Apoptosis restores cellular density by eliminating a physiologically or genetically induced excess of enterocytes in the Drosophila midgut.

Using pathogens or high levels of opportunistic bacteria to damage the gut, studies in Drosophila have identified many signaling pathways involved in gut regeneration. Dying cells emit signaling molecules that accelerate intestinal stem cell proliferation and progenitor differentiation to replace the dying cells quickly. This process has been named 'regenerative cell death'. Here, mimicking environmental conditions, we show that the ingestion of low levels of opportunistic bacteria was sufficient to launch an accelerated cellular renewal program despite the brief passage of bacteria in the gut and the absence of cell death and this is is due to the moderate induction of the JNK pathway that stimulates stem cell proliferation. Consequently, the addition of new differentiated cells to the gut epithelium, without preceding cell loss, leads to enterocyte overcrowding. Finally, we show that a couple of days later, the correct density of enterocytes is promptly restored by means of a wave of apoptosis involving Hippo signaling and preferential removal of old enterocytes.

The DH31/CGRP enteroendocrine peptide triggers intestinal contractions favoring the elimination of opportunistic bacteria.

The digestive tract is the first organ affected by the ingestion of foodborne bacteria. While commensal bacteria become resident, opportunistic or virulent bacteria are eliminated from the gut by the local innate immune system. Here we characterize a new mechanism of defense, independent of the immune system, in Drosophila melanogaster. We observed strong contractions of longitudinal visceral muscle fibers for the first 2 hours following bacterial ingestion. We showed that these visceral muscle contractions are induced by immune reactive oxygen species (ROS) that accumulate in the lumen and depend on the ROS-sensing TRPA1 receptor. We then demonstrate that both ROS and TRPA1 are required in a subset of anterior enteroendocrine cells for the release of the DH31 neuropeptide which activates its receptor in the neighboring visceral muscles. The resulting contractions of the visceral muscles favors quick expulsion of the bacteria, limiting their presence in the gut. Our results unveil a precocious mechanism of defense against ingested opportunistic bacteria, whether they are Gram-positive like Bacillus thuringiensis or Gram-negative like Erwinia carotovora carotovora. Finally, we found that the human homolog of DH31, CGRP, has a conserved function in Drosophila.

Hypochlorous Acid Staining with R19-S in the Drosophila Intestine upon Ingestion of Opportunistic Bacteria.

The intestine is endowed with an innate immune system that is required to fight any exogenous bacteria that are swallowed along with the food. The first line of defense that is mounted by the gut epithelium is the release of immune Reactive Oxygen Species (ROS), such as hypochlorous acid (HOCl), into the lumen. HOCl is produced within 1.5 h of bacterial ingestion and is very labile once released. Therefore, to monitor HOCl production upon ingestion of allochthonous bacteria, one needs a detection system that can quickly and efficiently detect HOCl production in the intestine. While most of the ROS-sensitive probes available in the market detect all kinds of ROS without any distinction, the R19-S fluorescent probe has been developed to specifically detect HOCl. Here, we describe a protocol to monitor HOCl production using this probe in the gut lumen of adult Drosophila upon ingestion of the opportunistic bacteria Bacillus thuringiensis.