Introduction

Parasitic nematodes, both gut- and tissue-parasitic, are remarkable in the chronicity of their infections in the face of otherwise intact immune systems of their hosts that they are able to modify to their advantage1,2,3,4. Over recent years it has become increasingly apparent that their secretions are key to their immunomodulatory activities, the active principles ranging from proteins, adducts to proteins, extracellular vesicles, to lipids identical to those used by their hosts to regulate their own immune and inflammatory responses2,4,5,6,7,8,9,10,11,12,13,14,15,16. Experimentally, purified natural or recombinant forms of each of these classes of material can be shown to be active, but it is also clear that each species of parasite likely uses a combination of effectors1,2,4,5,6,7,8,9,10,11,12,13,14,15,16.

Here we examined the p43 protein of Trichuris muris, the intestinal whipworm of mice, which is by far the most abundant protein in the secretions of the adult parasites. It has been found to bind and compromise the activity of IL-13, the crucial cytokine in the immune rejection of this parasite17. Moreover, p43 binds glycosaminoglycans (mucopolysaccharides), thereby localising and tethering the protein to its presumed site of action proximal to mucosa. Remarkably, there is only a limited antibody response, and no discernible T cell response, to p43 during infection, even in chronic infections with the parasite17. This may reflect its confinement largely to the intestinal niche despite its interaction with crucial components in the immune system. The anterior of Trichuris species is burrowed into the gut epithelium17,18 of the colon, which could expose the immune system to the protein, but it is not known from where in the worm that p43 is released, or whether there is some mechanism of induction of immune tolerance. Parasites of the Trichuris genus such as T. muris, Trichuris trichiura of humans, and Trichuris suis of pigs, share similar life cycles and morphological similarities such that it seems likely that they use similar mechanisms by which to modulate the immune systems of their hosts, and that their p43 orthologues may therefore play similar roles.

We show here that both the p43 protein of T. muris and its orthologue (p47) of T. trichiura bind lipids including fatty acids that can be precursors of a wide range of signalling lipids involved in cellular interactions, including immune and inflammatory responses. Moreover, p43 is the major protein in the pseudocoelomic fluid of T. muris, where it probably acts as a bulk mobiliser and transporter of lipids internal to the worms as a functional equivalent of the major pseudocoelomic proteins of other clades of nematodes19,20,21,22,23,24. In Trichuris parasites at least, p43 has been additionally devoted to a function external to the parasite. A lipid-binding protein released by a gut parasite might therefore modify local immune responses by delivering active lipids, or sequestering host signalling lipids. The indications are hence that the p43 protein of Trichuris whipworms is a major internal lipid transporter of a type common to Dorylaimia nematodes, but additionally adapted for immunomodulation of hosts.

Results

Fatty acid binding by T. muris p43

The non-specific hydrophobic probe ANS (8-anilino-1-naphthalenesulfonic acid) was used as an initial test for binding of apolar or hydrophobic ligands, which is poorly fluorescent in water but increases its fluorescence emission dramatically upon interaction with exposed apolar sites on proteins. It has been widely used to follow folding dynamics in proteins, as a measure of the degree of unfolded protein in a preparation, or the presence of apolar pockets or binding sites. p43 bound ANS yielding a substantial increase in its emission (Fig. 1A). Moreover, progressive addition of a fatty acid, oleic acid, caused a stepwise reduction in emission intensity consistent with displacement of ANS by this natural lipid. The degree of displacement was not complete, however, potentially due to the existence of apolar binding sites other than for fatty acids, or a proportion of the protein being misfolded resulting in exposure of hydrophobic internal structures.

Fig. 1
figure 1

Worm-derived Trichuris muris p43 protein binds hydrophobic ligands. Binding was investigated by spectrofluorometry. (A) p43 binding of the non-specific fluorescent hydrophobic probe 8-anilino-1-naphthalenesulfonic acid (ANS) to p43 and its progressive displacement with tenfold (here and elsewhere) increasing concentrations of a natural fatty acid (oleic). (B) p43 binding of a fluorophore-tagged fatty acid analogue 11-((5-dimethylaminonaphthalene-1-sulfonyl)amino)undecanoic (DAUDA) and its near complete displacement by arachidonic acid. (C) p43 binding of a unmodified, naturally fluorescent fatty acid, cis-parinaric acid (cPnA), and its competitive displacement by oleic acid. (D) Fluorescent titration of increasing amounts of p43 added to 1.48 μM DAUDA yielding a dissociation constant (Kd) of 0.35 μM with stoichiometry indicative of two binding sites per protein molecule. Fluorescence intensity counts were converted to relative arbitrary units. The small sharp peaks at shorter wavelengths in B are from water Raman scatter. See Materials and Methods for excitations wavelengths for each fluorophore. 

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A more specific probe, DAUDA, was then used, which is a short chain fatty acid linked to a dansyl fluorophore used to examine fatty acid binding sites in proteins with more specificity than with ANS20,25. The fluorophore is environmentally sensitive, poorly fluorescent in contact with water, but highly fluorescent when removed from a polar solvent in protein binding site. Upon addition of p43 to DAUDA in PBS there was a dramatic increase in fluorescence emission and, significantly, a considerable shift in peak fluorescence intensity towards the blue end of the spectrum from 532 to 485 nm (Fig. 1B), and to a peak at 482 nm when the emission by free DAUDA is subtracted (Supplementary Fig. S1A). A shift of this degree is usually taken as being indicative of the probe entering a highly apolar environment such as an organic solvent20, or, as here an apolar protein binding site environment or pocket. This change in DAUDA’s emission was reversible upon addition of arachidonic acid (an ω n-6 polyunsaturated fatty acid precursor to prostaglandins, prostacyclin, thromboxanes; Fig. 1B), oleic acid (Supplementary Fig. S1B), or linoleic (another precursor to active eicosanoids; Supplementary Fig. S1C) acids, indicating that it was displaced from a binding site with preference for natural fatty acids. We found essentially the same observations with another fluorescent probe, dansyl aminocaprylic acid (DACA), in which the dansyl fluorophore is fixed adjacent to the carboxy end of a fatty acid rather than to the methyl/omega end as in DAUDA (Supplementary Fig. S1D). That DAUDA and DACA undergo similar shifts in fluorescence intensity indicated that the whole fatty acid ligand is internalised within the protein. Also, the blue shift in emission seen with DACA is even more extreme than with DAUDA (475 nm versus 485 nm, respectively), indicating that the dansyl fluorophore is taken into an even more strongly apolar environment or orientation within the binding site. The blue shift in peak emission with DAUDA and DACA is comparable to that found with other lipid-binding proteins found only in nematodes—for DAUDA, from 532 to 482 nm for p43, to around 472 nm with nematode polyprotein allergens (NPAs) (e.g. ABA-1, Dv-NPA-1)20,21, and to about 480 nm for a nematode fatty acid and retinol-binding (FAR) proteins (e.g. Ov-FAR-1, Gp-FAR-1, Na-FAR-1)26,27,28.

To check whether the dansyl fluorophore adduct itself contributed to DAUDA and DACA’s binding, two simpler dansyl compounds—dansylglycine and dansylamide ((5-(dimethylamino)-1-naphthalenesulfonamide)—were examined. These compounds showed only trace changes in their fluorescence spectra with p43 (Supplementary Figs. S1E and F) indicative of the fatty acid parts of DAUDA and DACA being the essential principles for binding to p43 rather than the dansyl group.

As a final test for specificity in fatty acid binding by p43, cis-parinaric acid (cPnA) was used. This is a highly unsaturated, unmodified, naturally fluorescent fatty acid with no attached fluorophore. As with DAUDA, p43 bound cPnA strongly and was displaceable with oleic acid (Fig. 1C). The drop in fluorescence with oleic acid additions is much greater than due to the decay in water that cPnA undergoes with time, and the experiment was done using a concentration range of competitor that avoids confounding micelle accumulation that can occur with cPnA. The fact that p43 bound a natural fatty acid directly, and was displaced by another, is, again, strong indication of a lipid binding site or sites in p43 that are specialised for fatty acids.

Dissociation constant and stoichiometry

A fluorescence titration experiment was carried out with increasing concentrations of p43 added to a 1.48 μM solution of DAUDA. This yielded a binding curve that reached saturation, providing an estimate of dissociation constant (Kd) of 0.35 μM (Fig. 1D). Carrier proteins such as human intracellular fatty acid-binding proteins that acquire then release their cargo at a destination have dissociation constants typically in this micromolar range29,30,31,32. The calculated stoichiometry was 0.69. Under ideal conditions, a stoichiometry of 1 would indicate a 1:1 interaction, and a value of 0.5 would indicate a 1:2 protein:ligand interaction. A value of 0.69 would be consistent with two binding sites per p43 molecule in which a proportion of the protein is not binding competently, and/or that the protein contains resident ligands that are not displaced by DAUDA in the titration.

Other hydrophobic ligands

In our previous work on nematode-specific lipid-binding proteins, retinol (vitamin A) was also shown to bind20,21,26,27,28. Like the above probes, retinol is poorly fluorescent in water (in which it rapidly decays unless protected in, for instance, specialised proteins) and of enhanced fluorescence emission in specific, protective protein binding sites. Here, p43 bound retinol, from which it was displaceable by oleic acid (Fig. 2A), indicative of a shared or overlapping binding site for retinol and fatty acids. Protein binding sites that can accommodate both fatty acids or retinol are well known in nematodes and vertebrates20,21,26,27,28,33,34,35.

Fig. 2
figure 2

Hydrophobic ligand binding by Trichuris muris p43 and its orthologue from Trichuris trichiura p47. (A) T. muris p43 binds retinol in a binding site also occupied by oleic acid. (B) composite tryptophan intrinsic fluorescence spectrum of T. muris p43’s seven tryptophans with affect of addition of oleic acid; data were Savitsky-Golay 25 point smoothed and arithmetically brought to same peak value. (C) p47 of T. trichiura, the whipworm parasite of humans, binds fatty acids. (D) subtraction spectrum of the spectra ((DAUDA + p47)—(DAUDA alone)) yielding peak fluorescence emission at 482 nm, essentially identical to that for T. muris’s p43. The small sharp peaks at shorter wavelengths in the retinol and DAUDA specra are from water Raman scatter. 

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Using DAUDA, other lipids were checked for binding capacity to p43, with no evidence that cholesterol displaced the probe, although this does not exclude binding in a site into which DAUDA does not partition. Similarly, the intrinsically fluorescent steroid, dehydroergosterol, did not bind p43.

To examine how the nature of the head group of modified fatty acids may influence binding, such as those that have known pharmacological/physiological proclivities, DAUDA was used in further competitive displacement experiments. Neither platelet activating factor nor L-α-lysophosphatidylcholine had more than trivial effects on fluorescence emission by a pre-formed p43:DAUDA complex (not shown). Prostaglandin E2 (PGE2) has several potent physiological effects and is secreted in abundance by Trichuris suis adult worms, with consequent effects on macrophage activation12. However, we found very slight evidence that PGE2 could displace DAUDA from p43 (Supplementary Fig. S1G) at the pH of the buffer we used. An endocannabinoid, anandamide (arachidonylethanolamide), that has several pharmacological effects, including in the gut36,37,38,39,40, showed evidence of displacing DAUDA but to only a limited degree (Supplementary Fig. S1H). Anandamide comprises arachidonic acid with a relatively small change in the size of its head group—from a carboxylate to an ethanolamine, ethanolamide head group—yet arachidonic acid itself displaces DAUDA from p43 highly effectively (Fig. 1B). So, despite the fact that several (polyunsaturated) fatty acids can bind p43, there is a degree of specificity defined by the headgroup. Again, both PGE2 and anandamide may, however, bind to a subset of binding sites in p43 into which DAUDA may not enter.

Intrinsic tryptophan fluorescence

Proteins contain a few intrinsically fluorescent amino acids, the most useful of which is tryptophan (Trp), the emission spectrum of which provides information on the environment of its indole side chain41, changes in which has been used to measure the affinity of lipid-binding proteins for their ligands42,43. Thus, a Trp that is fully exposed to solvent water in a fully unfolded protein will exhibit a peak fluorescence emission at approximately 356 nm, and 318 nm in one that is fully buried. The peak Trp emission of p43 in buffer occurred at 342 nm, which will be a composite of the emissions of its seven Trps in their diverse environments (Fig. 2B). When oleic acid was added to p43 in buffer there was a slight blue shift that may occur upon a ligand entering a binding site where a Trp were present, or that ligand binding causes an alteration in the conformation of the protein that impinges on the local environment of one or more distal tryptophan sidechains.

Lipid binding by the p43 orthologue of the human parasite, Trichuris trichiura

Given the close phylogenetic relationship between T. muris and the similar parasite of humans, T. trichiura, we examined lipid binding using a recombinant version of the latter’s p43 orthologue, p47. The amino acid sequences of the proteins absent of their cleavable N-terminal signal sequences (cleavage site between position 17 and 18 (as predicted by Signalp 6.0) and terminating at the same place, are 84% identical. The T. trichiura p47 bound DAUDA in an essentially identical manner to T. muris p43 in terms of fluorescence enhancement, blue shift in peak fluorescence emission, and displacement by oleic acid (Fig. 2C). A subtraction spectrum to isolate emission by DAUDA when within the protein yielded a peak of fluorescence emission at 482 nm (Fig. 2D), which is essentially indistinguishable from T. muris p43 (Supplementary Fig. S1A).

p43 dominance in T. muris pseudocoelomic fluid

In multicellular animals, abundant extracellular lipid-binding proteins are often associated with resource distribution systems such as the blood of vertebrates (e.g. serum albumin, the components of lipoprotein complexes such as LDL, HDL, chylomicrons), and the haemocoel of arthropods (e.g. lipophorins of insects. Very little is known about the PCF of parasitic nematodes, except in the few large species from which it can be easily extracted such as Ascaris suum, the large roundworms of pigs, Toxocara canis, the roundworm of dogs, and Dioctophyme renale, the giant kidney worm (many host species)20,22,44. Of these, the latter is in the same sub-clade of the initially genomically-designated Clade I of nematodes (the Dorylaimia) as Trichuris45,46,47. The PCF of D. renale has two proteins that are abundant, P17 (a bright red, haem-bearing protein and presumed globin), and P44 (a fatty acid-binding orthologue of T. muris p43). SDS-PAGE comparison of the PCF of T. muris and D. renale showed a similar dominance of p43 and P44 in the respective parasites, but with p43 seemingly being considerably the more abundant relative to a ~ 17 kDa protein (the possible globin orthologue of P17 of D. renale) than in D. renale PCF (Fig. 3). p43 migrates more slowly in the electrophoresis gels than does P44, presumably because of the absence of a histidine-rich C-terminal sequence in the latter and possible differences in glycosylation. Both p43 and P44 migrated more slowly when reduced, which is also seen in mammalian serum albumin, and is presumably due, in the case of both p43 and P44, to the cleavage of the unusually high number of internal disulphide bonds causing unravelling of the molecule and thereby impeding its migration through the gel.

Fig. 3
figure 3

p43 of Trichuris muris is the dominant protein of the worm’s pseudocoelomic fluid (PCF), as is its orthologue in a related parasite. The two species are in a subbranch of Clade I nematodes, the Dorylaimia45. Protein electrophoretic comparison of the PCF of T. muris and the giant kidney worm, Dioctophyme renale, along with purified, worm-derived T. muris p43. The reduction in mobility of p43 and p47 under reducing conditions is typical of proteins with extensive internal cysteine-cysteine cross-links that are lost upon reduction, likely resulting is unravelling of the protein that impedes migration through a gel. 4–12% gradient SDS-PAGE. Coomassie blue stained. Mr, relative mobility of standard reference proteins (R) expressed in kiloDaltons (kDa). PCF, pseudocoelomic fluid; F, female; M, male; Tm, Trichuris muris; Dr, Dioctophyme renale.

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Potential ligand-binding cavities within the structure of p43 and their diversity

The only published structure of a dorylipophorin is that of T. muris p43. The protein’s amino acid sequence clearly indicates an internal duplication (see alignment in Supplementary Fig. S2; and such that it comprises two highly integrated sub-domains that are further linked by a disulphide bond, at the junction of which is predicted from molecular simulations to be the IL-13-binding site17. The protein’s structure is novel, and does not resemble the structures of the two other nematode-unique classes of lipid-binding proteins found in Clades III, IV, and V (the Chromadoria), namely the nematode polyprotein allergens (NPAs)48 and the fatty acids and retinol-binding proteins (FARs)27. p43 possesses unusual features such as a remarkably high proportion of Cys, Lys, Pro, and His residues, even excluding the extensive His-rich tail (Supplementary Fig. S3). The network of internal disulphide bonds potentially stabilises the structural integrity of a protein that comprises an extensive set of internal cavities that vary in the charges of their walls (Fig. 4). Notably, the size, shape, and surface charge of the equivalent cavities can be seen to differ between the two sub-domains. If the x-ray diffraction structure of p43 is shown absent of the protein structure itself, many of the cavities can be seen to be occupied by components of the crystallisation buffer, indicative of accessibility to the outside of the protein’s structure (Supplementary Fig. S4). Some of the cavities have features reminiscent of lipid-binding cavities in other proteins such as serum albumin of vertebrates49,50, and the FAR and NPA proteins of nematodes27,48,51, namely an elongated tunnel with predominately apolar walls and sometimes a charged end that may act to tether amphipathic compounds such as fatty acids and retinol. Other cavities in p43, however, have quite different surface charges so may bind different ligand types with quite different charge characteristics.

Fig. 4
figure 4

Trichuris muris p43 protein possesses extensive surface-connected internal cavities. Ribbon representation of the X-ray crystal structure of p43 showing a mix of alpha helices (coils) and extended/β structures (arrows) along with unstructured chains. (A) Two molecules as in crystal structure asymmetric unit (PDB 6QIX); cartoon ribbon for chains, surfaces coloured for electrostatic potential of their walls, culled to show only internal cavities, charges indicated red for negative, blue positive, white neutral. Cavities exhibit a complex mix of internal surface charges and shapes, and the cavities of the duplicated/repeat domains appear to have diverged. (B) Rotation of one molecule, again showing the diversity of cavities and the lack of repetition of types. Molecular display using PyMOL.

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Amino acid sequence diversity of Clade 1 p43 orthologues

There are currently complete amino acid sequences for DLPs from three Clade I nematode genera, namely Trichuris, Trichinella, and Dioctophyme, along with several incomplete sequences from Soboliphyme baturini. An alignment of the three complete sequences reveals distinct regions of similarity, notably around the highly conserved cysteines, interspersed with regions of divergence (Supplementary Fig. S5). If the regions of conservation are mapped onto p43’s structure, they tend to associate with extended/β structure and external loops, whereas the helical regions tend to be less conserved (Supplementary Fig. S6). This may indicate that surface features of DLPs are involved in interactions with entities such as other proteins, membranes, or membrane receptors that may be common to species within Clade I nematodes. That the DLPs of Trichuris and Trichinella possess long histidine-rich tails, but that of Dioctophyme renale does not, hints at some differences in function of DLPs that may include, in the case of parasites, animal or plant, how they interact with their hosts.

Discussion

We have shown that the major secreted protein of the whipworm T. muris, the highly immunomodulatory p43 protein, is a lipid-binding protein. It can bind polyunsaturated fatty acids such as arachidonic and linoleic acids that can themselves be pharmacologically active or be the precursors of biologically active eicosanoid lipids that include prostaglandins, leukotrienes, thromboxanes, and several other classes of signalling lipid52,53,54,55,56. p43’s lipid binding function may therefore add to controlling immune and inflammatory processes at the infection site and beyond either by delivering, or sequestering, specific lipids in order to compromise and modify the host’s own cellular signalling pathways. Notably, we also found that a recombinant form of the p43 orthologue (p47) in the human-parasitic whipworm T. trichiura has similar binding properties. p43 also binds retinol, which is a precursor to a wide range of retinoids that are important signals in numerous immune, cellular activation, differentiation, and repair responses56,57,58,59,60,61.

As can be seen from Fig. 4, T. muris p43 has numerous internal cavities that communicate with its surface. Some that do not so communicate visibly in the x-ray structure may do so in solution when the protein’s structure may be dynamic. The shapes of some these cavities, and the charge distributions of their walls, could be consistent with occupancy with hydrophobic ligands, or of binding compounds with different charge characteristics to lipids. A superficial visual comparison of the cavities and pockets of p43’s two repeat domains indicates that the domains may have diverged in their binding functions (Fig. 4). The same can be said of vertebrate serum albumin, which is also constructed of repeat domains (three) with several lipid-binding pockets, and, like p43, is held together by an unusually large number of disulphide bridges for a protein of its size49,50.

The first p43-like protein was defined from Trichinela spiralis and termed poly-cysteine and histidine-tailed protein (PCHTP) because of its unusual richness in these amino acids, as is p43 (Supplementary Fig. S3A)17,62. The His-richness derives from both their main sequence and an extended His-rich carboxy terminal tail. The proposed collective systematic name for these orthologues, “dorylipophorins” (DLP), is a portmanteau reflecting their confinement to the Dorylaimia (Clade I) nematodes, and their now-known biochemical activity. Histidine-richness is not always excessive in these proteins – the DLP of the giant kidney worm, D. renale, for instance, does not exhibit a His-rich carboxy terminal tail, or such a His-rich main sequence as does p43 (Supplementary Fig. S3A & B). Differences in His-richness of these proteins could be relevant to the differing biologies and parasitologies of this group of nematodes. Histidines can be associated with proteins that bind divalent cations (e.g. zinc, nickel, copper, iron) or haem, as in histidine-rich glycoprotein63,64, an abundant protein in human plasma, haemoglobin65, and the histidine-rich, haemozoin-binding protein of Plasmodium malaria parasites66,67. Notably, for p43, zinc is contingent to the protein’s interactions with glycosaminoglycans such as heparin sulphate17, which may be an aspect of its biological activity that may apply to other parasites. Curiously, His-rich regions in major pseudocoelomic lipid-binding proteins of nematodes distantly related to Trichuris are apparent (e.g. Dictyocaulus viviparus’s Dv-NPA-124; and Caenorhabditis elegans’s NPA (gene CELE_VC5.3)), so metal- (and possibly heme-) binding may be a common feature of some internal transport proteins in nematodes.

The extraordinary abundance of cysteines in p43 and other DLPs is also of particular note17,62; Supplementary Fig. S3) and may relate to the need to hold together a protein structure with such extensive intramolecular spaces and external loops, particularly if it were to be a dynamic structure. The elevated content of prolines in DLPs (Supplementary Fig. S3), p43 in particular, may also relate to this. The possibility also exists that binding of different lipids could modulate the structure of p43 to materially alter its biological activity, of which there are examples such as the surface spike protein of the SARS-CoV-2 virus68,69.

The dominance of p43 in the PCF of T. muris, along with that of its orthologue in D. renale, argues that the original function of DLPs was as internal bulk lipid transporters in the body fluid of this clade of nematodes, akin to serum albumin and the lipoproteins of vertebrates, and the lipophorins of insect haemolymph. DLPs do not appear to exist in Clades III, IV, and V nematodes (the Chromadorea of the original DNA-based classification70, subsequently refined45,46,47), their functions possibly replaced by other internally abundant lipid-binding protein classes found only in those nematodes, specifically, the NPA and FAR proteins, that are in turn absent in the Dorylaimia. There is currently no information on pseudocoelomic lipid binding proteins in the remaining branch of the nematodes, Clade II, the Enoplea, and we have so far found no signs of DLP-, NPA-, or FAR-like protein sequences in searching the albeit limited genomic information on this clade. Intriguingly, therefore, two, and perhaps three, branches of the Phylum Nematoda evolved structurally distinctive proteins, likely with no common ancestor protein, to perform a critical function in multicellular animals, that of distributing water-insoluble, and sometimes chemically unstable, lipids in bulk internally. The separation of the three main groups of nematodes is ancient, recently estimated to be have occurred nearly half a billion years ago in the Cambrian or early Ordovician71. In some of the Dorylaimia parasitic forms at least, here exemplified by T. muris, the DLPs may have been further adapted for external roles such as interactions with a parasite’s host’s tissues. Intriguingly, in addition to all larval and adult stages, p43 is also found in embryonated eggs of T. muris72, so it may play a part in lipid storage and mobilisation for the developing embryos, and maintenance of eggshell integrity such as is postulated for a fatty acid binding protein found in the perivitelline fluid of Ascaris73,74. It is of course likely that dorylipophorins could play a similar role in free-living members of the clade.

It remains to be seen whether the lipid-binding activity of p43 has any bearing on its immunomodulatory activity. However, one eicosanoid lipid, PGE2, has been demonstrated to be released in large quantities by adult worms of another Trichuris species, T. suis and the worm-derived PGE2 can suppress proinflammatory properties in human dendritic cells12. We did not, however, find good evidence that p43 binds PGE2 in our particular fluorescence-based competition assays, so it may not need a carrier protein for delivery to host tissue. The water-solubility and chemical stability of PGE2 is pH-dependent75, however, so it may partition into a carrier protein at some stage in its synthesis and export. Meanwhile, there is increasing evidence that lipids are important in affecting immune activities in the gut, cases of which are relevant to anti-helminth immunity36,56,76,77,78,79.

T. muris p43 (and presumably also p47 of T. trichiura) has joined a widening array of protein types that are secreted by parasitic nematodes to control and alter their environments. A curiosity is that these include lipid-binding proteins with similar binding propensities—p43 for T. muris at the very least for Dorylaimia species, and the FAR proteins in Chromadoria—FAR proteins have been found to be influential in both plant and animal parasitism80. p43 orthologues may similarly be important in plant, vertebrate, and arthropod (for example the Mermithids of insects) parasitism by other members of the Dorylaimia. For the moment, our findings of p43’s lipid-binding activities, and a future better understanding of its binding sites for lipids and perhaps other ligands, opens the way to test mutated versions of the protein in immunomodulation and control of the infection site, by, for example, investigating the effects of modified p43 on immune cells in vitro, or the success or otherwise of p43-genetically modified parasites in vivo.

Materials and methods

Ethics statement

All experimental procedures were performed under the regulations of the UK Home Office Scientific Procedures Act (1986) and were subject to local ethical review by the University of Manchester Animal Welfare and Ethical Review Body and followed all ARRIVE guidelines for animal research.

Protein nomenclature

The p43 protein of Trichuris spp. is an orthologue of the poly-cysteine and histidine-tailed protein (PCHTP) originally described from Trichinella spiralis62, and the P44 pseudocoelomic fluid protein of the giant kidney worm Dioctophyme renale44. This family of proteins appears to be confined to Clade 1 nematodes, the Dorylaimia45,47, hence the proposed name of doryliphorins (DLP), given their here-defined biochemical activity and clade of nematodes which have them. The systematic name of T. muris p43 would therefore be Tm-DLP-1, and of Trichuris trichiura would be Tt-DLP-1, but we use the terms p43 and p47 (based on their apparent molecular masses) here for simplicity and continuity.

Mice

Severe Combined Immunodeficient (SCID) mice (C.B-17cr scid/scid originally obtained from the Fox Chase Cancer Centre, Philadelphia) were bred in-house at the Biological Services Facility (BSF) at the University of Manchester. Experiments were performed under the regulations of the Home Office Scientific Procedures Act (1986), (Licences P043A082 and PP0172300) and were subject to local ethical review by the University of Manchester Animal Welfare and Ethical Review Body (AWERB) and followed ARRIVE 2.0 guidelines.

Worm-derived materials—T. muris p43 and pseudocoelomic fluid (PCF)

T. muris p43 protein was produced from adult worms that were cultured in vitro to obtain their excretory/secretory (E/S) material from which purified p43 was produced, all as described in17. SCID mice were infected with 200 T. muris eggs by oral gavage. Forty two days later, mice were killed by CO2 inhalation, and the caecum and proximal colon were removed. The caecum was split and washed in RPMI-1640 plus 500 U/ml penicillin and 500 μg/ml streptomycin (all from Sigma-Aldrich, UK). Worms were removed using fine forceps and cultured for 4 h in RPMI-1640 plus penicillin/streptomycin as above at 37 °C. Medium was collected from adult worms cultured for 4 h and also overnight. This E/S was centrifuged to removed eggs and filtered through a 0.22 \(\mu\)m syringe filter. The medium was incubated overnight with Ni–NTA Agarose (Qiagen, Manchester, UK) at 4 °C on a rotator. Following washing the p43 protein was eluted from the Ni–NTA beads with 250 mM imidazole and further purified using a size-exclusion column (Superdex 75; GE Healthcare, UK) and the relevant fractions were concentrated, filtered through a 0.22 \(\mu\)m membrane, aliquoted, and stored frozen.

Pseudocolemic fluid (PCF) was collected from adult T. muris by placing worms that had been rinsed in culture medium in a small amount of RPMI with 500 U/ml penicillin and 500 μg/ml streptomycin and cut open using a scalpel to release the pseudocolemic fluid. The resulting fluid was centrifuged to remove any worm eggs, cells, and debris.

Recombinant T. trichiura p47 protein

Recombinant T. trichiura p47 was commercially produced in insect cells (Peak Proteins (now Sygnature Discovery), Macclesfield, UK).

Spectrofluorimetry and lipid-binding

Lipid binding by proteins was detected spectrofluorometrically in a PerkinElmer (Beaconsfield, Buckinghamshire, UK) instrument, using all-trans retinol, or the fluorescent fatty acid analogue 11-((5-dimethylaminonaphthalene-1-sulfonyl)amino)undecanoic (DAUDA) or dansyl aminocaprylic acid (DACA) which bear the environment-sensitive dansyl fluorophore, the intrinsically fluorescent natural fatty acid cis-parinaric acid (cPnA), or the non-specific hydrophobic probe 8-anilino-1-naphthalenesulfonic acid (ANS). DAUDA and cPnA were obtained from Molecular Probes/Invitrogen (Renfrew, UK) and all other compounds were obtained from Sigma-Aldrich (Poole, Dorset, UK). The excitation wavelengths were 345 nm, 350 nm, 319 nm, and 390 nm for DAUDA and DACA, retinol, cPnA, and ANS respectively, which were at concentrations of approximately 1 μM, 1 μM, 4 μM, 4 μM, and 10 μM, respectively, in 2 ml phosphate buffered saline (PBS) pH 7.2 in a quartz cuvette. Intrinsic tryptophan fluorescence spectra were recorded for T. muris p43 protein in 2 ml PBS with excitation wavelength of 290 nm. Emission spectra were recorded over wavelength ranges appropriate for each fluorophore to encompass peak emission in water and any shift upon entry into a binding site. Competitive displacement of fluorescent lipids was detected by a reversal of fluorescence enhancement upon addition of the ligand to a preformed complex of protein and fluorescent probe. In the fluorescence-based titration with DAUDA, its concentration checked as measured using its extinction coefficient in methanol of 4400 M-1 cm-1 at 335 nm. The fluorescence spectra are uncorrected and were analyzed using Micocal/OriginLab ORIGIN software (https://www.originlab.com/). All the fluorescence spectra presented were the results of repeated experiments that showed identical results. All proteins were at a stock concentration of approximately 1 mg/mL, and added to the 2 ml in amounts of 20 μl or usually less. Previous publications using these fluorescence-based experiments indicate that control proteins known not to bind lipids such as fatty acids, or retinol, do not interact with our fluorescent lipid probes, producing no change in the emission spectra15,20. Oleic, arachidonic acids, and other competitors were added in 10 μl quantities to the cuvettes to yield approximate starting concentrations in the micromolar range in the cuvette, and a series of tenfold increasing concentration increments in competitive displacement experiments.

Protein gel electrophoresis

One-dimensional vertical sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out using the Invitrogen (Thermo Scientific, Paisley, UK) NuPAGE system with precast 4–12% gradient acrylamide gels, and β2-mercaptoethanol as reducing agent when required. Pre-stained molecular mass/relative mobility (Mr) standard proteins were obtained from New England Biolabs, Ipswich, MA, USA (Cat. No. P7706S). Gels were stained for protein using colloidal Coomassie Blue (InstantBlue; Expedion, Harston, UK), backlit with white light and images recorded using a Nokia C32 smartphone. Colour images were converted to greyscale using IrfanView (https://www.irfanview.com/).

Database searches and molecular structure display

Protein sequence BLAST searches were carried out through the NCBI server set to restrict to Nematoda. Sequence alignments were carried out using MultAlin (http://multalin.toulouse.inra.fr/) set for the Blossum62 substitution matrix. Amino acid content comparison plots were produced using ORIGIN from protein sequences with any cleavable secretory peptide removed as predicted by SignalP (https://services.healthtech.dtu.dk/services/SignalP-6.0/) set for Eukarya, and percent compositions calculated by ProtParam (https://web.expasy.org/protparam/). The latter were plotted against protein amino acid composition statistics from the complete UniProtKB/Swiss-Prot database release 2024_05 (https://web.expasy.org/docs/relnotes/relstat.html). PyMOL (https://pymol.org/) was used to display the T. muris p43 x-ray crystal structure using the RSCB PDB coordinates 6QIX.