The transcriptome of the salivary glands of the female western black-legged tick Ixodes pacificus (Acari: Ixodidae)

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Abstract

Sequencing of an Ixodes pacificus salivary gland cDNA library yielded 1068 sequences with an average undetermined nucleotide of 1.9% and an average length of 487 base pairs. Assembly of the expressed sequence tags yielded 557 contigs, 138 of which appear to code for secreted peptides or proteins based on translation of a putative signal peptide. Based on the BLASTX similarity of these contigs to 66 matches of Ixodes scapularis peptide sequences, only 58% sequence identity was found, indicating a rapid divergence of salivary proteins as observed previously for mosquito and triatomine bug salivary proteins. Here we report 106 mostly full-length sequences that clustered in 16 different families: Basic-tail proteins rich in lysine in the carboxy-terminal, Kunitz-containing proteins (monolaris, ixolaris and penthalaris families), proline-rich peptides, 5-, 9.4- and 18.7-kDa proteins of unknown functions, in addition to metalloproteases (class PIII-like) similar to reprolysins. We also have found a family of disintegrins, named ixodegrins that display homology to variabilin, a GPIIb/IIIa antagonist from the tick Dermacentor variabilis. In addition, we describe peptides (here named ixostatins) that display remarkable similarities to the cysteine-rich domain of ADAMST-4 (aggrecanase). Many molecules were assigned in the lipocalin family (histamine-binding proteins); others appear to be involved in oxidant metabolism, and still others were similar to ixodid proteins such as the anticomplement ISAC. We also identified for the first time a neuropeptide-like protein (nlp-31) with GGY repeats that may have antimicrobial activity. In addition, 16 novel proteins without significant similarities to other tick proteins and 37 housekeeping proteins that may be useful for phylogenetic studies are described. Some of these proteins may be useful for studying vascular biology or the immune system, for vaccine development, or as immunoreagents to detect prior exposure to ticks. Electronic version of the manuscript can be found at http://www.ncbi.nlm.nih.gov/projects/omes/.

Introduction

Lyme disease is the most prevalent vector-borne disease in the US and is transmitted by the tick vectors Ixodes scapularis and I. pacificus in eastern and western North America, respectively (Barbour, 1998). Humans usually acquire Lyme disease when an infected nymphal-stage Ixodes sp. tick attaches and transmits the spirochete Borrelia burgdorferi (Burgdorfer et al., 1985). I. scapularis and I. pacificus transmit other zoonotic agents besides the Lyme disease spirochete, such as Anaplasma phagocytophilum (both species) or Babesia microti (I. scapularis only) (Barbour, 1998). Transmission is facilitated by tick saliva that operates not only as a carrier for Borrelia sp. but also contains a large repertoire of molecules that counteract the host response to injury (Ribeiro and Francischetti, 2003), allowing ticks to feed for days (Sonenshine, 1985). Accordingly, many biologic activities have been described in tick saliva, including molecules that impair platelet aggregation or neutrophil function (Ribeiro et al., 1985) in addition to coagulation inhibitors such as ixolaris and penthalaris that block Factor VIIa/tissue factor complex (Francischetti et al., 2002a, Francischetti et al., 2004a) and SALP 14, which targets Factor Xa (Narasimhan et al., 2002). Enzymes such as a kininase that degrades bradykinin (Ribeiro and Mather, 1998), an apyrase that destroys ADP (Ribeiro et al., 1985), and a metalloprotease with fibrin(ogen)olytic activity (Francischetti et al., 2003) also have been reported. Tick saliva is also rich in small molecules such as prostacyclin, a potent inhibitor of platelet activation and strong inducer of vasodilation (Ribeiro et al., 1988).

As for the immune system, an inhibitor of the alternative complement pathway exists in ixodid tick saliva (Valenzuela et al., 2000). Immunomodulators affecting NK cell function (Kopecky and Kuthejlova, 1998)—in addition to inhibitors of the proliferation of T lymphocytes and an IL-2 binding activity—also are present in this secretion (Ramachandra and Wikel, 1992; Gillespie et al., 2001). Finally, saliva is important in transmission of tick-borne pathogens, as it may enhance pathogen transmission (for a review, see Wikel, 1999).

The pace of discovery of tick salivary proteins has been greatly increased by novel molecular biology techniques and bioinformatics analysis (Ribeiro and Francischetti, 2003). Our goal here has been to further study the complexity of I. pacificus salivary glands. We report the full-length clone of 87 novel sequences and discuss their potential role in modulating host inflammatory and immune responses.

Section snippets

Reagents

All water used was of 18 MΩ quality and was produced using a MilliQ apparatus (Millipore, Bedford, MA, USA). Organic compounds were obtained from Sigma (St. Louis, MO, USA) or as stated otherwise.

Salivary gland cDNA library construction and sequencing

Ticks were collected in northern California by dragging low vegetation with a tick-drag. Salivary glands were excised and kept at −80 °C until use. The mRNA from two pairs of I. pacificus salivary glands was obtained using a Micro-Fast Track mRNA isolation kit (Invitrogen, San Diego, CA, USA) according

Results and discussion

I. scapularis and I. pacificus are the respective vectors for B. burgdorferi in the eastern and western US (Fig. 1). After attachment to the host, infected ticks transmit B. burgdoferi after 1–2 days of blood-feeding (Barbour, 1998) via saliva, a secretion that contains a cocktail of bioactive molecules (Ribeiro and Francischetti, 2003). Actually, the identification of the transcripts and proteins present in the salivary gland of ticks such as I. scapularis (Valenzuela et al., 2002), Boophilus

Acknowledgements

We thank Drs. Thomas E. Wellems, Robert W. Gwadz, and Thomas J. Kindt for encouragement and support. We are thankful to Brenda Rae Marshal for editorial assistance.

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