Novel antiinflammatory biologics shaped by parasite-host coevolution.

Parasitic helminth infections, while a major cause of neglected tropical disease burden, negatively correlate with the incidence of immune-mediated inflammatory diseases such as inflammatory bowel diseases (IBD). To evade expulsion, helminths have developed sophisticated mechanisms to regulate their host's immune responses. Controlled experimental human helminth infections have been assessed clinically for treating inflammatory conditions; however, such a radical therapeutic modality has challenges. An alternative approach is to harness the immunomodulatory properties within the worm's excretory-secretory (ES) complement, its secretome. Here, we report a biologics discovery and validation pipeline to generate and screen in vivo a recombinant cell-free secretome library of helminth-derived immunomodulatory proteins. We successfully expressed 78 recombinant ES proteins from gastrointestinal hookworms and screened the crude in vitro translation reactions for anti-IBD properties in a mouse model of acute colitis. After statistical filtering and ranking, 20 proteins conferred significant protection against various parameters of colitis. Lead candidates from distinct protein families, including annexins, transthyretins, nematode-specific retinol-binding proteins, and SCP/TAPS were identified. Representative proteins were produced in mammalian cells and further validated, including ex vivo suppression of inflammatory cytokine secretion by T cells from IBD patient colon biopsies. Proteins identified herein offer promise as novel, safe, and mechanistically differentiated biologics for treating the globally increasing burden of inflammatory diseases.

The house dust mite allergen Der p 5 binds lipid ligands and stimulates airway epithelial cells through a TLR2-dependent pathway.

BACKGROUND: Protein crystallographic studies suggest that the house dust mite (HDM) allergen Der p 5 potentially interacts with hydrophobic ligands. Der p 5, in association with its ligand(s), might therefore trigger innate immune signalling pathways in the airway epithelium and influence the initiation of the HDM-allergic response. OBJECTIVE: We investigated the lipid binding propensities of recombinant (r)Der p 5 and characterized the signalling pathways triggered by the allergen in airway epithelial cells. METHODS: rDer p 5 was produced in Pichia pastoris and characterized by mass spectrometry, multi-angle light scattering and circular dichroism. Its interactions with hydrophobic ligands were investigated in fluorescence-based lipid binding assays and in-silico docking simulations. Innate immune signalling pathways triggered by rDer p 5 were investigated in airway epithelial cell activation assays in vitro. RESULTS: Biophysical analysis showed that rDer p 5 was monomeric and adopted a similar α-helix-rich fold at both physiological and acidic pH. Spectrofluorimetry experiments showed that rDer p 5 is able to selectively bind lipid ligands, but only under mild acidic pH conditions. Computer-based docking simulations identified potential binding sites for these ligands. This allergen, with putatively associated lipid(s), triggered the production of IL-8 in respiratory epithelial cells through a TLR2-, NF-kB- and MAPK-dependent signalling pathway. CONCLUSIONS AND CLINICAL RELEVANCE: Despite the fact that Der p 5 represents a HDM allergen of intermediate prevalence, our findings regarding its lipid binding and activation of TLR2 indicate that it could participate in the initiation of the HDM-allergic state.

Adsorption of the natural protein surfactant Rsn-2 onto liquid interfaces.

To stabilize foams, droplets and films at liquid interfaces a range of protein biosurfactants have evolved in nature. Compared to synthetic surfactants, these combine surface activity with biocompatibility and low solution aggregation. One recently studied example is Rsn-2, a component of the foam nest of the frog Engystomops pustulosus, which has been predicted to undergo a clamshell-like opening transition at the air-water interface. Using atomistic molecular dynamics simulations and surface tension measurements we study the adsorption of Rsn-2 onto air-water and cyclohexane-water interfaces. The protein adsorbs readily at both interfaces, with adsorption mediated by the hydrophobic N-terminus. At the cyclohexane-water interface the clamshell opens, due to the favourable interaction between hydrophobic residues and cyclohexane molecules and the penetration of cyclohexane molecules into the protein core. Simulations of deletion mutants showed that removal of the N-terminus inhibits interfacial adsorption, which is consistent with the surface tension measurements. Deletion of the hydrophilic C-terminus also affects adsorption, suggesting that this plays a role in orienting the protein at the interface. The characterisation of the interfacial behaviour gives insight into the factors that control the interfacial adsorption of proteins, which may inform new applications of this and similar proteins in areas including drug delivery and food technology and may also be used in the design of synthetic molecules showing similar changes in conformation at interfaces.

Frog foams and natural protein surfactants.

Foams and surfactants are relatively rare in biology because of their potential to harm cell membranes and other delicate tissues. However, in recent work we have identified and characterized a number of natural surfactant proteins found in the foam nests of tropical frogs and other unusual sources. These proteins, and their associated foams, are relatively stable and bio-compatible, but with intriguing molecular structures that reveal a new class of surfactant activity. Here we review the structures and functional mechanisms of some of these proteins as revealed by experiments involving a range of biophysical and biochemical techniques, with additional mechanistic support coming from more recent site-directed mutagenesis studies.

Diversity in the structures and ligand-binding sites of nematode fatty acid and retinol-binding proteins revealed by Na-FAR-1 from Necator americanus.

Fatty acid and retinol-binding proteins (FARs) comprise a family of unusual α-helix rich lipid-binding proteins found exclusively in nematodes. They are secreted into host tissues by parasites of plants, animals and humans. The structure of a FAR protein from the free-living nematode Caenorhabditis elegans is available, but this protein [C. elegans FAR-7 (Ce-FAR-7)] is from a subfamily of FARs that does not appear to be important at the host/parasite interface. We have therefore examined [Necator americanus FAR-1 (Na-FAR-1)] from the blood-feeding intestinal parasite of humans, N. americanus. The 3D structure of Na-FAR-1 in its ligand-free and ligand-bound forms, determined by NMR (nuclear magnetic resonance) spectroscopy and X-ray crystallography respectively, reveals an α-helical fold similar to Ce-FAR-7, but Na-FAR-1 possesses a larger and more complex internal ligand-binding cavity and an additional C-terminal α-helix. Titration of apo-Na-FAR-1 with oleic acid, analysed by NMR chemical shift perturbation, reveals that at least four distinct protein-ligand complexes can be formed. Na-FAR-1 and possibly other FARs may have a wider repertoire for hydrophobic ligand binding, as confirmed in the present study by our finding that a range of neutral and polar lipids co-purify with the bacterially expressed recombinant protein. Finally, we show by immunohistochemistry that Na-FAR-1 is present in adult worms with a tissue distribution indicative of possible roles in nutrient acquisition by the parasite and in reproduction in the male.

Core and body surface temperatures of nesting leatherback turtles (Dermochelys coriacea).

Leatherback turtles (Dermochelys coriacea) are the largest species of marine turtle and the fourth most massive extant reptile. In temperate waters they maintain body temperatures higher than surrounding seawater through a combination of insulation, physiological, and behavioural adaptations. Nesting involves physical activity in addition to contact with warm sand and air, potentially presenting thermal challenges in the absence of the cooling effect of water, and data are lacking with which to understand their nesting thermal biology. Using non-contact methods (thermal imaging and infrared thermometry) to avoid any stress-related effects, we investigated core and surface temperature during nesting. The mean±SE core temperature was 31.4±0.05°C (newly emerged eggs) and was not correlated with environmental conditions on the nesting beach. Core temperature of leatherbacks was greater than that of hawksbill turtles (Eretmochelys imbricata) nesting at a nearby colony, 30.0±0.13°C. Body surface temperatures of leatherbacks showed regional variation, the lateral and dorsal regions of the head were warmest while the carapace was the coolest surface. Surface temperature increased during the early nesting phases, then levelled off or decreased during later phases with the rates of change varying between body regions. Body region, behavioural phase of nesting and air temperature were found to be the best predictors of surface temperature. Regional variation in surface temperature were likely due to alterations in blood supply, and temporal changes in local muscular activity of flippers during the different phases of nesting. Heat exchange from the upper surface of the turtle was dominated by radiative heat loss from all body regions and small convective heat gains to the carapace and front flippers.

Useable diffraction data from a multiple microdomain-containing crystal of Ascaris suum As-p18 fatty-acid-binding protein using a microfocus beamline.

As-p18 is a fatty-acid-binding protein from the parasitic nematode Ascaris suum. Although it exhibits sequence similarity to mammalian intracellular fatty-acid-binding proteins, it contains features that are unique to nematodes. Crystals were obtained, but initial diffraction data analysis revealed that they were composed of a number of `microdomains'. Interpretable data could only be collected using a microfocus beamline with a beam size of 12 × 8 µm.

Two crystal forms of a helix-rich fatty acid- and retinol-binding protein, Na-FAR-1, from the parasitic nematode Necator americanus.

Na-FAR-1 is an unusual α-helix-rich fatty acid- and retinol-binding protein from Necator americanus, a blood-feeding intestinal parasitic nematode of humans. It belongs to the FAR protein family, which is unique to nematodes; no structural information is available to date for FAR proteins from parasites. Crystals were obtained with two different morphologies that corresponded to different space groups. Crystal form 1 exhibited space group P432 (unit-cell parameters a = b = c = 120.80 Å, α = ß = γ = 90°) and diffracted to 2.5 Šresolution, whereas crystal form 2 exhibited space group F23 (unit-cell parameters a = b = c = 240.38 Å, α = ß = γ = 90°) and diffracted to 3.2 Šresolution. Crystal form 2 showed signs of significant twinning.

Latherin and other biocompatible surfactant proteins.

Horses and other equids are unusual in producing protein-rich sweat for thermoregulation, a major component of which is latherin, a highly surface-active, non-glycosylated protein that is a member of the PLUNC (palate, lung and nasal epithelium clone) family. Latherin produces a significant reduction in water surface tension at low concentrations (≤1 mg/ml), and probably acts as a wetting agent to facilitate evaporative cooling through a thick, waterproofed pelt. Latherin binds temporarily to hydrophobic surfaces, and so may also have a disruptive effect on microbial biofilms. It may consequently have a dual role in horse sweat in both evaporative cooling and controlling microbial growth in the pelt that would otherwise be resourced by nutrients in sweat. Latherin is also present at high levels in horse saliva, where its role could be to improve mastication of the fibrous diet of equids, and also to reduce microbial adherence to teeth and oral surfaces. Neutron reflection experiments indicate that latherin adsorbs to the air/water interface, and that the protein undergoes significant conformational change and/or partial unfolding during incorporation into the interfacial layer.

The polyprotein allergens of nematodes (NPAs) - structure at last, but still mysterious.

We are engaged in structural and functional studies of several types of lipid binding protein that are only found in nematodes. Amongst these are the nematode polyprotein allergens (NPAs) and we now report the solution structure of ABA-1A (As-NPA-A1), the most repeated unit within the NPA array of Ascaris suum, which is almost identical in amino acid sequence to that of Ascaris lumbricoides. The protein forms a slightly flattened, compact, globular fold consisting of a long central helix that participates in two flanking helical bundles. Two pockets lined with apolar amino acid sidechains are apparent, one in the carboxy-terminal region of the protein, and another smaller one in the amino-terminal region. The former appears to be the main site of fatty acid binding, and the latter may have different, though possibly overlapping, ligand binding propensities. The structure of the binding sites indicates that lipid ligands are anchored within them with their hydrophobic tails oriented towards the core of the protein and their polar headgroups bound to charged sidechains at the mouth of the pockets. The three-dimensional architectures of the amino- and carboxy-terminal halves of ABA-1A are closely similar, thereby strengthening the long-suspected idea that the repeated units of NPAs themselves originate from an ancient duplication event.

Frog nest foams exhibit pharmaceutical foam-like properties.

Foams have frequently been used as systems for the delivery of cosmetic and therapeutic molecules; however, there is high variability in the foamability and long-term stability of synthetic foams. The development of pharmaceutical foams that exhibit desirable foaming properties, delivering appropriate amounts of the active pharmaceutical ingredient (API) and that have excellent biocompatibility is of great interest. The production of stable foams is rare in the natural world; however, certain species of frogs have adopted foam production as a means of providing a protective environment for their eggs and larvae from predators and parasites, to prevent desiccation, to control gaseous exchange, to buffer temperature extremes, and to reduce UV damage. These foams show great stability (up to 10 days in tropical environments) and are highly biocompatible due to the sensitive nature of amphibian skin. This work demonstrates for the first time that nests of the túngara frog (Engystomops pustulosus) are stable ex situ with useful physiochemical and biocompatible properties and are capable of encapsulating a range of compounds, including antibiotics. These protein foam mixtures share some properties with pharmaceutical foams and may find utility in a range of pharmaceutical applications such as topical drug delivery systems.

Building a home from foam--túngara frog foam nest architecture and three-phase construction process.

Frogs that build foam nests floating on water face the problems of over-dispersion of the secretions used and eggs being dangerously exposed at the foam : air interface. Nest construction behaviour of túngara frogs, Engystomops pustulosus, has features that may circumvent these problems. Pairs build nests in periodic bursts of foam production and egg deposition, three discrete phases being discernible. The first is characterized by a bubble raft without egg deposition and an approximately linear increase in duration of mixing events with time. This phase may reduce initial over-dispersion of foam precursor materials until a critical concentration is achieved. The main building phase is marked by mixing events and start-to-start intervals being nearly constant in duration. During the final phase, mixing events do not change in duration but intervals between them increase in an exponential-like fashion. Pairs joining a colonial nesting abbreviate their initial phase, presumably by exploiting a pioneer pair's bubble raft, thereby reducing energy and material expenditure, and time exposed to predators. Finally, eggs are deposited only in the centre of nests with a continuously produced, approximately 1 cm deep egg-free cortex that protectively encloses hatched larvae in stranded nests.

Anisakis simplex: from obscure infectious worm to inducer of immune hypersensitivity.

Infection of humans with the nematode worm parasite Anisakis simplex was first described in the 1960s in association with the consumption of raw or undercooked fish. During the 1990s it was realized that even the ingestion of dead worms in food fish can cause severe hypersensitivity reactions, that these may be more prevalent than infection itself, and that this outcome could be associated with food preparations previously considered safe. Not only may allergic symptoms arise from infection by the parasites ("gastroallergic anisakiasis"), but true anaphylactic reactions can also occur following exposure to allergens from dead worms by food-borne, airborne, or skin contact routes. This review discusses A. simplex pathogenesis in humans, covering immune hypersensitivity reactions both in the context of a living infection and in terms of exposure to its allergens by other routes. Over the last 20 years, several studies have concentrated on A. simplex antigen characterization and innate as well as adaptive immune response to this parasite. Molecular characterization of Anisakis allergens and isolation of their encoding cDNAs is now an active field of research that should provide improved diagnostic tools in addition to tools with which to enhance our understanding of pathogenesis and controversial aspects of A. simplex allergy. We also discuss the potential relevance of parasite products such as allergens, proteinases, and proteinase inhibitors and the activation of basophils, eosinophils, and mast cells in the induction of A. simplex-related immune hypersensitivity states induced by exposure to the parasite, dead or alive.

Stockpiling by pups and self-sacrifice by their fasting mothers observed in birth to weaning serum metabolomes of Atlantic grey seals.

During the uniquely short lactations of true seals, pups acquire a greater proportion of maternal body resources, at a greater rate, than in any other group of mammals. Mothers in many species enter a period of anorexia but must preserve sufficient reserves to fuel hunting and thermoregulation for return to cold seas. Moreover, pups may undergo a period of development after weaning during which they have no maternal care or nutrition. This nutritionally closed system presents a potentially extreme case of conflict between maternal survival and adequate provisioning of offspring, likely presenting strains on their metabolisms. We examined the serum metabolomes of five mother and pup pairs of Atlantic grey seals, Halichoerus grypus, from birth to weaning. Changes with time were particularly evident in pups, with indications of strain in the fat and energy metabolisms of both. Crucially, pups accumulate certain compounds to levels that are dramatically greater than in mothers. These include compounds that pups cannot synthesise themselves, such as pyridoxine/vitamin B6, taurine, some essential amino acids, and a conditionally essential amino acid and its precursor. Fasting mothers therefore appear to mediate stockpiling of critical metabolites in their pups, potentially depleting their own reserves and prompting cessation of lactation.

Correction to 'Buried treasure-marine turtles do not "disguise" or "camouflage" their nests but avoid them and create a decoy trail'.

[This corrects the article DOI: 10.1098/rsos.200327.].

Buried treasure-marine turtles do not 'disguise' or 'camouflage' their nests but avoid them and create a decoy trail.

After laying their eggs and refilling the egg chamber, sea turtles scatter sand extensively around the nest site. This is presumed to camouflage the nest, or optimize local conditions for egg development, but a consensus on its function is lacking. We quantified activity and mapped the movements of hawksbill (Eretmochelys imbricata) and leatherback (Dermochelys coriacea) turtles during sand-scattering. For leatherbacks, we also recorded activity at each sand-scattering position. For hawksbills, we recorded breathing rates during nesting as an indicator of metabolic investment and compared with published values for leatherbacks. Temporal and inferred metabolic investment in sand-scattering was substantial for both species. Neither species remained near the nest while sand-scattering, instead moving to several other positions to scatter sand, changing direction each time, progressively displacing themselves from the nest site. Movement patterns were highly diverse between individuals, but activity at each sand-scattering position changed little between completion of egg chamber refilling and return to the sea. Our findings are inconsistent with sand-scattering being to directly camouflage the nest, or primarily for modifying the nest-proximal environment. Instead, they are consistent with the construction of a series of dispersed decoy nests that may reduce the discovery of nests by predators.

Ranaspumin-2: structure and function of a surfactant protein from the foam nests of a tropical frog.

Ranaspumin-2 (Rsn-2) is a monomeric, 11 kDa surfactant protein identified as one of the major foam nest components of the túngara frog (Engystomops pustulosus), with an amino acid sequence unlike any other protein described so far. We report here on its structure in solution as determined by high-resolution NMR analysis, together with investigations of its conformation and packing at the air-water interface using a combination of infrared and neutron reflectivity techniques. Despite the lack of any significant sequence similarity, Rsn-2 in solution adopts a compact globular fold characteristic of the cystatin family, comprising a single helix over a four-stranded sheet, in a motif not previously associated with surfactant activity. The NMR structure of Rsn-2 shows no obvious amphiphilicity that might be anticipated for a surfactant protein. This suggests that it must undergo a significant conformational change when incorporated into the air-water interface that may involve a hinge-bending, clamshell opening of the separate helix and sheet segments to expose hydrophobic faces to air while maintaining the highly polar surfaces in contact with the underlying water layer. This model is supported by direct observation of the relative orientations of secondary structure elements at the interface by infrared reflection absorption spectroscopy, and by protein packing densities determined from neutron reflectivity profiles.

Foam nest components of the túngara frog: a cocktail of proteins conferring physical and biological resilience.

The foam nests of the túngara frog (Engystomops pustulosus) form a biocompatible incubation medium for eggs and sperm while resisting considerable environmental and microbiological assault. We have shown that much of this behaviour can be attributed to a cocktail of six proteins, designated ranaspumins (Rsn-1 to Rsn-6), which predominate in the foam. These fall into two discernable classes based on sequence analysis and biophysical properties. Rsn-2, with an amphiphilic amino acid sequence unlike any hitherto reported, exhibits substantial detergent-like surfactant activity necessary for production of foam, yet is harmless to the membranes of eggs and spermatozoa. A further four (Rsn-3 to Rsn-6) are lectins, three of which are similar to fucolectins found in teleosts but not previously identified in a land vertebrate, though with a carbohydrate binding specificity different from previously described fucolectins. The sixth, Rsn-1, is structurally similar to proteinase inhibitors of the cystatin class, but does not itself appear to exhibit any such activity. The nest foam itself, however, does exhibit potent cystatin activity. Rsn-encoding genes are transcribed in many tissues of the adult frogs, but the full cocktail is present only in oviduct glands. Combinations of lectins and cystatins have known roles in plants and animals for defence against microbial colonization and insect attack. Túngara nest foam displays a novel synergy of selected elements of innate defence plus a specialized surfactant protein, comprising a previously unreported strategy for protection of unattended reproductive stages of animals.

Identification of a secreted fatty acid and retinol-binding protein (Hp-FAR-1) from Heligmosomoides polygyrus.

Hp-FAR-1 is a major, secreted antigen of the parasitic nematode Heligmosomoides polygyrus, a laboratory mouse model frequently used to study the cellular mechanisms of chronic helminth infections. The DNA encoding Hp-FAR-1 was recovered by screening a fourth larval (L4) H. polygyrus cDNA expression library using antibodies raised against L4 stage excretory/secretory (E/S) proteins. Predictions of secondary structure based on the Hp-FAR-1 amino acid sequence indicated that an alpha-helix predominates in Hp-FAR-1, possibly with some coiled-coil conformation, with no beta-structure. Fluorescence-based ligand binding analysis confirmed that the recombinant Hp-FAR-1 (rHp-FAR-1) binds the fluorescent fatty acid analog 11-((5-[dimethylaminoaphthalene-1-sulfonyl)amino)undecanoic acid (DAUDA), and by competition oleic acid. RT-PCR amplification of the hp-far-1 gene indicated that the gene is transcribed in all parasitic stages of the organism's life cycle. The presence of a secreted FAR protein in the well-defined laboratory model of H. polygyrus provides an excellent model for the further study and analysis of the in vivo role of secreted FAR proteins in parasitism, and supports the mounting evidence that secreted FAR proteins play a major role in nematode parasitism.

Structure and ligand binding of As-p18, an extracellular fatty acid binding protein from the eggs of a parasitic nematode.

Intracellular lipid-binding proteins (iLBPs) of the fatty acid-binding protein (FABP) family of animals transport, mainly fatty acids or retinoids, are confined to the cytosol and have highly similar 3D structures. In contrast, nematodes possess fatty acid-binding proteins (nemFABPs) that are secreted into the perivitelline fluid surrounding their developing embryos. We report structures of As-p18, a nemFABP of the large intestinal roundworm Ascaris suum, with ligand bound, determined using X-ray crystallography and nuclear magnetic resonance spectroscopy. In common with other FABPs, As-p18 comprises a ten ß-strand barrel capped by two short α-helices, with the carboxylate head group of oleate tethered in the interior of the protein. However, As-p18 exhibits two distinctive longer loops amongst ß-strands not previously seen in a FABP. One of these is adjacent to the presumed ligand entry portal, so it may help to target the protein for efficient loading or unloading of ligand. The second, larger loop is at the opposite end of the molecule and has no equivalent in any iLBP structure yet determined. As-p18 preferentially binds a single 18-carbon fatty acid ligand in its central cavity but in an orientation that differs from iLBPs. The unusual structural features of nemFABPs may relate to resourcing of developing embryos of nematodes.