The Drosophila amidase PGRP-LB modulates the immune response to bacterial infection

The Drosophila host defense against gram-negative bacteria is mediated by the Imd pathway upon sensing of peptidoglycan by the peptidoglycan recognition protein (PGRP)-LC. Here we report a functional analysis of PGRP-LB, a catalytic member of the PGRP family. We show that PGRP-LB is a secreted protein regulated by the Imd pathway. Biochemical studies demonstrate that PGRP-LB is an amidase that specifically degrades gram-negative bacteria peptidoglycan. In agreement with its amidase activity, PGRP-LB downregulates the Imd pathway. Hence, activation of PGRP-LB by the Imd pathway provides a negative feedback regulation to tightly adjust immune activation to infection. Our study also reveals that PGRP-LB controls the immune reactivity of flies to the presence of ingested bacteria in the gut. Our work highlights the key role of PGRPs that encode both sensors and scavengers of peptidoglycan, which modulate the level of the host immune response to the presence of infectious microorganisms.

PIMS modulates immune tolerance by negatively regulating Drosophila innate immune signaling

Bruno Lemaitre

Metazoans tolerate commensal-gut microbiota by suppressing immune activation while maintaining the ability to launch rapid and balanced immune reactions to pathogenic bacteria. Little is known about the mechanisms underlying the establishment of this threshold. We report that a recently identified Drosophila immune regulator, which we call PGRP-LC-interacting inhibitor of Imd signaling (PIMS), is required to suppress the Imd innate immune signaling pathway in response to commensal bacteria. pims expression is Imd (immune deficiency) dependent, and its basal expression relies on the presence of commensal flora. In the absence of PIMS, resident bacteria trigger constitutive expression of antimicrobial peptide genes (AMPs). Moreover, pims mutants hyperactivate AMPs upon infection with Gram-negative bacteria. PIMS interacts with the peptidoglycan recognition protein (PGRP-LC), causing its depletion from the plasma membrane and shutdown of Imd signaling. Therefore, PIMS is required to establish immune tolerance to commensal bacteria and to maintain a balanced Imd response following exposure to bacterial infections.

2008

The contribution of the host stress response to the pathogenesis of Pseudomonas entomophila in the Drosophila intestine

Sveta Chakrabarti

Recently, several studies done in Drosophila have revealed that efficient and rapid recovery from bacterial infection in the gut is possible only when bacterial clearance by the immune system is coordinated with repair through renewal of the epithelium damaged by infection. In this thesis, I have analyzed how the entomopathogenic bacterium Pseudomonas entomophila affects the immune response and epithelium renewal. A microarray analysis first showed that P. entomophila is recognized by the Drosophila immune system since ingestion of this bacterium stimulated the expression of antimicrobial peptide genes by the Imd pathway. In addition, stress and damage related pathways were strongly induced by P. entomophila, which correlate with its capacity to inflict severe damage. While antibacterial genes were induced in the gut following infection with P. entomophila, the immune response was not productive due to a general inhibition of translation that affected all the newly synthesized transcripts. Furthermore, the blockage of translation also inhibited the repair program by blocking the production of JAK-STAT ligands (upd3, upd2) and growth factors, which stimulate the intestinal stem cells proliferation and differentiation. I next analyzed the pathways that link cellular damage to reduction of translation. Using a genetic approach, I showed that inhibition of translation was induced by two signaling pathways: i) the phosphorylation of elongation initiation factor 2α (eIF2α) by the stress kinase GCN2 and ii) the inhibition of the TOR pathway by the AMPK kinase. Both kinases sense metabolic deprivation, suggesting that cellular damages induced by P. entomophila induce a state of “starvation”. Inhibition of translation is usually an adaptive cellular response to adjust the metabolism to the energy status of the cells. The observation that GCN2-deficient flies survived better than wild-type flies to P. entomophila indicates that pathogenesis is linked to an over-activation of stress pathways that usually help to endure the consequence of an infection. In this in vivo model of infection, I also showed that the reduction of translation was a consequence of cellular damage to the intestine caused by host-derived reactive oxygen species (through the activity of Duox) and by the direct action of a pore-forming toxin produced by the pathogen. As a consequence of this translational arrest, flies succumbed P. entomophila infection because they were unable to repair gut damage. Finally, I showed that inhibition of translation also had a strong influence on innate immune responses observed upon P. entomophila infection. The specific activation of a systemic immune response (antimicrobial peptides produced by the fat body) observed upon P. entomophila infection could be recapitulated by feeding flies with a non-lethal pathogen and an inhibitor of translation. Hence, I showed translation inhibition could be an important feature that shapes the immune response. The p38 MAPK family is involved in stress and immunity in both mammals and Drosophila. In Drosophila, three p38-MAPK-encoding genes, p38a, p38b and p38c, have been identified but their function in the gut immune response is poorly characterized. In the second part of my thesis, I have analyzed the role of these three p38 MAPKs in Drosophila intestinal immunity, especially p38c, which is strongly enriched in the gut. I first confirmed that the three p38 are involved kinases are involved in the defense to oral infection with pathogenic (but non-lethal) bacteria such as Erwinia carotovora 15. Surprisingly, p38c mutant flies were more resistant to P. entomophila and did not show the reduction in translation observed in wild-type flies. Interestingly, the transcription of the ROS producing enzyme Duox was reduced in p38c raising the hypothesis that p38 contributes to P. entomophila pathogenicity by activating Duox. Furthermore, I have obtained preliminary data indicating that p38c and the transcription factor ATF-3 act in the same pathway contributing the host immune response and lipid metabolism. Together, my thesis reveals the complex cross-talks between stress and immune pathways during microbial infection. While, it shows that stress pathways usually contribute to endure the damage caused by infection, excessive activation of these pathways can contribute to pathogenesis.

EPFL2013

The regulatory isoform rPGRP-LC induces immune resolution via endosomal degradation of receptors

Bruno Lemaitre, Fanny Schüpfer

The innate immune system needs to distinguish between harmful and innocuous stimuli to adapt its activation to the level of threat. How Drosophila mounts differential immune responses to dead and live Gram-negative bacteria using the single peptidoglycan receptor PGRP-LC is unknown. Here we describe rPGRP-LC, an alternative splice variant of PGRP-LC that selectively dampens immune response activation in response to dead bacteria. rPGRP-LC-deficient flies cannot resolve immune activation after Gram-negative infection and die prematurely. The alternative exon in the encoding gene, here called rPGRP-LC, encodes an adaptor module that targets rPGRP-LC to membrane microdomains and interacts with the negative regulator Pirk and the ubiquitin ligase DIAP2. We find that rPGRP-LC-mediated resolution of an efficient immune response requires degradation of activating and regulatory receptors via endosomal ESCRT sorting. We propose that rPGRP-LC selectively responds to peptidoglycans from dead bacteria to tailor the immune response to the level of threat.

Nature Publishing Group2016

The contribution of the host stress response to the pathogenesis of Pseudomonas entomophila in the Drosophila intestine

Sveta Chakrabarti

EPFL2013