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.     

Identification of Trichinella spiralis early antigens at the pre-adult and adult stages

Three expression cDNA libraries from Trichinella spiralis worms 14 h, 20 h and 48 h post-infection (p.i.) were screened with serum from pigs experimentally infected with 20,000 T. spiralis muscle larvae. Twenty-nine positive clones were isolated from the 14 h p.i. cDNA library, corresponding to 8 different genes. A putative excretory-secretory protein similar to that of T. pseudospiralis was identified. Three clones corresponded to a T. spiralis serine proteinase inhibitor known to be involved in diverse functions such as blood coagulation and modulation of inflammation. Screening of the 20 h p.i. cDNA library selected 167 positive clones representing 12 different sequences. The clone with the highest redundancy encoded a small polypeptide having no sequence identity with any known proteins from Trichinella or other organisms. Fourteen clones displayed sequence identity with the heat shock protein (HSP) 70. HSPs are produced as an adaptive response of the parasite to the hostile environment encountered in the host intestine but their mechanism of action is not yet well defined. From the 48 h p.i. T. spiralis cDNA library, 91 positive clones were identified representing 7 distinct sequences. Most of the positive clones showed high similarity with a member of a putative T. spiralis serine protease family. This result is consistent with a possible major role for serine proteases during invasive stages of Trichinella infection and host-parasite interactions.     

Proteomic analysis of the tegument and excretory-secretory products of adult Schistosoma bovis worms

Schistosoma bovis is a ruminant pathogen that is poorly known at a molecular level. With an aim of identifying the parasite proteins involved in host-parasite interplay, we studied two protein extracts that contain, respectively, the proteins excreted/secreted by the adult worm (ES) and the tegumental proteins exposed to the host (TG). The 2-DE, 2-D immunoblot and MS were employed to separate and identify the antigenic proteins and the most abundant non-antigenic proteins in each extract. There were some 400 and 600 spots detected in the ES and the TG extracts, respectively. Ninety-six spots were subjected to MS analysis and 64 of them were identified. Overall, we identified 18 S. bovis proteins located at the host-parasite interface, 16 of which have not been identified previously in this parasite, and one of which -lysozyme- has never been reported in a Schistosoma species. Of the proteins identified, at least 4 can counteract host defence mechanisms. The other proteins are also likely to play some role in the host-parasite relationships. Therefore, studies in grater depth on all these proteins will provide a better understanding of how this parasite interacts with its host and new strategies for anti-schistosome drug or vaccine design.     

Trichinella spp.: differential expression of acid phosphatase and myofibrillar proteins in infected muscle cells

Major alterations are induced in muscle cells infected by either Trichinella spiralis or Trichinella pseudospiralis. To investigate the response of muscle to these infections we have analyzed the expression of acid phosphatase (ACP, EC 3.1.3.2), adult skeletal muscle myosin heavy chain, and muscle tropomyosin proteins in infected mouse skeletal muscle cells. Using T. spiralis-infected cells, we provide strong evidence that the tartrate-sensitive ACP of these cells was synthesized by the infected cell and localized in lysosomes. Isoenzyme analysis indicated that the ACP activity was of host muscle cell origin and the specific activity of this ACP was 2.5 times greater than that in associated inflammatory cells. Increased ACP activity was also demonstrated in muscle cells infected by T. pseudospiralis. In synchronized muscle infections, increased ACP activity was detected at 5 days post-muscle infection for both parasites. ACP activity was further increased in infected muscle cells at later times tested. This increased infected cell ACP activity represents the earliest positive enzyme marker yet described indicating expression of the infected cell phenotype. In contrast, myofibrillar proteins were not detected in muscle cells chronically infected by T. spiralis but were detected in muscle cells infected by T. pseudospiralis. Decrease in myofibrillar protein levels was detected by 10 days post-muscle infection by T. spiralis. The data presented demonstrate significant differences and similarities in the phenotypes of muscle cells infected by these two parasites and establish criteria that could facilitate identification of parasite factors that may be involved in these phenomena.     

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.     

Analysis of a 43-kDa glycoprotein from the intracellular parasitic nematode Trichinella spiralis.

The L1 larvae of the parasitic nematode Trichinella spiralis invade skeletal muscle and initiate a process that has been interpreted to represent skeletal muscle dedifferentiation. In this process, the infected region of the muscle cell is converted into a unique structure, called the Nurse cell. The nematode T. spiralis can survive for tens of years within the cytoplasm of the Nurse cell and secretes proteins into the cytoplasm that are believed to play a role in mediating the Nurse cell formation or maintenance. We have cloned a cDNA encoding the T. spiralis-derived, 43-kDa secreted protein. Structural analysis of the predicted 344-amino acid sequence revealed an N terminally located signal peptide and a potential helix-loop-helix motif in the main body of the protein. Antibodies raised against the 43-kDa recombinant protein were used in immunocytolocalizations of T. spiralis-infected skeletal muscle sections. These antibodies strongly stained the Nurse cell nuclei and the nematode itself. Specific, though slightly weaker staining also occurred in the Nurse cell cytoplasm. In Western blots, the antibodies react with the 43-kDa protein but also detected at least two other T. spiralis-secreted proteins. DNA hybridizations reveal at least one additional 43-kDa-related sequence encoded in the T. spiralis genome. We conclude that either the 43-kDa protein and/or a closely related 43-kDa family member is secreted into the muscle and translocates to the muscle-derived nuclei. This model may provide insights into the mechanisms involved in Nurse cell formation.     

Red blood cell lysate modulates the expression of extracellular matrix proteins in dermal fibroblasts

Trichinella spiralis is a zoonotic nematode and food borne parasite and infection with T. spiralis leads to suppression of the host immune response and other immunopathologies. The excretory/secretory (ES) products of T. spiralis play important roles in the process of immunomodulation. However, the mechanisms and related molecules are unknown. Macrophages, a target for immunomodulation by the helminth parasite, play a critical role in initiating and modulating the host immune response to parasite infection. In this study, we examined the effect of ES products from different stages of T. spiralis on modulating J774A.1 macrophage activities. ES products from different stages of T. spiralis reduced the capacity of macrophages to express pro-inflammatory cytokines (tumor necrosis factor α, interleukin-1β , interleukin-6 , and interleukin-12) in response to lipopolysaccharide (LPS) challenge. However, only ES products from 3-day-old adult worms and 5-day-old adult worms/new-born larvae significantly inhibited inducible nitric oxide synthase gene expression in LPS-induced macrophages. In addition, ES products alone boosted the expression of anti-inflammatory cytokines interleukin-10 and transforming growth factor-β and effector molecule arginase 1 in J774A.1 macrophages. Signal transduction studies showed that ES products significantly inhibited nuclear factor-κB translocation into the nucleus and the phosphorylation of both extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase in LPS-stimulated J774A.1 macrophages. These results suggest that ES products regulate host immune response at the macrophage level through inhibition of pro-inflammatory cytokines production and induction of macrophage toward the alternative phenotype, which maybe important for worm survival and host health.     

Infectivity, persistence, and antibody response to domestic and sylvatic Trichinella spp. in experimentally infected pigs

Groups of pigs were inoculated with genotypes of Trichinella belonging to: Trichinella spiralis, Trichinella nativa, Trichinella britovi, Trichinella pseudospiralis (from Caucasus), T. pseudospiralis (from USA), Trichinella murrelli, Trichinella sp. (from North America), and Trichinella nelsoni. The pigs were sacrificed between 5 and 40weeks p.i., and the number of muscle larvae per gram (l.p.g.) of tissue was determined as an average of 18 muscles. All Trichinella genotypes were infective for pigs, but both their infectivity and persistence varied: 5weeks p.i., T. spiralis muscle larvae were present in high numbers (mean=427l.p.g.), while T. britovi, T. nelsoni, and T. pseudospiralis larvae were present in moderate numbers (means=24-52l.p.g.); larvae of the remaining genotypes were recovered only in low numbers (means=0.05-5. 00l.p.g.). The total larval burden (live weight of pigxl.p.g.) was constant over time for T. spiralis, T. britovi, and T. nelsoni, but declined significantly (P<0.05) for the other genotypes. Antibody responses could be detected 3-4weeks p.i. by seven different Trichinella ES antigens, but the antibody levels and dynamics differed significantly among the experimental groups. In pigs inoculated with T. spiralis, T. britovi, or T. nelsoni, the antibody level increased rapidly between weeks 3 and 5 p.i. and was stable or increased slightly throughout the experimental period. In pigs inoculated with T. nativa, T. murrelli, or Trichinella (T6) (from North America), a rapid increase was detected between weeks 3 and 5 p.i., but for these genotypes a reduction in the antibody levels was seen thereafter. In the pigs inoculated with T. pseudospiralis, the antibody level increased more gradually over a period from week 3 p. i. to weeks 15-20 p.i., and decreased thereafter. In general, all species of Trichinella were detected by any of the seven ES antigens, which points to the potential use of one common antigen for surveillance and epidemiological studies on both domestic and sylvatic Trichinella in pigs. Homologous ES antigens were slightly more sensitive in detecting antibodies to the corresponding Trichinella species.     

Normal mouse intestinal epithelial cells as a model for the in vitro invasion of Trichinella spiralis infective larvae

It has been known for many years that Trichinella spiralis initiates infection by penetrating the columnar epithelium of the small intestine; however, the mechanisms used by the parasite in the establishment of its intramulticellular niche in the intestine are unknown. Although the previous observations indicated that invasion also occurs in vitro when the infective larvae are inoculated onto cultures of intestinal epithelial cells (e.g., human colonic carcinoma cell line Caco-2, HCT-8), a normal readily manipulated in vitro model has not been established because of difficulties in the culture of primary intestinal epithelial cells (IECs). In this study, we described a normal intestinal epithelial model in which T. spiralis infective larvae were shown to invade the monolayers of normal mouse IECs in vitro. The IECs derived from intestinal crypts of fetal mouse small intestine had the ability to proliferate continuously and express specific cytokeratins as well as intestinal functional cell markers. Furthermore, they were susceptible to invasion by T. spiralis. When inoculated onto the IEC monolayer, infective larvae penetrated cells and migrated through them, leaving trails of damaged cells heavily loaded with T. spiralis larval excretory-secretory (ES) antigens which were recognized by rabbit immune sera on immunofluorescence test. The normal intestinal epithelial model of invasion mimicking the natural environment in vivo will help us to further investigate the process as well as the mechanisms by which T. spiralis establishes its intestinal niche.     

Differentiation between Trichinella spiralis and T. pseudospiralis infective larvae by a monoclonal antibody

Crude saline extracts of Trichinella spiralis and T. pseudospiralis infective larvae were studied by Western blot analysis using a monoclonal antibody, named ES/TA2 and produced against T. spiralis larvae. This monoclonal antibody recognized seven major antigenic components in T. spiralis larvae with apparent Mr: 45, 48, 50, 68, 70, 92 and 105 kDa and five in T. pseudospiralis larvae: 38, 50, 70, 72 and 92 kDa. SDS-PAGE of both extracts did not reveal appreciable differences in the range of molecular weights recognized by ES/TA2. These facts show the existence of immunological differences among proteins with apparently identical molecular weights.     

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.     

Serine protease inhibitors of parasitic helminths

Serine protease inhibitors (serpins) are a superfamily of structurally conserved proteins that inhibit serine proteases and play key physiological roles in numerous biological systems such as blood coagulation, complement activation and inflammation. A number of serpins have now been identified in parasitic helminths with putative involvement in immune regulation and in parasite survival through interference with the host immune response. This review describes the serpins and smapins (small serine protease inhibitors) that have been identified in Ascaris spp., Brugia malayi, Ancylostoma caninum Onchocerca volvulus, Haemonchus contortus, Trichinella spiralis, Trichostrongylus vitrinus, Anisakis simplex, Trichuris suis, Schistosoma spp., Clonorchis sinensis, Paragonimus westermani and Echinococcus spp. and discusses their possible biological functions, including roles in host-parasite interplay and their evolutionary relationships.     

Trichinella spiralis shares epitopes with human autoantigens

Like other helminths, Trichinella spiralis has evolved strategies to allow it to survive in the host organism, including the expression of epitopes similar to those present in either expressed or hidden host antigens. To identify T. spiralis-derived antigens that are evolutionarily conserved in the parasite and its host and that could be responsible for its evasion of the host immune response, we examined the reactivity of six different types of autoantibodies to T. spiralis larvae from muscle. T. spiralis antigens that share epitopes with human autoantigens were identified by assessing the cross-reactivity of autoantibody-containing serum samples with T. spiralis antigens in the absence of specific anti-parasite antibodies. Of the 55 autoantibody-containing human serum samples that we analysed by immunohistological screening, 24 (43.6%) recognised T. spiralis muscle larvae structures such as the subcuticular region, the genital primordium or the midgut. Using Western blots, we demonstrated that the same sera reacted with 24 protein components of T. spiralis muscle larvae excretory-secretory L1 antigens. We found that the human autoantibodies predominantly bound antigens belonging to the TSL1 group; more specifically, the autoantibody-containing sera reacted most frequently with the 53-kDa component. Thus, this protein is a good candidate for further studies of the mechanisms of T. spiralis-mediated immunomodulation.     

Trichinella spiralis: modulation of experimental autoimmune encephalomyelitis in DA rats

Helminth infection has a potent systemic immunomodulatory effect on the host immune response, which also affects the development of autoimmune diseases. We investigated the dose-dependent influence of Trichinella spiralis infection on experimental autoimmune encephalomyelitis (EAE). Our model of concomitant T. spiralis infection and EAE demonstrates that established infection of Dark Agouti (DA) rats with the parasite causes amelioration of the clinical course of induced EAE in a dose-dependent way. Infection with T. spiralis L1 stage muscle larvae (TSL1) reduced the severity of the autoimmune disease as judged by lower maximal clinical score, cumulative index, duration of illness and degree of mononuclear cell infiltration in T. spiralis infected animals compared to control, EAE-induced group. This study provides a valuable model of worm infection to investigate helminth-induced regulatory mechanisms for optimal benefit to the host.     

How does Trichinella spiralis make itself at home?

The nurse cell-parasite complex of Trichinella spiralis is unlike anything else in Nature. It is derived from a normal portion of striated skeletal muscle cell and develops in a matter of 15 to 20 days after the larva invades that cell type. What are the molecular mechanisms at work that result in this unique relationship? Here, Dickson Despommier presents a hypothesis to account for its formation, in which secreted tyvelosylated proteins of the larva play a central role. These proteins are always present in the intracellular niche of the larva from Day 7 after infection and may be responsible for redirecting host genomic expression, leading to nurse cell formation.     

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.     

Stimulated chemotactic response in neutrophils from Trichinella pseudospiralis-infected mice and the neutrophilotactic potential of Trichinella extracts.

During infection with Trichinella pseudospiralis a strong neutrophil response is evident in the peripheral circulation of the mouse. This study compared the chemotactic response of neutrophils from uninfected, T. pseudospiralis-infected and Trichinella spiralis-infected mice to extracts from adult worms, newborn larvae and muscle-stage larvae of both species of parasite. The chemotactic response of neutrophils from T. pseudospiralis-infected mice to Zymosan-activated mouse serum (ZAMS) was significantly greater than that seen with neutrophils from either uninfected or T. spiralis-infected mice. Unstimulated chemotactic response of neutrophils from these three groups of animals to medium alone was similar. The chemotactic response of neutrophils from the three groups of animals was unaffected by either the concentration or source of serum. The chemotactic response of neutrophils from T. pseudospiralis-infected mice was significantly greater than that observed with cells from uninfected or T. spiralis-infected mice. Among parasite extracts, those from newborn larvae displayed the strongest chemotactic potential for neutrophils. Extracts from muscle larvae of T. spiralis and T. pseudospiralis and extracts of T. spiralis adult worms showed the weakest attraction for neutrophils. Extracts from adult T. pseudospiralis and from newborn larvae of both species elevated the chemotactic response of uninfected mouse neutrophils to a significantly greater level than that seen with ZAMS alone, while a significant reduction in this response was evident only when ZAMS was presented to neutrophils with 500 micrograms of extract from muscle larvae of T. pseudospiralis or T. spiralis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conservation of diagnostic antigen epitopes among biologically diverse isolates of Trichinella spiralis

Crude and immunoaffinity-purified excretory-secretory antigens derived from a domestic pig isolate of Trichinella spiralis were used in an enzyme-linked immunosorbent assay to test serum from mice infected with 25 different pig and wild animal isolates of T. spiralis sspp. All of the sera were found positive by ELISA using either of the antigen preparations, indicating all isolates shared certain antigen epitopes. Excretory-secretory antigens were prepared from 3 distinct isolates of T. spiralis sspp.--Trichinella spiralis spiralis (pig isolate), Trichinella spiralis nativa (polar bear isolate), and Trichinella spiralis pseudospiralis--and compared by electrophoresis and monoclonal antibody binding. While protein profiles varied among the isolates, a monoclonal antibody recognizing a major immunodiagnostic antigen epitope bound all 3 antigen preparations. However, this antigen epitope occurred on different molecular weight excretory-secretory proteins from the different isolates.