Drosophila melanogaster has emerged as a powerful model system to study innate immunity. Insects employ multilayered innate immune defenses including antimicrobial peptide responses and phagocytosis. In Drosophila, phagocytosis is carried out by plasmatocytes, a blood cell type similar to mammalian macrophages and neutrophils. The scavenger receptor Eater is expressed by larval and adult plasmatocytes and mediates recognition of a broad range of bacterial pathogens. Eater is required for fly survival after infection with Gram-positive and Gram-negative bacteria. However, the bacterial ligands of Eater, and the mechanisms by which this receptor recognizes these different types of bacteria, remain poorly understood.
To address this problem, I generated a soluble, Fc-tagged receptor variant of Eater comprising the N-terminal 199 amino acids (including four N-terminal EGF-like repeats) and raised antibodies against Eater. Using these tools, I established (i) that Eater is expressed on the surface of macrophage-like Drosophila S2 cells, (ii) that it interacts with broad, yet distinct classes of heat- and ethanol-inactivated microbes and (iii) that it binds peptidoglycan from Gram-negative Proteobacteria (E. coli) and Gram-positive Firmicutes (E. faecalis and S. aureus), but not Gram-positive Actinobacteria (M. luteus). In order to identify genes involved in the phagocytosis of M. luteus, I screened 39 candidate genes by RNA interference-mediated knock down in S2 cells.
A longstanding question was whether Eater recognizes live, naïve bacteria. I found that Eater-Fc bound equally well to naïve or heat-inactivated S. aureus or E. faecalis, suggesting that in vivo Eater directly targets live Gram-positive bacteria, enabling their phagocytic clearance and destruction. By contrast, Eater-Fc was unable to interact with live Gram-negative bacteria (E. coli, S. marcescens and P. aeruginosa). Eater binding required prior membrane-disrupting treatments. Cecropin A, a prototypic cationic, membrane-disrupting antimicrobial peptide could promote Eater-Fc binding to live E. coli, even at sublethal concentrations. These results suggest a previously unrecognized mechanism by which antimicrobial peptides cooperate with phagocytic receptors
