The melanization reaction induced by activated phenoloxidase in arthropods must be tightly controlled because of excessive formation of quinones and excessive systemic melanization damage to the hosts. However, the molecular mechanism by which phenoloxidase-induced melanin synthesis is regulated in vivo is largely unknown. It is known that the Spätzle-processing enzyme is a key enzyme in the production of cleaved Spätzle from pro-Spätzle in the Drosophila Toll pathway. Here, we provide biochemical evidence that the Tenebrio molitor Spätzle-processing enzyme converts both the 79-kDa Tenebrio prophenoloxidase and Tenebrio clip-domain SPH1 zymogen to an active melanization complex. This complex, consisting of the 76-kDa Tenebrio phenoloxidase and an active form of Tenebrio clip-domain SPH1, efficiently produces melanin on the surface of bacteria, and this activity has a strong bactericidal effect. Interestingly, we found the phenoloxidase-induced melanization reaction to be tightly regulated by Tenebrio prophenoloxidase, which functions as a competitive inhibitor of melanization complex formation. These results demonstrate that the Tenebrio Toll pathway and the melanization reaction share a common serine protease for the regulation of these two major innate immune responses.

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Background: During the Drosophila immune response, both Imd and Relish are cleaved in a manner dependent on the caspase-8 homolog Dredd. Results: Dredd cleaves Imd and Relish, but not the caspase inhibitor p35, without interdomain autoprocessing. Conclusion: Imd and Relish are direct substrates for full-length Dredd. Significance: Dredd is similar to some mammalian initiator caspases, which can function without interdomain cleavage.In Drosophila, the Imd pathway is activated by diaminopimelic acid-type peptidoglycan and triggers the humoral innate immune response, including the robust induction of antimicrobial peptide gene expression. Imd and Relish, two essential components of this pathway, are both endoproteolytically cleaved upon immune stimulation. Genetic analyses have shown that these cleavage events are dependent on the caspase-8 like Dredd, suggesting that Imd and Relish are direct substrates of Dredd. Among the seven Drosophila caspases, we find that Dredd uniquely promotes Imd and Relish processing, and purified recombinant Dredd cleaves Imd and Relish in vitro. In addition, interdomain cleavage of Dredd is not required for Imd or Relish processing and is not observed during immune stimulation. Baculovirus p35, a suicide substrate of executioner caspases, is not cleaved by purified Dredd in vitro. Consistent with this biochemistry but contrary to earlier reports, p35 does not interfere with Imd signaling in S2* cells or in vivo.The Imd pathway is one of two NF-B signaling pathways controlling antimicrobial peptide (AMP) 4 induction in the Drosophila immune response. Diaminopimelic acid-type peptidoglycan (PGN), from Gram-negative and some Gram-positive bacteria, stimulates the Imd pathway through receptors PGRP-LC and PGRP-LE, and leads to the activation of Relish, a NF-B precursor protein similar to mammalian p100 and p105. Unlike p100 or p105 processing, Relish is activated by endoproteolytic cleavage, and then translocates to the nucleus where it drives robust (100-fold or more) AMP gene expression (recently reviewed in Refs. 1 and 2).In this pathway, the Imd protein plays a central role as receptor proximal adapter (3). Imd interacts directly with both PGRP-LC or -LE receptors (4), as well as with the Drosophila FADD homolog, which in turn recruits the caspase-8-like Dredd (5, 6). Upon PGN stimulation, Imd is endoproteolytic cleaved, at aspartate residue 30 within a caspase recognition site. Genetic studies demonstrated that this signal-induced cleavage is dependent on Fadd and Dredd. Once cleaved, Imd exposes a new N terminus that contains inhibitor of apoptosis binding motif. The E3 ubiquitin ligase Iap2 associates with cleaved Imd through the newly exposed inhibitor of apoptosis binding motif, and rapidly conjugates Imd with K63-linked polyubiquitin. K63 polyubiquitin chains are suggested to function as a scaffold to activate Tak1 and IKK kinases (Ird5 and Kenny), which are critical for Relish activation and AMP gene induction (7).In the absence of immune stimulation, Relish is found in the c...

Legionella pneumophila, a human intracellular pathogen, encodes about 290 effector proteins that are translocated into host cells through a secretion machinery. Some of these proteins have been shown to manipulate or subvert cellular processes during infection, but functional roles of a majority of them remain unknown. Lpg0393 is a newly identified Legionella effector classified as a hypothetical protein. Through X-ray crystallographic analysis, we show that Lpg0393 contains a Vps9-like domain, which is structurally most similar to the catalytic core of human Rabex-5 that activates the endosomal Rab proteins Rab5, Rab21 and Rab22. Consistently, Lpg0393 exhibited a guanine-nucleotide exchange factor activity toward the endosomal Rabs. This work identifies the first example of a bacterial guanine-nucleotide exchange factor that is active towards the Rab5 sub-cluster members, implying that the activation of these Rab proteins might be advantageous for the intracellular survival of Legionella.