Pathogen entrapment by transglutaminase--a conserved early innate immune mechanism.

Clotting systems are required in almost all animals to prevent loss of body fluids after injury. Here, we show that despite the risks associated with its systemic activation, clotting is a hitherto little appreciated branch of the immune system. We compared clotting of human blood and insect hemolymph to study the best-conserved component of clotting systems, namely the Drosophila enzyme transglutaminase and its vertebrate homologue Factor XIIIa. Using labelled artificial substrates we observe that transglutaminase activity from both Drosophila hemolymph and human blood accumulates on microbial surfaces, leading to their sequestration into the clot. Using both a human and a natural insect pathogen we provide functional proof for an immune function for transglutaminase (TG). Drosophila larvae with reduced TG levels show increased mortality after septic injury. The same larvae are also more susceptible to a natural infection involving entomopathogenic nematodes and their symbiotic bacteria while neither phagocytosis, phenoloxidase or-as previously shown-the Toll or imd pathway contribute to immunity. These results firmly establish the hemolymph/blood clot as an important effector of early innate immunity, which helps to prevent septic infections. These findings will help to guide further strategies to reduce the damaging effects of clotting and enhance its beneficial contribution to immune reactions.

Fondue and transglutaminase in the Drosophila larval clot.

Hemolymph coagulation is vital for larval hemostasis and important in immunity, yet the molecular basis of coagulation is not well understood in insects. Of the larval clotting factors identified in Drosophila, Fondue is not conserved in other insects, but is notable for its effects on the clot's physical properties, a possible function in the cuticle, and for being a substrate of transglutaminase. Transglutaminase is the only mammalian clotting factor found in Drosophila, and as it acts in coagulation in other invertebrates, it is also likely to be important in clotting in Drosophila. Here we describe a Fondue-GFP fusion construct that labels the cuticle and clot, and show that chemical inhibition and RNAi knockdown of the Drosophila transglutaminase gene affect clot properties and composition in ways similar to knockdown of the fon gene. Thus, Fondue appears to be incorporated into the cuticle and is a key transglutaminase substrate in the clot. This is also the first direct functional confirmation that transglutaminase acts in coagulation in Drosophila.

Clotting factors and eicosanoids protect against nematode infections.

We show that hemolymph clotting protects Drosophila melanogaster against infections with an entomopathogenic nematode and its symbiotic bacterium. We also provide biochemical and genetic evidence for an involvement of eicosanoids in the same infection model. Taken together, our results confirm the conserved nature of the immune function of clot formation.