. Then we probed salivary gland extracts from engorged nymphal ticks with an anti-horseradish peroxidase (HRP) antibody that recognizes the sugar epitope of core α1,3-fucose to assess whether N-glycan structures carrying core α1,3-fucose were present. We detected several proteins carrying core α1,3-fucose residues in I. scapularis salivary glands and midguts (Fig. 1C – panel B; lanes SG; MG). Core α1,3-fucose residues were also observed in Drosophila (Fig. 1C – panel B; lane Dros). However, core α1,3-fucose residues were not detected in the human promyelocytic cell line HL-60 (Fig. 1C – panel B; lane HL60).

Fig. 1. α1,3-Fucose is present in ticks.

Fig. 1

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A. N-glycan structures isolated from I. scapularis salivary glands (n = 100) were released by N-glycosidase A and N-glycan analysis was performed using MALDI–TOF-MS. The relevant structures carrying fucose residues are marked with arrowheads. Glycan m/z values and abbreviations are also shown.

B. N-glycan structures previously identified in (A) and found in a range of invertebrate organisms are shown according to the glycan symbol nomenclature established by the Consortium for Functional Glycomics (http://www.functionalglycomics.org).

C. Protein extracts (1 µg) from salivary glands (SG) and midguts (MG) of fed I. scapularis nymphs, human HL60 (HL60) cells and adult Drosophila (Dros) were electrophoresed on 4–20% gradient SDS-PAGE followed by staining with Coomassie Blue R250 (A). Protein extracts were then immunoblotted with anti-HRP antibody. (B) Anti-HRP antibodies recognized core α1,3-fucose residues in arthropods but not in human cells. A minimum of two independent experiments were performed.

α1,3-Fucose is required for A. phagocytophilum colonization of ticks

To determine whether A. phagocytophilum would use α1,3-fucosylated glycans to colonize I. scapularis ticks, we first searched for putative α1,3-fucosyltransferase homologues in the expressed sequence tag (EST) database of vectorbase. VectorBase (http://www.vectorbase.org) is a bioinformatics resource centre focused on invertebrate vectors of human pathogens (Lawson et al., 2009). Bioinformatics analysis revealed the occurrence of one I. scapularis EST (ti|1681614834) displaying homology to insect α1,3-fucosyltransferases and encoding conserved motifs for α1,3-fucosyltransferases (Fig. 2A; Fig. S1). This α1,3-fucosyltransferase sequence was later annotated as the corresponding isft7 gene. To assess whether silencing of α1,3-fucosyltransferases would affect A. phagocytophilum colonization of ticks, a corresponding dsRNA was synthesized. The dsRNA covered the core and the conserved motif II region of the I. scapularis EST displaying homology to insect α1,3-fucosyltransferases. It was assumed that a dsRNA targeting this ‘conserved’ region would affect the expression of multiple fucosyltransferases. We placed control and α1,3-fucosyltransferase dsRNA-injected I. scapularis nymphs on A. phagocytophilum-infected mice and allowed naïve ticks to feed for 66–72 h. Changes in fucosylation at the protein level were observed when fucosyltransferases were silenced (Fig. 2B; anti-HRP; lane 2). These results were specific for α1,3-fucosyltransferase silencing because we did not observe any changes in fucosylation when a control salp25d dsRNA was used (Fig. 2B; anti-HRP; lane 1). Further, immunoblots of salivary gland protein extracts with anti-Salp25D showed that Salp25D was silenced in the control group (Fig. 2B; anti-Salp25D; lane 1) but not in the α1,3-fucosyltransferase dsRNA treatment (Fig. 2B; anti-Salp25D; lane 2). Immunoblot with rabbit anti-tick immune serum served as a loading control and showed that the protein amounts were similar in control and α1,3-fucosyltransferase dsRNA groups (Fig. 2B; anti-tick; lanes 1 and 2). Engorgement was not affected by silencing of α1,3-fucosyltransferases (P = 0.78) (Fig. 2C).

Fig. 2. α1,3-Fucose is required for A. phagocytophilum colonization of I. scapularis ticks.

Fig. 2

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A. Partial sequence from the I. scapularis expressed sequence tag (ti|1681614834). The 245 bp fragment from I. scapularis encoding the signature motif for α1,3-fucosyltransferase was cloned into the L4440 RNAi vector and the dsRNA was produced. Grey shade – 245 bp amplicon; grey underlined shade – amplicon primers; green underlined – motif I; red – core motif; yellow – motif II. All motifs for α1,3-fucosyltransferase were identified as previously discussed (Oriol et al., 1999; Rendić et al., 2007b).

B. Extracts from salivary glands injected with control salp25d dsRNA (lane 1) and α1,3-fucosyltransferase dsRNA (lane 2). Salivary glands were immunoblotted with anti-Salp25D, anti-HRP and anti-tick antibodies.

C. Average weight of ticks in mock and α1,3-fucosyltransferase dsRNA treatments is shown.

D and E. (D) A. phagocytophilum load in the salivary glands and (E) B. burgdorferi presence in the tick midguts were measured during acquisition (66–72 h post attachment). 16s rDNA and flab were used as reference genes to measure bacterial load.

F–K. Confocal microscopy in α1,3-fucosyltransferase siRNA-injected and control ticks fed on naïve mice for 66–72 h. Salivary glands (SG; F–H) and the midgut (MG; I–K) were stained with an antibody that recognizes core α1,3-fucose (anti-HRP). Salivary glands and the midgut of ticks showed reduced presence of core α1,3-fucose when a combination of siRNAs for isft1, isft2 and isft3 (combo) was injected in ticks (H and K). Non-injected (F and I) and control injected siRNA (G and J) showed abundant presence of core α1,3-fucose in the tissues. Acquisition experiments were repeated four times. Acquisition experiments used 14 mice per group and 10–12 ticks per mouse. Statistical analysis was performed using the Student’s t-test (P < 0.05).

We then determined A. phagocytophilum load in mock and dsRNA-injected nymphs. A. phagocytophilum load in the salivary glands of dsRNA-injected nymphs was lower when compared with the control treatment (fold difference = 1934; P = 0.02) (Fig. 2D). This effect seemed specific for A. phagocytophilum because acquisition of the unrelated pathogen Borrelia burgdorferi – the agent of Lyme disease – was not dependent on core α1,3-fucosylation (P = 0.42) (Fig. 2E). With the data from the ongoing I. scapularis genome project now available on vectorbase (Lawson et al., 2009), we discovered using tblastn that a total of 10 putative fucosyltransferases are present in the I. scapularis genome (including the afore-mentioned homologue first identified as an EST and used as a basis for the dsRNA analysis) (Fig. S1B). Therefore, we designed specific siRNAs for the 5′ region of isft1, isft2 and isft3 (Fig. S2). We chose this region because the N-terminal non-catalytic domain is dissimilar among α1,3-fucosyltransferases (Ma et al., 2006). Confocal microscopy indicated that silencing of these α1,3-fucosyltransferases reduced core α1,3-fucosylation in the salivary gland and the midgut of I. scapularis ticks when the three siRNAs were used in combination (Fig. 2H and K). Importantly, core α1,3-fucosylation was not affected when an unrelated gene – salp25d – was silenced (Fig. 2G and J).

Transfection of the dsRNA targeting the conserved α1,3-fucosyltransferase sequence into cells originating from Ixodes ricinus also decreased the expression of α1,3-fucosyltransferases in the absence (fold difference = 17; P < 0.01) and presence of A. phagocytophilum – (fold difference = 12; P < 0.01) (Fig. 3A). Reduction of α1,3-fucosyltransferase expression significantly reduced the capacity of A. phagocytophilum to colonize I. ricinus cells (fold difference = 3; P < 0.05) (Fig. 3B). These results were confirmed using confocal microscopy, in which A. phagocytophilum load was reduced post infection (Fig. 3E). Core 1,3-fucosylation is present in I. ricinus (Fig. 3F; lane B); and α1,3-fucosyltransferases in I. scapularis and I. ricinus share high levels of homology (Fig. 3G).

Fig. 3. A. phagocytophilum colonizes I. ricinus cells by using core α1,3-fucosylation.

Fig. 3

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A. I. ricinus IRE/CTVM19 (2 × 105) cells were transfected with the α1,3-fucosyltransferase dsRNA and the silencing of α1,3-fucosyltransferase was examined in the presence (+) and absence (−) of A. phagocytophilum.

B. A. phagocytophilum load is shown 72 h post infection in mock and dsRNA-transfected I. ricinus cells.

C–E. Mock and dsRNA-transfected I. ricinus cells were visualized under the microscope and the presence of A. phagocytophilum is shown (arrows).

F. Protein extracts (1 µg) from I. ricinus cells (Iri) were electrophoresed and stained with Coomassie Blue R250 (lane A). Protein extracts were immunoblotted with the anti-HRP antibody (lane B) showing that anti-HRP antibodies recognized core α1,3-fucose residues in I. ricinus cells.

G. α1,3-Fucosyltransferase 7 (ft7) was amplified from I. ricinus cDNA originated from the IRE/CTVM19 cell line. A fragment of 110 bp was cloned into the plasmid pGEM-T Easy Vector and sequenced. This sequence was 99% identical to the conserved region (nucleotide position 452–561) of I. scapularis fucosyltransferase 7 (XM_023318). α1,3-Fucosyltransferase 2 (ft2) was amplified from I. ricinus cDNA originated from the IRE/CTVM19 cell line. A fragment of 189 bp was cloned into the plasmid pGEM-T Easy Vector and sequenced. This sequence shared 96% identity (nucleotide position 1–189) with the fucosyltransferase I. scapularis 2 (isft2) (XM_002401196). Cell culture experiments were repeated twice. Statistical analysis was performed using the Student’s t-test (P < 0.05).

Transmission of A. phagocytophilum does not require α1,3-fucosylation

As α1,3-fucosylation was important for A. phagocytophilum acquisition in ticks, we determined if pathogen transmission was also affected. Nymphal ticks were injected with dsRNA targeting either salp25d (as control) or the conserved sequence of α1,3-fucosyltransferases. Ticks attached on mice for 24 h displayed low bacterial replication within salivary glands and were not able to transmit A. phagocytophilum (data not shown). On the other hand, ticks placed on mice for 72 h had higher levels of A. phagocytophilum within the salivary glands and transmitted the pathogen to the mammalian host (Fig. 4B). A. phagocytophilum bacterial load during tick transmission was not dependent on core α1,3-fucosylation. Bacterial load in mice was similar at days 5 (P = 0.26), 10 (P = 0.57) and 15 post infection (P = 0.29) (Fig. 4B). Similar to the acquisition studies, B. burgdorferi transmission was not dependent on α1,3-fucosylation (Fig. 4C).

Fig. 4. Transmission of A. phagocytophilum does not require α1,3-fucosylation. I. scapularis nymphs infected with A. phagocytophilum were injected with dsRNA-salp25d or dsRNA-α1,3-fucosyltransferase. Ticks were then placed on C57Bl/6 mice for transmission experiments.

Fig. 4

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A. Ticks were removed at 24 or 72 h post feeding and A. phagocytophilum load within ticks was measured.

B. A. phagocytophilum-infected ticks were removed at 72 h post feeding and transmission of A. phagocytophilum at days 5, 10 and 15 in the mouse peripheral blood was measured.

C. I. scapularis nymphs infected with B. burgdorferi were injected with mock or dsRNA-α1,3-fucosyltransferase. Infected ticks were placed on Balb/c mice during transmission experiments. B. burgdorferi infection was measured at day 18 post infection in multiple organs by qPCR using the flab gene as a reference. Transmission experiments were repeated twice. Transmission experiments used six mice per group and 8–10 ticks per mouse. Statistical analysis was performed using the Student’s t-test (P < 0.05).

A. phagocytophilum increases the expression of specific α1,3-fucosyltransferases in ticks

As a total of 10 α1,3-fucosyltransferase homologues could be defined by tblastn searching, we were prompted to investigate which of these genes were important for A. phagocytophilum infection. We hypothesized that A. phagocytophilum would modulate the expression of target genes for its own benefit. This hypothesis was based on our previous analysis showing that the expression of the tick salivary gland protein gene salp16 (Sukumaran et al., 2006) – an essential gene for A. phagocytophilum survival within I. scapularis – was dramatically increased in A. phagocytophilum-infected ticks during feeding. We did not detect any expression of the tick α1,3-fucosyltransferases isft5, isft6 and isft8 during larval acquisition of A. phagocytophilum or isft1, isft5, isft6 and isft8 during nymphal transmission of A. phagocytophilum. Interestingly, isft2 and isft3 at 24 h and isft1 and isft2 at 48 h showed increased levels of transcription during A. phagocytophilum acquisition by I. scapularis (Fig. 5A and B). The increase in transcription for the genes isft1, isft2 and isft3 was also observed during A. phagocytophilum infection of I. ricinus cell culture (Fig. 5E). This effect was specific for acquisition, because during transmission of A. phagocytophilum by infected nymphs to naïve mice, isft1, isft2 and isft3 expression levels were comparable to that in uninfected nymphs (Fig. 5C and D).

Fig. 5. A. phagocytophilum acquisition by Ixodes ticks increases isft1, isft2 and isft3 expression.

Fig. 5

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A–D. Naïve (n = 90) tick larvae were placed on infected and non-infected C57Bl/6 mice for 24 and 72 h. A. phagocytophilum load was measured in ticks by qRT-PCR and the data normalized to actin. The expression of α1,3-fucosyltransferases was measured in the presence and absence of A. phagocytophilum at (A) 24 and (B) 72 h post attachment (isft5, isft6 and isft8 were not detected). Infected (n = 90) and non-infected nymphal (n = 90) ticks were placed on naïve C57Bl/6 mice for 24 and 72 h. The expression profile of α1,3-fucosyltransferases was measured in the salivary glands of nymphal I. scapularis at (C) 24 and (D) 72 h post attachment (isft1, isft5, isft6 and isft8 were not detected).

E. The transcription of α1,3-fucosyltransferases isft1, isft2 and isft3 was measured in infected and non-infected I. ricinus cells. A. phagocytophilum load is measured using the expression of the p44 gene as a reference. One representative experiment is shown. Results are expressed as mean plus standard deviation. Statistical analysis was performed using the Student’s t-test (P < 0.05). Statistical significance is demonstrated by an asterisk.

Silencing of isft1, isft2 and isft3 reduces A. phagocytophilum acquisition during tick infection

As A. phagocytophilum infection significantly influenced isft1, isft2 and isft3 expression, we determined the relative importance of each of these genes for infection of ticks. We silenced each individual gene. The combination of siRNAs for isft1, isft2 and isft3 was most effective in reducing A. phagocytophilum colonization of tick cells (Fig. 6C and G). However, silencing of isft1, isft2 and isft3 individually also reduced A. phagocytophilum colonization (Fig. 6D–F and G). Importantly, the transfection of siRNA specific for isft4 (Fig. S2B) – an α1,3-fucosyltransferase that did not show altered expression during A. phagocytophilum infection – did not have any effect on A. phagocytophilum colonization of ticks (Fig. 6H).

Fig. 6. Silencing of isft1, isft2 and isft3 by specific siRNAs decreases A. phagocytophilum colonization of tick cells. Six hundred ηg of siRNAs specific for α1,3-fucosyltransferase 1 (FT1), α1,3-fucosyltransferase 2 (FT2) and α1,3-fucosyltransferase 3 (FT3) were transfected into I. ricinus (1 × 107) cells. Twenty-four hours later, cells were infected with A. phagocytophilum [multiplicity of infection (moi) 5].

Fig. 6

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A–F. Confocal microscopy showing A. phagocytophilum in I. ricinus cells using a nuclear-specific stain (Topro-3) and an A. phagocytophilum-specific antibody. (A) Non-transfected and non-infected cells (B) cells infected with A. phagocytophilum and transfected with the salp25d control siRNA, and (C) cells infected with A. phagocytophilum and transfected with a combination of siRNAs specific for isft1, isft2 and isft3 as well as (D–F) individual transfections are shown. Arrows show the presence of A. phagocytophilum.

G. A. phagocytophilum 16s rDNA was measured by qRT-PCR 72 h post infection and compared with the expression of the control siRNA salp25d (25d) or a combination of siRNAs for isft1, isft2 and isft3 (Mix).

H. siRNAs specific for isft4 did not show any effect on A. phagocytophilum infection in I. ricinus cells. Results are expressed as mean plus standard deviation. Statistical analysis was performed using the Student’s t-test (*P < 0.05).

Discussion

A number of human pathogens colonize mammalian cells through fucosylated structures. However, it has been poorly defined how fucosylation contributes to pathogen colonization of arthropods. We studied how the tick-borne pathogen A. phagocytophilum colonizes arthropods. We showed that the salivary glands of ticks carry core α1,3-fucosylated structures in N-glycans. Fucosylated N-glycans were analysed as opposed to fucosylated O-glycans structures because system-wide analysis for O-glycoproteins remains a challenge due to their structural heterogeneity and the lack of specific tools (Schwientek et al., 2007; Hanisch et al., 2009). Our results revealed a contrast of fucosylation patterns in mammalian versus arthropod cells. While core α1,3-fucosylation – the common pentasaccharide consisting of two N-acetylglucosamine and three mannose residues present in N-linked oligosaccharides – was commonly present in arthropods, this sugar structure was not detected in human cells. Further, our mass spectrometry analysis with ticks only revealed the presence of oligosaccharides of lower complexity, as opposed to the presence of complex sugar structures commonly observed in mammalian cells.

In our studies, we reasoned that A. phagocytophilum would serve as a valuable model to investigate the contribution of fucosylation for microbial pathogenesis in arthropods because: (i) A. phagocytophilum resides in two different locations during its life cycle – neutrophils and I. scapularis salivary glands, and (ii) A. phagocytophilum uses sialylation and fucosylation to colonize mammalian cells (Goodman et al., 1999; Herron et al., 2000; Yago et al., 2003; Reneer et al., 2006; 2008; Sarkar et al., 2007). Here we provided strong evidence that A. phagocytophilum (i) modulates the expression of three α1,3-fucosyltransferases and (ii) uses α1,3-fucose to colonize ticks during acquisition. These findings seem specific for A. phagocytophilum because colonization by B. burgdorferi was not affected when α1,3-fucosylated structures were reduced in ticks.

Our findings were somewhat surprising as independent groups have demonstrated that A. phagocytophilum uses the tetrasaccharide sialyl LewisX to colonize mammalian cells; and sialyl LewisX is usually attached to O-glycans (Goodman et al., 1999; Herron et al., 2000; Yago et al., 2003; Reneer et al., 2006; 2008; Sarkar et al., 2007). We suggest that this discrepancy may simply be an evolutionary adaptation acquired by A. phagocytophilum to retain optimal cell interaction in both arthropods and mammals. At least two lines of evidence point towards this hypothesis. First, we could not detect the presence of sialic acid in the N-glycome of tick salivary glands; such residues are generally rare in invertebrates. On the contrary, our results only revealed the presence of oligosaccharides of lower complexity. A second line of evidence pointing towards A. phagocytophilum plasticity is the presence of another type of fucosylation in ticks that is not generally present in mammals – core α1,3-fucosylation. Core α1,3-fucosylation has been described in arthropods, plants and parasites, but it is absent in mice and humans (Ma et al., 2006).

In humans, A. phagocytophilum uses platelet selectin glycoprotein-1 (PSGL1) to infect neutrophils, thereby, ‘hijacking’ a molecule involved in the inflammatory response. Both α-2,3-sialylation and α1,3-fucosylation of PSGL1 are essential for successful entry (Goodman et al., 1999; Herron et al., 2000). Interestingly, A. phagocytophilum readily infects psgl1-deficient mice demonstrating that murine PSGL1 does not play a role similar to its human counterpart (Carlyon et al., 2003). Mice lacking α1,3-fucosylation, however, are refractory to A. phagocytophilum infection. A. phagocytophilum adhesins required for binding of α1,3-fucosylated receptors during colonization of ticks and mammals have not been identified. The current model of pathogen–cell interaction suggests that A. phagocytophilum expresses at least two adhesins to bind human and mouse receptors (Carlyon et al., 2003; Yago et al., 2003; Reneer et al., 2006; 2008; Sarkar et al., 2007; Sarkar et al., 2008; Troese et al., 2009). One adhesin might bind to α1,3-fucosylated and α2,3-sialylated glycans; the second adhesin may bind to an N-terminal peptide component of human PSGl-1 or a mouse orthologue. It was hypothesized that A. phagocytophilum adhesins function cooperatively to achieve maximal binding and tyrosine sulfate residues as well as sialyl Lewisx on a particular O-glycan are of utmost importance to retain optimal cell interaction. Our findings add new features to this model. Our results suggest that A. phagocytophilum exploits core α1,3-fucose on N-glycans of ticks, rather than Lewis-type antennal fucosylation. Despite all this evidence indicating the importance of N-glycan fucosylation for A. phagocytophilum colonization of ticks, our results cannot completely rule out the use of O-glycans during infection of ticks. The Mucin-type O-glycan core 1 structure has been found in the model organism Drosophila (Aoki et al., 2008). Therefore, this glycan or a variant of this glycan may be present and used by A. phagocytophilum during vector infection.

The fucosylated receptor that A. phagocytophilum uses to colonize ticks remains unknown. Interestingly, the phenotype related to another important tick protein during A. phagocytophilum infection – Salp16 (Sukumaran et al., 2006) – may be linked to fucosylation. Salp16 shows potential sites for O- and N-glycosylation in its sequence and glycans may be modified with fucose residues during glycoprotein processing. Similar to the phenotype observed with α1,3-fucosyltransferases in ticks, salp16 shows upregulation during infection and the silencing of salp16 reduced A. phagocytophilum colonization in tick salivary glands. Future studies should elucidate the intricate mechanisms of A. phagocytophilum colonization in ticks, and provide similarities and contrasts with infection of mammalian cells; but fucosylation is certainly a critical element for A. phagocytophilum infection of mammals and arthropods, and it would be most interesting to understand these mechanisms.

A better understanding of how A. phagocytophilum interferes with α1,3-fucosylation may lead to the development of novel therapeutic interventions against human granulocytic anaplasmosis. The disruption of the A. phagocytophilum–α1,3-fucosylation interaction in ticks, the inhibition of tick α1,3-fucosyltransferases, and the development of vaccination against core α1,3-fucosylated proteins based on antibody-mediated killing mechanisms are just a few important strategies to consider when developing novel prophylaxis against A. phagocytophilum infection. Our work adds a new facet to the understanding of tick-borne diseases. We provide a better understanding of the glycoconjugates required for A. phagocytophilum colonization of the tick I. scapularis. Glycans participate in many biological processes such as bacterial cell adhesion, molecular trafficking, receptor activation, signal transduction and endocytosis. These data may be applicable to other tick-borne infections and help to uncover molecular mechanisms that govern the physiology and pathology of rickettsial infections.

Experimental procedures

Ethics statement

Animals were housed in the Animal Resources Facility at Yale University School of Medicine according to the guidelines described under the federal Animal Welfare Regulations Act. Food and water were provided and all animal procedures were approved by the Institutional Animal Care and Use Committee of Yale University.

Tick acquisition and transmission, pathogens and I. ricinus cell line

Ixodes scapularis was obtained from the Connecticut Agricultural Experimental Station (New Haven, CT). Ticks were reared at 26°C with 85% relative humidity and a daily photoperiod of 14 h light and 10 h dark. The I. ricinus (L.) cell line IRE/CTVM19 (Bell-Sakyi et al., 2007) was maintained in L-15 (Leibovitz) medium supplemented with 10% tryptose phosphate broth, 20% fetal calf serum, 2 mM l-glutamine, 100 units ml−1 penicillin and 1 µg ml−1 streptomycin in ambient air at 30°C. A. phagocytophilum strain NCH1 and B. burgdorferi strain N40 were used.

To isolate I. ricinus α1,3-fucosyltransferases orthologous to those of I. scapularis, primers flanking the conserved region of these genes were used. α1,3-Fucosyltransferase 7 PCR products were ligated into pGEM-T Easy vector and 15 clones sequenced. For I. ricinus α1,3-fucosyltransferase 2 isolation, isft2 forward and reverse primers were used to obtain the amplicon. The PCR product was cloned into pGEM-T Easy vector and four clones were sequenced. α1,3-Fucosyltransferase 7 and α1,3-fucosyltransferase 2 were amplified from I. ricinus cDNA originated from the IRE/CTVM19 cell line. I. ricinus α1,3-fucosyltransferase 7 was 99% identical to the conserved region (nucleotide position 452–561) of isft7 (XM_023318). α1,3-Fucosyltransferase 2 from I. ricinus shared 96% identity (nucleotide position 1–189) with the fucosyltransferase I. scapularis 2 (isft2) (XM_002401196). All primers used throughout this study are available on Table S1.

Acquisition and transmission studies were carried out as previously described (Pedra et al., 2006; Narasimhan et al., 2007). Experiments to address acquisition were performed four times with 14 mice used per group and 10–12 ticks per mouse. Pathogen-free I. scapularis nymphs were injected through the integument with control or α1,3-fucosyltransferase dsRNA. Ticks were placed on infected mice and allowed to feed for 66–72 h. Salivary glands were analysed in pools of three, and midguts were analysed in pools of two. Results of a representative experiment are shown in the figures. For transmission experiments, naïve larvae were fed on infected C57Bl/6 (A. phagocytophilum transmission) or C3H/HeN (B. burgdorferi transmission) for 3–5 days and tested for pathogen acquisition – typically 90%. Infected larvae molted to nymphs and nymphs were used for transmission experiments. In experiments to address transmission, six mice were used per group and 8–10 ticks per mouse. Transmission experiments were repeated twice. Infected ticks injected with either salp25d or α1,3-fucosyltransferase dsRNA were placed on C57Bl/6 (A. phagocytophilum transmission) or C3H/HeN (B. burgdorferi transmission) mice. Ticks were allowed to feed for 24 or 72 h. Ticks were gently removed, and midguts and salivary glands were dissected and processed in pools of three salivary glands or two midguts for quantitative RT-PCR (qRT-PCR) analysis. A representative experiment is shown in the figures. A. phagocytophilum transmission was measured in the peripheral blood of mice at days 5, 10 and 15. B. burgdorferi transmission was measured in indicated organs at day 18 post infection.

dsRNA synthesis and injection

The 245 bp fragment of the α1,3-fucosyltransferase EST from I. scapularis with similarity to a signature motif for α1,3-fucosyltransferase (described in this study as corresponding to isft7) was cloned into the L4440 double T7 Script II vector (Fire et al., 1998). Gene-specific primers containing SacI and KpnI restriction sites were used in the PCR reaction. dsRNA complementary to the isfucot sequence was synthesized using the Megascript RNAi kit (Ambion, TX) and tick injections were performed as described (Narasimhan et al., 2007). Control ticks were injected with dsRNA isolation buffer (Ambion, TX) (mock), salp25d dsRNA or salp25d siRNA (Narasimhan et al., 2007).

siRNA construction and synthesis

siRNA target sites were chosen based on the guidelines available on http://www.ambion.com/techlib/misc/siRNA_design.html. The sequences for salp25d, isft1, isft2, isft3 and isft4 were compared with the I. scapularis genome (http://www.vectorbase.org) and the NCBI database using the blast program (http://www.ncbi.nlm.nih.gov/BLAST) to eliminate sequences that had homology to unrelated coding sequences. The sequences of two sets of siRNAs for salp25d, isft1, isft2, isft3 and isft4 are described in Table S1. siRNAs were synthesized by in vitro transcription using the silencer siRNA construction kit (Ambion, TX). siRNAs were injected into the body of ticks as described for dsRNAs. Transfection of dsRNA or siRNAs in I. ricinus cells was performed according to RNAiFect transfection guidelines (Qiagen, CA).

dsRNA and siRNA transfection of I. ricinus cell lines

Ixodes ricinus cells were cultured in vitro as described earlier (Bell-Sakyi et al., 2007). Confluent cells were harvested and maintained at 30°C overnight into 12-well cell culture plates at a density of 106 cells per well. Tick α1,3-fucosyltransferase dsRNA or siRNA (600 µg) was added to 6 µl of RNAiFect (Qiagen, CA) in 100 µl of L15 (Leibowitz) cell culture medium. This solution was incubated at room temperature for 10 min and diluted to 400 µl of fresh medium. This mixture was added into each well to bring the final concentration of dsRNA or siRNA to 2 µg µl−1. Control cells received equivalent concentration of salp25d dsRNA and plates were maintained at 37°C for 66 h. Cells were then harvested and processed for RNA extraction and microscopy.

RNA isolation and PCR

cDNA from ticks, cells and mouse tissues were prepared as described (Pedra et al., 2006). Tick samples were normalized to the I. scapularis actin gene (AF426178). The genes B. burgdorferi N40 flagellin (flab) (X75200) and A. phagocytophilum 16s rDNA (M73224) were then quantified. Gene-specific primers are described in Table S1. Quantitative PCR (qPCR) was performed using the iQ SYBR green supermix (Bio-Rad, CA).

N-glycan profile in ticks

N-glycan structures were isolated from 60 µg of I. scapularis salivary gland extract using a previously described method (Rendić et al., 2007a). Briefly, salivary gland proteins were loaded onto a SDS-PAGE gel and stained with Coomassie Blue. Stained proteins were trypsinized and glycopeptides were extracted using 5% formic acid. N-glycans were released using N-glycosidase A digestion and then purified. Dried N-glycans were resuspended in deionized water and used for MALDI–TOF-MS (matrix-assisted laser-desorption ionization–time-of-flight mass spectrometry) analysis. N-glycan structures were identified according to the glycan symbol nomenclature established by the Consortium for Functional Glycomics (http://www.functionalglycomics.org). Mass spectra of released glycans were interpreted on the basis of knowledge about invertebrate glycans, their probable adduct status (Na+ conferring an m/z difference of 22) and the different contributions of monosaccharide units to the overall mass of these glycans (203 for N-acetylhexosamine, 162 for hexose and 146 for fucose).

Confocal microscopy and Western blot

Confocal microscopy and tick-immune rabbit serum were used as described earlier (Narasimhan et al., 2007). Topro-3 iodide (Invitrogen, MD) was used to stain the nucleus, rabbit polyclonal anti-HRP Sigma and rabbit polyclonal anti-A. phagocytophilum antibodies were used to stain core α1,3-fucose and A. phagocytophilum respectively. Immunoblots to detect core α1,3-fucose, Salp25D and immunogenic tick proteins were performed as described (Narasimhan et al., 2007; Rendić et al., 2007b).

Bioinformatics analysis

The whole genome shotgun sequences of I. scapularis were searched in vectorbase using the NCBI tblastn program for homology to that encoded by the previously identified EST (ti|1681614834). This EST sequence was described as the sequence corresponding to isft7. Ten putative fucosyltransferase reading frames were assembled in silico and the sequences 5′ to the homologous regions were examined for an ATG start codon followed by a sequence potentially encoding a transmembrane domain. These ‘self-assembled’ putative open reading frames were used to design primers for RT-PCR. No obvious start codon could be identified for isft1. Accession ID/gene numbers for isft1 (ISCW004192); isft2 (ISCW003590); isft3/9 (ISCW004236); isft4 (ISCW024758); isft5 (ISCW024943); isft6 (ISCW024303); isft7 (ISCW023318); isft8 (ISCW024461); isft10 (ISCW05151) are given.

Statistical analysis

P-values were calculated using experimental and control data using the Student’s t-test. Statistical significance was denoted by an asterisk and set at P < 0.05. Normal distribution was observed throughout our analysis.

Supplementary Material

Figures

Acknowledgements

We thank Swapna Samanta and Nancy Marcantonio for technical assistance and Felix Guerrero (United States Department of Agriculture) for providing us with the Rhipicephalus (Boophilus) microplus α1,3-fucosyltransferase plasmid. This work was supported by grants from the National Institutes of Health (E.F.), a cooperative agreement number K01 CK000101 from the Centers for Disease Control and Prevention (J.H.F.P.) and by the Austrian Fonds zur Förderung der wissenschaftlichen Forschung (L314 to I.B.H.W.).

Footnotes

Supporting information

Additional Supporting Information may be found in the online version of this article:

Fig. S1. α1,3-Fucosyltransferases present in the I. scapularis genome.

A. Comparison of signature motifs among several α1,3-fucosyltransferases: Bm {EST777694} Rhipicephalus (Boophilus) microplus (cattle tick); Am {CAK50252; CAK50261} Apis mellifera (honeybee); Dm {NP_648754; NP_001036320} Drosophila melanogaster; Aa {XP_001661902} Aedes aegypti (yellow fever mosquito); Mm {NP_034372} Mus musculus (mouse) and the expressed sequence tag isfucot (isft7) from I. scapularis database {ti|1681614834}.

B. Ten α1,3-fucosyltransferases were detected in the genome of the tick I. scapularis. Bioinformatics analysis showed the presence of highly conserved α1,3-fucosyltransferase signature motifs (blue) across all tick α1,3-fucosyltransferases – isft1 (ISCW004192); isft2 (ISCW003590); isft3/9 (ISCW004236); isft4 (ISCW024758); isft5 (ISCW024943); isft6 (ISCW024303); isft7 (ISCW023318); isft8 (ISCW024461); isft10 (ISCW05151).

Fig. S2. siRNA specificity for α1,3-fucosyltransferases associated with A. phagocytophilum colonization of ticks.

A. Two siRNAs for isft1 (blue), isft2 (red) and isft3 (green) were designed to determine the importance of these genes during A. phagocytophilum acquisition. The siRNAs shown above do not have any similarity with any other gene in the I. scapularis genome and are highly specific for isft1, isft2 and isft3. Asterisks represent nucleotide similarity between sequences.

B. Two overlapping specific siRNAs for isft4 (green box) were designed to determine the importance of this gene during A. phagocytophilum acquisition. Bold sequence differentiates siRNA-1 from siRNA-2. Yellow highlight indicates primers used for amplication of the isft4 gene. Red highlight indicates primer used for qPCR detection during experimental analysis.

Table S1. List of primers.

Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

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