Fig 1.
C. elegans ilys-3 is expressed in the pharynx, intestine and coelomocytes.
(A-C) GFP expression directed by the 1 Kb ilys-3 promoter (upstream the start codon). (A) Example of expression of ilys-3p in L1. (B) The transcriptional reporter expresses strongly in the pharyngeal muscle pm7 (grinder), in the marginal cell mc2 and in the intestine of an adult worm. (C) Details of the variation in ilys-3 transcription in intestinal int8 and int9 cells in an adult worm. (D-I) Expression of the ily-3p::ILYS-3::mCherry translational fusion reporter. (D) Distribution of ILYS-3-associated vesicles in the cytoplasm of intestinal cells in an adult hermaphrodite. The intestinal lumen is also visible. (E) Detail of a late adult (8 day after L4) showing mCherry in the intestinal lumen, pharynx and coelomocytes with copious mCherry positive puncta in pharynx and diffuse distribution in intestinal lumen. (F & I) Detail of a late adult worm fed with 0.5 μm yellow-green microspheres 1 hour before imaging showing accumulation of fluorescent beads (arrowhead) in the gut lumen together with faint ILYS-3::mCherry. (F) Fluorescence image and (I) overlay of a DIC image and the epifluorescence image shown in F. (G) Enlarged image showing coelomocyte expression of ILYS-3::mCherry. (H) Image of a starvation-induced-dauer showing high luminal gut expression of ILYS-3::mCherry. (J-K) Representative images of the 4.5 Kb ilys-3 long promoter GFP construct in adult hermaphrodites. (J) Promoter activity in pharynx and intestine and (K) in the coelomocytes. ph:pharynx; int: intestine; cc: coelomocytes. Scale bars as indicated.
Fig 2.
Intestinal ILYS-3::mCherry associates with endocytic and recycling endosomes and LROs.
(A-C) Confocal images of the middle focal plane of the intestine showing that ILYS-3::mCherry partially colocalizes with a subpopulation of GFP::RAB-7 in the median cytoplasm in late endosomes, labeled by both ILYS-3::mCherry (A) and GFP:RAB-7 (B). (C) Overlay of the corresponding red and green channel, magnified 3x region in inset. (D-F) ILYS-3::mCherry occasionally appears in RAB-11 positive vesicles residing in the middle focal plane of the intestine. No double labeling is seen on the apical side of the intestine. (D) ILYS-3::mCherry. (E) GFP::RAB-11. (F) Overlay of the corresponding red and green channel, magnified 3x region in inset. (G-I) ILYS-3::mCherry colocalizes with LysoTracker Green in the intestinal lysosome-related organelles (LROs). (G) ILYS-3::mCherry. (H) LysoTracker Green is internalized apically by the intestinal cells and accumulates in the acidic LROs (green granules). Inset in (I) shows the doubly marked LROs. No red signal is seen in the apical side of the intestine. (J-K) Images of the top focal plane showing the basolateral compartment of the intestine. (J) Micrograph acquired underneath the basolateral membrane showing a population of ILYS-3::mCherry positive vesicles in the cytosol in a wild-type control. (K) ILYS-3::mCherry accumulates in the recycling endosome tubular network in the rme-1 mutant. (L) ILYS-3::mCherry accumulates apically in the plasma membrane and in puncta in rab-10 mutants. Micrograph acquired in the middle focal plane of the intestine. Asterisks depicts the lumen of the intestine. Arrows point to the apical membrane. Arrowheads point to intestinal vesicles. Dashed arrow marks basolateral tubular network mCherry-labeled. (M) Pearson's correlation coefficient for colocalization of ILYS-3::mCherry signal with GFP::RAB-7, GFP::RAB-11 and LysoTracker Green. Confocal images were from deconvolved 3D stacks acquired in living adults expressing mCherry- and GFP-tagged proteins in the intestinal epithelial cells. Autofluorescence was corrected using Leica LAS X core software. Values for each group represent n = 17/21 areas from 8 animals. Error bars represent SEM.
Fig 3.
During dauer arrest ILYS-3 is secreted in the lumen but returns to its steady state cytosolic expression upon dauer recovery.
(A-I) Time course of ILYS-3 intestinal distribution in dauers. (A-C) Fluorescence images of ILYS-3::mCherry (A), GFP::RAB-11 labeling apical recycling endosomes in plasma membrane (B) and an overlay (C) of the two images acquired with red and green channels. Images are representative of 1-week old dauers yielded from nutrient depleted NGM plates. Arrow and arrowhead depict the pharyngeal and the intestinal lumens, respectively. (D) Micrograph of a dauer animal recovering after 1 hour on OP50. Red depicts ILYS-3::mCherry and green depicts GFP::RAB-7 that marks early endosomes near PM and late endosomes in cytoplasm. Animal shows luminal (arrowhead) and cytosolic (arrow) ILYS-3 at the anterior and posterior ends, respectively. (E-F) Micrographs of a dauer animal recovering after 3 hours on OP50. Red depicts ILYS-3::mCherry in lumen and green depicts fluorescent beads added to the bacterial lawns. (F) overlay of the two channels. (G-I) Micrographs of post-dauer animal recovering after overnight on OP50. (G) Red signal is only detected in vesicles in the cytosol. (H) GFP::RAB-11 in puncta scattered in the cytoplasm. (I) overlay. (J) The mean fluorescence intensity profile corresponding to the animal shown in images A-C. mCherry and GFP containing regions have little overlap and the red signal is extracellular. The green dashed line in (C) indicates the cross section used to quantify fluorescence.
Fig 4.
The C. elegans ilys-3 is transcriptionally activated in the intestine upon Gram-positive exposure and is a readout for monitoring danger/hunger signals.
(A) Fluorescence images of representative animals carrying the 1 Kb ilys-3p::GFP transgene following exposure to Gram-positive bacteria. (i) The basal expression of ilys-3 in the intestine of E. coli (OP50) fed worms is hardly detected. (ii) Enhanced expression of GFP is observed in animals grown on 100% lawns of the virulent (swelling) and (iii) the attenuated strains of M. nematophilum, CBX102 and UV336, respectively. (iv) High GFP signal is also detected in the gut of animals exposed to M. luteus. (v) ilys-3 reporter transgenic young adults grown on OP50. (vi) Activation of ilys-3 transcription in the gut of young adults off food for 24 hours. (vii) Expression of the ilys-3 reporter in L1 larvae at hatching and (viii) in arrested-L1s obtained from nutrient-depleted plates 24 hours after hatching. (ix) and (x) Representative images of the ilys-3 expression in an L1 at hatching and an one-day-old arrested L1. (B) Quantification of the ilys-3p::GFP fluorescence in the intestinal cell int8 of the ilys-3 reporter in animals grown on OP50, CBX102 and UV336. Shown are box plots distributions for the GFP expression in the intestinal cell of L1 animals maintained on the three bacteria for 48 hours at 25°C. The focal plane with the highest GFP signal was used to measure fluorescence intensity within a region of interest (ROI) set to 40 μ diameter and 0.4 μ thickness. Graph is representative of two independent experiments. Asterisks indicate the results of Mann Whitney test of fluorescence values, 95% confidence interval relative to OP50 of worms on CBX102 (** p = 0.0012), and on UV336 (*** p < 0.0001). NS: not significant. N = 15 per group. (C) qRT-PCR analysis of ilys genes from L1 larvae propagated on OP50, CBX102, UV336 for 48 hours, showing high levels of ilys-2 and ilys-3. Expression levels were normalized to OP50, and to the endogenous control gene rla-1. ilys-2 and ilys-3 transcripts were clearly responsive to Gram-positive bacteria. In contrast, ilys-4 and ilys-5 mRNA levels remained mainly unchanged. Data were analyzed with two-way Anova, Holm-Sidak's multiple comparison tests (99% CI) and showed that the increased levels of induction of ilys-3 mRNA by CBX102 and M. luteus were significantly different (** p = 0.0059) but there was no statistically significant change (NS) between animals on CBX102 and UV337 (p = 0.4644). Gene expression was analyzed using the comparative ΔΔCt method. Data are representative of four independent experiments. Error bars are SEM. (D) Quantification of the ilys-3p::GFP fluorescence in the intestinal cell int2 of the ilys-3 reporter in animals subjected to nutrient depletion. Shown are box plots distributions for the GFP expression in the intestinal cell of hatched L1 larvae, one day-old arrested L1s, and young adults 24 hours after they were removed from food. Graph is representative of two independent experiments. Mean values for one-day-old adults off food differ significantly from their sibling controls on OP50 (**** p < 0.0001). Statistically significant differences were also seen in one-day-old arrested larvae (off OP50) when compared to naïve animals hatched overnight (* p = 0.0237). N = 12/group. Asterisks indicate the results of Mann Whitney test of fluorescence values, 95% confidence interval.
Fig 5.
ilys-3 is required in the pharynx and in the intestine to prevent bacterial burden in the gut lumen and to protect against M. nematophilum.
2. (A) Images of a wild-type, ilys-3 and ilys-3; eEx752 one-day old adults fed for 24 hour on E. coli expressing GFP. Live bacteria cells are seen in the pharyngeal (arrow) but not intestinal lumen (arrowhead). (i-ii) N2. (i) Composite DIC and GFP fluorescence image. (ii) Green channel. (iii-iv) ilys-3 deletion mutants accumulate live bacteria in the gut lumen and exhibit impaired ability to disrupt bacteria. (iii) Composite DIC and GFP fluorescence image (iv) Green channel. (v-vi) Overexpression of ILYS-3 in ilys-3 with eEx752 array rescues luminal bacterial accumulation in an animal of the same chronological age. (v) Composite DIC and GFP fluorescence image. (vi) Green channel. (B) Overlays of DIC and epifluorescence images of one-day-old adults of WT, ilys-3 or ilys-3; eEx752 exposed to SYTO 13-labeled CBX102 cells for 2 hours. (i) Fluorescence image of an N2 animal showing few stained CBX102 cells, indicative of non-viable bacteria. (ii) Gut lumen of an ilys-3 animal with high accumulation of live CBX102 cells that fluoresced bright green due to SYTO 13. (iii) ilys-3; eEx752 transgenic displaying reduced luminal bacterial accumulation. (C) The effect of ilys-3 knockout on passage of live bacteria into the gut lumen. Bacterial load was calculated using a colony-forming units (CFU) count assay. N2 and ilys-3 mutants were exposed as L4 larvae to E. coli::GFP for 24 hours. Each symbol represents the average bacterial load obtained from pools of 10 animals. Thick horizontal bars represent the median of three independent experiments (n = 270 animals/ group analyzed). Asterisk indicates the results of a two-tailed unpaired t-test, with Welch's correction, comparing values of colony forming units/10 worms on ilys-3 versus N2 (* p = 0.0338, 95% CI). (D- E) Effect of ILYS-3 overexpression on survival rates of OP50-fed N2, ilys-3(ok3222), ilys-3; eEx752, ilys-3; eEx754, and +; eEx754 cultured at 20°C. P value vs control calculated with the Mantel-Cox log-rank test (95% CI). Results are the mean of 3 independent experiments with an average of 100 animals analyzed each time. Data in bar graphs depict means ± standard deviation. (D) Lifespan analysis showing that ILYS-3 overexpression extends lifespan in ilys-3 mutants. (E) Average lifespan plot showing that the decreased average lifespan of ilys-3 deletion mutants is restored to WT levels in ILYS-3 overexpressing animals carrying eEx752 or eEx754 arrays (*** p < 0.0001). (F) Counts of CFU isolated from one-day old adult animals, fed for 24 hour on CBX102. Each symbol represents the average bacterial load obtained from three biological replicates. Asterisk indicates the results of a two-tailed unpaired t-test, with Welch's correction, comparing values of CFU/10 worms on ilys-3 versus N2 (* p = 0.0232) and ilys-3 versus eEx752 (* p = 0.0269), with a statistical confidence p value of <0.05 for each of the three repeats. (G-H) Effect of ILYS-3 overexpression on survival rates of N2 and ilys-3, upon exposure to CBX102. Transgenes used were eEx752 or eEx754. P value vs control calculated with the Mantel-Cox log-rank test (95% CI). Results are the mean of 3 independent trials. Data in bar graphs depict means ± standard deviation. (G) Lifespan curves. (H) Loss of ilys-3 decreases lifespan in animals exposed to CBX102, but ILYS-3 overexpression enhances their survival during infection by this pathogen.
Fig 6.
Induction of ilys-3 requires ERK.
(A) mpk-1 mutants show reduced or no ilys-3 transcription in the intestine and cannot induce ilys-3 when exposed to M. nematophilum CBX102 (arrow). Images show representative fluorescence of the intestine of adults carrying the ilys-3p::GFP transgene grown on CBX102 in the strains: (i) ilys-3p::GFP in the wild-type background and (ii) mpk-1(ku1); ilys-3p::GFP. (B) Post-developmental inhibition of the MEK activity using the chemical inhibitor U0126 that mimics mpk-1(ku1) loss-of function mutants. (i) Detail of animal exposed to DMSO (control) and (ii) U0126 treated animal showing reduced GFP signal in the gut after exposure to CBX102. Note the decreased fluorescence in the intestine of an animal treated with U0126 (arrow) (ii) and the reduced swelling in contrast to the DMSO-treated control (i). (C) Expression of ilys-3p::GFP, measured by fluorescence intensity in the intestinal cell int8 in WT and mutant backgrounds, expressed in arbitrary fluorescence units. ROI was set to 40 μ diameter and 0.4 μ thickness. Asterisks indicate the results of a Mann–Whitney Unpaired test statistical comparisons of the fluorescence intensity for mpk-1(ku1); ilys-3p::GFP_OP50 vs ilys-3p::GFP_OP50 and mpk-1(ku1); ilys-3p::GFP_CBX102 vs ilys-3p::GFP_CBX102 (*** p < 0.0001). Mean values for mpk-1 mutants on CBX102 were not significantly different from their sibling controls on OP50 (p = 0.6098). N = 15 per group. (D) Confocal quantification of the mean fluorescence intensity in the intestinal cell int8 of L4 expressing ilys-3p::GFP treated with 50 µM of the MEK inhibitor U0126 or DMSO as control. ROI was set to 40 µ diameter and 0.4 µ thickness. Asterisks indicate the results of a Mann–Whitney Unpaired test statistical comparisons of the fluorescence intensity for worms treated with U0126 relative to the DMSO control. Mean values of U0126 on HB101 and on CBX102 all differed significantly from their respective controls (*** p < 0.0001). The mean values for DMSO treated worms on CBX102 were marginally different from those for OP50 controls (p = 0.022). 95% confidence interval. N = 15 per group. (E) Activation of ERK MAPK kinase in the gpb-2 mutant leads to overexpression and ectopic expression of ilys-3 in (i) epidermis (ep) but not seam cells, (ii) whole pharynx (ph) (iii) intestine (int) and rectal epithelium (rep). (F) Representative images of gpb-2 mutants on OP50. (G) Induction of ilys-3 in the gut of gpb-2 mutants after 24 hours on 100% CBX102. Gain was set to the brightest samples i.e. gpb-2 in CBX102. (H) Representation of the ERK-1/2 MAPK genetic pathway that is activated by EGL-30 (Gαq) in the context of the morphological changes that take place for the rectal epithelial cell swelling in response to M. nematophilum. Presumably in this pathway, GPB-2 (Gα2) negatively regulates EGL-30 (Gαq), which in turn, is required for activation of MEK-2 and its downstream effector MPK-1. The network that regulates the transcriptional induction of ILYS-3 upon M. nematophilum infection (this work) is under the control of MPK-1 and the negative regulation by GPB-2. This signaling cascade can be blocked by the MEK inhibitor U0126, corresponding to mpk-1 loss-of-function.
Fig 7.
The activation of ILYS-3 does not require MPK-1 activity in the gut.
(A) Images of single and double transgenic animals carrying the ilys-3p::GFP reporter without or with the transgene mtl-2p::MPK-1 in the WT (N2) and in the mpk-1(ku1) backgrounds. The construct mtl-2p::MPK-1 drives MPK-1 expression in the intestine (int). In the mpk-1 mutants, ilys-3 expression was blocked. This phenotype was not rescued when MPK-1 is restored in the intestine. (B) Quantification of fluorescence intensity in the intestinal cell int2. Asterisks indicate the results of a Mann–Whitney Unpaired test statistical comparisons of the fluorescence intensity for mpk-1(ku1); ilys-3p::GFP; mtl-2p::MPK-1vs ilys-3p::GFP; mtl-2p::MPK-1(*** p = 0.0005) and mpk-1(ku1); ilys-3p::GFP vs ilys-3p::GFP (*** p = 0.0002). Mean values for mpk-1 mutants with the double transgene were not significantly different (NS) from their sibling controls harbouring the ilys-3p::GFP reporter only (p = 0.0934). N = 10-15/group.
Fig 8.
The activation of ILYS-3 is cell non-autonomous and requires MPK-1 activity in the pharynx.
(A) Images of single and double transgenic animals expressing ilys-3p::GFP only or in combination with the myo-2p::MPK-1 in mpk-1(ku1) and WT (N2) backgrounds. The myo-2p::MPK-1 construct drives MPK-1 expression in the pharynx and restored ilys-3 expression in the intestine (int) of mpk-1 mutants. (B) Quantification of fluorescence intensity (after background subtraction) in the intestinal cell int2 of single and double transgene reporter strains. Data analyzed with Mann–Whitney Unpaired test, 95% confidence level. Fluorescence intensity values for mpk-1(ku1); ilys-3p::GFP; myo-2p::MPK-1 vs ilys-3p::GFP; myo-2p::MPK-1 and mpk-1(ku1); ilys-3p::GFP; myo-2p::MPK-1 vs ilys-3p::GFP were not significantly different (NS). Mean values for mpk-1 mutants with the double transgene differ significantly from their sibling controls harbouring the ilys-3p::GFP reporter only (*** p = 0.0003). N = 10-15/group.
Fig 9.
Recombinant ILYS-3 possesses hydrolytic activity on M. luteus and M. nematophilum.
(A) SDS-PAGE analysis of the purified rILYS-3 fusion proteins under non-reducing conditions. Lanes M: protein marker; MBP::ILYS-3: Purified MBP::ILYS-3 fusion protein (4 µg); MBP: purified MBP migrates as 45 kDa (10 µg); GST::ILYS-3: solubilized inclusion bodies harboring GST::ILYS-3 signal less fusion protein; Hen LYS.: Hen egg-white lysozyme migrates as 14 kDa (1 µg). Gel was visualized by Coomassie staining. The target protein is indicated by arrowhead. (B) Zymogram analysis of recombinant ILYS-3 fused to MBP or GST on an SDS-polyacrylamide gel with M. luteus cells. The hydrolytic activity was assayed by methylene blue staining. Samples appear in the same order as in (A). Non-stained zones indicate peptidoglycan degradation. The recombinant MBP::ILYS-3 fusion protein produced a band of clearing at the expected position (arrowhead), indicating peptidoglycan-hydrolytic activity. Purified Hen LYS and MBP alone were used as positive and negative controls, respectively. No gel clearing was detected with MBP alone. Solubilized inclusion bodies recovered from IPTG-induced E. coli BL21 harboring the recombinant GST::ILYS-3 signal peptide less construct produced a clear band resolved at 41 kDa (expected size), and denotes enzymatic activity. The target protein is indicated by arrowhead. (C) Zymogram analysis of recombinant ILYS-3 signal less peptide fused to GST on an SDS-polyacrylamide gel with M. nematophilum cells. The target protein is indicated by arrowhead.