Comparative analysis of the excretory-secretory proteome of the muscle larva of Trichinella pseudospiralis and Trichinella spiralis

The nematodes Trichinella spiralis and Trichinella pseudospiralis are both intracellular parasites of skeletal muscle cells and induce profound alterations in the host cell resulting in a re-alignment of muscle-specific gene expression. While T. spiralis induces the production of a collagen capsule surrounding the host-parasite complex, T. pseudospiralis exists in a non-encapsulated form and is also characterised by suppression of the host inflammatory response in the muscle. These observed differences between the two species are thought to be due to variation in the proteins excreted or secreted (ES proteins) by the muscle larva. In this study, we use a global proteomics approach to compare the ES protein profiles from both species and to identify individual T. pseudospiralis proteins that complement earlier studies with T. spiralis. Following two-dimensional gel electrophoresis, tandem mass spectrometry was used to identify the peptide spots. In many cases identification was aided by the determination of partial peptide sequence from selected mass ions. The T. pseudospiralis spots identified included the major secreted glycoproteins and the secreted 5'-nucleotidase. Furthermore, two major groups of T. spiralis-specific proteins and several T. pseudospiralis-specific proteins were identified. Our results demonstrate the value of proteomics as a tool for the identification of ES proteins that are differentially expressed between Trichinella species and as an aid to identifying key parasite proteins that are involved in the host-parasite interaction. The value of this approach will be further enhanced by data arising out the current T. spiralis genome sequencing project.     

A freeze-fracture study of muscle fibres infected with Trichinella spiralis

The muscle fibres of mice containing the infective-stage larvae of the nematode Trichinella spiralis have been studied by means of the freeze-fracturing technique. The larva lies in what appears to be a fluid-filled cavity within the cytoplasm of an altered muscle fibre. There is no membrane separating the cytoplasm of the nurse cell from the cavity surrounding the larva which is therefore truly intracellular, unlike many parasites that reside within a membrane-lined parasitophorous vacuole within the host cell. This altered muscle fibre, known as a nurse cell, lacks myofilaments but does contain extensive cisternae of endoplasmic reticulum; membrane-bound vesicles are budded off from the endoplasmic reticulum and traverse the cytoplasm towards the cavity containing the nematode where they apparently pass into the cavity. It is suggested that the contents of these vesicles are used to sustain the nematode. Attention is drawn to the similarity to giant cells that have been induced by the plant-parasitic nematode Meloidogyne in the roots of host plants and which sustain the nematode. The conversion of the muscle fibre into a nurse cell is probably brought about by the presence of a metabolic sink, the larval nematode, within the cell. This take-over of the control of a metazoan cell by another metazoan organism is most unusual and warrants further study.     

Immunocytolocalization of two protection-inducing antigens of Trichinella spiralis during its enteral phase in immune and non-immune mice

Monoclonal antibodies (mAb) recognizing epitopes on the 48K (beta stichocyte specific) and the 50/55K antigen (alpha stichocyte specific) were used as first ligands for immunocytolocalization on de-paraffinized sections of infected gut tissue of non-immune and immune CFW strain mice. The enteral phase was studied at 6, 14, 23, 30 hr and 7 days after initiation of infection via the oral route, times corresponding in worm development to the first (L1), second (L2), and third (L3) stage larva and adult. No change in the intensity of the immune reaction with either mAb was noted in parasites developing within immune or non-immune mice for any of the time-points studied. The 48K and the 50/55K antigens were present within the stichocytes at 6 hr. Enterocytes adjacent to some worms also stained positive for both epitopes at this time. Throughout worm development, the amount of each antigen within the worm diminished, until almost none was left at 30 hr. At day 7, the 48K antigen was present within a few stichocyte cells, the canalicular tree, and within the lumen of the midgut. The 50/55K antigen at this time point was localized within only a few stichocyte granules and on the lining of the worm's gut. Embryo stages did not possess either the 48K or 50/55K epitopes. A marked increase in cells bearing IgG in the lamina propria was noted in immune mice when compared with their non-immune counterparts.     

Proteomic analysis of the excretory-secretory proteins of the Trichinella spiralis L1 larva, a nematode parasite of skeletal muscle

Trichinella spiralis is an intracellular nematode parasite of mammalian skeletal muscle. Infection of the muscle cell leads to the formation of a host-parasite complex that results in profound alterations to the host cell and a re-alignment of muscle-specific gene expression. The role of parasite excretory-secretory (ES) proteins in mediating these effects is currently unknown, largely due to the difficulty in identifying and assigning function to individual proteins. In this study, a global proteomics approach was used to analyse the ES proteins from T. spiralis muscle larvae. Following 2-DE of ES proteins,MALDI-TOF-MS and LC-MS/MS were used to identify the peptide spots. Specific Trichinella EST databases were assembled and used to analyse the data. Despite the current absence of a Trichinella genome-sequencing project, 43 out of 52 protein spots analysed were identified and included the major secreted glycoproteins. Other novel proteins were identified from matches with sequences in the T. spiralis database. Our results demonstrate the value of proteomics as a tool for the identification of Trichinella ES proteins and in the study of the molecular mechanism underpinning the formation of the host-parasite complex during Trichinella infections.     

Trichinella spiralis: recognition of muscle larva antigens during experimental infection of swine and its potential use in diagnosis

Longitudinal studies with Trichinella spiralis experimentally infected pigs were carried out to identify muscle larva antigens recognized during infection. This was approached using Western blot analysis and ELISA assays. Immunoblots of sera from experimentally infected pigs using total parasite extracts revealed five principal parasite antigens throughout infection. A similar pattern of antigen recognition was given by sera from backyard pigs in areas of Mexico, some of them endemic for Trichinella. Four of the five antigens recognized (MW 47, 52, 67, and 72 kDa) corresponded to surface/stichosomal antigens purified by monoclonal antibody NIM-M1. In addition, Western blots of excretions-secretions of muscle larva contained three (MW 52, 67, and 72 kDa) of the four surface/stichosomal components recognized by NIM-M1. Affinity-purified surface/stichosomal components, total soluble extracts, and excretory-secretory antigens of muscle larva were then evaluated in ELISA for detection of T. spiralis infections in experimentally infected, noninfected control, and 295 backyard pigs. These assays showed that purified surface/stichosomal components and excretory-secretory antigens increased the specificity of ELISA. These results suggest that muscle larva components purified by monoclonal antibody NIM-M1 are the major antigens recognized during infection of pigs with T. spiralis and therefore potentially useful for diagnosis of swine trichinellosis.     

Trichinella spiralis: light microscope monoclonal antibody localization and immunochemical characterization of phosphorylcholine and other antigens in the muscle larva

A panel of monoclonal antibodies was used to examine the structure of the muscle larva of Trichinella spiralis under the light microscope. Immunofluorescence and, in some cases, immunoperoxidase staining were used. All four antibodies reacted with the cuticle of the organism, although differences in the staining pattern were observed for some of these. Interestingly, all the antibodies also reacted with the stichosome. One of the antibodies (Ts2Ab) is specific for the hapten, phosphorylcholine. In a binding assay, this antibody also reacted with extracts of Trichuris suis, Ascaris suum, and Fasciolopsis buski, but not with extracts derived from Cysticercus cellulosae, Candida albicans, Salmonella typhi, or Escherichia coli. This crossreactivity was confirmed microscopically in which the cuticle, oviduct and eggs of T. suis, the cuticle, muscle cells, and eggs of A. suum, and the cuticle and vitelline glands of F. buski were seen to be clearly stained by the antibody. In addition, Ts2Ab also reacted with the cuticle and stichosome of the adult T. spiralis worm. In Western blot analysis, Ts2Ab recognized a 43-kDa antigen from T. spiralis muscle larvae extracts, while a previously studied antibody (7C2C5Ab) identified four major antigens (48.5, 47, 43, and 39 kDa) in this preparation. Similar results were obtained when the 24-hr excretory-secretory (ES) antigens of T. spiralis were immunoblotted with the antibodies, although the reactivity shown by Ts2Ab was relatively weak. With the 72-hr ES material, on the other hand, major antigens of lower mol wt (44, 28, and 25 kDa) were revealed by 7C2C5Ab, and no reactivity was seen with Ts2Ab. However, this antigen preparation reacted well with both antibodies in an enzyme-linked immunoassay. Taken together, the findings suggest that the 72-hr ES antigens probably result from extensive degradation of material originally secreted or excreted by the worm. Similar binding studies on the 24-hr ES preparation indicated that this source may be relatively rich in 7C2C5Ab-reactive epitopes and relatively poor in the antigen identified by Ts2Ab. Other studies performed demonstrated that the antigens recognized by these two antibodies were distinct and physically unassociated.     

Expressed sequence tags of Trichinella spiralis muscle stage larvae

In order to obtain greater insight into the relevant genomic expression patterns of Trichinella spiralis, 992 expressed sequence tags (ESTs) were collected from a cDNA library of T. spiralis muscle stage larvae and assembled into 60 clusters and 385 singletons. Of them, 445 (44.7%) ESTs were annotated to their homologous genes, and small fractions were matched to known genes of nematodes. The annotated ESTs were classified into 25 eukaryotic orthologous groups (KOG). Cytochrome C oxidase (34 clones) was found to be most frequent species.     

Antigen production by encysted muscle larvae of Trichinella spiralis

The production of excretory-secretory antigens by encysted muscle larvae of Trichinella spiralis has been investigated immuno-histochemically using an antiserum raised by infection in rabbits and purified both before and after conjugation by ion-exchange chromatography. The specificity of the antibody for excretory-secretory products was demonstrated by the pattern of staining of live worms in vitro and the failure of the labelled antibody to stain dead, non-metabolizing worms. Using this labelled antibody, and unlabelled antibody in the immunoperoxidase system, the presence of parasite antigen-bearing cells in close proximity to encysted muscle larvae has been demonstrated. This is believed to be the first demonstration of antigen production by encysted muscle larvae in vivo. The implications of this observation to current concepts of immunity to Trichinella spiralis are discussed.     

Trichinella spiralis: site selection by the larva during the enteral phase of infection in mice

Mice, belonging to two strains, were infected by the oral route with muscle larvae of Trichinella spiralis. Host animals were killed at various times up to 48 hr after administration of larvae, and the infected small intestines were fixed immediately in 10% neutral formalin. Sections of infected gut, embedded in paraffin and cut at 5 μm, or in methacrylate and cut at 0.5 μm, revealed that all stages (i.e., 1 to 4) of T. spiralis were embedded between the lamina propria and the columnar epithelium. First-stage muscle larvae occupied this niche as early as 10 min after introducing them into the host by the oral route.     

The role of malic enzyme in the carbohydrate metabolism of Trichinella spiralis spiralis and Trichinella spiralis pseudospiralis

The role of malic enzyme in the carbohydrate metabolism of Trichinella spiralis spiralis and Trichinella spiralis pseudospiralis. International Journal for Parasitology16: 435–440. The activities, intracellular localization and some regulatory properties of malic enzymes from homogenates of T.s. spiralis and T.s. pseudospiralis larvae have been studied. The malate saturation curves exhibit sigmoidicity. With increasing pH a ‘double sigmoidicity’ was observed in both NAD- and NADP-specific malic enzyme from T.s.spiralis and Trichinella malic enzymes resemble the Ascaris enzyme in their nondecarboxylation of oxaloacetate and in nucleotide and ammonium sulphate sensitivity, but the enzyme from T.s. pseudospiralis differs in its equal specificity for NAD and NADP. The cytoplasmic localizations and some properties of phosphoenolpyruvate carboxykinase in both Trichinella species are similar to the characteristics of the same enzyme in Ascaris.     

Trichinella spiralis: Subversion of differentiated mammalian skeletal muscle cells

Infection by Trichinella spiralis initiates changes in terminally differentiated mammalian skeletal muscle cells that lead to host cell cycle re-entry, cell cycle arrest in apparent G2/M phase, the repression of differentiated skeletal muscle characteristics and the expression of a phenotype unlike other stages in the muscle lineage. Although the parasite must be involved in initiating these changes, its precise role is unclear. Based on regulatory mechanisms which control myogenesis Douglas Jasmer proposes that host cell regulatory products, expressed as a consequence of cell cycle repositioning may determine aspects of the infected host cell phenotype. Distinguishing between host- and parasite-regulated changes should aid in identifying pertinent parasite molecules and uncovering the significance of host cell changes to parasite growth, developments and survival.     

Trichinella spiralis: modifications of the cuticle of the newborn larva during passage through the lung.

A scintigraphic method was developed to study the distribution of radioactivity after iv injection of 131I-labeled Trichinella spiralis newborn larvae into normal rats. It was found that the radioactivity was immediately retained in the lungs and thereafter slowly released, with a mean transit time in excess of 9 hr, as calculated by image analysis. At various times after iv injection of newborn larvae into normal mice, the lungs were removed and parasites were recovered and counted. Fifty to seventy percent of the larvae injected were recovered after 30 sec, between 10 and 30% after 1 min, and less than 4% at 15 min. These results indicate that during the very rapid passage of newborn larvae through the lungs, labeled components of the cuticle are detached and retained. It is suggested that the modifications produced in the cuticle of the newborn larva during its passage through the lung may increase its resistance to the nonspecific defense mechanisms of the host.     

Comparison of cholinesterase activities in the excretion-secretion products of Trichinella pseudospiralis and Trichinella spiralis muscle larvae

The presence of cholinesterases (ChE) is reported in T. pseudospiralis excretion-secretion products (ESP) by spectrophotometric method, using acetylthiocholine (ATCI) and butyrilthiocholine (BTCI) as substrates. By inhibition assays, we found that T. pseudospiralis release both acetyl- and butiryl-cholinesterases (AchE and BchE, respectively). The sedimentation coefficientes of these enzymes were determined by sucrose density gradient. We studied the in vivo ChE secretion by immunoblot assays using AchE from Electrophorus (electric eel) and sera from normal or infected mice with T. pseudospiralis or T. spiralis. The presence of anti-AchE antibodies was only demonstrated in the sera from T. pseudospiralis infected mice. Moreover the in vivo secretion was corroborated by the high difference determinate between the ChE activity of the immuno complexes from T. pseudospiralis infected sera and the immunocomplexes from T. spiralis infected sera as well as normal sera. Finally, we analyzed the effect of the organophosphate Neguvón (metrifonate) on the ChE activity from the T. pseudospiralis ESP. The drug inhibits in part this activity. Moreover Neguvón (metrifonate) showed a high activity against the T. pseudospiralis viability.     

Trichinella spiralis: altered expression of muscle proteins in trichinosis

Mammalian muscle undergoes significant alterations morphologically, ultrastructurally, and biochemically following infection by Trichinella spiralis larvae. To investigate this host/parasite relationship in more detail, a new method to isolate T. spiralis-infected cells (nurse cells) in preparative quantities was developed. Nurse cells isolated by sequential protease treatments contain larvae and retain many of the characteristics of those in situ. When analyzed by SDS-PAGE, a wide range of proteins detected in nurse cells were not apparent in muscle by the methods employed. Proteins associated with the nurse cell capsule and organellar fractions appear to account for most of the dominant nurse cell proteins. In contrast, most major muscle proteins were either reduced in abundance or undetectable in nurse cells. The myofibrillar proteins myosin heavy chain, alpha-actin, and alpha- and beta-tropomyosin were identified using antibody reagents and two-dimensional PAGE analysis. None of these proteins were detectable in nurse cells and except for beta-tropomyosin, the relative abundance of these proteins was a minimum 100-fold lower compared to muscle. The data indicate that the reduction of muscle products in the nurse cell is much greater than previously reported. The inability to detect myofibrillar proteins raises the possibility that the nurse cell is not blocked in a regenerating muscle phenotype as previously suggested. Availability of isolated nurse cells in large quantity should facilitate resolution of this and other issues regarding the T. spiralis/skeletal muscle relationship.