Abstract
BackgroundTsetse flies (Glossina sp.), the African trypanosome vectors, rely on anti-hemostatic compounds for efficient blood feeding. Despite their medical importance, very few salivary proteins have been characterized and functionally annotated.Methodology/Principal FindingsHere we report on the functional characterisation of a 5′nucleotidase-related (5′Nuc) saliva protein of the tsetse fly Glossina morsitans morsitans. This protein is encoded by a 1668 bp cDNA corresponding at the genomic level with a single-copy 4 kb gene that is exclusively transcribed in the tsetse salivary gland tissue. The encoded 5′Nuc protein is a soluble 65 kDa glycosylated compound of tsetse saliva with a dual anti-hemostatic action that relies on its combined apyrase activity and fibrinogen receptor (GPIIb/IIIa) antagonistic properties. Experimental evidence is based on the biochemical and functional characterization of recombinant protein and on the successful silencing of the 5′nuc translation in the salivary gland by RNA interference (RNAi). Refolding of a 5′Nuc/SUMO-fusion protein yielded an active apyrase enzyme with Km and Vmax values of 43±4 µM and 684±49 nmol Pi/min×mg for ATPase and 49±11 µM and 177±37 nmol Pi/min×mg for the ADPase activity. In addition, recombinant 5′Nuc was found to bind to GPIIb/IIIa with an apparent KD of 92±25 nM. Consistent with these features, 5′Nuc potently inhibited ADP-induced thrombocyte aggregation and even caused disaggregation of ADP-triggered human platelets. The importance of 5′Nuc for the tsetse fly hematophagy was further illustrated by specific RNAi that reduced the anti-thrombotic activities in saliva by approximately 50% resulting in a disturbed blood feeding process.Conclusions/SignificanceThese data show that this 5′nucleotidase-related apyrase exhibits GPIIb/IIIa antagonistic properties and represents a key thromboregulatory compound of tsetse fly saliva.
Highlights
Efficient acquisition of a blood meal by hematophagous arthropods relies on a broad repertoire of physiologically active saliva components inoculated at the blood feeding site
These are primarily anti-hemostatic components that interfere with host responses such as vasoconstriction [1,2], primary hemostasis through the adherence and aggregation of thrombocytes [3,4] and a secondary hemostatic cascade mainly relying on serine proteases such as thrombin [5,6]
Salivary apyrases of hematophagous arthropods could inhibit several aspects of hemostasis and inflammation thereby promoting the blood feeding process and possibly facilitating pathogen transmission, a feature that we have described for the saliva of tsetse flies, blood feeding insects that transmits the protozoan agents of African trypanosomiasis [18]
Summary
Efficient acquisition of a blood meal by hematophagous arthropods relies on a broad repertoire of physiologically active saliva components inoculated at the blood feeding site. ATP binds onto the cationgated P2X1 receptor that is present on thrombocytes resulting in cytoskeletal reorganisation and higher responsiveness in terms of aggregation and degranulation towards other platelet activating triggers such as collagen [reviewed in [7,8]] To overcome these ATP- and ADP-related host responses, blood sucking insects have ATP/ADP-hydrolysing [ATP(D)ase] enzymes present in their salivary secretions [9]. These enzymes are often called apyrases (nucleosidetriphosphate diphosphohydrolases) and have been described in the saliva of a variety of blood feeding arthropods such as Cimex lectularius bed bugs [10], Ixodes dammini ticks [11], Aedes aegypti and Anopheles gambiae mosquitoes [12,13], Phlebotomus papatasi sand flies [14] and Triatoma infestans reduviids [15]. Very few salivary proteins have been characterized and functionally annotated
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