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

Abstract

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.

Fig 1. Ingestion of opportunistic bacteria rapidly induces visceral muscle spasms.

(A) Pictures of a midgut dissected from a fly having ingested a blue food dye. The lower image shows the method to measure the length of the midgut between the proventriculus and the hindgut. (B) Measurement of midgut length upon intoxication by 10⁴, 10⁶ and 10⁸ CFU of Btk. The length is expressed in function of the control (Ctrl, 100%). Flies were fed for 30 min and the measurements were taken 30 min, 1 h, 2 h and 4 h after the initiation of feeding. (C) Midgut length upon intoxication by 10⁸ CFU of the commensal bacterium L. plantarum. (D) Immunolabelling of posterior midgut 2 h post ingestion of 5% sucrose (Ctrl) or Btk (10⁸ CFU/fly were provided). DAPI labels the nuclei (Blue), Arm marks the basolateral compartment (Red) and Dlg::GFP marks the apical compartment. (E) Caspase3 staining (Red, Casp3) of posterior midgut 2 h after feeding with 5% sucrose (Ctrl) or Btk (10⁸ CFU/fly were provided). Green marks the enterocytes (EC) and blue the nuclei (DAPI). Note the very few presence of apoptotic enterocytes (red arrow) in both conditions. (F) Visceral muscle fibers were labelled by Phalloidin 2 h after feeding with 5% sucrose (Ctrl) or Btk (10⁸ CFU/fly were provided). Note that only the longitudinal fibers are contracted in presence of Btk. Because the images are more apparent in the posterior midgut, this region was chosen to illustrate the visceral contractions in this figure and in all the following images. The vertical bars on the pictures indicate the proportion of intestines with (red) or without (yellow) contractions. (G) Measure of the width of the longitudinal visceral muscle fibers. Data are represented as a function of the control (Ctrl, 100%). Objective is 40X in D and 20X in E and F.

Fig 2. Visceral muscle spasms helps to evict opportunistic bacteria.

(A) Measurement of the length or (B) phalloidin staining (red, 20X objective) of midguts from flies 2 h post-intoxication with sucrose (Ctrl), 10⁸ CFU of Btk, sucrose + loperamide or 10⁸ CFU of Btk + loperamide. (C) Monitoring of Btk persistence in the midgut of flies fed with 10⁸ CFU of Btk (grey bars) or 10⁸ CFU of Btk + loperamide (blue bars). Note that after 30 min of feeding, the same amount of Btk is recovered in the midgut (about 2.10⁵ CFU compared to the 10⁸ CFU/fly deposited on the medium). (D) Labelling of HOCl in anterior midgut by the R19-S fluorescent probe (Orange). Flies were fed with 10⁸ CFU of Btk or 10⁸ CFU of Btk + loperamide. 20X objective. The proportion of R19-S-positive (orange bars) intestines are presented in the graph below the pictures.

Fig 3. HOCl signaling is required to promote visceral spasms.

(A) CFU counting in midguts of WT flies fed with 10⁸ CFU of Btk or 10⁸ CFU of Btk + DTT. (B) Measure of midgut length from flies 2 h post-intoxication with DTT (left) or 10⁸ CFU of Btk + DTT (right). (C) Phalloidin staining of posterior midgut 2 h after feeding with 5% sucrose (Ctrl) or Btk + DTT. 20X objective. (D) CFU counting in midguts of WT (grey bars) or myo1A^ts>DUOX^RNAi (where DUOX expression was silenced in enterocytes, blue bars) flies fed with 10⁸ CFU of Btk. (E) Measure of midgut length of myo1A^ts>DUOX^RNAi flies 2 h post-intoxication with sucrose (left) or 10⁸ CFU of Btk (right). Note that the silencing of DUOX in enterocytes does not affect the length of midguts compared to those from WT flies fed with 5% sucrose (Ctrl). (F) Phalloidin staining of posterior midgut of myo1A^ts>DUOX^RNAi flies after feeding with 5% sucrose (left panel) or 10⁸ CFU of Btk (right panel). 20X objective.

Fig 4. TRPA1 is involved in visceral spasms.

(A) CFU counting in the midgut of WT (grey bars) or TrpA1¹ homozygote (blue bars) flies fed with 10⁸ CFU of Btk. (B) Measure of midgut length of TrpA1¹ homozygote flies 2 h post-intoxication with sucrose (Ctrl) or 10⁸ CFU of Btk. (C) Phalloidin staining of posterior midgut of TrpA1¹ homozygote flies 2 h after feeding with 5% sucrose (Ctrl) or 10⁸ CFU of Btk. 20X objective. (D) Upper picture: reconstructed image of a WT midgut (10X objective). The midgut was stained for DH31 (Green) and Prospero (Red, Pros) that marks all the EECs. DAPI (blue) marks the nuclei. Anterior is to the left and posterior to the right. Lower insets: magnifications (40X objective) of the R2a (left), R2b (middle) and R4c (right) regions (http://flygut.epfl.ch/). (E-G and I-K) R2b anterior midgut region of WT flies (E and I-K) and TrpA1¹ homozygote flies (F and G) fed either with sucrose (E and F) or with 10⁸ CFU of Btk (G and I-K). (G and I-K) midguts were dissected and fixed 15 min, 30 min and 1 h PI as indicated on the pictures. Bright green arrows point EECs with a strong DH31 labelling and dark green arrows point EECs with a lower labelling. (H) Counting of high intensity DH31-positive EECs in the anterior R2b domain in conditions described in (E-G). (L) Counting of all DH31-positive EECs in the anterior R2b domain in conditions described in (I-K). (M and N) Fast AiryScan imaging of one representative EEC in the anterior R2b midgut domain of WT flies 1h PI of sucrose (M) or 10⁸ CFU of Btk (N).

Fig 5. DH31 peptide is required to promote visceral contractions.

(A) DH31^ts>GCaMP6s midguts were scored for GFP-positive EECs in the R2b region in control flies (light blue) or in flies fed with 10⁸ CFU of Btk (dark blue). Error bars correspond to the SEM. (B and E) CFU counting in the midguts of WT (grey bars) or DH31^KG09001 homozygote flies (B, blue bars) or mef2^ts>DH31-R^RNAi flies (E, blue bars) fed with 10⁸ CFU of Btk. (C and F) Measure of midgut length of DH31^KG09001 homozygote flies (C) or mef2^ts>DH31-R^RNAi flies (F) 2 h post-intoxication with sucrose (Ctrl) or 10⁸ CFU of Btk. (D and G) Phalloidin staining of posterior midgut of DH31^KG09001 homozygote flies (D) and mef2^ts>DH31-R^RNAi flies (G) 2 h after feeding with 5% sucrose (left) or 10⁸ CFU of Btk (right). 20X objective.

Fig 6. DH31 peptide is sufficient to promote visceral contractions.

(A) Phalloidin labelling of posterior midgut of DH31^ts>reggie flies maintained at 18°C (control, left) or raised for 24 h at 29°C (right). 20X objective. (B) Measurement of midgut length in conditions described in (A). (C) DH31^ts>DH31^#9 posterior midguts maintained at 18°C for control (left) or shifted for 48 h at 29°C (right) and stained with phalloidin. 20X objective. Overexpression of DH31 in all EECs induced strong visceral contractions. (D) Measurement of midgut length in the conditions described in (C). (E) mef2^ts>t-DH31^P2-B9 posterior midguts maintained at 18°C for control (left) or shifted for 24H at 29°C (right) and stained with phalloidin. 20X objective. Ectopic expression of the membrane-tethered form of DH31 in the visceral muscle induced autonomous visceral contractions. (F) Measurement of midgut length in the conditions described in (E).

Fig 7. The human DH31 homolog CGRP promotes visceral contractions.

(A) Midgut length of WT flies fed continuously either with sucrose (Ctrl) or with CGRP. (B) Phalloidin labelling of posterior midgut of WT flies fed continuously either with sucrose (Ctrl) or with CGRP. In (A and B) flies were dissected 1h30 after the onset of feeding. (C) Midgut length of mef2^ts>DH31-R^RNAi flies raised at 18°C (the two left bars, Gal80^ts on/RNAi off) or shifted for 7 days at 29°C (the two right bars, Gal80^ts off/RNAi on) and fed with sucrose (first and third bars) only or with CGRP for 1h30 (second and fourth bars). The length is expressed in % to the control (Ctrl) (D) Phalloidin labelling of mef2^ts>DH31-R^RNAi midguts from flies fed with sucrose (left) or CGRP (middle and right) and maintained at 18°C (left and middle) or shifted for 7 days at 29°C (right). (E) CFU counting in the midgut of WT flies fed with 10⁸ CFU of Btk (grey bars) or 10⁸ CFU of Btk + DTT (blue bars) or 10⁸ CFU of Btk + DTT + CGRP (turquoise bars). Note that the ingestion of CGRP can rescue the absence bacterial eviction caused by the inhibition of ROS production. (F) Model summarizing the sequence of events following the ingestion of opportunistic bacteria. Left: In unchallenged flies, only a little DH31 is secreted by EECs, probably involved in peristalsis. Middle: Upon ingestion of opportunistic bacteria, enterocytes (EC) release immune ROS (HOCl) in a DUOX-dependent manner to damage the bacteria. EECs sense the presence of luminal HOCl thanks to the TRPA1 receptor. Right: large amounts of DH31 are secreted by EECs. DH31 reaches the visceral muscle where it binds its receptor DH-31R, promoting spasms of the visceral muscles (VM) to expel bacteria.