Function Identification And Drug Target Screening Of Cathepsin F In Trichinella Spiralis

Trichinellosis is a re-emerging infectious disease, caused by Trichinella spp. Trichinella spiralis has its unique characteristics, when invading the muscle fiber, a cyst that are adaptive to anaerobic environment within host was formed, the cyst formation may be results of positive sellected genes and divergent comparative genome constitution. Genes that experience positive selection are reckoned to have an evolutionary feature such that the nonsynonymous substitution is higher than that of synonymous substitutions. Random genetic mutation may confer selective advantages that can alter amino acids, thus acquire function that has positive effect on the survival of the parasite to be adaptive to host. And the comparative genome analysis between T. spiralis and different species may explain another adaptive mechanism. Thus the partial comparative genome analysis between T. spiralis and different helminth and positive selected genes within T.spiralis were studied.Parasite proteases may play key roles as virulence factors in many parasite infections. Some of them have been revealed to be potential drug targets for antihelminth agents, chemotherapy or immunoprophylaxis, vaccine antigen candidate. Mechanical action and hydrolysis mechanism were used to invade the muscle fiber of host, whether cysteine protease can participated in invasion or not is unknown. Cathepsin F belongs to cysteine protease family, cathpsin F in T. spiralis was multi-gene family, but its characterization is unknown, we carried out the identification of cathepsin F genes in T. spiralis.By comparing five published genome sequences of T. spiralis, B. malayi, T. suis, A. ceylanicum, C.elegan, positive selected genes within T. spiralis were screened. CODEML was used to identified the positive selected genes and 986 positive selected genes was identified, the genes that under positive selection may explain biological adaptation to anaerobic environment within host, the identified genes may have the potential use in drug and vaccine development. Blastn was used for comparative genome study, by respectively comparing cysteine protease between T. spiralis and H. sapiens, Ancylostoma ceylanicum, Angiostrongylus cantonensis, Loa loa, Ascaris suum, Necator americanus, Brugia malayi, Brugia pahangi, T. canis, C. briggsae, C. elegans, Trichuris muris, Dirofilaria immitis, Trichuris suis, H. contortus, Trichuris trichiura, Echinicoccus granulosus and Schistosoma mansoni, the results revealed that the constitution of cathepsin F, cathepsin B, cathepsin L, gut-specific cysteine protease and dipeptidyl peptidase were variable in different species. The full-length of TsCF1,TsCF2 and TsCF3 genes were cloned, the ORF of TsCF1, TsCF2 and TsCF3 encodes 366 aa,496 aa and 365 aa respectively. After delete the peptide signal sequence, TsCF1,TsCF2 and TsCF3 ligated with pET-30 a vector to acquire the recombinant plasmid, and then the rTsCF1, rTsCF2 and rTsCF3 protein were expressed by prokaryotic expression system, the molecular weight of recombinant proteins was 45.00 kDa, 58.29 kDa and 43.94 kDa. The purified rTsCF1, rTsCF2 and rTsCF3 was used to immunize rabbit and the immune serum could recognize respective band in soluble protein of adult, muscle larvae and ES product of muscle larvae. Immunolocalization analysis showed that TsCF1 localized in the cuticle and stichosome, TsCF2 localized in the genital primordium and TsCF3 localized in the cuticle, stichosome and hindgut. Antibody blocking experiments demonstrates that by comparing with the control group, the groups that challenge with T. spiralis that treated with anti-rTsCF1, anti-rTsCF2 and anti-rTsCF3 rabbit serum,the muscle larvae burden reduction was 54.38%, 23% and 48% respectively in muscle larvae of mice after 35 days challenge with T. spiralis,the adult larvae burden reduction was 62.5%, 50.6% and 55.67% in adult larvae of mice after 5 days challenge with T.spiralis. the above experiments demonstrated that TsCF1, TsCF2 and TsCF3 play vital role in the survival of T.spiralis and suggested that TsCFs family were embryonic lethal. The purified TsCF1, TsCF2, TsCF3 and the mixture of TsCF1, TsCF2, TsCF3 were used as protein vaccine to immunize BALB/c mice, at 2 weeks after the last immunization, 200 muscle larvae of T.spiralis were challenged, comparing with the health control, the muscle larvae burden reduction was 23.24%, 21.28%, 23.29% and 51.72% respectively. After renaturation, rTsCF1 and rTsCF2 demonstrated substantial enzyme activity to Z-Phe-Arg-AMC substrate with the optimal pH 5.5 and this activity could be inhibited by cysteine protease inhibitor E-64. Further analysis showed the kinetic parameters of rTsCF1 to be Km = 0.5091 μM and Vmax = 6.12 RFU/s μM at pH 5.5, and the IC50 value of E64 was 135.50 ± 16.90 nM, while the kinetic parameters of rTsCF2 to be Km=2.016 μM and Vmax=5.72 RFU/s μM at pH 5.5, and the IC50 value of E64 was 112.50±6.16 nM, however, rTsCF3 has no enzyme activity. Virtual docking experiments were carried out to study the interactions between TsCF1,TsCF2,TsCF3 with E64 and K11777, Virtual docking experiments were carried out to study the interactions between cathepsin Fs and cystatin family and the best binding conformation were obtained, which revealed that cystatin can bind with cathesin Fs, this can partially explain cystatin family could inhibit cathespin Fs. Virtual screening was carried out by T. spiralis cathepsin Fs with database of FDA approved drugs and database of natural products, 200 chemical compounds were obtained respectively by virtual screening with T. spiralis cathepsin Fs. SPRi technique was used to screen FDA approved drugs that rTsCF1 interacts with, cefdinir, furosemide, ketoprofen, deferasirox, suprofen, tranilast, candesartan, valsartan, tilmicosin, betaxolol hydrochloride, azithromycin, quinine HCl dihydrate and azithromycin dihydrate can specifically bind with rTsCF1, according the results we suggested that the above drugs can be used as in vivo and in vitro candidate drugs for curing trichinellosis. The above results revealed that TsCFs can be used as a potential drug target for treating trichinellosis.