The invertebrate lysozyme effector ILYS-3 is systemically activated in response to danger signals and confers antimicrobial protection in C. elegans

Little is known about the relative contributions and importance of antibacterial effectors in the nematode C. elegans, despite extensive work on the innate immune responses in this organism. We report an investigation of the expression, function and regulation of the six ilys (invertebrate-type lysozyme) genes of C. elegans. These genes exhibited a surprising variety of tissue-specific expression patterns and responses to starvation or bacterial infection. The most strongly expressed, ilys-3, was investigated in detail. ILYS-3 protein was expressed constitutively in the pharynx and coelomocytes, and dynamically in the intestine. Analysis of mutants showed that ILYS-3 was required for pharyngeal grinding (disruption of bacterial cells) during normal growth and consequently it contributes to longevity, as well as being protective against bacterial pathogens. Both starvation and challenge with Gram-positive pathogens resulted in ERK-MAPK-dependent up-regulation of ilys-3 in the intestine. The intestinal induction by pathogens, but not starvation, was found to be dependent on MPK-1 activity in the pharynx rather than in the intestine, demonstrating unexpected communication between these two tissues. The coelomocyte expression appeared to contribute little to normal growth or immunity. Recombinant ILYS-3 protein was found to exhibit appropriate lytic activity against Gram-positive cell wall material

During dauer arrest ILYS-3 is secreted in the lumen but returns to its steady state cytosolic expression upon dauer recovery.

<p>(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.</p

<i>ilys-3</i> is required in the pharynx and in the intestine to prevent bacterial burden in the gut lumen and to protect against <i>M</i>. <i>nematophilum</i>.

<p><b>2.</b> (A) Images of a wild-type, <i>ilys-3</i> and <i>ilys-3; eEx752</i> one-day old adults fed for 24 hour on <i>E</i>. <i>coli</i> 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) <i>ilys-3</i> 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 <i>ilys-3</i> with <i>eEx752</i> 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, <i>ilys-3</i> or <i>ilys-3; eEx752</i> 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 <i>ilys-3</i> animal with high accumulation of live CBX102 cells that fluoresced bright green due to SYTO 13. (iii) <i>ilys-3; eEx752</i> transgenic displaying reduced luminal bacterial accumulation. (C) The effect of <i>ilys-3</i> knockout on passage of live bacteria into the gut lumen. Bacterial load was calculated using a colony-forming units (CFU) count assay. N2 and <i>ilys-3</i> mutants were exposed as L4 larvae to <i>E</i>. <i>coli</i>::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 <i>ilys-3</i> versus N2 (* <i>p</i> = 0.0338, 95% CI). (D- E) Effect of ILYS-3 overexpression on survival rates of OP50-fed N2, <i>ilys-3(ok3222)</i>, <i>ilys-3; eEx752</i>, <i>ilys-3; eEx754</i>, and +; <i>eEx754</i> cultured at 20°C. <i>P</i> value <i>vs</i> 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 <i>ilys-3</i> mutants. (E) Average lifespan plot showing that the decreased average lifespan of <i>ilys-3</i> deletion mutants is restored to WT levels in ILYS-3 overexpressing animals carrying <i>eEx752</i> or <i>eEx754</i> arrays (*** <i>p <</i> 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 <i>ilys-3</i> versus N2 <i>(* p</i> = 0.0232) and <i>ilys-3</i> versus <i>eEx752</i> (* <i>p</i> = 0.0269), with a statistical confidence <i>p</i> value of <0.05 for each of the three repeats. (G-H) Effect of ILYS-3 overexpression on survival rates of N2 and <i>ilys-3</i>, upon exposure to CBX102. Transgenes used were <i>eEx752</i> or <i>eEx754</i>. <i>P</i> value <i>vs</i> 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 <i>ilys-3</i> decreases lifespan in animals exposed to CBX102, but ILYS-3 overexpression enhances their survival during infection by this pathogen.</p

The activation of ILYS-3 does not require MPK-1 activity in the gut.

<p>(A) Images of single and double transgenic animals carrying the <i>ilys-3p</i>::<i>GFP</i> reporter without or with the transgene <i>mtl-2p</i>::<i>MPK-1</i> in the WT (N2) and in the <i>mpk-1(ku1)</i> backgrounds. The construct <i>mtl-2p</i>::<i>MPK-1</i> drives MPK-1 expression in the intestine (int). In the <i>mpk-1</i> mutants, <i>ilys-3</i> 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 <i>mpk-1(ku1); ilys-3p</i>::<i>GFP; mtl-2p</i>::<i>MPK-1vs ilys-3p</i>::<i>GFP; mtl-2p</i>::<i>MPK-1</i>(*** <i>p</i> = 0.0005) and <i>mpk-1(ku1); ilys-3p</i>::<i>GFP vs ilys-3p</i>::<i>GFP</i> (*** <i>p</i> = 0.0002). Mean values for <i>mpk-1</i> mutants with the double transgene were not significantly different (NS) from their sibling controls harbouring the <i>ilys-3p</i>::<i>GFP</i> reporter only (<i>p</i> = 0.0934). N = 10-15/group.</p

Recombinant ILYS-3 possesses hydrolytic activity on <i>M</i>. <i>luteus</i> and <i>M</i>. <i>nematophilum</i>.

<p>(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 <i>M</i>. <i>luteus</i> 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 <i>E</i>. <i>coli</i> 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 <i>M</i>. <i>nematophilum</i> cells. The target protein is indicated by arrowhead.</p

Intestinal ILYS-3::mCherry associates with endocytic and recycling endosomes and LROs.

<p>(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 <i>rme-1</i> mutant. (L) ILYS-3::mCherry accumulates apically in the plasma membrane and in puncta in <i>rab-10</i> 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.</p

<i>C</i>. <i>elegans ilys-3</i> is expressed in the pharynx, intestine and coelomocytes.

<p>(A-C) GFP expression directed by the 1 Kb <i>ilys-3</i> promoter (upstream the start codon). (A) Example of expression of <i>ilys-3p</i> 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 <i>ilys-3</i> transcription in intestinal int8 and int9 cells in an adult worm. (D-I) Expression of the <i>ily-3p</i>::<i>ILYS-3</i>::<i>mCherry</i> 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 <i>ilys-3</i> 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.</p

The activation of ILYS-3 is cell non-autonomous and requires MPK-1 activity in the pharynx.

<p>(A) Images of single and double transgenic animals expressing <i>ilys-3p</i>::<i>GFP</i> only or in combination with the <i>myo-2p</i>::<i>MPK-1</i> in <i>mpk-1(ku1)</i> and WT (N2) backgrounds. The <i>myo-2p</i>::<i>MPK-1</i> construct drives MPK-1 expression in the pharynx and restored <i>ilys-3</i> expression in the intestine (int) of <i>mpk-1</i> 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 <i>mpk-1(ku1); ilys-3p</i>::<i>GFP</i>; <i>myo-2p</i>::<i>MPK-1 vs ilys-3p</i>::<i>GFP; myo-2p</i>::<i>MPK-1</i> and <i>mpk-1(ku1); ilys-3p</i>::<i>GFP; myo-2p</i>::<i>MPK-1 vs ilys-3p</i>::<i>GFP</i> were not significantly different (NS). Mean values for <i>mpk-1</i> mutants with the double transgene differ significantly from their sibling controls harbouring the <i>ilys-3p</i>::<i>GFP</i> reporter only (*** <i>p</i> = 0.0003). N = 10-15/group.</p

The <i>C</i>. <i>elegans ilys-3</i> is transcriptionally activated in the intestine upon Gram-positive exposure and is a readout for monitoring danger/hunger signals.

<p>(A) Fluorescence images of representative animals carrying the 1 Kb <i>ilys-3p</i>::<i>GFP</i> transgene following exposure to Gram-positive bacteria. (i) The basal expression of <i>ilys-3</i> in the intestine of <i>E</i>. <i>coli</i> (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 <i>M</i>. <i>nematophilum</i>, CBX102 and UV336, respectively. (iv) High GFP signal is also detected in the gut of animals exposed to <i>M</i>. <i>luteus</i>. (v) <i>ilys-3</i> reporter transgenic young adults grown on OP50. (vi) Activation of <i>ilys-3</i> transcription in the gut of young adults off food for 24 hours. (vii) Expression of the <i>ilys-3</i> 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 <i>ilys-3</i> expression in an L1 at hatching and an one-day-old arrested L1. (B) Quantification of the <i>ilys-3p</i>::<i>GFP</i> fluorescence in the intestinal cell int8 of the <i>ilys-3</i> 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 (** <i>p</i> = 0.0012), and on UV336 (*** <i>p</i> < 0.0001). NS: not significant. N = 15 per group. (C) qRT-PCR analysis of <i>ilys</i> genes from L1 larvae propagated on OP50, CBX102, UV336 for 48 hours, showing high levels of <i>ilys-2</i> and <i>ilys-3</i>. Expression levels were normalized to OP50, and to the endogenous control gene <i>rla-1</i>. <i>ilys-2</i> and <i>ilys-3</i> transcripts were clearly responsive to Gram-positive bacteria. In contrast, <i>ilys-4</i> and <i>ilys-5</i> 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 <i>ilys-3</i> mRNA by CBX102 and <i>M</i>. <i>luteus</i> were significantly different (** <i>p</i> = 0.0059) but there was no statistically significant change (NS) between animals on CBX102 and UV337 (<i>p</i> = 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 <i>ilys-3p</i>::<i>GFP</i> fluorescence in the intestinal cell int2 of the <i>ilys-3</i> 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 (**** <i>p</i> < 0.0001). Statistically significant differences were also seen in one-day-old arrested larvae (off OP50) when compared to naïve animals hatched overnight (* <i>p</i> = 0.0237). N = 12/group. Asterisks indicate the results of Mann Whitney test of fluorescence values, 95% confidence interval.</p