The Hedgehog Signaling Pathway Emerges as a Pathogenic Target

The Hedgehog (Hh) signaling pathway plays an essential role in the growth, development, and homeostatis of many tissues in vertebrates and invertebrates. Much of what is known about Hh signaling is in the context of embryonic development and tumor formation. However, a growing body of evidence is emerging indicating that Hh signaling is also involved in postnatal processes such as tissue repair and adult immune responses. To that extent, Hh signaling has also been shown to be a target for some pathogens that presumably utilize the pathway to control the local infected environment. In this review, we discuss what is currently known regarding pathogenic interactions with Hh signaling and speculate on the reasons for this pathway being a target. We also hope to shed light on the possibility of using small molecule modulators of Hh signaling as effective therapies for a wider range of human diseases beyond their current use in a limited number of cancers

Processing of the Drosophila Hedgehog Signaling Effector Ci-155 to the Repressor Ci-75 Is Mediated by Direct Binding to the SCF Component Slimb

SummarySignaling by extracellular Hedgehog (Hh) molecules is crucial for the correct allocation of cell fates and patterns of cell proliferation in humans and other organisms [1, 2]. Responses to Hh are universally mediated by regulating the activity and the proteolysis of the Gli family of transcriptional activators such that they induce target genes only in the presence of Hh [1, 3]. In the absence of Hh, the sole Drosophila Gli homolog, Cubitus interruptus (Ci), undergoes partial proteolysis to Ci-75, which represses key Hh target genes [4]. This processing requires phosphorylation of full-length Ci (Ci-155) by protein kinase A (PKA), casein kinase 1 (CK1), and glycogen synthase kinase 3 (GSK3), as well as the activity of Slimb [5–7]. Slimb is homologous to vertebrate β-TRCP1, which binds as part of an SCF (Skp1/Cullin1/F-box) complex to a defined phosphopeptide motif to target proteins for ubiquitination and subsequent proteolysis [8–10]. Here, we show that phosphorylation of Ci at the specific PKA, GSK-3, and CK1 sites required in vivo for partial proteolysis stimulates binding to Slimb in vitro. Furthermore, a consensus Slimb/β-TRCP1 binding site from another protein can substitute for phosphorylated residues of Ci-155 to direct conversion to Ci-75 in vivo. From this, we conclude that Slimb binds directly to phosphorylated Ci-155 to initiate processing to Ci-75. We also explore the phosphorylated motifs in Ci that are recognized by Slimb and provide some evidence that silencing of Ci-155 by phosphorylation may involve more than binding to Slimb

Ubiquitin-Mediated Response to Microsporidia and Virus Infection in <i>C. elegans</i>

<div><p>Microsporidia comprise a phylum of over 1400 species of obligate intracellular pathogens that can infect almost all animals, but little is known about the host response to these parasites. Here we use the whole-animal host <i>C. elegans</i> to show an <i>in vivo</i> role for ubiquitin-mediated response to the microsporidian species <i>Nematocida parisii</i>, as well to the Orsay virus, another natural intracellular pathogen of <i>C. elegans</i>. We analyze gene expression of <i>C. elegans</i> in response to <i>N. parisii</i>, and find that it is similar to response to viral infection. Notably, we find an upregulation of SCF ubiquitin ligase components, such as the cullin ortholog <i>cul-6</i>, which we show is important for ubiquitin targeting of <i>N. parisii</i> cells in the intestine. We show that ubiquitylation components, the proteasome, and the autophagy pathway are all important for defense against <i>N. parisii</i> infection. We also find that SCF ligase components like <i>cul-6</i> promote defense against viral infection, where they have a more robust role than against <i>N. parisii</i> infection. This difference may be due to suppression of the host ubiquitylation system by <i>N. parisii</i>: when <i>N. parisii</i> is crippled by anti-microsporidia drugs, the host can more effectively target pathogen cells for ubiquitylation. Intriguingly, inhibition of the ubiquitin-proteasome system (UPS) increases expression of infection-upregulated SCF ligase components, indicating that a trigger for transcriptional response to intracellular infection by <i>N. parisii</i> and virus may be perturbation of the UPS. Altogether, our results demonstrate an <i>in vivo</i> role for ubiquitin-mediated defense against microsporidian and viral infections in <i>C. elegans</i>.</p></div

Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Pfam domain (PF) enrichment analysis of <i>C. elegans</i> genes upreguated by <i>N. parisii</i> infection.

<p>GO term, KEGG pathway, and Pfam domain enrichment was analyzed using online DAVID Bioinformatics Resources 6.7. To eliminate redundancy, each term had at least 30% of associated genes not associated with any other term with a more significant P-value. *For each gene group the number of genes included in the analysis out of the total differentially expressed genes is indicated.</p

Model for SCF E3 ligases and ubiquitin-mediated responses to intracellular infection in <i>C. elegans</i>.

<p>Intracellular microsporidia or viral infection triggers the expression of SCF ligase components in <i>C. elegans</i>, including a large number of F-box genes, the cullin <i>cul-6</i>, and Skp1-related genes, <i>skr-3</i>, <i>-4</i>, and <i>-5</i>. Due to the modularity of the SCF ligase complex, many SCF ligases with vast substrate recognition potential may be formed, which could recognize pathogen-derived proteins or host proteins. Ubiquitylation of substrates leads to their degradation by the proteasome or by autophagy, with large substrates such as microsporidia cells, potentially viral particles, and protein aggregates (not shown), targeted by autophagy, and individual pathogen or host proteins by the proteasome. <i>N. parisii</i> parasite cells may be able to suppress or evade ubiquitylation. Both intracellular infection and UPS stress can induce SCF ligase components, and greater demand on the UPS during intracellular infection may contribute to upregulation of SCF ligase components. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004200#s3" target="_blank">Discussion</a> for more details.</p

UPS perturbation induces similar gene expression responses to <i>N. parisii</i> infection.

<p>A) Expression of <i>C17H1.6p::gfp</i> and <i>F26F2.1p::gfp</i> in the intestine (pharyngeal <i>myo-2p::mCherry</i> expression is a marker for the presence of the transgene) following infection with <i>N. parisii</i> (8 and 24 hpi). Scale bars  = 100 µm. B) Expression of <i>C17H1.6p::gfp</i> and <i>F26F2.1p::gfp</i> following RNAi against <i>ubq-1, ubq-2</i>, <i>pas-5</i> and <i>rpn-2</i> in the absence of infection. C) Expression of endogenous mRNA of <i>C17H1.6</i> and <i>F26F2.1</i>, as well as the SCF ligase components <i>skr-1</i>, <i>skr-3</i>, <i>skr-4</i>, <i>skr-5</i> and <i>cul-6</i> following RNAi against <i>ubq-1, ubq-2</i>, <i>pas-5</i> and <i>rpn-2</i> in the absence of infection, as assessed by qRT-PCR. Due to the very large changes in expression of <i>C17H1.6</i> and <i>F26F2.1</i> genes, these are presented on a separate graph to allow for expansion of the y-axis and easier observation of expression changes in SCF ligase components. Mean +/- SEM of two to three independent experiments.</p

<i>C. elegans</i> gene expression during infection with <i>N. parisii</i>.

<p>A) Diagram of <i>N. parisii</i> infection stages in <i>C. elegans</i> intestinal cells. B) Synchronized populations of <i>fer-15;fem-1</i> sterile animals were inoculated with <i>N. parisii</i> spores and collected for RNA extraction at timepoints corresponding to specific stages of infection. Uninfected controls were included for each timepoint. C) Number of significantly (FDR<0.05) up- or downregulated <i>C. elegans</i> genes during infection with <i>N. parisii</i>. D) Proportion of intestine- and germline-associated <i>C. elegans</i> genes with significantly altered expression at each timepoint. The “reference” bars indicate intestine or germline associated genes as a percentage of the <i>C. elegans</i> genome (20,404 genes). At each timepoint, 39% to 56% of all highly regulated genes were associated with the intestine, which represents a significant enrichment (chi-squared test, p<1.03E-26, all comparisons), while germline genes were significantly underrepresented (chi-squared test, p<1.51E-05, all comparisons) (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004200#ppat-1004200-g001" target="_blank">Figure 1D</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004200#ppat.1004200.s011" target="_blank">Table S2</a>). E) Correlations between genes regulated by <i>N. parisii</i> infection and genes upregulated by other pathogens, stressors and immunity pathways. Gene sets were compared using the GSEA software (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004200#ppat.1004200.s014" target="_blank">Table S5</a> for detailed summary of results) and normalized enrichment scores (NESs) with a relaxed significance threshold (FDR<0.25, p<0.05) are reported in the figure. A positive NES (yellow) indicates a correlation with genes upregulated in response to <i>N. parisii</i> infection, while a negative NES (blue) indicates a correlation with genes downregulated in response to <i>N. parisii</i> infection (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004200#s4" target="_blank">Materials and Methods</a> for analysis details). Black indicates no significant (FDR<0.25, p<0.05) correlation, and an NES with FDR<0.05 is indicated with an asterisk.</p

<i>N. parisii</i> cells are almost never targeted by ubiquitin later during infection, but ubiquitin forms clusters that accumulate in the <i>C. elegans</i> intestine.

<p>A,B) <i>C. elegans</i> intestines stained an anti-conjugated-ubiquitin antibody FK2 (green), and DAPI for DNA (blue): Panel B also includes a FISH probe against <i>N. parisii</i> rRNA (red). A) Sections of uninfected and <i>N. parisii</i>-infected <i>C. elegans</i> intestines. In the infected intestine an <i>N. parisii</i> spore labeled with the anti-conjugated-ubiquitin antibody is enlarged in box inset in upper left, and meront (arrowhead), and host nucleus (N) are indicated. Scale bar  = 10 µm. B) Sections of uninfected and <i>N. parisii</i>-infected <i>C. elegans</i> intestines (30 hpi) shown with ubiquitin cluster (arrow) and ubiquitin staining within an <i>N. parisii</i> meront (arrowhead) indicated. Scale bar  = 10 µm. C) Intestines of uninfected and infected animals expressing an intestinal GFP::ubiquitin transgene (48 hpi, grown at 20°C to prevent construct aggregation) are shown. Small GFP::ubiquitin aggregates are sometimes observed in uninfected animals (arrow). Scale bar  = 20 µm. D) Enlarged portion of box in panel C. Oblong <i>N. parisii</i> meronts are visible through the absence of green (arrowhead) and ubiquitin clusters associating with the meronts (arrow) are indicated. Scale bar  = 10 µm. E) Animals expressing the intestinal GFP::ubiquitin construct were infected with <i>N. parisii</i> and, together with control uninfected animals, fixed at the indicated times. Fixed animals were stained with a FISH probe against <i>N. parisii</i> rRNA to mark the infection and their intestinal cells were inspected for visible GFP::ubiquitin aggregates (30 transgenic animals were inspected per timepoint and condition). F) Animals expressing the intestinal control GFP::ubiquitinΔGG construct were treated and analyzed as in E.</p

Ubiquitin-mediated host response and defense against Orsay viral infection in <i>C. elegans</i>.

<p>A) Viral pathogen load in nematodes treated with RNAi against the SCF ligase components <i>skr-3</i>, <i>skr-4, skr-5</i>, and <i>cul-6</i> compared to vector control RNAi (L4440), as assessed by qRT-PCR for viral transcript. Mean +/− SEM of three independent experiments shown. B) Viral pathogen load in nematodes treated with RNAi against <i>ubq-2</i>, <i>pas-5</i> and <i>rpn-2</i> analyzed as above. Mean +/− SEM of three independent experiments shown. C) Intestines of <i>F26F2.1p::gfp</i> transgenic animals were stained with the FK2 antibody against conjugated ubiquitin (red), and DAPI for DNA (blue). Intestines outlined with white dotted line. Animals infected with virus have increased ubiquitin clustering in some intestinal cells compared to uninfected animals (arrowhead), and also express the GFP reporter. Scale bar  = 20 µm. ** p<0.01.</p

<i>N. parisii</i> cells are targeted by host ubiquitin early during infection.

<p>A–C) <i>C. elegans</i> intestines stained with a FISH probe against <i>N. parisii</i> rRNA (red), and DAPI for DNA (blue). A section of the image outlined by a dotted square is enlarged on the right and shows both an <i>N. parisii</i> parasite cell that colocalizes with ubiquitin (arrow), and one that does not (arrowhead). A) Animals were stained with an anti-conjugated-ubiquitin antibody, FK2 (green), and B, C) Transgenic <i>C. elegans</i> intestines expressing wild-type (B) or conjugation-defective mutant (C) GFP::ubiquitin (green). For A–C, scale bar  = 10 µm in main images and 2 µm in enlarged sections. D) Quantification of parasite cell colocalization at 12 hpi (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004200#s4" target="_blank">Materials and Methods</a> for timepoint information) with FK2 antibody in the presence of increasing doses of fumagillin. Difference is significant: chi-squared test, p<6.3E-28, all comparisons. E) Quantification of parasite cell colocalization at 15 hpi with wild-type or mutant GFP::ubiquitin in the presence of increasing doses of fumagillin. Mean +/− SEM of two independent experiments is shown. F) Quantification of parasite cell colocalization at 15 hpi with wild-type GFP::ubiquitin following knockdown of <i>cul-6</i> RNAi compared to L4440 vector control. Targeting of ubiquitin to parasite cells was less robust and more variable in animals feeding on HT115 RNAi bacteria compared to OP50-1 <i>E. coli</i>, ranging from 10.6% to 2.2% in control animals, and 2.1% to 1.8% in <i>cul-6</i> RNAi treated animals and the data presented are normalized for the average level of targeting in each independent experiment. Mean +/− SEM of three independent experiments is shown. Number of individual parasite cells assessed for colocalization with ubiquitin is indicated.</p