α2-macroglobulin function of thioester-containing proteins guards <i>Drosophila</i> from a bacterial protease via two immune-induced peptides

RNAseq analysis of the <i>Drosophila</i> genome has revealed further immune-induced genes. Two genes initially annotated as lncRNAs, <i>CG44404</i>(<i>yulü</i>) and <i>CG45045</i>(<i>shenshu</i>), are strongly induced. We report here that these two genes actually encode highly related secreted peptides found in the Sophophora subgenus of <i>Drosophila</i> species. We have generated single and double null mutants of these loci and found that the double mutant line did not display any enhanced susceptibility to an immune challenge with a panel of bacterial and fungal pathogens, except for <i><i>Pseudomonas aeruginosa</i></i>. We did not observe any increased <i><i>P. aeruginosa</i></i> burden in <i>yulü-shenshu</i> mutants, suggesting that the two peptides may not be required for resistance to infection. Rather, we find that they provide a level of protection against Outer Membrane Vesicles (OMVs) purified from either <i><i>P. aeruginosa</i></i> or <i><i>Serratia marcescens</i></i> culture supernatants. We have recently reported that <i>S. marcescens</i> OMVs induce the paralysis of flies through the induction of apoptosis in at least some neurons. Much of the virulence of these OMVs is mediated by the metalloprotease PrtA. While Yulü/Shenshu do not display any protease inhibition activity, the detection of an association between Yulü and the <i>Drosophila</i> complement thioester-containing protein 2 (Tep2) led to the finding that both <i>Tep2</i> and <i>Tep4</i> mutants are sensitive to the injection of PrtA while their overexpression significantly protects wild-type flies from the effects of this protease. Both Tep2 and Tep4 are able to inhibit the activity of PrtA in a thioester- and <i>yulü/shenshu</i>-dependent manner. Thus, these Teps may also function as α2-macroglobulins.