Date Published: July 26, 2013
Publisher: Public Library of Science
Author(s): Mathilde Gendrin, Anna Zaidman-Rémy, Nichole A. Broderick, Juan Paredes, Mickaël Poidevin, Alain Roussel, Bruno Lemaitre, François Leulier.
PeptidoGlycan Recognition Proteins (PGRPs) are key regulators of the insect innate antibacterial response. Even if they have been intensively studied, some of them have yet unknown functions. Here, we present a functional analysis of PGRP-LA, an as yet uncharacterized Drosophila PGRP. The PGRP-LA gene is located in cluster with PGRP-LC and PGRP-LF, which encode a receptor and a negative regulator of the Imd pathway, respectively. Structure predictions indicate that PGRP-LA would not bind to peptidoglycan, pointing to a regulatory role of this PGRP. PGRP-LA expression was enriched in barrier epithelia, but low in the fat body. Use of a newly generated PGRP-LA deficient mutant indicates that PGRP-LA is not required for the production of antimicrobial peptides by the fat body in response to a systemic infection. Focusing on the respiratory tract, where PGRP-LA is strongly expressed, we conducted a genome-wide microarray analysis of the tracheal immune response of wild-type, Relish, and PGRP-LA mutant larvae. Comparing our data to previous microarray studies, we report that a majority of genes regulated in the trachea upon infection differ from those induced in the gut or the fat body. Importantly, antimicrobial peptide gene expression was reduced in the tracheae of larvae and in the adult gut of PGRP-LA-deficient Drosophila upon oral bacterial infection. Together, our results suggest that PGRP-LA positively regulates the Imd pathway in barrier epithelia.
Drosophila, in contrast to mammals, lacks adaptive immunity and therefore relies entirely on innate immunity for defense against invading pathogens , . Microorganisms are recognized through the interaction between microbial compounds and host pattern-recognition receptors. In insects, the peptidoglycan recognition proteins (PGRPs) are a major class of pattern-recognition receptors that sense bacteria by interacting with peptidoglycan and regulate host antibacterial defenses. In Drosophila, the Toll and Imd pathways are the two major signaling cascades regulating the massive expression of antimicrobial peptide genes and other immune genes by the fat body following a systemic infection –. The Toll pathway is strongly induced by Gram-positive bacteria and fungi, and controls the expression of several genes, notably the antifungal peptide gene Drosomycin; the Imd pathway is strongly induced by Gram-negative and bacillus-shaped Gram-positive bacteria and regulates the expression of genes such as Diptericin, encoding an antibacterial peptide . Activation of both pathways by bacteria is achieved through the sensing of specific forms of peptidoglycan by PGRPs. Peptidoglycan is an essential cell wall component of bacteria, composed of long glycan chains with alternating N-acetylglucosamine and N-acetylmuramic acid residues that are cross-linked to each other by short peptide bridges. The third residue of these stem peptides differs between bacteria: it is a lysine in Gram-positive cocci and a meso-diaminopimelic acid (DAP) in both Gram-negative bacteria and Gram-positive bacilli, such as Bacillus and Listeria species . Studies using highly purified bacterial compounds have shown that the highest Toll pathway activity is observed upon injection of Lysine-type peptidoglycan, while the Imd pathway is activated by DAP-type peptidoglycan . Further studies have shown that both polymeric and monomeric DAP-type peptidoglycan can activate the Imd pathway. A specific monomer, the GlcNAc-MurNAc(anhydro)-L-Ala-γ-D-Glu-meso-DAP-D-Ala, also known as tracheal cytotoxin (TCT), has been identified as the minimal peptidoglycan motif capable of efficient induction of the Imd pathway , .
In this manuscript, we present a first detailed analysis of PGRP-LA function. Our structural study predicts that the PGRP domain of PGRP-LA is unlikely to bind peptidoglycan by itself. We next show that over-expression of PGRP-LAD isoform, but not of PGRP-LAC and PGRP-LAF, leads to the activation of Diptericin expression in absence of infection. Our experiments placed PGRP-LAD upstream of the Dredd caspase and of the Tak1 MAP3K. The intracellular domain of PGRP-LAD contains a RHIM motif similar to that observed in PGRP-LC and PGRP-LE for which it is essential for Imd pathway activation . This suggests that the RHIM motif confers to PGRP-LAD the capacity to induce the Imd pathway. Studies involving short mutations in PGRP-LC and PGRP-LE reported that their RHIM motifs are not involved in any physical interaction with Imd, the downstream adaptor of the Imd pathway, but bind with Pirk, a negative regulator of the Imd pathway , . Further analysis will be required to test whether the different PGRP-LA isoforms physically interacts with Pirk and/or with PGRP-LC. Collectively, this initial molecular characterization of PGRP-LA suggests a modulatory role of this PGRP in the Imd pathway.