Justin R. Pool
Recently, defensins have been isolated in several species of ticks (Hynes et. al. 2005, Todd et. al. 2007, Zhou et. al. 2007). Defensins are small arginine rich proteins, usually containing six cysteine residues that form disulfide bridges (Hynes et. al. 2005). They have been found in both invertebrates and vertebrates, functioning as host defense proteins (Hoffmann and Hetru 1992, Ganz 2003). These peptides kill invading microorganisms by forming membrane-penetrating channels in the cells of the microorganisms (Gillespie et. al. 1997, Ganz 2003). Recently, a defensin, called scapularisin, was found in I. scapularis, but its role as an antimicrobial peptide remains unclear (Hynes et. al. 2005).
The American dog tick, Dermacentor variabilis, has a defensin, varisin, that is similar to the defensins found in insects (Johns et. al. 2001). The amino acid sequences of varisin and scapularisin are 78.9%, and the six cysteine residues are located in that same relative positions (Hynes et. al. 2005). Therefore, it is unclear why scapularisin is not effective against B. burgdorferi.
In this study scapularisin and varisin genes were isolated using RT-PCR from I. scapularis and D. variabilis ticks. The cDNA was then amplified and sent out for sequencing. Once the genes were confirmed, the cDNA was placed in a eukaryotic in-vitro transcription/translation system to see if both sequences could be translated. The structure of the translated proteins were also studied.
Materials and Methods
Adult I. scapularis were collected at The Louis Calder Center, the biological station of Fordham Univeristy. Adult Dermacentor variabilis ticks were donated by Dr. Daniel Sonenshine from Old Dominion University in Norfolk, Virginia. Ticks were placed in 100% ethanol twenty-four hours prior to RNA extraction. Ticks were homogenized using Lysing Matrix A tubes (MPBio) with a sterile garnet matrix and one 1/4 inch ceramic sphere, which were placed in a FastPrep24 (MPBio) set at a speed of 4.0 m/s for 40 seconds. Immediately following homogenization, the total RNA extraction was performed using RNeasy® Plus Minikit (QIAGEN). Primers specific for both scapularisin and varisin were designed. RT-PCR was performed using QIAGEN® One-Step RT-PCR Kit following the kit instructions. Temperature cycles for both scapularisin and varisin were: one cycle each of 50°C for 30 minutes and 95°C for 15 minutes; 50 cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds; followed by a final extension at 72°C for 10 minutes; and a final hold at 4°C. The results of the RT-PCR were visualized on a gel. Bands representative of scapularisin and varisin were extracted from the gel. Following gel extraction, ligation and transformation were performed using pGEM-T and pGEM-T Easy Vector Systems (Promega). Once scapularisin and varisin were confirmed, in-vitro transcription/translation was performed using TnT® Quick Coupled Transcription/Translation Systems Kit following the kit instructions. Results were run on an SDS polyacrylamide gel.
The RT-PCR successfully amplified both scapularisin and varisin (Fig. 1). Each defensin was expected to be around 225 base pairs and this was confirmed when the agarose gel was visualized using a UV trans-illuminator. Both RT-PCR products for the defensins showed the presence of primer-dimer, therefore the bands representing scapularisin and varisin had to be cut out of the gel and purified. Following gel extraction, the products were placed into a plasmid vector for amplification. Both scapularisin and varisin produced numerous white colonies. Sequencing confirmed the presence of both scapularisin and varisin (Figs. 2 and 3). There were a few point mutations in the sequence of scapularisin from the reported sequence for scapularisin; however, there were no premature stop codons because of the mutations, and all six cysteine residues were in the same relative position. After confirming the presence of both scapularisin and varisin, in-vitro transcription/translation was performed. When the products were run on an SDS polyacrylamide gel in the presence of β-mercaptoethanol, both scapularisin and varisin produced one band just about 10 kDa (Fig. 4). However, when the same products were run on an SDS polyacrylamide gel without β-mercaptoethanol, both scapularisin and varisin produced one band just above 20 kDa, suggesting that both defensins form dimers.
Scapularisin and varisin were also shown to form dimers when analyzed using SDS-PAGE without the presence of β-mercaptoethanol. Dimers and multimers have been reported in the defensins of other species (Ganz 2003). This is the first time dimerization has been shown in a tick defensin. All defensins act by similar mechanisms, however it is possible that defensins that form dimers and multimers have evolved to target different types of bacteria (Ganz 2003).
In the future, it would be beneficial to study the stability of scapularisin and varisin to monitor the decay rates of these peptides. One hypothesis as to why scapularisin does not defend the tick against B. burgdorferi is the peptide immediately decays upon synthesis. The antimicrobial activity of scapularisin also needs to be clarified to determine whether or not this peptide can be effective against vector-borne diseases.
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