Preotection againstTrichinella spiralis induced by purified stage-specific surface antigens of infective larvae

The stage-specific surface antigens of the infective larvae ofTrichinella spiralis, isolated using an affinity column of monoclonal antibody (Mab) NIM-M1, consisted of four components with molecular weights of 72, 65, 52, and 47 kDa, respectively. These four components may have unique as well as shared structural features and appear to be products of the stichosome. When injected i.p. as an emulsion in complete Freund's adjuvant, the purified antigens induced protection against infection of BALB/c mice withT. spiralis, as assessed by reductions in both the muscle larvae load and the number of adult intestinal worms.     

A mouse monoclonal antibody that binds to an α-stichocyte of Trichinella spiralis

Monoclonal antibodies were generated for the isolation of specific antigens from Trichinella spiralis. A monoclonal antibody (TS32D12) of the Igg₁ class was selected according to its reactivity and specificity by enzyme-linked immunosorbent assay and immunofluorescent technique. The TS32D12 antibody was purified from ascites by fast protein liquid chromatography. The purified antibody showed a sensitive reaction to the T. spiralis antigen, but not to any other heterologous parasite antigens so far examined. Western blot analysis showed that the monoclonal antibody bound to epitopes present on the 160-kDa molecule. The antigen molecule was fragmented into 56-kDa molecules by heat treatment. The epitopes seemed to be destroyed since the antibody could not bind to the 56-kDa molecule. Staining with the periodic acid-Schiff (PAS) reagent suggested that the two molecules of 160 kDa and 56 kDa were glycoproteins. The 160-kDa molecule was detected only in the -stichocyte of T. spiralis muscle larvae.     

T cell responses to fractionated Mycobacterium leprae antigens in leprosy. The lepromatous nonresponder defect can be overcome in vitro by stimulation with fractionated M. leprae components

Protective immunity against Mycobacterium leprae is dependent on M. leprae-reactive T lymphocytes. M. leprae-directed T cell reactivity is high in the localized tuberculoid form of leprosy but specifically absent in the disseminated lepromatous type of the disease. Two important questions that are relevant for the understanding of the immune response in leprosy as well as for the design of rational immunoprophylaxis and -therapy strategies are: (a) what are the antigens that trigger T cell responses in tuberculoid patients and thus protect these individuals from developing lepromatous leprosy and (b) is it possible to restore T cell responsiveness to M. leprae in lepromatous patients by rechallenging the immune system with selected antigens that will trigger help but not suppression? We have addressed these question by directly probing the peripheral T cell repertoire of 10 tuberculoid and 18 lepromatous patients with large numbers of different M. leprae and BCG antigenic components that had been separated on the basis of their relative molecular mass (M_r) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto nitrocellulose. This technique allows the identification of T cell-stimulating antigens independent of the expression of B cell epitopes by these antigens. So far T cell epitopes have only been mapped on M. leprae proteins that had previously been defined by antibodies. Our results show that: (a) tuberculoid patients' T cells responded preferentially to M. leprae and BCG antigens in the lower (i.e. < 70 kDa) M_r range with a peak in the 10–25 kDa range; (b) 6 out of 18 lepromatous patients that did not respond to whole M. leprae responded strongly to isolated M. leprae components; antigens in the lower M_r. range were recognized by five out of these six patients and thus commonly seen by both tuberculoid and lepromatous patients' T cells; however, antigens in the higher M_r range, in particular > 150 kDa, were only recognized by lepromatous patients' T lymphocytes; (c) furthermore, the T and B cell repertoires in leprosy patients are skewed towards different antigenic fractions.     

The occurrence and synthesis of octopamine and catecholamines in the cestode Hymenolepis diminuta

We have defined the surface protein antigens on Plasmodium gallinaceum zygotes using radioiodination methods and rabbit anti-zygote serum which blocks transmission of the parasite to Aedes aegypti mosquitoes. Fifteen protein bands (1–15) in the molecular weight range of 40 000–240 000 and one band at the bromophenol blue dye marker were labelled by the lactoperoxidase and IODOGEN (1,3,4,6-tetrachloro-3α,6α-diphenylglycoluril) methods. The localization of these radioiodinated components on the cell surface was confirmed in two ways: (1) They were completely degraded by trypsin or Streptomyces griseus protease treatment of intact viable zygotes. (2) They were immunoprecipitated following incubation of intact zygotes with antibody prior to detergent solubilization. Reactivity with immune rabbit serum demonstrated that the major surface immunogens were components with M_r 240 000, 200 000, 180 000, 80 000, 55 000, 50 000 and the band at the dye marker. A band of M_r 180 000 was shown immunologically to be a host serum protein selectively adsorbed to the zygote surface. The identity of this protein and the significance of its adsorption to the surface of the zygote are unknown.     

Tritiation of protein antigens of Plasmodium knowlesi schizont-infected erythrocytes using pyridoxal phosphate-sodium boro[3H]hydride

The protein antigens of Plasmodium knowlesi schizont-infected red blood cells (SI-RBCs) and normal RBCs were compared using the pyridoxal phosphate/NaB³H₄ method. Permeation of the outer SI-RBC membrane by the pyridoxal phosphate anion was enhanced since unlike normal RBCs it was not possible to exclusively label surface membrane proteins without concurrent haemoglobin labelling. Under conditions of minimal haemoglobin labelling a subset of total susceptible SI-RBC proteins (M_r 125 000, 50 000, 45 000 and 30 000) were labelled that were absent from normal RBCs and which may be surface proteins. The M_r 125 000 band labels much more readily than Band 3, the normal anion transporter, suggesting that it may be a new anion transporter in the SI-RBC membrane. At higher pyridoxal phosphate concentrations additional bands (M_r 230 000, 180 000, 165 000, 145 000, 107 000 and 72 000) were labelled exclusively with SI-RBCs. The new pyridoxal phosphate-labelled proteins had altered electrophoretic mobility and reduced Coomassie Blue staining, both properties assisting in their identification. Antigen analysis using Protein A-Sepharose and sera from infected monkeys demonstrated that all new ³H-labelled proteins were SI-RBC-specific antigens. One very high M_r antigen (> 250 000) recognized only by homologous strain antisera may represent a strain-specific antigen.     

Protein change of intestinal epithelial cells induced in vitro by <Emphasis Type="Italic">Trichinella spiralis</Emphasis> infective larvae

The aim of this study was to observe the protein changes of intestinal epithelial cells induced in vitro by Trichinella spiralis infective larvae and their excretory-secretory (ES) or surface antigens and identity the proteins related with invasion. HCT-8 cells were incubated for 2 h in the culture medium contained ES or surface antigens of infective larvae, and observed by Immunofluorescent test (IFT). The infective larvae were inoculated into culture of HCT-8 cells to incubate for 18 h, and the lysates of HCT-8 cells were analyzed by SDS-PAGE and Western blot. IFA showed that normal HCT-8 cells had positively reactions with sera of the infected mice and mice immunized with ES or surface antigens. However, after incubating with ES or surface antigens, HCT-8 cells had stronger positively reaction with the above sera. On Western blot, after cultured with infective larvae, additional seven protein bands (66, 61, 57, 45, 34, 21, and 17 kDa) of HCT-8 cells were recognized by sera of the infected or immunized mice, but three protein bands (48, 43, and 23 kDa) of HCT-8 cells were not recognized by the above sera, compared with normal HCT-8 cells. Our results showed that after cultured with infective larvae the protein components of HCT-8 cell changed, suggesting that additional seven proteins recognized by sera of the infected or immunized mice may be related with invasion of intestinal epithelial cells by infective larvae, these proteins might mediate or facilitate entry into the cells, while the three proteins not recognized by the above sera may be the specific mediators released from the cells which permit invasion.     

Partial chemical characterization of the carbohydrate moieties in Leishmania adleri glycoconjugates

Promastigotes of Leishmania adleri were submitted to an extraction procedure providing different carbohydrate-containing extracts. The purified aqueous extract showed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis a complex peptide pattern but carbohydrate was present only in bands of M_r ≈ 45 000–50 000 and 13 500. Methylated derivatives of the hexose components in this extract, analysed by mass spectrometry, suggest the presence of short sugar chains of α-d-mannopyranose and a branched α-d-mannan. The phenol extract, released in the aqueous layer a chloroform/methanol/water soluble complex contained 25% protein, 17% phosphate, 11% glucosamine, uronic acid and 61% neutral carbohydrate, and a chloroform/methanol/water insoluble fraction consisting of a glycoprotein M_r ≈ 22 000 and a proteic doublet M_r ≈ 58 000–66 000. A polysaccharide, showing galactose as predominant sugar, was released through alkaline extraction corresponding to a branched, mainly 1→3 linked galactan associated with α-d-mannopyranosyl units.     

Radioiodination of new protein antigens on the surface of Plasmodium knowlesi schizont-infected erythrocytes

Schizont-infected red blood cells (SI-RBCs) from Plasmodium knowlesi-infected rhesus monkeys (Macaca mulatta) have been radioiodinated and the ¹²⁵I-proteins and ¹²⁵I-antigens compared with those of uninfected RBCs. New ¹²⁵I-proteins of M_r 230 000, 180 000, 165 000, 155 000, 135 000, 107 000, 72 000 and 65 000 were shown to be parasite-dependent components of SI-RBCs, the M_r 230 000 and 72 000 bands being quantitatively the major new components. New ¹²⁵I-antigens of SI-RBCs that were absent from uninfected cells and were only immunoprecipitated by sera from infected monkeys had M_r 230 000, 200 000, 180 000, 165 000, 155 000, 135 000, 130 000, 107 000, 72 000, 65 000 and 47 000. The following approaches were used to test which new radioiodinated components are on the SI-RBC outer membrane: analysis of radioactivity in haemoglobin; electron microscopic analysis of the integrity and purity of the SI-RBCs; treatment of intact ¹²⁵I-SI-RBCs with trypsin to ascertain the sensitivity of new proteins to exogenous protease; immunoprecipitation of antigen-antibody complexes after addition of antibody to intact ¹²⁵I-SI-RBCs. Although each test has inherent disadvantages, the accumulated evidence suggests that many, if not all of the new antigens identified for the first time in this report are on the SI-RBC outer membrane. The SICA variant antigen on P. knowlesi SI-RBCs was not identified by this approach.     

Protein Phosphorylation Pattern and Role of Products of c-erbB-1 and c-abl Proto-oncogenes in Murine Preimplantation Embryonic Development

PROBLEM: To investigate the protein phosphorylation pattern and role of products of c-erbB-1 and c-abl proto-oncogenes with known tyrosine kinase activity in preimplantation embryonic development in mice. METHOD: The protein phosphorylation pattern was studied by in vitro ³²P metabolic labeling of murine ova/embryos as well as by in vitro kinase assay performed directly on various ova/embryos extracts. The role of products of c-erbB-1 (170 kDa, receptor for epidermal growth factor [EGF]) and c-abl proto-oncogenes (150 kDa) was examined by in vitro culturing murine embryos in the presence of monoclonal antibodies to respective protein products and by co-culturing with EGF, the ligand for EGF receptor (EGF-R). RESULTS: In vitro metabolic labeling of murine ova/embryos showed ³²P incorporation into at least two protein bands of murine ova (M_r 81 and 36 kDa), six protein bands of two-cell (M_r 81, 36; and 97, 52, 22 and 19 kDa, respectively), six protein bands of morula (M_r 81, 36; 97, 22, and 19; and 33 kDa, respectively), and eight protein bands of blastocyst (81, 36; 97, 22, 19; and 115, 58, and 15 kDa, respectively), stage embryos; there were some specific bands in each stage. Prolonged labeling from 2 to 4 h not only resulted in a relative increase in ³²P incorporation into these proteins but also revealed additional bands in morula (M_r 133 and 115 kD) and blastocyst (M_r 49, 33, and 31 kD) stage embryos. In vitro kinase assays performed directly on various ova/embryos extracts revealed at least three phosphoproteins (M_r 58, 36 and 33, respectively) that were common to ova, two-cell, morula, and early/late blastocyst stage embryos. Additionally, three protein bands each in murine ova and two-cell embryos (M_r 108, 81, 73 kDa, respectively), and four protein bands of late blastocyst (M_r 108, 73; 133 and 18 kDa, respectively) stage embryos were also revealed. Culture of two-cell embryos in the presence of EGF, the ligand for EGF-receptor, resulted in a concentration dependent increase (P< .001) in the number of cells per blastocyst. Monoclonal antibody to c-erbB-1 170 kDa protein (receptor for EGF) did not affect development of in vitro cultured murine embryos from two-cell to morula, but significantly (P<.001) inhibited the in vitro development of morula to late blastocyst stage. Monoclonal antibody to c-abl protein inhibited the development of murine embryos from two-cell to morula (P<.017), as well as, from morula to late blastocyst stage (P<.002 to .01). CONCLUSIONS: These results suggest that the stage-specific protein phosphorylation pattern and specific products of c-erbB-1 and c-abl proto-oncogenes may have a role in preimplantation embryonic development in mice.     

Plasminogen activator inhibitor induced by lipopolysaccharide injection in the rat Zymographic analysis

It has been shown that endotoxin injection induces formation of Plasminogen Activator Inhibitor (PAI). The present study shows that a decrease in euglobulin fibrinolytic activity 4 h after lipopolysaccharide (LPS) injection is associated with an increase in urokinase neutralisation, either by euglobulin or plasma. After 30 μg/kg LPS this inhibitor reaches 6 U/ml in euglobulin and 41 U/ml in plasma. Zymographic analysis of post LPS euglobulin shows evidence of an increased PAI resulting in a reduction of tissue plasminogen activator (tPA) (M_r 68 000) and formation of higher complexes (M_r, 105 000), but development time is critical for detecting these modifications simultaneously. Increase in a urokinase binding factor in post-LPS plasma or euglobulin is clearly demonstrated from reduction in urokinase lytic bands (M_r, 34 000 and 54 000) with a parallel generation of a large complex (M_r 80 000 to 105 000). From this shift, the mol. weight of PAI may be estimated to be 46 000–51000. Combination of Polymyxin B or Colimycin with LPS could prevent the decrease in euglobulin fibrinolytic activity in low endotoxemia.     

Characterization of TsDAF-21/HSP90 protein from the parasitic nematode Trichinella spiralis

The Tsdaf-21 cDNA was cloned into pET-32a and subsequently expressed in Escherichia coli. The recombinant protein TsDAF-21 was purified and evaluated as an antigen in Western blot. The serum from mouse 14, 21, and 28 days after being infected with Trichinella spiralis recognized rTsDAF-21, but the serum from mouse 7 days post infection of T. spiralis could not react with rTsDAF-21. It implied that the antibody of TsDAF-21 did appear in the host 14 days after infection of T. spiralis. Western blot analysis revealed that the expression of TsDAF-21 in the muscle larvae incubated at room temperature for 8 h and at 37 °C for 4 and 8 h was increased compared to the muscle larvae incubated at 4 °C for 4 and 8 h. The results imply that the expression of TsDAF-21 could be induced at high temperature, not by the cold stress. The expression of TsDAF-21 could be attenuated under the different concentrations of geldanamycin (GA) treatment. Western blot showed that the expression of TsDAF-21 was reduced in the muscle larvae of T. spiralis treated with GA compared to Ampicillin and Gentamycin sulfate treated as control, and this inhibition effect was GA dependent. Other antibiotic treatments did not show any inhibition effects. Immunolocalization showed that TsDAF-21 was expressed in the different stages of T. spiralis including newborn larvae, muscle larvae, and adult worms. TsDAF-21 was mostly localized in nuclei of the cells in the worm. These results suggest that the expression of TsDAF-21 could be induced by the heat stress and attenuated by GA treatment, and TsDAF-21 might be a diagnostic marker for Trichinellosis.     

Ultrastructural colocalization of phosphorylcholine and a phosphorylcholine-associated epitope in first-stage larvae ofTrichinella spiralis

Although the presence of phosphorylcholine (PC) inTrichinella is well established, the structures of the TSL-4 antigens that bear this epitope are unknown. A subset of TSL-4 antigens (TSL-8 antigens) has been reported to be absent from the surface of first-stageT. spiralis larvae. We report experiments with a monoclonal antibody (mAb US2) developed in mice with a relative inability to produce antibodies to PC. In immunoblotting, mAb US2 and anti-PC mAb (BH8) showed apparently identical binding patterns. In addition, we used an immunogold double-labeling technique to study the anatomical distribution of the epitopes recognized by these mAbs; the results obtained indicate close colocalization of epitopes for BH8 and US2 in tissues ofT. spiralis first-stage larvae. On the basis of these results, we suggest that US2 probably binds to allT. spiralis TSL-4 antigens, including TSL-8 antigens. We also clarify some conflicting previous reports on the distribution of PC immunoreactivity in first-stage larvae ofT. spiralis.     

Participation of Parasite Surface Glycoproteins in Antibody-Mediated Protection of Epithelial Cells against Trichinella spiralis

The L1 stage of the parasitic nematode Trichinella spiralis displays on its surface glycoproteins that are immunologically cross-reactive with several larval excretory-secretory (ES) products. The basis for the cross-reactivity is tyvelose, the terminal residue on the complex glycans shared by these surface and ES glycoproteins. In neonatal rats, tyvelose-specific monoclonal antibodies mediate the expulsion of larvae from the intestine. The aim of the studies described in this report was to determine how antibody binding to larval surfaces contributes to expulsion. In these experiments, which involve an in vitro assay of epithelial cell invasion, surface proteins on living larvae were biotinylated to distinguish them from ES products. Biotinylated and nonbiotinylated larvae were cocultured with avidin, biotin-specific antibodies, or anti-tyvelose monoclonal antibodies. Biotinylated larvae cultured with avidin or biotin-specific antibodies invaded Madin-Darby canine kidney (MDCK) cells equally as well as biotinylated larvae cultured with medium alone. Anti-tyvelose monoclonal antibodies were highly protective in this assay; however, biotinylation of larval surfaces hindered the ability of anti-tyvelose monoclonal antibodies to prevent larval invasion of epithelial cells. This correlated with a reduction in the binding of anti-tyvelose antibody to biotinylated larval surfaces. Our results indicate that antibody binding to surface glycoproteins contributes to protection against T. spiralis invasion but that surface binding alone is not sufficient for protection. Our findings support the notion that protection is effected by cross-linking of ES products to surface antigens.     

Analysis of bovine herpesvirus 4 (DN 599) major antigens with monoclonal antibodies and polyclonal immune serum

Summary Monoclonal antibodies (MAbs) and polyclonal immune sera were produced and used to identify the major antigens of bovine herpesvirus type 4 (BHV-4). SDS-polyacrylamide gel electrophoresis of immunoprecipitates of radiolabeled lysates from infected cells resolved 24 peptide bands varying from 12kDa to over 300kDa. Six peptides were identified as major viral antigens by immunoprecipitation. Based on the pattern of radioimmunoprecipitation, MAbs were assigned into four groups. Group 1 precipitated a tunicamycinsensitive glycoprotein complex which contained six components (245, 190, 152, 123, and 48/46kDa). Deglycosylation with endoglycosidase F revealed two peptides with M_r of 93 and 38kDa as the basic peptides of the glycoprotein complex. In addition, a 115kDa glycopeptide containing glycan-peptide bonds of mixed type was identified. Group 2 precipitated a non-glycosylated protein complex consisting of three monomers (33/31/30kDa). Groups 3 and 4 reacted with single monomeric non-glycosylated peptides with M_r of 48 and 14kDa, respectively. Although none of the MAbs exhibited significant neutralizing activity, some reacted strongly in immunosorbent and/or immunohistochemical assays, suggesting they may be good candidates for use in diagnostic assays.     

Identification of diagnostic antigens fromTrichinella spiralis

The Western blotting technique was used to determine the antigens ofTrichinella spiralis muscle larvae that were recognized by antibodies in sera from humans and pigs displayingT. spiralis infections. This resulted in the identification of several antigens that were recognized by all sera. Some of these antigens, notably those that were recognized during the early stage of infection, cross-reacted with antibodies to other parasites. This cross-reactivity was caused by the presence of phosphorylcholine on these antigens. A large portion of the antigens that were recognized by antibodies from infected humans and pigs were found to share a singleTrichinella-specific determinant. TheTrichinella-specific antigen population could be isolated from phosphorylcholine-containing antigens by a simple two-step affinity chromatography procedure using monoclonal antibodies to both determinants. The resulting preparation consisted primarily of a single antigen showing an apparent molecular weight of 45 kDa that corresponded to a mamor constituent of excretory-secretory (ES) products of muscle larvae. When tested in an enzyme-linked immunosorbent assay (ELISA), this antigen displayed diagnostic specificity that was comparable with the ES fraction and diagnostic sensitivity comparable with the crude muscle-larvae extract.     

Antibodies in sera from multiple sclerosis patients recognize Trichinella spiralis muscle larvae excretory–secretory antigens

Helminths, as complex pathogens, possess a large number of different epitopes, some of which may be similar to the epitopes of the host. Besides being the cause for the activation of self-reactive immune cells, molecular mimicry may also be the cause for the expansion of regulatory T cells, crucial for the host tolerance of self-antigens. Amelioration of experimental autoimmune encephalomyelitis (EAE), animal model of multiple sclerosis (MS), caused by Trichinella spiralis infection or application of its muscle larvae excretory-secretory products (ES L1), was achieved through activation of Th2 and regulatory responses. The present study aimed to reveal whether the cause of observed immunomodulation could be the existence of shared epitopes between ES L1 antigens and auto-antigens. Serum samples from 92 MS patients were tested in Western blot for the reactivity toward components of ES L1. Immunoglobulins from the sera of MS patients recognized several ES L1 components, but 45, 49 and 58 kDa proteins dominated others by the frequency of interaction. According to the logistic regression analysis, these interactions were statistically significantly associated with MS, regardless of the disease phenotype or severity. Selected molecules might share homology with self-antigens and as such are worthy of further investigation in terms of potential immunomodulatory capacity and involvement in the parasite’s provoked amelioration of EAE.     

Activation of complement, the induction of antibodies to the surface of nematodes and the effect of these factors and cells on worm survival in vitro

This report describes the effect on various stages in the life cycle of the nematodes T. spiralis and N. brasiliensis of complement, antibodies from rats infected with these parasites and several different cell types. The cuticle of the infective larvae and adult worms of both nematode species activates complement via the alternative pathway, but the cuticle of newborn T. spiralis lacks this property initially. As newborn larvae grow, however, the newly formed cuticle in the midregion of their body is able to activate complement. Rats infected with either nematode species produce antibodies to the cuticle of all life cycle stages which show marked specificity to each stage in the life cycle. Whereas the cuticle of T. spiralis reacts evenly over the entire surface both to complement and to antibodies, the reaction of the cuticle of N. brasiliensis to either reagent is patchy. Infective larvae of N. brasiliensis were killed in vitro in the presence of complement, by neutral red-positive peritoneal macrophages which were nonadherent to plastic. The infective and newborn larvae of T. spiralis were killed by eosinophil-enriched cell populations and antibodies. The speed of eosinophil killing of the T. spiralis larvae was enhanced when the serum was freshly collected and when the eosinophil suspension also contained neutral red-positive nonadherent macrophages. Newborn larvae of T. spiralis and infective larvae of N. brasiliensis assumed a rigid appearance at death. Infective larvae of T. spiralis burst, extruding their internal organs through their cuticle weakened by antibodies and the cells.     

Identification and characterisation of proteins associated with the rhoptry organelles of Plasmodium falciparum merozoites

We describe antigens of Plasmodium falciparum recognised by murine monoclonal antibodies which by immunofluorescence react with the rhoptry organelles of the extracellular merozoite stage. Immunoblotting shows that the antibodies recognise two major parasite antigens of M_r 82 and 65 kilodaltons (kDa). Immunoprecipitations from detergent extracts of [³⁵S]-methioninelabelled parasites show that the 82-kDa and 65-kDa antigens are parasite proteins. Pulse-chase experiments on synchronous parasite cultures show that the 82-kDa protein is synthesised during early schizogony and is later processed into the 65-kDa antigen in segmenting schizonts. In Nonidet P-40, these antigens are non-covalently associated with two other proteins of 40 kDa and 42 kDa. The 40/42-kDa doublet is synthesised in parallel with the 82 kDa antigen and persists, apparently unchanged, till the end of the cell cycle.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycolbis-(aminoethylether) tetraacetic acid - PMSF phenylmethylsulphonylfluoride - TLCK tosyl-L-lysine chloromethyl ketone - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - M _r relative molecular mass     

Construction and use of a Trichinella spiralis phage display library to identify the interactions between parasite and host enterocytes

Although it has been known for many years that Trichinella spiralis initiates infection by penetrating the columnar epithelium of the small intestine, the mechanisms by which T. spiralis infective larvae recognize and invade the intestinal epithelial cells (IECs) are unknown. It is speculated that the molecular interactions between the parasite and host enterocytes may mediate the recognition and invasion of IECs by T. spiralis. However, no Trichinella proteins that interact with the enterocytes have been identified previously. The aim of this study was to identify Trichinella proteins that bind to IECs by screening a T7 phage display cDNA library constructed using messenger RNA from T. spiralis intestinal infective larvae. Following five rounds of biopanning, sequencing, and bioinformatics analysis, ten T. spiralis proteins (Tsp1–Tsp10) with significant binding to normal mouse IECs were identified. The results of the protein classification showed that six proteins (Tsp1, calcium-transporting ATPase 2 protein; Tsp4, ovochymase-1; Tsp6, T-complex protein 1 subunit eta; Tsp7, glycosyl hydrolase family 47; Tsp8, DNA replication licensing factor MCM3; and Tsp10, nudix hydrolase) of these T. spiralis proteins were annotated with putative molecular functions. Out of the six proteins, five have catalytic activity, four have binding activity, and one has transporter activity. Anti-Tsp10 antibodies prevented the in vitro partial invasion of IECs by infective larvae and the mice immunized with the recombinant phage T7-Tsp10 showed a 62.8 % reduction in adult worms following challenge with T. spiralis muscle larvae. Although their biological functions are not yet fully known, these proteins might be related to the larval invasion of host enterocytes. Future experiments will be necessary to ascertain whether these proteins play important roles in the recognition and invasion of host enterocytes. The construction and biopanning of Trichinella phage display libraries provide a novel approach for searching for candidate genes that are related to invasion and for identifying protein interactions between parasite and host.