Expression of a toll signaling regulator serpin in a mycoinsecticide for increased virulence

Serpins are ubiquitously distributed serine protease inhibitors that covalently bind to target proteases to exert their activities. Serpins regulate a wide range of activities, particularly those in which protease-mediated cascades are active. The Drosophila melanogaster serpin Spn43Ac negatively controls the Toll pathway that is activated in response to fungal infection. The entomopathogenic fungus Beauveria bassiana offers an environmentally friendly alternative to chemical pesticides for insect control. However, the use of mycoinsecticides remains limited in part due to issues of efficacy (low virulence) and the recalcitrance of the targets (due to strong immune responses). Since Spn43Ac acts to inhibit Toll-mediated activation of defense responses, we explored the feasibility of a new strategy to engineer entomopathogenic fungi with increased virulence by expression of Spn43Ac in the fungus. Compared to the 50% lethal dose (LD50) for the wild-type parent, the LD50 of B. bassiana expressing Spn43Ac (strain Bb::S43Ac-1) was reduced ~3-fold, and the median lethal time against the greater wax moth (Galleria mellonella) was decreased by ~24%, with the more rapid proliferation of hyphal bodies being seen in the host hemolymph. In vitro and in vivo assays showed inhibition of phenoloxidase (PO) activation in the presence of Spn43Ac, with Spn43Ac-mediated suppression of activation by chymotrypsin, trypsin, laminarin, and lipopolysaccharide occurring in the following order: chymotrypsin and trypsin>laminarin>lipopolysaccharide. Expression of Spn43Ac had no effect on the activity of the endogenous B. bassianaderived cuticle-degrading protease (CDEP-1). These results expand our understanding of Spn43Ac function and confirm that suppression of insect immune system defenses represents a feasible approach to engineering entomopathogenic fungi for greater efficacy.