Abstract
Ticks successfully feed and transmit pathogens by injecting pharmacological compounds in saliva to thwart host defenses. We have previously used LC-MS/MS to identify proteins that are present in saliva of unfed Amblyomma americanum ticks that were exposed to different hosts. Here we show that A. americanum serine protease inhibitor (serpin) 27 (AAS27) is an immunogenic saliva protein that is injected into the host within the first day of tick feeding and is an anti-inflammatory protein that might act by blocking plasmin and trypsin functions. Although AAS27 is injected into the host throughout tick feeding, qRT-PCR and western blotting analyses indicate that the respective transcript and protein are present in high amounts within the first 24 h of tick feeding. Biochemical screening of Pichia pastoris-expressed recombinant (r) AAS27 against mammalian proteases related to host defense shows it is an inhibitor of trypsin and plasmin, with stoichiometry of inhibition indices of 3.5 and 3.8, respectively. Consistent with typical inhibitory serpins, rAAS27 formed heat- and SDS-stable irreversible complexes with both proteases. We further demonstrate that rAAS27 inhibits trypsin with ka of 6.46 ± 1.24 x 104 M-1 s-1, comparable to serpins of other tick species. We show that native AAS27 is part of the repertoire of proteins responsible for the inhibitory activity against trypsin in crude tick saliva. AAS27 is likely utilized by the tick to evade the hosts inflammation defense since rAAS27 blocks both formalin and compound 48/80-induced inflammation in rats. Tick immune sera of rabbits that had acquired resistance against tick feeding following repeated infestations with A. americanum or Ixodes scapularis ticks reacts with rAAS27. Of significant interest, antibody to rAAS27 blocks this serpin inhibitory functions. Taken together, we conclude that AAS27 is an anti-inflammatory protein secreted into the host during feeding and may represent a potential candidate for development of an anti-tick vaccine.
Highlights
The lone star tick Amblyomma americanum is a hard tick species of medical and veterinary importance in the United States and Mexico [1,2,3]
We report that Amblyomma americanum ticks secrete an antiinflammatory serpin into the host during feeding
This work emphasizes the importance of understanding the functional roles of tick saliva proteins to tick feeding physiology to identify new targets in development of novel strategies for tick and tick-borne diseases control and to search and find new potentially pharmacological active compounds
Summary
The lone star tick Amblyomma americanum is a hard tick species of medical and veterinary importance in the United States and Mexico [1,2,3]. This tick species is a known vector of a number of tick-borne diseases (TBD) agents including Borrelia lonestari, the causative agent of southern tick-associated rash illness (STARI) [4]; Francisella tularensis, the causative agent of tularemia in humans [5,6]; Ehrlichia chaffeensis and Ehrlichia ewingii, the causative agents of human ehrlichiosis [7,8]; and Rickettsia amblyommii, the causative agent of rickettsiosis of the spotted fever group [9,10]. In the effort to find effective targets for an anti-tick vaccine development, understanding tick-feeding physiology could lead to the discovery of important tick saliva proteins that can be targeted for anti-tick vaccine development
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