Selective autophagy tolerates symbiotic bacteria in the Drosophila intestine.

Intestinal epithelium functions as a barrier to protect the host from environmental microbes. Defects in macroautophagy/autophagy combined with intestinal microbes cause a disruption of homeostasis of the tissue, which is associated with the etiology of Crohn disease, an inflammatory bowel disease. However, the molecular mechanism of how autophagy interacts with microbes in the pathology are mostly unrevealed. Our recent findings using Drosophila as a model system showed that autophagy in enterocytes suppresses a regenerative response triggered by reactive oxygen species (ROS) secreted by the host epithelia toward commensal bacteria in the intestine. Without this suppression, accumulation of a receptor protein of selective autophagy, ref(2)P, continuously acts as a signaling platform to cause excessive regeneration via cytokine production by yki (yorkie) activation. This chronic response leads to the acceleration of age-dependent barrier dysfunction, systemic inflammation, and shorter lifespan. These results uncover a novel regulatory network linking commensal bacteria, autophagy, and gut homeostasis, represented by ROS, ref(2)P, and the hippo pathway.

Homeostatic Regulation of ROS-Triggered Hippo-Yki Pathway via Autophagic Clearance of Ref(2)P/p62 in the Drosophila Intestine.

Homeostasis of intestinal epithelia is maintained by coordination of the proper rate of regeneration by stem cell division with the rate of cell loss. Regeneration of host epithelia is normally quiescent upon colonization of commensal bacteria; however, the epithelia often develop dysplasia in a context-dependent manner, the cause and underlying mechanism of which remain unclear. Here, we show that in Drosophila intestine, autophagy lowers the sensitivity of differentiated enterocytes to reactive oxygen species (ROS) that are produced in response to commensal bacteria. We find that autophagy deficiency provokes ROS-dependent excessive regeneration and subsequent epithelial dysplasia and barrier dysfunction. Mechanistically, autophagic substrate Ref(2)P/p62, which co-localizes and physically interacts with Dachs, a Hippo signaling regulator, accumulates upon autophagy deficiency and thus inactivates Hippo signaling, resulting in stem cell over-proliferation non-cell autonomously. Our findings uncover a mechanism whereby suppression of undesirable regeneration by autophagy maintains long-term homeostasis of intestinal epithelia.

A Receptor Guanylate Cyclase, Gyc76C, Mediates Humoral, and Cellular Responses in Distinct Ways in Drosophila Immunity.

Innate immunity is an evolutionarily conserved host defense system against infections. The fruit fly Drosophila relies solely on innate immunity for infection defense, and the conservation of innate immunity makes Drosophila an ideal model for understanding the principles of innate immunity, which comprises both humoral and cellular responses. The mechanisms underlying the coordination of humoral and cellular responses, however, has remained unclear. Previously, we identified Gyc76C, a receptor-type guanylate cyclase that produces cyclic guanosine monophosphate (cGMP), as an immune receptor in Drosophila. Gyc76C mediates the induction of antimicrobial peptides for humoral responses by a novel cGMP pathway including a membrane-localized cGMP-dependent protein kinase, DG2, through downstream components of the Toll receptor such as dMyD88. Here we show that Gyc76C is also required for the proliferation of blood cells (hemocytes) for cellular responses to bacterial infections. In contrast to Gyc76C-dependent antimicrobial peptide induction, Gyc76C-dependent hemocyte proliferation is meditated by a small GTPase, Ras85D, and not by DG2 or dMyD88, indicating that Gyc76C mediates the cellular and humoral immune responses in distinct ways.

Immunoglobulin superfamily beat-Ib mediates intestinal regeneration induced by reactive oxygen species in Drosophila.

Reactive oxygen species (ROS) often injure intestinal epithelia that cause loss of damaged cells, which is mainly repaired by proliferation of intestinal stem cells (ISCs). To maintain the homeostatic state, coordination of sensing of the ROS injury and the subsequent epithelial cell loss with the replenishment by cell renewal is crucial. However, little is known about how gut epithelial cells initiate regenerative responses against ROS to maintain the tissue integrity. Here, we carried out a genome-wide screen, by which we identify immunoglobulin superfamily beaten path Ib (beat-Ib) as an essential gene for provoking ISC proliferation against ROS in Drosophila intestine. Interestingly, the beat-Ib function is required in differentiated enterocytes, the main targeted cells by ROS in the intestinal tract, but is dispensable in the stem cells. Moreover, beat-Ib is not involved in enterocyte apoptosis at ROS injury. These findings indicate the essential role of beat-Ib in Drosophila midgut enterocytes for initiating the non-cell-autonomous induction of ISC division in response to environmental ROS stresses.

Organ identity specification factor WGE localizes to the histone locus body and regulates histone expression to ensure genomic stability in Drosophila.

Over-expression of Winged-Eye (WGE) in the Drosophila eye imaginal disc induces an eye-to-wing transformation. Endogenous WGE is required for organ development, and wge-deficient mutants exhibit growth arrest at the larval stage, suggesting that WGE is critical for normal growth. The function of WGE, however, remains unclear. Here, we analyzed the subcellular localization of WGE to gain insight into its endogenous function. Immunostaining showed that WGE localized to specific nuclear foci called the histone locus body (HLB), an evolutionarily conserved nuclear body required for S phase-specific histone mRNA production. Histone mRNA levels and protein levels in cytosolic fractions were aberrantly up-regulated in wge mutant larva, suggesting a role for WGE in regulating histone gene expression. Genetic analyses showed that wge suppresses position-effect variegation, and that WGE and a HLB component Mute appears to be synergistically involved in heterochromatin formation. Further supporting a role in chromatin regulation, wge-deficient mutants showed derepression of retrotransposons and increased γH2Av signals, a DNA damage marker. These findings suggest that WGE is a component of HLB in Drosophila with a role in heterochromatin formation and transposon silencing. We propose that WGE at HLB contributes to genomic stability and development by regulating heterochromatin structure via histone gene regulation.

Intracellular recognition of pathogens and autophagy as an innate immune host defence.

Pathogen recognition is the first and crucial step in innate immunity. Molecular families involved in the recognition of pathogens and activation of the innate immune responses in immunoreactive cells include the Toll-like receptor family in mammals and the peptidoglycan recognition protein (PGRP) family in Drosophila, which sense microorganisms in an extracellular or luminal compartment. Other emerging families are the intracellular recognition molecules for bacteria, such as nucleotide binding and oligomerization domain-like receptors in mammals and PGRP--LE in Drosophila, several of which have been shown to detect structures of bacterial peptidoglycan in the host cell cytosol. Exciting advances in recent studies on autophagy indicate that macroautophagy (referred to here as autophagy) is selectively induced by intracellular recognition molecules and has a crucial role in the elimination of intracellular pathogens, including bacteria, viruses and parasites. This review discusses recent studies related to intracellular recognition molecules and innate immune responses to intracellular pathogens, and highlights the role of autophagy in innate immunity.

Cooperative regulation of the induction of the novel antibacterial Listericin by peptidoglycan recognition protein LE and the JAK-STAT pathway.

Intracellular bacteria cause serious infectious diseases such as tuberculosis, shigellosis, and listeriosis. The Drosophila peptidoglycan recognition protein (PGRP)-LE functions as an important host pattern recognition receptor against intracellular bacteria such as Listeria monocytogenes. One PGRP-LE-mediated intracellular response against L. monocytogenes infection is the induction of autophagy, a conserved intracellular degradation system. Here, to further elucidate PGRP-LE-mediated intracellular innate immune responses, we performed a strategic microarray analysis and identified the Listericin gene, whose expression is induced in response to L. monocytogenes infection in a PGRP-LE-dependent manner. RNA interference and overexpression experiments demonstrated that Listericin gene induction is cooperatively regulated by PGRP-LE and the JAK-STAT (Janus kinase-signal transducers and activators of transcription) pathway. An in vitro cell culture assay showed that Listericin is secreted as processed forms and suppresses the growth of L. monocytogenes and Gram-negative bacteria. A colony formation unit assay clearly demonstrated that induction of the Listericin gene suppresses not only the growth of L. monocytogenes but also the growth of Gram-negative bacteria in vivo. Based on these findings, we propose that the Listericin gene encodes a novel antibacterial peptide-like protein whose induction is cooperatively regulated by PGRP-LE and the JAK-STAT pathway.

Induction of autophagy via innate bacterial recognition.

Macroautophagy (referred to hereafter as autophagy) functions not only in self-digestion, but also in the killing and degradation of infectious pathogens in in vitro-cultured cells. Based on genetic manipulations of both the host, Drosophila and pathogen, Listeria monocytogenes, we recently reported that L. monocytogenes-induced autophagy is dependent on the recognition of the pathogen by the Drosophila pattern recognition protein, PGRP-LE. Autophagy and PGRP-LE are crucial for inhibition of the intracellular growth of bacteria in hemocytes, the target cells of L. monocytogenes infection in vivo. The importance of autophagy in the resistance of Drosophila against L. monocytogenes is further indicated in in vivo survival experiments. The signaling pathway(s) that induces autophagy by PGRP-LE is independent of the known immune signaling pathways, suggesting that another unidentified pathway(s) is involved. The results of the present study demonstrate that the induction of autophagy, as an innate immune response targeting intracellular pathogens, is activated by intracellular sensors through unidentified pathways.

Autophagic control of listeria through intracellular innate immune recognition in drosophila.

Autophagy, an evolutionally conserved homeostatic process for catabolizing cytoplasmic components, has been linked to the elimination of intracellular pathogens during mammalian innate immune responses. However, the mechanisms underlying cytoplasmic infection-induced autophagy and the function of autophagy in host survival after infection with intracellular pathogens remain unknown. Here we report that in drosophila, recognition of diaminopimelic acid-type peptidoglycan by the pattern-recognition receptor PGRP-LE was crucial for the induction of autophagy and that autophagy prevented the intracellular growth of Listeria monocytogenes and promoted host survival after this infection. Autophagy induction occurred independently of the Toll and IMD innate signaling pathways. Our findings define a pathway leading from the intracellular pattern-recognition receptors to the induction of autophagy to host defense.

PGRP-LC and PGRP-LE have essential yet distinct functions in the drosophila immune response to monomeric DAP-type peptidoglycan.

Drosophila rely entirely on an innate immune response to combat microbial infection. Diaminopimelic acid-containing peptidoglycan, produced by Gram-negative bacteria, is recognized by two receptors, PGRP-LC and PGRP-LE, and activates a homolog of transcription factor NF-kappaB through the Imd signaling pathway. Here we show that full-length PGRP-LE acted as an intracellular receptor for monomeric peptidoglycan, whereas a version of PGRP-LE containing only the PGRP domain functioned extracellularly, like the mammalian CD14 molecule, to enhance PGRP-LC-mediated peptidoglycan recognition on the cell surface. Interaction with the imd signaling protein was not required for PGRP-LC signaling. Instead, PGRP-LC and PGRP-LE signaled through a receptor-interacting protein homotypic interaction motif-like motif. These data demonstrate that like mammals, drosophila use both extracellular and intracellular receptors, which have conserved signaling mechanisms, for innate immune recognition.

Structural basis for preferential recognition of diaminopimelic acid-type peptidoglycan by a subset of peptidoglycan recognition proteins.

Drosophila peptidoglycan recognition protein (PGRP)-LCx and -LCa are receptors that preferentially recognize meso-diaminopimelic acid (DAP)-type peptidoglycan (PGN) present in Gram-negative bacteria over lysine-type PGN of gram-positive bacteria and initiate the IMD signaling pathway, whereas PGRP-LE plays a synergistic role in this process of innate immune defense. How these receptors can distinguish the two types of PGN remains unclear. Here the structure of the PGRP domain of Drosophila PGRP-LE in complex with tracheal cytotoxin (TCT), the monomeric DAP-type PGN, reveals a buried ionic interaction between the unique carboxyl group of DAP and a previously unrecognized arginine residue. This arginine is conserved in the known DAP-type PGN-interacting PGRPs and contributes significantly to the affinity of the protein for the ligand. Unexpectedly, TCT induces infinite head-to-tail dimerization of PGRP-LE, in which the disaccharide moiety, but not the peptide stem, of TCT is positioned at the dimer interface. A sequence comparison suggests that TCT induces heterodimerization of the ectodomains of PGRP-LCx and -LCa in a closely analogous manner to prime the IMD signaling pathway, except that the heterodimer formation is nonperpetuating.

Peptidoglycan recognition protein (PGRP)-LE and PGRP-LC act synergistically in Drosophila immunity.

In innate immunity, pattern recognition molecules recognize cell wall components of microorganisms and activate subsequent immune responses, such as the induction of antimicrobial peptides and melanization in Drosophila. The diaminopimelic acid (DAP)-type peptidoglycan potently activates imd-dependent induction of antibacterial peptides. Peptidoglycan recognition protein (PGRP) family members act as pattern recognition molecules. PGRP-LC loss-of-function mutations affect the imd-dependent induction of antibacterial peptides and resistance to Gram-negative bacteria, whereas PGRP-LE binds to the DAP-type peptidoglycan, and a gain-of-function mutation induces constitutive activation of both the imd pathway and melanization. Here, we generated PGRP-LE null mutants and report that PGRP-LE functions synergistically with PGRP-LC in producing resistance to Escherichia coli and Bacillus megaterium infections, which have the DAP-type peptidoglycan. Consistent with this, PGRP-LE acts both upstream and in parallel with PGRP-LC in the imd pathway, and is required for infection-dependent activation of melanization in Drosophila. A role for PGRP-LE in the epithelial induction of antimicrobial peptides is also suggested.

Hrp48, a Drosophila hnRNPA/B homolog, binds and regulates translation of oskar mRNA.

Establishment of the Drosophila embryonic axes provides a striking example of RNA localization as an efficient mechanism for protein targeting within a cell. oskar mRNA encodes the posterior determinant and is essential for germline and abdominal development in the embryo. Tight restriction of Oskar activity to the posterior is achieved by mRNA localization-dependent translational control, whereby unlocalized mRNA is translationally repressed and repression is overcome upon mRNA localization. Here we identify the previously reported oskar RNA binding protein p50 as Hrp48, an abundant Drosophila hnRNP. Analysis of three hrp48 mutant alleles reveals that Hrp48 levels are crucial for polarization of the oocyte during mid-oogenesis. Our data also show that Hrp48, which binds to the 5' and 3' regions of oskar mRNA, plays an important role in restricting Oskar activity to the posterior of the oocyte, by repressing oskar mRNA translation during transport.

Overexpression of a pattern-recognition receptor, peptidoglycan-recognition protein-LE, activates imd/relish-mediated antibacterial defense and the prophenoloxidase cascade in Drosophila larvae.

In Drosophila, microbial infection activates an antimicrobial defense system involving the activation of proteolytic cascades in the hemolymph and intracellular signaling pathways, the immune deficiency (imd) and Toll pathways, in immune-responsive tissues. The mechanisms for microbial recognition are largely unknown. We report that, in larvae, the imd-mediated antibacterial defense is activated by peptidoglycan-recognition protein (PGRP)-LE, a PGRP-family member in Drosophila. Consistent with this, PGRP-LE binds to the diaminopimelic acid-type peptidoglycan, a cell-wall component of the bacteria capable of activating the imd pathway, but not to the lysine-type peptidoglycan. Moreover, PGRP-LE activates the prophenoloxidase cascade, a proteolytic cascade in the hemolymph. Therefore, PGRP-LE acts as a pattern-recognition receptor to the diaminopimelic acid-type peptidoglycan and activates both the proteolytic cascade and intracellular signaling in Drosophila immunity.