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

Regulation of transcription termination in the nematode Caenorhabditis elegans

The current predicted mechanisms that describe RNA polymerase II (pol II) transcription termination downstream of protein expressing genes fail to adequately explain, how premature termination is prevented in eukaryotes that possess operon-like structures. Here we address this issue by analysing transcription termination at the end of single protein expressing genes and genes located within operons in the nematode Caenorhabditis elegans. By using a combination of RT-PCR and ChIP analysis we found that pol II generally transcribes up to 1 kb past the poly(A) sites into the 3′ flanking regions of the nematode genes before it terminates. We also show that pol II does not terminate after transcription of internal poly(A) sites in operons. We provide experimental evidence that five randomly chosen C. elegans operons are transcribed as polycistronic pre-mRNAs. Furthermore, we show that cis-splicing of the first intron located in downstream positioned genes in these polycistronic pre-mRNAs is critical for their expression and may play a role in preventing premature pol II transcription termination

Glycosylation Genes Expressed in Seam Cells Determine Complex Surface Properties and Bacterial Adhesion to the Cuticle of Caenorhabditis elegans

The surface of the nematode Caenorhabditis elegans is poorly understood but critical for its interactions with the environment and with pathogens. We show here that six genes (bus-2, bus-4, and bus-12, together with the previously cloned srf-3, bus-8, and bus-17) encode proteins predicted to act in surface glycosylation, thereby affecting disease susceptibility, locomotory competence, and sexual recognition. Mutations in all six genes cause resistance to the bacterial pathogen Microbacterium nematophilum, and most of these mutations also affect bacterial adhesion and biofilm formation by Yersinia species, demonstrating that both infection and biofilm formation depend on interaction with complex surface carbohydrates. A new bacterial interaction, involving locomotory inhibition by a strain of Bacillus pumilus, reveals diversity in the surface properties of these mutants. Another biological property—contact recognition of hermaphrodites by males during mating—was also found to be impaired in mutants of all six genes. An important common feature is that all are expressed most strongly in seam cells, rather than in the main hypodermal syncytium, indicating that seam cells play the major role in secreting surface coat and consequently in determining environmental interactions. To test for possible redundancies in gene action, the 15 double mutants for this set of genes were constructed and examined, but no synthetic phenotypes were observed. Comparison of the six genes shows that each has distinctive properties, suggesting that they do not act in a linear pathway

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

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

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