Preliminarily Analysis Of Heat Shock Protein Superfamily Of Schistosoma Japonicum And Its Stress Response During The Skin Invasion By Cercariae

From ancient times, epidemic of schistosomiasis endanger human health and its pathogenic organisms has a special life cycle. Screw and mammals are its intermediate host and final host respectively. Schistosomiasis infection and development require recognition, invasion and anti-stress ability.Schistosomiasis japonica is a multicellular animal, affiliated to the trematode. In the process of parasitic, host can produce resistance to parasite. Research shows that in epidemic area population has certain immune protection; The east voles is resistance to schistosomiasis completely; Animal infection with ultraviolet-attenuated cercariae can produce resistance to challenge infection. The host’s immune survival is a challenge to Schistosomiasis japonica.Schistosomiasis japonica anti-stress and the host immune attack are important factors to study different outcomes, this paper will help to further understand the relationship between the host and the schistosomiasis.Chapterl:The expression pattern of main HSPs of Schistosoma japonicum after ultraviolet-attenuated cercariae penetrating into BABL/c mice skinObjective:Ultraviolet-attenuated cercariae (UVAC) from Schistosoma japonicum can generate high levels of protection. UVAC have a difference in its structure and gene expression compared with normal cercariae (NC).Heat Shock Proteins (HSPs) play a key role in cercariae invasion into host and transformation to schistosomula. In the present study, the comparison of HSPs expressional profiling between UVAC and NC during their invasion and transformation in BABL/c mice skin, can help to create a better understanding of the molecular mechanisms for host high-protection induced by UVAC.Methods:1. Searching KEGG database and finding the main pathway that heat shock proteins associated with.2. Dipping out the heat shock proteins in NCBI.3. The heat shock proteins were categorized into different superfamily according to their molecular weight and function difference in different cell part. The heat shock proteins of the same superfamily were aligned by DNAMAN. Phylogenetic tree was constructed by MEGA6.0.4.15 female BALB/c mice with the same age and weight were randomly assigned into group A, B, C, D and E. Group E was taken as normal control group without infection. Group A and C were percutaneously infected with 5000 NC through the skin of abdomen while group B and D with 5000 UVAC. The infected skins were then acquired after 1h post-infection in group A, B and 2.5h post-infection in group C, D, E respectively.5. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) were used to detect the expression levels of heat shock proteins.6. HE staining was used to observe the morphologic changes, Immunohistochemistry were utilized for analysis the expression pattern of Sjp40 in different groups.7. Statistical analysis was used by software SPSS 19.0. It was considered significant when P<0.05.Results:1. Through bioinformation analysis there are many heat shock proteins in Schistosoma japonicum. It can divide into HSP70 superfamily, HSP90 family, HSP40 family, small heat shock protein family, HSP60 and HSP10. It also contain one HSF.2. HSP70 superfamily contain HSPA5, HSPA8, HSPA9 and HSP110; HSP90 family contain HSP83, Gp96 and TRAP.3. Phylogenetic tree and alignment study shows that Schistosoma japonicum is near with SM, followed by human and mouse.4. RT-qPCR results illustrated the lower expression level of HSPs in UVAC group compared with NC group.5. Immunohistochemistry indicated a lower fluorescence intensity in UVAC along with some irregular changes in its morphology.Conclusion:1. S.japonicum owns an integrate HSPs system. This system can help the parasite cope with difficult situation as it goes a complicated life cycle in different organism and environment.2. HSPs are crucial for parasites self-protection against host immune attack. The disabled heat shock response in UVAC could be one of the main factors affecting its structure, migration and growth, thus UVAC being more easily recognized and attacked by the host immune system and induced host high-protection.Chapter 2 The gene structure and alternative splicing analysis of heat shock factor of Schostosoma japonicumObjective:Heat shock factor (HSF) reacts as a transcription factor through binding heat shock element (HSE) domain in heat shock protein gene. It plays an important role in regulating the stress response. But there is little report about HSF of Schostosoma japonicum. Then, in the present study,SjHSF was firstly cloned and identified. The genetic diversity and expressional profiling of SjHSF were comprehensively analyzed.Methods:1. Adult worms and rabbits livers were harvested from the New Zealand rabbits 42 days post-infection with S. japonicum cercariae.2. Sj-hsf open reading frame (ORF) and the splicing fragments were amplified by RT-PCR from the female, male and egg samples, then cloned and identified by enzyme digestion and sequencing3. DNAMAN8.0, InterPro, Mega6 combined with the internet databases were utilized to analyze the gene structure, alternative splicing pattern, structure domain, sequences alignment and phylogenetic tree of HSFs.Results:1. The positive recombinant plasmids pBSjHSFf-F, pBSjHSFf-M, pBSjHSFf-E containing Sj-hsf ORF, pBSjHSFs-F, pBSjHSFs-M, pBSjHSFs-E with Sj-hsf splicing fragments were cloned and identified from the female, male and egg samples of S.japonicum..2. We observed three alternative splicing Sj-hsf isoforms:Sj-hsf-isoforml (2050bp),Sj-hsf-isoform2(2086bp) and Sj-hsf-isoform3(2111bp), GenBank accession numbers were KU954546, KX119143 and KX119144, respectively. All three isoforms located in the same Contig SJC_S000780 of S.japonicum genome and expressed at female, male and egg stages, with Sj-hsf-isoforml expressed most among three stages.3. Sj-HSF-isoforml (671aa) and Sj-HSF-isoform2(683aa) had DBD(DNA Binding Domain), HR-A/B and HR-C domains, while Sj-HSF-isoform3(282aa) stopped in advance without HR-C domain.4. Phylogenetic tree analysis of HSFs shows that Sj-HSFs belong to HSF1 family. It also indicates the near phylogenetic relationship between S.japonicum and S. mansoni.Conclusion:1. Three alternative splicing isoforms of HSF existed in S. japonicum, with Sj-HSF-isoforml expressed most among male, female and egg stages.2. Sj-HSFs belong to HSF1 family and their structure have differences.