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

An image analysis toolbox for high-throughput C. elegans assays

We present a toolbox for high-throughput screening of image-based Caenorhabditis elegans phenotypes. The image analysis algorithms measure morphological phenotypes in individual worms and are effective for a variety of assays and imaging systems. This WormToolbox is available through the open-source CellProfiler project and enables objective scoring of whole-worm high-throughput image-based assays of C. elegans for the study of diverse biological pathways that are relevant to human disease.National Institutes of Health (U.S.) (U54 EB005149

GTPase Rac1 et ubiquitination

Cette thèse a été consacrée à l étude de la régulation par ubiquitination d une protéine de signalisation cellulaire, la GTPase Rac1. J ai montré que l ubiquitination dégradative de Rac1 affecte peu son variant d épissage Rac1b, et qu elle requiert l'activité JNK, stimulée par Rac1 mais non par Rac1b. En parallèle, j ai mis en évidence une ubiquitination non dégradative de Rac1 qui pourrait contribuer à l internalisation bactérienne lors de l invasion. En recherchant l enzyme responsable de l ubiquitination spécifique de Rac1, j ai pu identifier la protéine à domaine RING finger Unkempt comme un nouvel effecteur de Rac1. Cette ubiquitine ligase potentielle, activée par Rac1, serait impliquée dans l ubiquitination du facteur BAF60b appartenant au complexe chromatinien SWI/SNF. J ai par ailleurs observé que Rac1 stimule la mono-ubiquitination de l histone H2A. Ainsi, la GTPase Rac1 serait impliquée dans une ou plusieurs voie(s) de signalisation inédite(s) contrôlant le remodelage de la chromatineThis thesis has been dedicated to the study of the regulation by ubiquitination of a signaling protein, the Rac1 GTPase. I have shown that the degradative ubiquitination of Rac1 affects poorly its splice variant Rac1b, and requires JNK activity, which is stimulated by Rac1 but not by Rac1b. In addition, I have described a non-degradative ubiquitination of Rac1, which could participate in pathogen endocytosis during bacterial infection. Searching for the enzyme responsible for specific Rac1 ubiquitination, I have identified a RING finger protein, Unkempt, as a new effector of Rac1. I have shown that this potential ubiquitin ligase, which is activated by Rac1, could be involved in the ubiquitination of BAF60b, a component of the chromatin remodeling complex SWI/SNF. Moreover, I have observed that Rac1 stimulates histone H2A mono-ubiquitination. Thus, Rac1 GTPase could be involved in novel pathways by controlling chromatin remodelingPARIS-BIUP (751062107) / SudocSudocFranceF

Ubiquitin-related processes and innate immunity in C. elegans

International audienceInnate immunity is an evolutionary ancient defence strategy that serves to eliminate infectious 19 agents while maintaining host health. It involves a complex network of sensors, signaling proteins 20 and immune effectors that detect the danger, then relay and execute the immune programme. Post-21 translational modifications relying on conserved ubiquitin and ubiquitin-like proteins are an integral 22 part of the system. Studies using invertebrate models of infection, such as the nematode 23 Caenorhabditis elegans, have greatly contributed to our understanding of how ubiquitin-related 24 processes act in immune sensing, regulate immune signaling pathways, and participate to host 25 defence responses. This review highlights the interest of working with a genetically tractable model 26 organism and illustrates how C. elegans has been used to identify ubiquitin-dependent immune 27 mechanisms, discover novel ubiquitin-based resistance strategies that mediate pathogen clearance, 28 and unravel the role of ubiquitin-related processes in tolerance, preserving host fitness during 29 pathogen attack. Special emphasis is placed on processes that are conserved in mammals

An Evolutionarily Conserved PLC-PKD-TFEB Pathway for Host Defense

The mechanisms that tightly control the transcription of host defense genes have not been fully elucidated. We previously identified TFEB as a transcription factor important for host defense, but the mechanisms that regulate TFEB during infection remained unknown. Here, we used C. elegans to discover a pathway that activates TFEB during infection. Gene dkf-1, which encodes a homolog of protein kinase D (PKD), was required for TFEB activation in nematodes infected with Staphylococcus aureus. Conversely, pharmacological activation of PKD was sufficient to activate TFEB. Furthermore, phospholipase C (PLC) gene plc-1 was also required for TFEB activation, downstream of Gαq homolog egl-30 and upstream of dkf-1. Using reverse and chemical genetics, we discovered a similar PLC-PKD-TFEB axis in Salmonella-infected mouse macrophages. In addition, PKCα was required in macrophages. These observations reveal a previously unknown host defense signaling pathway, which has been conserved across one billion years of evolution

Group-I PAKs-mediated phosphorylation of HACE1 at serine 385 regulates its oligomerization state and Rac1 ubiquitination

International audienc

Decrease of Staphylococcus aureus Virulence by Helcococcus kunzii in a Caenorhabditis elegans Model

International audienceSocial bacterial interactions are considered essential in numerous infectious diseases, particularly in wounds. Foot ulcers are a common complication in diabetic patients and these ulcers become frequently infected. This infection is usually polymicrobial promoting cell-to-cell communications. Staphylococcus aureus is the most prevalent pathogen isolated. Its association with Helcococcus kunzii, commensal Gram-positive cocci, is frequently described. The aim of this study was to assess the impact of co-infection on virulence of both H. kunzii and S. aureus strains in a Caenorhabditis elegans model. To study the host response, qRT-PCRs targeting host defense genes were performed. We observed that H. kunzii strains harbored a very low (LT50: 5.7 days ± 0.4) or an absence of virulence (LT50: 6.9 days ± 0.5). In contrast, S. aureus strains (LT50: 2.9 days ± 0.4) were significantly more virulent than all H. kunzii (P < 0.001). When H. kunzii and S. aureus strains were associated, H. kunzii significantly reduced the virulence of the S. aureus strain in nematodes (LT50 between 4.4 and 5.2 days; P < 0.001). To evaluate the impact of these strains on host response, transcriptomic analysis showed that the ingestion of S. aureus led to a strong induction of defense genes (lys-5, sodh-1, and cyp-37B1) while H. kunzii did not. No statistical difference of host response genes expression was observed when C. elegans were infected with either S. aureus alone or with S. aureus + H. kunzii. Moreover, two well-characterized virulence factors (hla and agr) present in S. aureus were down-regulated when S. aureus were co-infected with H. kunzii. This study showed that H. kunzii decreased the virulence of S. aureus without modifying directly the host defense response. Factor(s) produced by this bacterium modulating the staphylococci virulence must be investigated

<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

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

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