Spatial and temporal coordination of Duox/TrpA1/Dh31 and IMD pathways is required for the efficient elimination of pathogenic bacteria in the intestine ofDrosophilalarvae

Abstract

ABSTRACTMultiple gut antimicrobial mechanisms are coordinated in space and time to efficiently fight foodborne pathogens. InDrosophila melanogaster, production of reactive oxygen species (ROS) and antimicrobial peptides (AMPs) together with intestinal cell renewal play a key role in eliminating gut microbes. An alternative protective mechanism would be to selectively prevent the penetration of the intestinal tract by pathogenic bacteria while allowing colonization by commensals. Using real-time imaging to follow the fate of ingested bacteria, we demonstrate that while commensalLactiplantibacillus plantarumfreely enter and cross the larval midgut, pathogenic strains such as.Erwinia carotovoraorBacillus thuringiensis, are actively locked down in the anterior midgut where they are rapidly eliminated by antimicrobial peptides. This sequestration of pathogenic bacteria in the anterior midgut requires the Duox enzyme in enterocytes, and both TrpA1 and Dh31 in enteroendocrine cells. Supplementing larval food with hCGRP, the human homolog of Dh31, is sufficient to trigger lockdown, suggesting the existence of a conserved mechanism. While the IMD pathway is essential for eliminating the trapped bacteria, it is dispensable for the lockdown. Genetic manipulations impairing bacterial lockdown results in abnormal colonization of posterior midgut regions by pathogenic bacteria. This ectopic colonization leads to bacterial proliferation and larval death, demonstrating the critical role of bacteria anterior sequestration in larval defense. Our study reveals a temporal orchestration during which pathogenic bacteria, but not innocuous, are compartimentalized in the anterior part of the midgut in which they are eliminated in an IMD pathway dependent manner.AUTHOR SUMMARYTypically, when considering the immune response of animals to infection, we focus on classical immunity, encompassing both innate and adaptive aspects such as antimicrobials and circulating immune cells. However, a broader perspective on immunity includes additional strategies that enhance host protection, such as behavioral avoidance and internal mechanisms that restrict pathogen propagation. In our study usingDrosophilalarvae as a model, we uncovered spatially and temporally interconnected events that are crucial for effectively combating intestinal infections. Our findings reveal a two-step defense mechanism: first, the larvae rapidly discriminate between bacterial strains, effectively confining hazardous ones in the anterior section of the intestine in a process we term ‘lockdown’. These locked down bacteria trigger the synthesis and release of antimicrobial peptides by the host, which ultimately eradicate the entrapped pathogens. Our experiments show that larvae capable of both initiating a lockdown and producing antimicrobial peptides withstand infections. In contrast, the absence of either one of these sequential defenses results in high mortality among the larvae, emphasizing the importance of each step and the necessity of their precise coordination in the immune response.