OGT-1 is required for the <i>C. elegans</i> response to <i>S. aureus</i>.

<p><i>O-</i>GlcNAc cycling null nematodes are similarly susceptible to <i>P. aeruginosa</i> exposure as N2 animals: (A) <i>ogt-1(ok1474)</i>, (B) <i>oga-1(tm3642)</i>, (C) <i>ogt-1(ok430)</i>, and (D) <i>oga-1(ok1207)</i>. (A–H) <i>pmk-1(km25)</i> animals are hypersensitive to <i>P. aeruginosa</i> and <i>S. aureus</i>. (A, C) <i>ogt-1; pmk-1</i> and (B, D) <i>oga-1; pmk-1</i> are equally susceptible to <i>P. aeruginosa</i> as <i>pmk-1(km25)</i> single mutants. (E, G) <i>ogt-1</i> animals are hypersensitive to <i>S. aureus</i> while (F, H) <i>oga-1</i> mutants maintain survival similar to N2. (E, G) <i>ogt-1; pmk-1</i> are more susceptible to <i>S. aureus</i> than either <i>ogt-1</i> or <i>pmk-1</i> single mutants and (F, H) <i>pmk-1; oga-1</i> are similarly susceptible to <i>S. aureus</i> as <i>pmk-1</i> single mutants. Results are representative of at least two independent assays and are represented by comprehensive plots. <i>n</i>≧162. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113231#pone.0113231.s008" target="_blank">Table S1</a> for individual assay statistical analysis.</p

Conserved Nutrient Sensor O-GlcNAc Transferase Is Integral to <i>C. elegans</i> Pathogen-Specific Immunity

<div><p>Discriminating pathogenic bacteria from bacteria used as a food source is key to <i>Caenorhabidits elegans</i> immunity. Using mutants defective in the enzymes of O-linked N-acetylglucosamine (O-GlcNAc) cycling, we examined the role of this nutrient-sensing pathway in the <i>C. elegans</i> innate immune response. Genetic analysis showed that deletion of O-GlcNAc transferase (<i>ogt-1</i>) yielded animals hypersensitive to the human pathogen <i>S. aureus</i> but not to <i>P. aeruginosa</i>. Genetic interaction studies revealed that nutrient-responsive OGT-1 acts through the conserved β-catenin (BAR-1) pathway and in concert with p38 MAPK (PMK-1) to modulate the immune response to <i>S. aureus</i>. Moreover, whole genome transcriptional profiling revealed that O-GlcNAc cycling mutants exhibited deregulation of unique stress- and immune-responsive genes. The participation of nutrient sensor OGT-1 in an immunity module evolutionarily conserved from <i>C. elegans</i> to humans reveals an unexplored nexus between nutrient availability and a pathogen-specific immune response.</p></div

OP50 <i>E. coli</i>-fed nematodes are generally healthy while pathogen-fed worms demonstrate a decrease in overall health.

<p>(A), (B), and (C) The average pumping rate in a 20 second interval for nematodes fed OP50 or a pathogen. (A) All pumping rates are similar in N2 and mutant animals. (B) The average pumping rate for nematodes fed <i>P. aeruginosa</i> is decreased when compared to N2 animals fed OP50. Worms with a <i>pmk-1(km25)</i> background exhibit the largest decrease in pumping rate. (C) The average pumping rate for nematodes fed <i>S. aureus</i> is decreased when compared to N2 animals fed OP50. Double mutant <i>ogt-1; pmk-1</i> animals exhibit the largest decrease in pumping rate. Statistics are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113231#pone.0113231.s009" target="_blank">Table S2</a> and error bars represent standard deviation. All experiments were performed at 18°C. <i>n</i> = 10–25 animals.</p

Double mutants ogt-1(ok1474); bar-1(ga80) and oga-1(ok1207) bar-1(ga80) are hypersensitive to S. aureus.

<p>(A) <i>ogt-1(ok1474)</i> and <i>bar-1(ga80)</i> are sensitive to <i>S. aureus.</i> (A–B) <i>ogt-1(ok1474); bar-1(ga80)</i> and <i>oga-1(ok1207) bar-1(ga80)</i> are more sensitive to <i>S. aureus</i> than N2 and <i>oga-1</i>. Results are representative of at least two independent assays represented by comprehensive plots. <i>n</i>≧57. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113231#pone.0113231.s008" target="_blank">Table S1</a> for individual assay statistical analysis and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113231#pone.0113231.s007" target="_blank">Figure S7</a> for <i>ogt-1(ok430); bar-1(ga80)</i> and <i>oga-1(tm3642) bar-1(ga80)</i> data. Animals were exposed to cdc25.1 RNAi for 24 hr (A) or 48 hr (B).</p

Distinct genes are deregulated in <i>C. elegans</i> mutants lacking active OGT-1, OGA-1, or PMK-1 after 6 hours of pathogen exposure.

<p>(A) Venn diagram of genes deregulated in <i>ogt-1(ok1474)</i> (red), <i>oga-1(ok1207)</i> (green), and <i>pmk-1(km25)</i> (blue) on OP50 vs. N2. (B) Categories of 387 commonly deregulated genes enriched according to DAVID analysis of microarray samples from A. (C) Heat map of relative levels of genes differentially regulated in mutant backgrounds compared to N2 on OP50 and those deregulated within individual mutants upon pathogen exposure. <i>cpr-2, fmo-2, ilys-3, clec-60,</i> and <i>clec-52</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113231#pone.0113231-Irazoqui1" target="_blank">[11]</a>, <i>mtl-1, nspe-5, vhp-1, abf-2,</i> and <i>cyp-35A4</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113231#pone.0113231-PukkilaWorley1" target="_blank">[55]</a>, <i>spp-20</i> and <i>cyp-35A3</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113231#pone.0113231-Alper1" target="_blank">[30]</a>, <i>ilys-2</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113231#pone.0113231-Irazoqui1" target="_blank">[11]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113231#pone.0113231-Simonsen1" target="_blank">[28]</a>, <i>cnc-7</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113231#pone.0113231-PukkilaWorley1" target="_blank">[55]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113231#pone.0113231-Zugasti1" target="_blank">[56]</a>, <i>cyp-34A2</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113231#pone.0113231-Studencka1" target="_blank">[57]</a>, <i>tir-1</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113231#pone.0113231-Aballay1" target="_blank">[58]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113231#pone.0113231-Couillault1" target="_blank">[59]</a>, <i>exc-5</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113231#pone.0113231-Irazoqui1" target="_blank">[11]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113231#pone.0113231-PukkilaWorley1" target="_blank">[55]</a>, <i>irg-2</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113231#pone.0113231-Dunbar1" target="_blank">[29]</a>, Y75B8A.28 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113231#pone.0113231-Cui1" target="_blank">[60]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113231#pone.0113231-Engelmann2" target="_blank">[61]</a>, Y41C4A.11 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113231#pone.0113231-Sun1" target="_blank">[62]</a>, <i>pgp-1</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113231#pone.0113231-MahajanMiklos1" target="_blank">[53]</a>.</p

<i>S. aureus</i> exposed <i>C. elegans</i> exhibit differential sensitivity to <i>S. aureus</i>.

<p>(A–B) Median survival values (hours after transfer to <i>S. aureus</i>) representing at least two independent biological replicates. Each circle represents the median survival for an individual assay done in technical duplicate or triplicate, statistics are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113231#pone.0113231.s008" target="_blank">Table S1</a>. <i>ogt-1; pmk-1</i> are more sensitive than N2 and single mutants to <i>S. aureus</i> while the <i>ogt-1; bar-1</i> mutants are similarly susceptible as the <i>bar-1</i> single mutant. (C) Simplified model showing how OGT-1 participates in the <i>C. elegans</i> immune response.</p

Lifespan of mutant strains in recovery after 30 days of ARD and summary model.

Lifespan of wild-type and mutant strains in control husbandry conditions (blue bars) compared to those recovered after 30 days of ARD (gray bars). Lifespan after recovery from ARD was significantly defective in aak-2(ok524), daf-16(mu86), and rsks-1(ok1255). The mutant skn-1(zj15) also had a significantly reduced lifespan post-ARD but not as strongly as the others. A minimum of 8 worms from independent experiments were analyzed for lifespan and statistics were performed in Graph Pad Prism using one-way ANOVA. P-value **** = nhr-49, oga-1, ogt-1, and sir-2.1 play a role in ARD initiation, daf-16, rsks-1, and skn-1 function primarily in recovery from ARD, with aak-2 playing a role in the regulation of both states. Conversely, none of the alleles that we explored significantly impacted maintenance of the ARD.</p

Changes in glycogen and trehalose levels before and after ARD.

(A) Carminic acid staining (indicative of glycogen and trehalose levels) varied greatly between strains. As we have previously reported [22], ogt-1(1474) showed slightly higher levels of carminic acid staining than other strains in standard husbandry conditions, though in this study this change did not reach significance. After 30 days of ARD (lower panels) we noted that nhr-49(nr2041) had a dramatic increase in staining, while wild type and ogt-1(ok1474) did not. These results did not correlate with the observed defect in ARD entry for these strains. (B) ImageJ based quantification of carminic acid fluorescence by pixel intensity. P-value **** = (TIF)</p

Fatty acid beta-oxidation, but not desaturation, plays a role in ARD entry.

(A) NHR-49 regulates both fatty acid beta-oxidation (ACS-2) and desaturation (FAT-7) via genetically separable pathways that are known to generate distinct phenotypic outputs. (B) ARD entry was significantly impaired with acs-2(ok2457) but not with the fat-7(wa86) mutant. The double mutant acs-1(ok2457); ogt-1(ok1474) was also significantly impaired for ARD entry. P-value **** = <0.0001 in comparison with N2 as determined using one-way ANOVA, NS = not significant. (C) Lifespan under standard conditions and with refeeding at day 30 of ARD was not significantly altered in these mutants as determined by one-way ANOVA. Error bars represent standard deviation.</p

Germline shrinkage during ARD maintenance and regrowth during recovery.

Representative images (with magnified panels for detail] showing the germline (outlined in yellow] of worms starved (left panel] at day 30 of ARD, with uterine embryos noted with white arrows. The right panel shows worms that have been re-feed for 48 hours after 30 days of ARD, with white dashed lines to indicate the germline, where healthy embryos and/or oocytes can be seen. All strains showed robust shrinkage of the germline during starvation and regrowth after feeding. The 50 μm scale bar shown in the top row applies to all images in each column.</p