Rudra Interrupts Receptor Signaling Complexes to Negatively Regulate the IMD Pathway

Abstract

Insects rely primarily on innate immune responses to fight pathogens. In Drosophila, antimicrobial peptides are key contributors to host defense. Antimicrobial peptide gene expression is regulated by the IMD and Toll pathways. Bacterial peptidoglycans trigger these pathways, through recognition by peptidoglycan recognition proteins (PGRPs). DAP-type peptidoglycan triggers the IMD pathway via PGRP-LC and PGRP-LE, while lysine-type peptidoglycan is an agonist for the Toll pathway through PGRP-SA and PGRP-SD. Recent work has shown that the intensity and duration of the immune responses initiating with these receptors is tightly regulated at multiple levels, by a series of negative regulators. Through two-hybrid screening with PGRP-LC, we identified Rudra, a new regulator of the IMD pathway, and demonstrate that it is a critical feedback inhibitor of peptidoglycan receptor signaling. Following stimulation of the IMD pathway, rudra expression was rapidly induced. In cells, RNAi targeting of rudra caused a marked up-regulation of antimicrobial peptide gene expression. rudra mutant flies also hyper-activated antimicrobial peptide genes and were more resistant to infection with the insect pathogen Erwinia carotovora carotovora. Molecularly, Rudra was found to bind and interfere with both PGRP-LC and PGRP-LE, disrupting their signaling complex. These results show that Rudra is a critical component in a negative feedback loop, whereby immune-induced gene expression rapidly produces a potent inhibitor that binds and inhibits pattern recognition receptors.

The Caspase-8 Homolog Dredd Cleaves Imd and Relish but Is Not Inhibited by p35

“…Transfection-Drosophila S2* cells and D.mel-2 cells were transfected using the calcium phosphate method as described previously (20). For experiments in Figs.…”

“…RNA Analysis-Total RNA was extracted and analyzed by Northern blotting or real-time RT-PCR as previously described (20).…”

“…Full-length ORFs of Dredd, six other Drosophila caspases, imd, Relish, and p35 were amplified by PCR using primers harboring indicated epitope tag sequences and cloned into pMT/V5-His vector (Invitrogen) or pRmHa-3 vector (19). Site-directed mutagenesis was performed to introduce point mutations or insertion using a QuikChange II Site-directed Mutagenesis Kit (Agilent Technologies).Transfection-Drosophila S2* cells and D.mel-2 cells were transfected using the calcium phosphate method as described previously (20). For experiments in Figs.…”

The Drosophila Imd Signaling Pathway

“…Pirk expression appears to be induced by the Ras/MAPK pathway as well, suggesting that Ras/MAPK signaling functions in immunity by negatively regulating the Imd pathway (92). Pirk interacts with the receptors PGRP-LC and PCRP-LE and the adaptor protein Imd, thus likely interrupting signal transduction at the receptor complex (91,93,94).…”

The multilayered innate immune defense of the gut

“…[92] At the intracellular level, the accessibility of both PGRP-LE and PGRP-LC receptors to the downstream components of the cascade is antagonized by Pirk (Poor IMD Response upon Knock in), also referred to as PIMS (PGRP-LC-interacting inhibitor of IMD signaling) or Rudra. [28,93,94] Indeed, Aggarwal et al, showed that the Pirk inhibitor interacts with PGRP-LC, PGRP-LE, and IMD, thus disrupting the signaling complex. Like PGRP-LB, Pirk is a critical component in the negative feedback loop that maintains a balanced IMD response following bacterial infection.…”