Complex Microflora

<div><p>Diverse bacterial and fungal cells found in association with a natural isolate of C. elegans in a decomposed apple, visualized by Nomarski microscopy. Long arrows mark filamentous fungi, short arrows mark yeast, barbed arrows mark round bacteria (cocci), and arrowheads mark rod-shaped bacteria.</p> <p>(Image courtesy of Marie-Anne Felix)</p></div

Where the Wild Worms Are

<div><p>Rotten apples and slugs, such as those shown here, are prime sources for finding new isolates of Caenorhabditis species, along with their natural pathogens.</p> <p>(Image courtesy of Marie-Anne Felix)</p></div

C. elegans and Its Enemies

<p>Diagram of C. elegans anatomy, indicating some of the pathogens under laboratory investigation and their modes of attack on the worm.</p

Increased expression of TPH-1 in ADF chemosensory neurons is caused by reduced contact with contaminated bacterial lawns.

<p>Wild type and <i>egl-30(ad805)</i> animals carrying an integrated <i>tph-1p</i>::DSRED transgene were infected with <i>M. nematophilum</i> or an avirulent form of <i>M. nematophilum</i> using standard (small lawn) or “big lawn” assay conditions. The mean <i>tph-1p</i>::DSRED fluorescence in ADF neurons was quantified. Expression of <i>tph-1p</i>::DSRED was significantly increased when wild type animals were grown on small lawns contaminated with virulent <i>M. nematophilum</i>. This increase in expression was not observed under conditions when animals were unable to leave the bacterial lawn; in <i>egl-30(ad805)</i> animals or when wild type animals were infected on “big lawns”.</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

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

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

Rectal epithelial EGL-10 acts downstream of serotonin to modify the immune response and affect pathogen clearance.

<p>Treatment of wild type animals with 3.8/ml 5-HT caused a decrease in the number of Dar animals following infection with <i>M. nematophilum</i> (A) and decreased the clearance of SYTO13 labeled pathogen from the rectal opening (B). Serotonin was unable to decrease the percentage of Dar animals (A) or the rate of pathogen clearance (B) when EGL-10 cDNA was overexpressed in the rectal epithelium of wild type animals suggesting that GOA-1(Gáo) signaling in the rectal epithelium is required for serotonin to suppress the immune response. Animals lacking <i>tph-1</i> have wild type levels of Dar response on lawns contaminated with 10% <i>M. nematophilum</i> (A) but are more able to clear pathogen infections than wild type (C). Conversely activation of GOA-1(Gáo) using <i>egl-10</i> loss-of-function mutants results in a decrease in the percentage of Dar animals (A) and infections clear more slowly than wild type animals (C). To determine whether GOA-1(Gáo) acts downstream of serotonin we combined <i>egl-10(n692)</i> with <i>tph-1(mg280)</i> or <i>tph-1(n4622)</i>. The percentage of Dar animals (A) and the rate of pathogen clearance was indistinguishable between <i>egl-10(n692)</i> and these double mutants (C). Thus GOA-1(Gáo) signaling acts downstream of serotonin synthesis to suppress the immune response to <i>M. nematophilum</i> infection.</p

Serotonin synthesis in chemosensory neurons inhibits the immune response by altering rectal epithelial G-protein signaling.

<p>In response to environmental cues, such as the presence or absence of food, serotonin, released from ADF chemosensory neurons acts, directly or indirectly, to regulate GOA-1(Gαo) signaling in the rectal epithelium. This signaling suppresses the Dar phenotype that forms part of the innate immune response and limits the rate of pathogen clearance from the rectal opening.</p