Final Report Summary - GUTDROSO (Gut immunity and homeostasis in Drosophila)
The gut is the major interface between microbes and their animal hosts and constitutes the main entry route for pathogens. Our project involved integrated approaches to dissect the gut immune response and homeostasis using the insect Drosophila as a model host.
Our studies revealed that the expression of antimicrobial peptide genes along the gut is patterned and that the immune response in the gut is highly complex; a high number of genes being induced upon infection. Our studies have shown that efficient and rapid recovery from bacterial infection is possible only when clearance of bacteria from the gut is coordinated with epithelium renewal to repair damage caused by infection. Epithelium renewal of the Drosophila gut is stimulated by the release of secreted factor Upd3 from damaged enterocytes, which then activates the JAK/STAT pathway in intestinal stem cells to promote both their division and differentiation, establishing a homeostatic regulatory loop. Thus repair of the gut through epithelium renewal can be considered as an essential facet of the Drosophila host defense in the gut.
We have also shown that the Drosophila indigenous microbiota affect at basal levels the immune and gut stem cell activities. The chronic stimulation of stem cell by the indigenous microbiota is stronger in old flies (due to higher bacterial count) and lead to defects in gut morphogenesis. We have analyzed mechanisms preventing the activation of an immune response to the gut microbiota. We focus our attention on attention on Peptidoglycan recognition proteins (PGRPs) that are key regulators of insect immune responses. In addition to recognition PGRPs, which activate the Toll and Imd pathways, the Drosophila genome encodes six catalytic PGRPs with the capacity to scavenge peptidoglycan. We have performed a systematic analysis of catalytic PGRP function using deletions, separately and in combination. Our findings show that flies lacking all six catalytic PGRPs were still viable but exhibited deleterious immune responses to the gut indigenous microbiota. Together with recent studies on mammalian PGRPs, our study uncovers a conserved role for PGRPs in gut homeostasis. In parallel, we have shown that pattern-recognition of bacteria in the Drosophila intestine is compartmentalized as fly used a combination of transmembrane and intracellular sensor to detect bacteria.
Using our microarray analysis as a starting point, we also investigated the function of several genes encoding proteins with a chitin-binding domain. One of them, Drosocrystallin, appears to be a component of the peritrophic matrix, a chitinous layer that lines the gut of insect and play a role similar to the mucus of mammals. The use of this mutation allows us to demonstrate for the first time a role of the peritrophic matrix against entomopathogenic bacteria, counteracting the action of a pore-forming toxin.
To uncover the strategy used by entomopathogens to disrupt gut homeostasis, we have performed a screen for virulence factors of Pseudomonas entomophila. This led to identification of a new pore forming toxin, named Monalysin, that participate to damage inflicted to the by P. entomophila. We also demonstrated that P. entomophila infection provokes a reduction of the level of protein translation impairing both immune and repair programs. Thus P. entomophila pathogenesis is linked to excessive activation of stress-responsive pathways as a consequence of damages; which then shut of translation as a metabolic adaptation.
Finally, Here, we provided a comprehensive atlas of Drosophila adult midgut. Specifically, we uncovered a fine-grained regional organization consisting of 14 subregions with distinct morphological, histological, and genetic properties. We also showed that this midgut compartmentalization is achieved through the interplay between pan-midgut and regionalized transcription factors, in concert with spatial activities of morphogens. Interestingly, disruption of the midgut compartmentalization leads to a loss of intestinal homeostasis characterized by an increase in stem cell proliferation and aberrant immune responses.
The work developed in this project has led highly innovative results as it combines both systematic genetic and genomic studies of the gut immune response in an in vivo model. The gut has become the focus of many studies by other groups. In particular, our results (confirmed by other labs) showing that a stem cell program can be triggered upon bacterial infection, had a strong impact in the field far beyond immunity.
Our studies revealed that the expression of antimicrobial peptide genes along the gut is patterned and that the immune response in the gut is highly complex; a high number of genes being induced upon infection. Our studies have shown that efficient and rapid recovery from bacterial infection is possible only when clearance of bacteria from the gut is coordinated with epithelium renewal to repair damage caused by infection. Epithelium renewal of the Drosophila gut is stimulated by the release of secreted factor Upd3 from damaged enterocytes, which then activates the JAK/STAT pathway in intestinal stem cells to promote both their division and differentiation, establishing a homeostatic regulatory loop. Thus repair of the gut through epithelium renewal can be considered as an essential facet of the Drosophila host defense in the gut.
We have also shown that the Drosophila indigenous microbiota affect at basal levels the immune and gut stem cell activities. The chronic stimulation of stem cell by the indigenous microbiota is stronger in old flies (due to higher bacterial count) and lead to defects in gut morphogenesis. We have analyzed mechanisms preventing the activation of an immune response to the gut microbiota. We focus our attention on attention on Peptidoglycan recognition proteins (PGRPs) that are key regulators of insect immune responses. In addition to recognition PGRPs, which activate the Toll and Imd pathways, the Drosophila genome encodes six catalytic PGRPs with the capacity to scavenge peptidoglycan. We have performed a systematic analysis of catalytic PGRP function using deletions, separately and in combination. Our findings show that flies lacking all six catalytic PGRPs were still viable but exhibited deleterious immune responses to the gut indigenous microbiota. Together with recent studies on mammalian PGRPs, our study uncovers a conserved role for PGRPs in gut homeostasis. In parallel, we have shown that pattern-recognition of bacteria in the Drosophila intestine is compartmentalized as fly used a combination of transmembrane and intracellular sensor to detect bacteria.
Using our microarray analysis as a starting point, we also investigated the function of several genes encoding proteins with a chitin-binding domain. One of them, Drosocrystallin, appears to be a component of the peritrophic matrix, a chitinous layer that lines the gut of insect and play a role similar to the mucus of mammals. The use of this mutation allows us to demonstrate for the first time a role of the peritrophic matrix against entomopathogenic bacteria, counteracting the action of a pore-forming toxin.
To uncover the strategy used by entomopathogens to disrupt gut homeostasis, we have performed a screen for virulence factors of Pseudomonas entomophila. This led to identification of a new pore forming toxin, named Monalysin, that participate to damage inflicted to the by P. entomophila. We also demonstrated that P. entomophila infection provokes a reduction of the level of protein translation impairing both immune and repair programs. Thus P. entomophila pathogenesis is linked to excessive activation of stress-responsive pathways as a consequence of damages; which then shut of translation as a metabolic adaptation.
Finally, Here, we provided a comprehensive atlas of Drosophila adult midgut. Specifically, we uncovered a fine-grained regional organization consisting of 14 subregions with distinct morphological, histological, and genetic properties. We also showed that this midgut compartmentalization is achieved through the interplay between pan-midgut and regionalized transcription factors, in concert with spatial activities of morphogens. Interestingly, disruption of the midgut compartmentalization leads to a loss of intestinal homeostasis characterized by an increase in stem cell proliferation and aberrant immune responses.
The work developed in this project has led highly innovative results as it combines both systematic genetic and genomic studies of the gut immune response in an in vivo model. The gut has become the focus of many studies by other groups. In particular, our results (confirmed by other labs) showing that a stem cell program can be triggered upon bacterial infection, had a strong impact in the field far beyond immunity.