Peptidoglycan molecular requirements allowing detection by the Drosophila immune deficiency pathway

Innate immune recognition of microbes is a complex process that can be influenced by both the host and the microbe. Drosophila uses two distinct immune signaling pathways, the Toll and immune deficiency (Imd) pathways, to respond to different classes of microbes. The Toll pathway is predominantly activated by Gram-positive bacteria and fungi, while the Imd pathway is primarily activated by Gram-negative bacteria. Recent work has suggested that this differential activation is achieved through peptidoglycan recognition protein (PGRP)-mediated recognition of specific forms of peptidoglycan (PG). In this study, we have further analyzed the specific PG molecular requirements for Imd activation through the pattern recognition receptor PGRP-LC in both cultured cell line and in flies. We found that two signatures of Gram-negative PG, the presence of diaminopimelic acid in the peptide bridge and a 1,6-anhydro form of N-acetylmuramic acid in the glycan chain, allow discrimination between Gram-negative and Gram-positive bacteria. Our results also point to a role for PG oligomerization in Imd activation, and we demonstrate that elements of both the sugar backbone and the peptide bridge of PG are required for optimum recognition. Altogether, these results indicate multiple requirements for efficient PG-mediated activation of the Imd pathway and demonstrate that PG is a complex immune elicitor.

Drosophila immunity: analysis of PGRP-SB1 expression, enzymatic activity and function

Bruno Lemaitre, Juan Camilo Paredes Escobar, David Welchman

Peptidoglycan is an essential and specific component of the bacterial cell wall and therefore is an ideal recognition signature for the immune system. Peptidoglycan recognition proteins (PGRPs) are conserved from insects to mammals and able to bind PGN (non-catalytic PGRPs) and, in some cases, to efficiently degrade it (catalytic PGRPs). In Drosophila, several non-catalytic PGRPs function as selective peptidoglycan receptors upstream of the Toll and Imd pathways, the two major signalling cascades regulating the systemic production of antimicrobial peptides. Recognition PGRPs specifically activate the Toll pathway in response to Lys-type peptidoglycan found in most Gram-positive bacteria and the Imd pathway in response to DAP-type peptidoglycan encountered in Gram-positive bacilli-type bacteria and in Gram-negative bacteria. Catalytic PGRPs on the other hand can potentially reduce the level of immune activation by scavenging peptidoglycan. In accordance with this, PGRP-LB and PGRP-SC1A/B/2 have been shown to act as negative regulators of the Imd pathway. In this study, we report a biochemical and genetic analysis of PGRP-SB1, a catalytic PGRP. Our data show that PGRP-SB1 is abundantly secreted into the hemolymph following Imd pathway activation in the fat body, and exhibits an enzymatic activity towards DAP-type polymeric peptidoglycan. We have generated a PGRP-SB1/2 null mutant by homologous recombination, but its thorough phenotypic analysis did not reveal any immune function, suggesting a subtle role or redundancy of PGRP-SB1/2 with other molecules. Possible immune functions of PGRP-SB1 are discussed.

Public Library of Science2011

The Drosophila immune system detects bacteria through specific peptidoglycan recognition

Bruno Lemaitre

The Drosophila immune system discriminates between different classes of infectious microbes and responds with pathogen-specific defense reactions through selective activation of the Toll and the immune deficiency (Imd) signaling pathways. The Toll pathway mediates most defenses against Gram-positive bacteria and fungi, whereas the Imd pathway is required to resist infection by Gram-negative bacteria. The bacterial components recognized by these pathways remain to be defined. Here we report that Gram-negative diaminopimelic acid-type peptidoglycan is the most potent inducer of the Imd pathway and that the Toll pathway is predominantly activated by Gram-positive lysine-type peptidoglycan. Thus, the ability of Drosophila to discriminate between Gram-positive and Gram-negative bacteria relies on the recognition of specific forms of peptidoglycan.

Nature Publishing Group2003

Analysis of a specific modular serine protease acting in the Drosophila Toll pathway

Valentin Sottas

In Drosophila, the Toll pathway plays an important role in the immune defense against Gram-positive bacteria and fungi. Molecular determinants coming from those pathogens are directly detected by pattern recognition receptors (PPRs) circulating in the hemolymph. It has been proposed that several serine protease cascades are activated by PRRs, leading to the activation of a cleaved form of the cytokine-like molecule Spätzle, the ligand of the Toll receptor. The results obtained during my master project demonstrate an essential role for ModSP, a modular serine protease acting in the activation of the Toll pathway upon Gram-positive and fungal infections. We demonstrate that ModSP integrates signals coming from GNBP3 and PGRP-SA recognition molecules and that ModSP sends this signal to Grass, a serine protease already known to activate SPE and thereby Spätzle. Further biochemical experiments show the interaction between ModSP and GNBP1 demonstrating an apical role of this serine protease in the proteolytic cascades leading to Toll pathway activation. We also find that ModSP is expressed in specific vesicles released from the fat bodies into the hemolymph. Also, preliminary studies suggest that ModSP does not participate in the melanization reaction, a secondary but important insect immune mechanism. Biochemical analysis done by some collaborators indicates that ModSP does not cleave Grass and that ModSP exhibits a high level of auto-proteolysis when this molecule is expressed. Our data reveal a conserved role of modular serine protease in the regulation of immune proteolytic cascade in insects

2009