EGL-9 Controls C. elegans Host Defense Specificity through Prolyl Hydroxylation-Dependent and -Independent HIF-1 Pathways

Understanding host defense against microbes is key to developing new and more effective therapies for infection and inflammatory disease. However, how animals integrate multiple environmental signals and discriminate between different pathogens to mount specific and tailored responses remains poorly understood. Using the genetically tractable model host Caenorhabditis elegans and pathogenic bacterium Staphylococcus aureus, we describe an important role for hypoxia-inducible factor (HIF) in defining the specificity of the host response in the intestine. We demonstrate that loss of egl-9, a negative regulator of HIF, confers HIF-dependent enhanced susceptibility to S. aureus while increasing resistance to Pseudomonas aeruginosa. In our attempt to understand how HIF could have these apparently dichotomous roles in host defense, we find that distinct pathways separately regulate two opposing functions of HIF: the canonical pathway is important for blocking expression of a set of HIF-induced defense genes, whereas a less well understood noncanonical pathway appears to be important for allowing the expression of another distinct set of HIF-repressed defense genes. Thus, HIF can function either as a gene-specific inducer or repressor of host defense, providing a molecular mechanism by which HIF can have apparently opposing roles in defense and inflammation. Together, our observations show that HIF can set the balance between alternative pathogen-specific host responses, potentially acting as an evolutionarily conserved specificity switch in the host innate immune response

Distinct Pathogenesis and Host Responses during Infection of C. elegans by P. aeruginosa and S. aureus

The genetically tractable model host Caenorhabditis elegans provides a valuable tool to dissect host-microbe interactions in vivo. Pseudomonas aeruginosa and Staphylococcus aureus utilize virulence factors involved in human disease to infect and kill C. elegans. Despite much progress, virtually nothing is known regarding the cytopathology of infection and the proximate causes of nematode death. Using light and electron microscopy, we found that P. aeruginosa infection entails intestinal distention, accumulation of an unidentified extracellular matrix and P. aeruginosa-synthesized outer membrane vesicles in the gut lumen and on the apical surface of intestinal cells, the appearance of abnormal autophagosomes inside intestinal cells, and P. aeruginosa intracellular invasion of C. elegans. Importantly, heat-killed P. aeruginosa fails to elicit a significant host response, suggesting that the C. elegans response to P. aeruginosa is activated either by heat-labile signals or pathogen-induced damage. In contrast, S. aureus infection causes enterocyte effacement, intestinal epithelium destruction, and complete degradation of internal organs. S. aureus activates a strong transcriptional response in C. elegans intestinal epithelial cells, which aids host survival during infection and shares elements with human innate responses. The C. elegans genes induced in response to S. aureus are mostly distinct from those induced by P. aeruginosa. In contrast to P. aeruginosa, heat-killed S. aureus activates a similar response as live S. aureus, which appears to be independent of the single C. elegans Toll-Like Receptor (TLR) protein. These data suggest that the host response to S. aureus is possibly mediated by pathogen-associated molecular patterns (PAMPs). Because our data suggest that neither the P. aeruginosa nor the S. aureus–triggered response requires canonical TLR signaling, they imply the existence of unidentified mechanisms for pathogen detection in C. elegans, with potentially conserved roles also in mammals

<i>egl-9</i> inactivation causes enhanced susceptibility to <i>S. aureus-</i>mediated killing.

<p><b>A..... </b><i>egl-9(sa307)</i> animals exhibited enhanced susceptibility, whereas <i>egl-9(sa307);hif-1(ia4)</i> mutants exhibited near wild-type susceptibility. Survival analysis: <i>egl-9</i> Kaplan-Meier Median Survival (MS) = 62 h, Time to 50% Death by nonlinear regression analysis (LT₅₀) = 48.78 h, Number of animals (N) = 142, <i>p</i><0.0001 (Log-Rank test, compared with wild type); <i>egl-9;hif-1</i> MS = 68 h, LT₅₀ = 62.10 h, N = 122/2, <i>p</i> = 0.0030 (compared with wild type). <b>B..... </b><i>vhl-1(ok161)</i> and <i>hif-1(ia4)</i> animals exhibited near wild-type susceptibility. Survival analysis: wild type MS = 74 h, LT₅₀ = 67.03 h, N = 117/5; <i>vhl-1</i> MS = 62 h, LT₅₀ = 61.86 h, N = 118, <i>p</i><0.0001 (compared with wild type); <i>hif-1</i> MS = 74 h, LT₅₀ = 64.77 h, N = 136, <i>p</i> = 0.0943 (compared with wild type). <b>C..... </b><i>egl-9(sa330)</i> animals and <b>D..... </b><i>egl-9(ok478)</i> animals exhibit wild type susceptibility. <b>E..... </b><i>egl-9(n586ts)</i> animals are hypersusceptible to <i>S. aureus</i>. Survival analysis: <i>egl-9(sa307)</i> MS = 43 h, N = 95/1, <i>p</i><0.0001 (compared with wild type); <i>egl-9;(n586ts)</i> MS = 43 h, N = 96/15, <i>p</i><0.0001 (compared with wild type); wild type MS = 50 h, N = 92/9. As all killing assays, this assay was performed at 25°C, which is the restrictive temperature of <i>n586ts</i>. <b>F.</b> Wild type, <i>egl-9(sa307);crp-1::egl-9</i> (Intestinal <i>egl-9</i>), and <i>egl-9(sa307);crp-1::gfp</i> (Intestinal <i>gfp</i>) animals show that intestinal expression of EGL-9, but not GFP, rescues the <i>egl-9(sa307)</i> enhanced susceptibility phenotype. Survival analysis: wild type MS = 70 h, N = 108/7; <i>Intestinal egl-9</i> MS = 61 h, N = 115/14, <i>p</i><0.0001 (compared with wild type), <i>p</i><0.0001 (compared with <i>Intestinal gfp</i>); <i>Intestinal gfp</i> MS = 48 h, N = 102/3, <i>p</i><0.0001 (compared with wild type). Results are representative of two independent trials, performed in triplicate. Animals were subjected to <i>cdc-25</i> RNAi to prevent reproduction, and subsequently transferred to <i>S. aureus</i> killing assay plates.</p

Methicillin resistance in Staphylococcus aureus requires glycosylated wall teichoic acids

Staphylococcus aureus peptidoglycan (PG) is densely functionalized with anionic polymers called wall teichoic acids (WTAs). These polymers contain three tailoring modifications: d-alanylation, α-O-GlcNAcylation, and β-O-GlcNAcylation. Here we describe the discovery and biochemical characterization of a unique glycosyltransferase, TarS, that attaches β-O-GlcNAc (β-O-N-acetyl-d-glucosamine) residues to S. aureus WTAs. We report that methicillin resistant S. aureus (MRSA) is sensitized to β-lactams upon tarS deletion. Unlike strains completely lacking WTAs, which are also sensitive to β-lactams, ΔtarS strains have no growth or cell division defects. Because neither α-O-GlcNAc nor β-O-Glucose modifications can confer resistance, the resistance phenotype requires a highly specific chemical modification of the WTA backbone, β-O-GlcNAc residues. These data suggest β-O-GlcNAcylated WTAs scaffold factors required for MRSA resistance. The β-O-GlcNAc transferase identified here, TarS, is a unique target for antimicrobials that sensitize MRSA to β-lactams

Repression of HIF-1-repressed host defense genes causes enhanced susceptibility to <i>S. aureus</i>.

<p><b>A.</b> Non-hierarchical cluster analysis of <i>egl-9-</i>induced gene expression changes in infected <i>hif-1(ia4)</i>, <i>swan-1(ok267)</i>, <i>swan-1(ok267);[hif-1^P621G] egl-9(sa307)</i>, <i>vhl-1(ok161)</i>, <i>hif-1(ia4);[hif-1^P621G]</i>, and <i>hif-1;[hif-1]</i> animals normalized to wild type. <b>B.</b> Non-hierarchical cluster analysis of <i>egl-9-</i>repressed gene expression changes in infected <i>hif-1(ia4)</i>, <i>swan-1(ok267)</i>, <i>swan-1(ok267);[hif-1^P621G] egl-9(sa307)</i>, <i>vhl-1(ok161)</i>, <i>hif-1(ia4);[hif-1^P621G]</i>, and <i>hif-1;[hif-1]</i> animals normalized to wild type. Blue indicates downregulation, red indicates upregulation. Color intensity reflects magnitude of change; darker colors correspond to larger changes. <b>C.</b> Enhanced-RNAi mutant <i>eri-1(mg366)</i> animals were subjected to feeding RNAi from hatching to L4 stage, and subsequently transferred to <i>S. aureus</i> pathogenesis assays. Vector, empty L4440 RNAi plasmid. Survival analysis: <i>vector</i> MS = 75 h, N = 83/12; <i>ilys-3</i>, <i>Y65B4BR.1</i>, <i>lys-5</i> RNAi MS = 48 h, N = 93/9, <i>p</i> = 0.0001 (compared with vector control). Results are representative of two independent trials, performed in triplicate.</p

<i>egl-9</i> is required to lift repression of host defense genes by <i>hif-1</i>.

<p><b>A, B, C.......... </b><i>egl-9(sa307)</i> and <i>egl-9(sa307);hif-1(ia4)</i> animals were fed heat-killed non-pathogenic <i>E. coli</i> for 8 h and gene expression, measured by qRT-PCR, was normalized to parallel wild type controls. Genes were divided into three groups, according to their expression in infected <i>egl-9</i> animals (see G, H, I): <b>A.......... </b><i>egl-9-</i>repressed genes, <b>B.......... </b><i>egl-9-</i>independent genes, and <b>C.......... </b><i>egl-9-</i>induced genes. <b>D, E, F.......... </b><i>vhl-1(ok161)</i> and <i>vhl-1(ok161);hif-1(ia4)</i> animals were fed heat-killed non-pathogenic <i>E. coli</i> and gene expression was normalized to wild type. Genes were grouped as in A, B, C. <b>G, H, I.......... </b><i>egl-9(sa307)</i> and <i>egl-9(sa307);hif-1(ia4)</i> animals were infected with <i>S. aureus</i> for 8 h and gene expression, measured by qRT-PCR, was normalized to wild type. Genes are divided into three groups: <b>G.......... </b><i>egl-9-</i>repressed genes, <b>H.......... </b><i>egl-9-</i>independent genes, and <b>I.......... </b><i>egl-9-</i>induced genes. <b>J, K, L.......... </b><i>vhl-1(ok161)</i> and <i>vhl-1(ok161);hif-1(ia4)</i> animals were infected and gene expression was normalized to wild type. Genes are grouped as in G, H, I. Data are means of 2–5 independent biological replicates, error bars are SEM. *, <i>p</i>≤0.05 (compared with wild type by two-sample <i>t</i> test).</p

Noncanonical signaling contributes to lifting <i>hif-1</i>-mediated repression of the host defense response.

<p><b>A... </b><i>hif-1</i> animals overexpressing wild type HIF-1 (<i>hif-1;[hif-1]</i>) or non-hydroxylatable HIF-1 (<i>hif-1;[hif-1^P621G]</i>) were infected with <i>S. aureus</i> for 8 h and gene expression, measured by qRT-PCR, was normalized to wild type. Data are means of 2 independent biological replicates, error bars are SEM. *, <i>p</i>≤0.05 (compared with wild type by two-sample <i>t</i> test); †, <i>p</i>≤0.05 (compared <i>hif-1;[hif-1]</i> with <i>hif-1;[hif-1^P621G]</i> by two-sample <i>t</i> test). <b>B... </b><i>swan-1(ok267)</i> mutants were infected with <i>S. aureus</i> for 8 h and gene expression, measured by qRT-PCR, was normalized to wild type. <i>egl-9(sa307)</i> data from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002798#ppat-1002798-g003" target="_blank">Figure 3</a> are included for comparison. Results are means of 3–5 independent biological replicates, error bars are SEM. *, <i>p</i>≤0.05 (compared with wild type by two-sample <i>t</i> test). <b>C.</b> Genes whose expression levels were intermediate in <i>swan-1; [hif-1^P621G]</i> animals compared with <i>swan-1</i> and <i>egl-9</i> animals. <i>swan-1</i> animals overexpressing non-hydroxylatable HIF-1 (<i>swan-1;hif-1^P621G</i>) were infected with <i>S. aureus</i> for 8 h and gene expression, measured by qRT-PCR, was normalized to wild type. Data are means of 2 independent biological replicates, error bars are SEM. *, <i>p</i>≤0.05 (compared with wild type by two-sample <i>t</i> test). Data for <i>swan-1</i> and <i>egl-9</i> mutants from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002798#ppat-1002798-g005" target="_blank">Figure 5A</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002798#ppat-1002798-g003" target="_blank">3</a> are included for comparison. <b>D.</b> Genes whose expression levels did not appear intermediate in <i>swan-1;hif-1^P621G</i> animals compared with <i>egl-9</i> and <i>swan-1</i> animals. Data for <i>swan-1</i> and <i>egl-9</i> mutants from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002798#ppat-1002798-g005" target="_blank">Figure 5A</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002798#ppat-1002798-g003" target="_blank">3</a> are included for comparison. <b>E... </b><i>swan-1(ok267)</i> mutants exhibit enhanced susceptibility to <i>S. aureus</i>. Survival analysis: wild type MS = 65 h, N = 110/4; <i>swan-1</i> MS = 48 h, N = 108/1, <i>p</i> = 0.0036 (compared with wild type); <i>egl-9</i> MS = 40 h, N = 87/2, <i>p</i><0.0001 (compared with wild type). Results are representative of two independent trials, performed in triplicate.</p

Working model for HIF-1-mediated inflammatory regulation.

<p><b>A.</b> Proposed model of noncanonical HIF-1 inhibition lifting HIF-1-mediated repression of host defense genes. Infection by <i>S. aureus</i> causes induction of host defense genes. Some of these genes are also induced by HIF, while others are repressed by HIF. Genes induced by HIF include genes that mediate resistance to pore-forming toxins (PFT) and <i>P. aeruginosa</i>. Genes that are repressed by HIF include genes that mediate resistance to <i>S. aureus</i>. Canonical HIF regulation mediated by VHL-1 requires EGL-9 catalytic activity and controls the gene-inductive activity of HIF. O₂ is a signal driving HIF inhibition by the canonical pathway. Noncanonical HIF regulation, which is independent of EGL-9 catalytic activity but requires SWAN-1, controls the gene-repressive activity of HIF. The signal(s) that regulate noncanonical signaling are currently not known. <b>B.</b> Diagram of predicted and observed phenotypes in mutants defective in canonical or noncanonical HIF signaling.</p