Characterization of an Ixodes Scapularis Serine Proteinase Inhibitor

Abstract

The blacklegged tick, Ixodes scapularis is a medically important tick species that transmits six of the fifteen human tick-borne disease agents in the United States. Without successful feeding, ticks cannot cause damage to the host nor transmit any disease agents. Thus, a deeper understanding of how ticks accomplish feeding could lead to the identification of proteins to target for development of innovative tick control methods. Members of the serpin superfamily (serine protease inhibitors) are among proteins thought to facilitate tick feeding. In order to complete feeding, ticks have to overcome serine protease mediated pathways that are controlled by serpins. This honors thesis describes the characterization process of I. scapularis tick Serpin 1A. In addition to providing qualitative and semi-quantitative RT-PCR analysis of Serpin 1A in the nymph and female adult development stages at different feeding intervals (unfed, 24h, 48h, 72h, 96h, and 120h). The Serpin 1A mature open reading frame (ORF1A) was successfully subcloned into the yeast recombinant protein expression plasmid, pPICZαC, which was effectively used to transfect Pichia pastoris strain X-33, yeast cells. Efforts to induce recombinant Serpin 1A protein failed, therefore halting the biochemical characterization of this protein. Further research into the expression of recombinant Serpin 1A is needed for follow-up experiments to characterize the role(s) of Serpin 1A in tick feeding. Even though, characterization of the protein was halted, the qualitative and semi-quantitative data revealed that Serpin 1A mRNA was expressed at different feeding intervals and highly expressed during the first day and second day of feeding in the salivary gland. These findings suggest that Serpin 1A is associated with the I. scapularis tick feeding physiology. Moreover, it implies that this serpin is mostly associated with the early stages of tick feeding in nymph and female adult tick organs. Furthermore, these results propose that Serpin 1A may play a role in pathogen development, since the salivary gland is considered to be the site for pathogen development in tick anatomy. Most tick borne pathogens spread by this species are transmitted after the tick has for more than one day by the early life states and female adult stage. Thus, this information on Serpin 1A could be utilized to target this serpin in an anti-tick vaccine that is aimed at preventing tick borne disease transmission.