Plasticity of the β-Trefoil Protein Fold in the Recognition and Control of Invertebrate Predators and Parasites by a Fungal Defence System

Discrimination between self and non-self is a prerequisite for any defence mechanism; in innate defence, this discrimination is often mediated by lectins recognizing non-self carbohydrate structures and so relies on an arsenal of host lectins with different specificities towards target organism carbohydrate structures. Recently, cytoplasmic lectins isolated from fungal fruiting bodies have been shown to play a role in the defence of multicellular fungi against predators and parasites. Here, we present a novel fruiting body lectin, CCL2, from the ink cap mushroom Coprinopsis cinerea. We demonstrate the toxicity of the lectin towards Caenorhabditis elegans and Drosophila melanogaster and present its NMR solution structure in complex with the trisaccharide, GlcNAcβ1,4[Fucα1,3]GlcNAc, to which it binds with high specificity and affinity in vitro. The structure reveals that the monomeric CCL2 adopts a β-trefoil fold and recognizes the trisaccharide by a single, topologically novel carbohydrate-binding site. Site-directed mutagenesis of CCL2 and identification of C. elegans mutants resistant to this lectin show that its nematotoxicity is mediated by binding to α1,3-fucosylated N-glycan core structures of nematode glycoproteins; feeding with fluorescently labeled CCL2 demonstrates that these target glycoproteins localize to the C. elegans intestine. Since the identified glycoepitope is characteristic for invertebrates but absent from fungi, our data show that the defence function of fruiting body lectins is based on the specific recognition of non-self carbohydrate structures. The trisaccharide specifically recognized by CCL2 is a key carbohydrate determinant of pollen and insect venom allergens implying this particular glycoepitope is targeted by both fungal defence and mammalian immune systems. In summary, our results demonstrate how the plasticity of a common protein fold can contribute to the recognition and control of antagonists by an innate defence mechanism, whereby the monovalency of the lectin for its ligand implies a novel mechanism of lectin-mediated toxicity

The Roles and Interactions of Symbiont, Host and Environment in Defining Coral Fitness

Background: Reef-building corals live in symbiosis with a diverse range of dinoflagellate algae (genus Symbiodinium) that differentially influence the fitness of the coral holobiont. The comparative role of symbiont type in holobiont fitness in relation to host genotype or the environment, however, is largely unknown. We addressed this knowledge gap by manipulating host-symbiont combinations and comparing growth, survival and thermal tolerance among the resultant holobionts in different environments.\ud Methodology/Principal Findings: Offspring of the coral, Acropora millepora, from two thermally contrasting locations, were experimentally infected with one of six Symbiodinium types, which spanned three phylogenetic clades (A, C and D), and then outplanted to the two parental field locations (central and southern inshore Great Barrier Reef, Australia). Growth and survival of juvenile corals were monitored for 31–35 weeks, after which their thermo-tolerance was experimentally assessed. Our results showed that: (1) Symbiodinium type was the most important predictor of holobiont fitness, as measured by growth, survival, and thermo-tolerance; (2) growth and survival, but not heat-tolerance, were also affected by local environmental conditions; and (3) host population had little to no effect on holobiont fitness. Furthermore, coral-algal associations were established with symbiont types belonging to clades A, C and D, but three out of four symbiont types belonging to clade C failed to establish a symbiosis. Associations with clade A had the lowest fitness and were unstable in the field. Lastly, Symbiodinium types C1 and D were found to be relatively thermo-tolerant, with type D conferring the highest tolerance in A. millepora.\ud Conclusions/Significance: These results highlight the complex interactions that occur between the coral host, the algal symbiont, and the environment to shape the fitness of the coral holobiont. An improved understanding of the factors affecting coral holobiont fitness will assist in predicting the responses of corals to global climate change

Refining the specificity of the CCL2 lectin.

<p>(A) The chemical and schematic structure of the fucosylated chitobiose (GlcNAcβ1,4[Fucα1,3]GlcNAc-spacer) that was used as ligand for binding studies and structure determination. Indicated is also the B face that is defined as the face on which the carbons are numbered in an anticlockwise order <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002706#ppat.1002706-Taylor1" target="_blank">[69]</a>. (B) Chemical shift deviations upon complex formation at a protein concentration of 0.4 mM at pH 5.7. Overlay of ¹⁵N-HSQC spectra of free CCL2 (blue) and CCL2 bound to one equivalent of fucosylated chitobiose (red). (C) Titration of the amide signal of T111 in CCL2 with fucosylated chitobiose using ¹⁵N-HSQC spectra. The protein∶ligand ratio is displayed on the left. (D) Plot of the chemical shift differences between free and bound CCL2 ( δ = [ δ_HN²+(δ_N/R_scale)² ]^1/2, R_scale = 5).</p

Sequence conservation among CCL2-like proteins and comparison to two typical representatives of fungi and plants.

<p>Sequence alignment of several fungal and plant R-type lectins. CCL2_A: CCL2 of <i>C. cinerea</i> strain AmutBmut; CCL2_O: CCL2 of <i>C. cinerea</i> strain Okayama7; CCL1_A: CCL1 of <i>C. cinerea</i> strain AmutBmut; CCL1_O: CCL1 of <i>C. cinerea</i> strain Okayama7; PP_L1: <i>Postia placenta</i> lectin 1 (Pospl1_130016); PP_L2: <i>Postia placenta</i> lectin 2 (Pospl1_121916); SL_L1: <i>Serpula lacrymans</i> lectin 1 (SerlaS7_144703); CP_L1: <i>Coniophora puteana</i> lectin 1 (Conpu1_119225); PO_L1: <i>Pleurotus ostreatus</i> lectin 1 (PleosPC9_89828); PO_L2: <i>Pleurotus ostreatus</i> lectin 2 (PleosPC15_1043947); PO_L3: <i>Pleurotus ostreatus</i> lectin 3 (PleosPC9_64199); PO_L4: <i>Pleurotus ostreatus</i> lectin 4 (PleosPC15_1065820); DS_L1: <i>Dicomitus squalis</i> lectin 1 (Dicsq1); AO_L1: <i>Arthrobotrys oligospora</i> lectin 1 (s00075g2); LB_L1: <i>Laccaria bicolor</i> lectin 1 (Lbic_330799); LB_L2: <i>Laccaria bicolor</i> lectin 2 (Lbic_327918); MOA: <i>Marasmius oreades</i> agglutinin; SNA-II: <i>Sambucus nigra</i> agglutinin/ribosome inactivating protein type II. The distantly related canonical R-type lectins MOA (fungal, 14% sequence identity) and SNA-II (plant, 13% sequence identity) were included in the alignment based on comparison of their 3D structures <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002706#ppat.1002706-Grahn2" target="_blank">[70]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002706#ppat.1002706-Maveyraud1" target="_blank">[71]</a>. The Clustal X color scheme was used. Residues involved in the carbohydrate recognition are indicated at the bottom for CCL2, MOA and SNA-II. The secondary structure of CCL2 and the conservation is indicated as well. The alignment was generated with Jalview <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002706#ppat.1002706-Waterhouse1" target="_blank">[72]</a>.</p

Carbohydrate-binding dependent biotoxicity of CCL2.

<p>(A) Schematic representation of N-glycan structures in plants, insects and nematodes. Upper panel, left: Typical paucimannosidic plant N-glycan, highly abundant in HRP. Upper panel right: Fucosylated paucimannosidic N-glycan present in <i>D. melanogaster</i>. Lower panel: Fucose biosynthesis and N-glycan structure in <i>C. elegans</i>. Genes coding for enzymes involved in the fucose biosynthesis (lower panel, left) and fucose transfer to the core of N-glycans in <i>C. elegans</i> (lower panel, right) are indicated in dashed boxes. (B) Toxicity of recombinant <i>E. coli</i> expressing CCL2 (black bars) towards <i>C. elegans</i> wildtype (N2) and various fucosylation mutants. Error bars indicate standard errors of the mean. Asterisks (*) show cases where all data were 0. Significant differences were observed between the vector control and CCL2 for N2 (n = 10, p = 0.013), <i>fut-1(ok892)</i> (n = 10, p = 0.013) and <i>fut-6(ok475)</i> (n = 10, p = 0.013) worms, but not for <i>bre-1(ye4)</i> (n = 10, p = 0.329) or <i>fut-6(ok475)fut-1(ok892)</i> (n = 10, p = 0.329). (C) Fluorescence microscopy of <i>C. elegans</i> feeding on <i>E. coli</i> expressing a dTomato-CCL2 fusion protein, showing the grinder and anterior part of the intestine. (D) Toxicity of purified CCL2 towards <i>D. melanogaster</i> quantified as number of developed pupae (gray bars) or flies (black bars). BSA was included as control. Error bars indicate standard errors of the mean. Development of pupae and flies treated with CCL2 were significantly different from the control (pupae: n = 10, p = 0.013; flies: n = 10, p = 0.013). (E) Toxicity of <i>E. coli</i> expressing different CCL2 variants with mutations in residues involved in carbohydrate binding towards <i>C. elegans</i> wildtype (N2). Vector control and CCL2 wildtype (WT) were included as controls. Asterisks (*) show cases where all data were 0. Error bars indicate standard error of the mean. W78A, Y92A and W94A were significantly different from WT control (n = 10, p = 0.013), whereas L87A, N91A, V93A were not (n = 10, p = 1.0).</p

Potential intermolecular protein–carbohydrate hydrogen bonds based on the orientations and positions of the carbohydrate in the complex structure.

<p>Note that no intermolecular hydrogen bond constraints were used during the calculations.</p

Solution structure of the CCL2 lectin in the absence of a ligand determined by NMR spectroscopy.

<p>The side (A) and top (B) view of the most representative structure out of 20 structures is shown. The three pseudo symmetric sections of the β-trefoil fold corresponding to residues S9–N60, S61–S100 and G101–V142 are colored green, yellow and orange, respectively. Characteristic regions are labeled according to Renko et al. for better orientation <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002706#ppat.1002706-Renko1" target="_blank">[26]</a>. (C) Chemical shift deviations mapped on the structure of CCL2 in the same orientation as in A. Chemical shifts of residues in red experience a combined NH chemical shift deviation >0.4 ppm, for residues in pink >0.15 ppm. (D) Secondary structure and subdomain borders displayed on the protein sequence. The same color code as in A and B is used. Bold residues are forming the hydrophobic core of the protein.</p