Preminary Biopanning Of Trichinella Spiralis Proteins Related With Invasion By Shotgun Approach

Trichinella spiralis is an intracellular parasitic nematode as both a larva and an adult can be found in the same host’s muscle and intestinal epithelial cell. The parasite can infect susceptible hosts and it can infect more than 150 kinds of animals. Trichinellosis caused by Trichinella spiralis is a major food-borne zoonosis. Following people or animals eat raw or undercooked meat which contain trichinella, the larvae release in the stomach by digestion of the contaminated meat, and then promptly occur within the intracellular niches composed of numerous epithelial cells. When the infective larvae invade small intestinal epithelium niches, they are called the first-stage infective larvae in intestinal phase (L1). After 29-36h, they molt four times (L1-L4) and develop to adults that mate and reproduce the next generation of larvae 96h later. The newborn larvae travel via the blood systems to striated muscle and keep the infectivity within muscle cells in several years. Hence, the infective larvae invade the intestinal mucosa develop or expel from the intestinal canal of the host is the key step which the larvae infect the hosts.Although it has been known for many years that T. spiralis larvae invade gut epithelium and the in vitro model of epithelial invasion by the larvae has been developed, the mechanisms by which infective T. spiralis larvae recognize, invade, and migrate within the intestinal epithelium are unknown. In this study, we imitate the natural environment of Trichinella infect host. Infective larvae and human colonic carcinoma cell line-8 (HCT-8) were cultured in vitro, the larvae proteins and cell proteins were separated by means of Sodium dodecyl sulfate-polycrylamide gel electr phoresis (SDS-PAGE) combination Western blot, the matched discrepant protein bands on the gel were excised and then in-gel digested with trypsin. Protemic analysis was performed by LC-MS/MS, then the proteins were identified by using SEQUEST software and protein database search. We classified all of the proteins identified in this study according to Gene Ontology Annotation (GOA) in aspects of molecular function, biological process and cellular location as well. The early expressed proteins may be related with the invasion. There were certain value to early diagnose trichinosis by the proteins of T. spiralis which co-culture with intestinal epithelium. The purpose of this study for future research of mechanism on the invasion by T. spiralis provide reference and data support.Materials and methods1. Trichinella spp, experimental animals and cell lines Trichinella spiralis isolate (ISS534) used in this study was obtained from domestic pigs in Nanyang city of Henan Province, China. This isolate was maintained by serial passage in Kunming mice at 6 weeks intervals in our laboratory. Animals were bought from the Experimental Animal Center of Henan province. Human colonic epithelial cell line HCT-8 was obtained from Cell resource Center of Shanghai Institute.2. Collection and Activation of T. spiralis muscle larvae T. spiralis muscle larvae were recovered from infected Kunming mice by digestion of carcasses with 0.33% pepsin and 1%HC1. These Kunming mice had been infected at least 42 days prior to larvae collection. For in vitro experiments, muscle larvae were activated by incubation in 5% bovine bile in phosphate-buffered saline (PBS) (pH 7.4) at 37℃in 5% CO2 for 2h. Then, bile was removed prior to analysis by exhaustively washing the worms in PBS supplemented with 100 U penicillin/ml and 100μg streptomycin/ml, and incubated in PBS at 37℃in 5% CO2 for additional 1 h.3. Cell culture HCT-8 cells were cultured in RPMI-1640 containing L-glutamine, nonessential amino acids and 10% fetal bovine serum (FBS) at 37℃in 5% CO2. The cell monolayer was dispersed by trypsinization (0.25% trypsin,0.02% ethylenediaminctetraacetic acid, EDTA). HCT-8 cells were grown to confluence for the invasion assay. The cell passaged no more than 15 times before being used in experiments.4. SDS-PAGE analysis and Western blot identification of T. spiralis proteins and HCT-8 cells proteins The activation of muscle larvae and the medium in accordance with 5000/ml added to HCT-8 cells, and they were incubated at 37℃in 5% CO2 for 18h. Larvae were collected from the monolayer. The HCT-8 cells were collected when they were digested with 0.25% trypsin-0.02%EDTA. The protein concentration was measured by the BCA method. SDS-PAGE analysis and Western blot identification of T. spiralis proteins and HCT-8 cells protein were done at last.5. LC-MS/MS After Western blot, the matched discrepant protein bands on the gel were identified by comparison with the molecular size ladder. Then, the discrepant protein bands were manually excised from Coomassie-stained gels and in-gel digested with trypsin. Peptide mixtures from the above extraction were separated by liquid chromatography (LC) followed by tandem MS analysis. LC-MS/MS experiments were performed with a LTQ linear ion trap mass spectrometer equipped with a microspray source. The mobile phases used for the reverse phase were (A):0.1% methanoic acid in water; (B):0.1% formic acid in acetonitrile. Peptides were eluted using a 2-80% linear gradient of solvent in 60 min, repeat count 3. The mass spectrometer was set so that one full MS scan was followed by ten MS/MS scans.6. Analysis of Mass Spectrometric data The date search engine were carried out on Bioworks version3.2 software (SEQUSET, Thermo Electron). By using SEQUEST algorithm, only the peptide which got high score can be retained. Then we got the result of protein identification. The protein identification criteria used in our study was based on Delta CN (≥0.1) and Xcorr (one charge≥1.9, two charges≥2.2, three charges≥3.75). At last, we got the profile of protein list from trypsinization.7. InterPro Annotation and Gene Ontology (GO) Categories Using EXPASY tool to analyse the fundamental physicochemical property of the protein, such as molecular weight, isoelectric point etc and show it in diagram. InterProscan software was used to perform protein sequences. We used the GO categories tool to clarify the different molecular functions, biological processes and cellular components on the GO based data in which the proteins identified in the infective larvae are involved. Meanwhile, using the online tool Web Gene Ontology Annotation Plot (WEGO) show the proteins distribution.Results1. Changes of protein components of T. spiralis infective larvae induced in vitro by HCT-8 SDS-PAGE result showed that the proteins of the infective larvae after culture in RPMI-1640 medium had 41 bands with the range of molecular weight from 12 to 135 kDa. However, we found that after infective larvae were incubated with HCT-8 cells, additional nine protein bands with 15,29,31,35,37,58,69,121 and 132 kDa were observed, and four protein bands with 12,17,40 and 51 kDa were disappeared, compared with the proteins of the larvae incubated without HCT-8 cells. Western blot of larval lysates analysed by sera of the mice infected with T. spiralis showed that 18 protein bands of the larvae incubated in RPMI-1640 were recognized by sera of the infected mice. The range of molecular weight is from 135 to 18 kDa. But after larvae were incubated with HCT-8 cells, the extra 7 protein bands (21,28, 30,36,58,77 and 123 kDa) were recognized by sera of the infected mice, but three protein bands (23,51,97kDa) were not recognized.2. LC-MS/MS analysis and identification of proteins of T. spiralis infective larvae To analyze the proteins of T. spiralis infective larvae, a shotgun LC-MS/MS analysis was applied. Differential protein bands were excised (the portion I, II and III contained the proteins of 21,36,58 kDa, respectively), digested and performed LC-MS/MS analysis. A total of 211 non-redundant proteins were identified. About 123 (58.3%) proteins were distributing in a range of pI 4-7,58 (28.9%) proteins were distributing in a range of pI 8-10. Less than 8.5% of proteins showed pI 7-8. Furthermore, approximately 10 (3.8%) proteins with higher pI (more than 10) were also identified by LC-MS/MS.3. Functional categories of proteins T. spiralis infective larvae by gene ontology We used the Blast2Go analysis tool to clarify the different molecular functions, biological processes and cellular components on the GO based data in which the proteins identified in the infective larvae are involved. Ten catalogues of molecular function were clustered and binding (110 proteins,25.6%) and catalytic activity (75,17.5%) were the two major molecular function categories. Most of the cellular and metabolic processes were related to synthesis and degradation of macromolecules, particularly carbohydrates, nucleotides and proteins that might be associated with the release and development of infective larvae. Proteins of T. spiralis were annotated as being associated with cellular components. Of these,139 occur in the cell and 105 occur in the organelle.4. Change of protein components of HCT-8 cells induced in vitro by T. spiralis infective larvae The lysates of HCT-8 cells were analyzed with SDS-PAGE and the results were showed that normal HCT-8 cells have 44 protein bands with a range of molecular weight from 12 to 134 kDa. But, we found that after HCT-8 cells were incubated with T. spiralis infective larvae, additional five protein bands with 19, 34,61,128 and 132 kDa were observed, and one protein band with 118 kDa was disappeared, compared with the proteins of normal HCT-8 cells incubated without the infective larvae. Western blot results showed that 5 protein bands (30,31,53,55 and 129 kDa) of normal HCT-8 cells were recognized by sera of the infected mice. But after HCT-8 cells were incubated with infective larvae, the additional four protein bands (24,35,61 and 115 kDa) of HCT-8 cells incubated with infective larvae were recognized by sera of the infected mice.5. LC-MS/MS analysis of proteins of HCT-8 cells The additional proteins (the portionⅠ,Ⅱand III contained the proteins of 61,35 and 24 kDa, respectively) of HCT-8 cells after culture with T. spiralis were excised, digested and analyzed by a shotgun LC-MS/MS. A total of 64 non-redundant proteins of T. spiralis were identified. MW ranged from 11.8 kDa to 336.4 kDa,43 (67.2%) proteins were distributing in a range of 10-70 kDa. The protein pi was between 4.47 and 10.92, with 26 proteins (40.6%) was distributed within the range of pI5-6.6. Functional categories of additional proteins of HCT-8 cells after culture with T. spiralis by gene ontology Gene Ontology (GO) signatures of 54 out of the 64 proteins identified were available. T. spiralis proteins in HCT-8 cells cultured with the infective larvae were annotated as being associated with cellular components. Of these proteins,46 occur in the cell and 35 occur in the organelle. For the molecular function ontology, seven subcategories were assigned, of which the activities of binding (43 proteins,79.6%) and catalytic activity (23,42.6%) were the two major molecular function categories. In the biological process category, a large proportion of 54 proteins of T. spiralis were related to cellular process, metabolism, and regulation of biological process, response to stimulus and signaling.Conclusions1. After the larvae incubated with HCT-8 cells, seven additional proteins (21,28, 30,36,58,77 and 123 kDa) recognized by sera of the infected mice may be the new proteins of Trhichella when it co-culture with cells, three proteins (23,51,97kDa) not recognized by sera of the infected mice may be related with enzymolysis by enzymes released from HCT-8 cells or the changed proteins in specific stage.2. After HCT-8 cell culture with the infective larvae, four additional protein bands (24,35,61,115 kDa) of HCT-8 cells were recognized by sera of the infected mice, which may be the larval secretory proteins which enter into the cell.3. Applying the shotgun strategy, we identified in total 211 unique proteins from infective larvae protein (21,36,58 kDa) and 64 unique proteins from HCT-8 cells proteins (24,35,61 kDa) when they co-culture in vitro, respectively. A T. spiralis protein profiles of related with invasion has been preliminary established, which establish the foundation for future screen and identify the invade protein of T. spiralis.