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

Reviewer #1 (Public Review):

Tleiss et al. demonstrate that while commensal Lactiplantibacillus plantarum freely circulate within the intestinal lumen, pathogenic strains such as Erwinia carotovora or Bacillus thuringiensis are blocked 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. This effect induces muscular muscle contraction, which is marked by the formation of TARM structures (thoracic ary-related muscles). This muscle contraction-related blocking happens early after infection (15mins). On the other side, the clearance of bacteria is done by the IMD pathway possibly through antimicrobial peptide production while it is dispensable for the blockage. Genetic manipulations impairing bacterial compartmentalization result in abnormal colonization of posterior midgut regions by pathogenic bacteria. Despite a functional IMD pathway, this ectopic colonization leads to bacterial proliferation and larval death, demonstrating the critical role of bacteria anterior sequestration in larval defense.

This important work substantially advances our understanding of the process of pathogen clearance by identifying a new mode of pathogen eradication from the insect gut. The evidence supporting the authors' claims is solid and would benefit from more rigorous experiments.

(1) The authors performed the experiments on Drosophila larvae. I wonder whether this model could extend to adult flies since they have shown that the ROS/TRPA1/Dh31 axis is important for gut muscle contraction in adult flies. If not, how would the authors explain the discrepancy between larvae and adults?

(2) The authors performed their experiments and proposed the models based on two pathogenic bacteria and one commensal bacterial at a relatively high bacterial dose. They showed that feeding Bt at 2X1010 or Ecc15 at 4X108 did not induce a blockage phenotype. I wonder whether larvae die under conditions of enteric infection with low concentrations of pathogenic bacteria. If larvae do not show mortality, what is the mechanism for resisting low concentrations of pathogenic bacteria? Why is this model only applied to high-dose infections?

(3) The authors claim that the lock of bacteria happens at 15 minutes while killing by AMPs happens 6-8 hours later. What happened during this period? More importantly, is IMD activity induced in the anterior region of the larval gut in both Ecc15 and Bt infection at 6 hours after infection? Are they mostly expressed in the anterior midgut in both bacterial infections? Several papers have shown quite different IMD activity patterns in the Drosophila gut. Zhai et al. have shown that in adult Drosophila, IMD activity was mostly absent in the R2 region as indicated by dpt-lacZ. Vodovar et al. have shown that the expression of dpt-lacZ is observable in proventriculus while Pe is not in the same region. Tzou et al. showed that Ecc15 infection induced IMD activity in the anterior midgut 24 hours after infection. Using TrpA1 and Dh31 mutant, the authors found both Ecc15 and Bt in the posterior midgut. Why are they not evenly distributed along the gut? Last but not least, does the ROS/TrpA1/Dh31 axis affect AMP expression?

(4) The TARM structure part is quite interesting. However, the authors did not show its relevance in their model. Is this structure the key-driven force for the blocking phenotype and killing phenotype? Is the ROS/TrpA1/Dh31 axis required to form this structure?

Reviewer #2 (Public Review):

This article describes a novel mechanism of host defense in the gut of Drosophila larvae. Pathogenic bacteria trigger the activation of a valve that blocks them in the anterior midgut where they are subjected to the action of antimicrobial peptides. In contrast, beneficial symbiotic bacteria do not activate the contraction of this sphincter, and can access the posterior midgut, a compartment more favorable to bacterial growth.

Strengths:

The authors decipher the underlying mechanism of sphincter contraction, revealing that ROS production by Duox activates the release of DH31 by enteroendocrine cells that stimulate visceral muscle contractions. The use of mutations affecting the Imd pathway or lacking antimicrobial peptides reveals their contribution to pathogen elimination in the anterior midgut.

Weaknesses:

- The mechanism allowing the discrimination between commensal and pathogenic bacteria remains unclear.

- The use of only two pathogens and one symbiotic species may not be sufficient to draw a conclusion on the difference in treatment between pathogenic and symbiotic species.

- We can also wonder how the process of sphincter contraction is affected by the procedure used in this study, where larvae are starved. Does the sphincter contraction occur in continuous feeding conditions? Since larvae are continuously feeding, is this process physiologically relevant?