Abstract
Ixodes scapularis is the main vector of Lyme disease in the eastern and central United States. Tick salivary secretion has been shown as important for both blood-meal completion and pathogen transmission. Here we report a duplication event of cystatin genes in its genome that results in a transcription-regulated boost of saliva inhibitory activity against a conserved and relatively limited number of vertebrate papain-like cysteine proteases during blood feeding. We further show that the polypeptide products of the two genes differ in their binding affinity for some enzyme targets, and they display different antigenicity. Moreover, our reverse genetic approach employing RNA interference uncovered a crucial mediation in tick-feeding success. Given the role of the targeted enzymes in vertebrate immunity, we also show that host immunomodulation is implicated in the deleterious phenotype of silenced ticks making I. scapularis cystatins attractive targets for development of anti-tick vaccines.
Highlights
Among the differences that make a relationship between two organisms parasitic rather than symbiotic is the lack of mutual benefit; the parasite manages to continuously receive valuable resources from the host without returning this favor; in addition, sometimes it triggers catastrophic conditions to the host such as disease transmission
The Two Cystatin Transcripts Are Encoded by Two Different Genes—Several I. scapularis transcripts were revealed to be of salivary origin during our most recent massive expressed sequence tags (ESTs) sequencing project [8] including a novel cystatin that shows 75% identity at the protein level to sialostatin L, a secreted cystatin previously characterized in our laboratory [9]
In this report we identify two different loci in the tick genome encoding for two I. scapularis cystatin transcripts
Summary
Protocols followed standard procedures [11], and all experiments were performed at room temperature (25 Ϯ 1 °C). CDNA was PCR-amplified using gene-specific primers; sialostatin L2, forward 5Ј-CTA TGC GGC TTC CTC GAA GGG GCT-3Ј, and reverse, 5Ј-GGC TAC AGC GAG AGG GCG AAC CAC CAA-3Ј; tick salivary gland Isac, forward, OCTOBER 5, 2007 VOLUME 282 NUMBER 40. Double-stranded RNA (dsRNA) Synthesis, Tick Injections, and Feeding—Sialostatin L2 RT-PCR product was joined to the Block-iT T7 TOPO linker This TOPO linking reaction was used in two PCR reactions with gene-specific and T7 PCR primers to produce sense and antisense linear DNA templates. The ears of the rabbits exposed to dsRNA sialostatin L2 or water-injected ticks were cleaned by the end of the experiment; the animals were kept for 14 days and re-exposed to normal unfed ticks, and feeding success evaluation was performed as described above. Statistical significance was determined by Student’s t test; differences in multiple comparisons among different experimental groups were determined by analysis of variance using the Tukey test
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