| Schistosomiasis is one of the top ten main serious tropical diseases in the world, which is a joint initiative of the United Nations development program/the World Bank Loan Project/the World Health Organization. It is a widely prevalent zoonotic parasitic disease and still pose a risk to public health. S.japonicum is the unique pathogen of schistosomiasis in China. Egg is the key pathogenic factor caused schistosomiasis. Mature eggs lodged in the host liver release soluble egg antigen (SEA), which stimulates immune infiltration, granuloma formation and eventual fibrosis. Although great progress has been made to prevent and control schistosomiasisover the past 50 years in China, as well as in the studies on genome, transcriptome and microRNAs of S. japonicum, while there is no effective solution to cure the hepatic egg granulomas. Therefore, in this study, we established New Zealand white rabbit animal-model of S. japonicum infection, major egg antigen p40 of Schistosoma japonicum (Sjp40) as a target gene, RNA interference (RNAi) was employed to observe the biological functions of Sjp40 in male and female pairing of male-female pairs 19 days post infection (19 dpi) which was a gametogenesis period and in egg laying of male-female pairs 45 dpi which was a egg production peak time. In addition, we examined the expressional profiling and immunofluorescence localization of Sjp40 at 29 dpi livers in which a few immaturate eggs deposited but no egg granulomatous lesions was found and 45 dpi livers in which a lot of mature egg deposited and acute liver granulomatous lesions were found. We also established BALB/c mice animal-model of S. japonicum infection for preliminary omics analysis during the development of granulomas formation after eggs trapped in the 29 dpi livers and 45 dpi livers. These works lay a foundation for the further study on the pathogenic molecular mechanism of Sjp40 during the pathological process of egg granulomas formation and development in the livers of infected New Zealand white rabbits and BALB/c mice.Chapter I small interference RNA-mediated Sjp40 knock-down in Schistosoma japonicum male and female worms for its preliminary function analysisObjective Study on the effect of small interference RNA(siRNA)-mediated Sjp40 knock-down in S.japonicum male and female worms and the subsequent preliminary function research on male and female pairing of male-female pairs(19 dpi group) and on egg laying of male-female pairs(45 dpi group).Methods1. Three different siRNAs targeting Sjp40 transcript were designed using the on-line RNAi Design Tool (http://rnaidesigner.lifetechnologies.com/rnaiexpress/). A common irrelevant siRNA was used as a negative control. All siRNAs were labeled with FAM fluorescence.2. Cercariae (Hunan strains) released from snails infected naturally with S. japonicum, then abdominal infected New Zealand white rabbits. Adult worms were harvested from the infected rabbits by portal perfusion at four time points: 19 dpi,29 dpi,34 dpi and 45 dpi.3. All worms were washed thrice with PBS (pH7.4) before cultured in complete schistosome medium (CSM) at 37℃ in an atmosphere of 5% CO2 in air.4. Females and males were harvested from the infected rabbits at 19 dpi,29 dpi,34 dpi and 45 dpi and those worms cultured in vitro for several days for subsequent total RNA extraction.5. Male-female pairing worms collected at 19 dpi were soaked with FAM labeled siRNA40 at a final concentration of 200 nmol/L. An irrelevant siRNA was used as a siRNA negative control and a group soaked without siRNA was acted as a blank control. Those worms were harvested after soaking for 3 days and 5 days at 37℃ in an atmosphere of 5% CO2 in air. Male and female worms were separated and washed thrice with PBS (pH7.4) for total RNA extraction. Number of male-female pairing worms was counted after soaking for 5 days.6. Male-female pairing worms collected at 45 dpi were electroporated individually with Sjp40-specific siRNA40, an irrelevant siRNA at a final concentration of 200 nmol/L. Those worms were harvested after electroporated for 48 h at 37℃ in an atmosphere of 5% CO2 in air. Male and female worms were separated and washed thrice with PBS (pH7.4) for total RNA extraction. The egg laying status of each group was compared using microscope after electroporated for 48 h.7. Total RNA was extracted using Trizol reagent following manufacturer’s instructions. After further DNase digestion, cDNA was synthesized. mRNA expression levels of Sjp40 were detected by quantitative reverse transcription-PCR (qRT-PCR).8. Female-male pairs collected from the infected rabbits 45 dpi were separated.Total RNA was extracted from each 20 female individual or 20 male individual. The full-length coding sequence (CDS) of Sjp40 from each individual was amplified from each female or male worm cDNA by RT-PCR.Then all the PCR products were cloned into pEasy-Blunt vector and picked at least 3 positive clones of each individual for sequencing.Sjp40 sexual dimorphism was analyzed using all the individuals’ Sjp40 squences by DNAMAN.9. Search for RNAi effectors in S.japonicum using ncbi, uniprot, interpro and pfam databases by refer to RNAi effectors complements gene names and their domain structures in caenorhabditis elegans (C. elegans).10. All data were analyzed using GraphPad Prism 5 and IBM SPSS 19.0. One Way ANO VA was used to compare means among three or more groups. Student’s t-test was used to compare the means between a target group and a control group. Kruskal-Wallis Test was used to compare Male-female pairing rates after RNAi. P<0.05 was considered statistically significant and P> 0.1 was considered no statistically significant.Results1. Sjp40 transcript levels of male worms tended to be low and stable, but it is exactly different for females giving an obvious fluctuation trend at all our measurement points during the development process after finishing female and male pairing. The transcript levels of Sjp40 of female worms stayed low the same as males at the two early measurement points (2 days and 3 days cultured in vitro after harvested at 19 dpi), meanwhile, it increased more than 3 folds from 3 days to 5 days cultured in vitro after collecting at 19 dpi.Sjp40 transcript levels of female worms were significantly higher than male worms collected from 29 dpi group,34 dpi group and 45 dpi group.2. Effects of RNA interference (RNAi) on Sjp40 of S.japonicum female-male pairs 19 dpi soaked with siRNA presented a sexual genetic dimorphism:females 19 dpi soaked for 5 days resulted in a significant reduction (～60%) in Sjp40 transcript level, but no significant degression was found in males, compared to negative controls. Both females and males 19 dpi soaked with siRNA for 3 days led to no significant reduction in Sjp40 transcript level. Rates of parasite pairing among siRNA40 group, negative control group and blank group after siRNAi for 5 days were considered to be no significant difference.3. Effects of RNA interference (RNAi) on Sjp40 of S. japonicum female-male pairs 45 dpi electroporated with siRNA also presented a sexual genetic dimorphism: females electroporated for 48 h resulted in a significant reduction (～55%) in Sjp40 transcript level, but no significant degression was found in males, compared to negative controls. Number of egg laying of siRNA40 group was reduced when observed with microscope after siRNAi, compared to negative controls.4. Identity of Sjp40 CDS among the 20 female/male individual is 100%.5. Compared to C. elegans, S. japonicum posses all functional sub-grouping RNAi effectors (Small RNA biosynthesis, dsRNA uptake and spread, Amplification proteins, Argonautes and RISC components, RNAi inhibitors and Nuclear RNAi effectors), but deficiencies in the diversity of these proteins are found.Conclusion1. Sjp40 transcript levels of female worms increased rapidly when female-male pairs 19 dpi cultured for 5 days, and stay higher than males with some fluctuations thereafter all measurement points. Besides, the preliminary observations showed that number of egg laying was obviously reduced when Sjp40 transcript levels were suppressed by RNAi. It demonstrates that Sjp40 is closely related to female egg laying.2. The initial observations indicated that there were no obvious correlation between Sjp40 gene and female and male pairing after Sjp40 of females during the process of gamete formation (19 dpi group) suppressed by siRNA40.3. Sexual diomorphism of S. japonicum was found answering Sjp40 siRNAi on female-male pairs both in 19 dpi group and 45 dpi group.4. Sexual diomorphism of Sjp40 RNA interference of S. japonicum was not a result of sex-specific genetic dimorphism of Sjp40.ChapterⅡ Expressional profiling, immunofluorescence localization of p40 of Schistosoma japonicum during the development of eggs trapped in the livers and preliminary omics analysis of the livers of infected animal-modelsObjective In this study we examined the expressional profiling and immunofluorescence localization of Sjp40 during the development of granulomas formation after eggs trapped in the livers of infected infected New Zealand white rabbits. Preliminary omics analysis of livers during the development of granulomas formation of infected BALB/c mice.Methods1. Cercariae (Hunan strains) released from snails infected naturally with S. japonicum, then abdominal infected New Zealand white rabbits and BALB/c mice. Livers were harvested at 29 dpi and 45 dpi, and the uninfected rabbits’or mice’livers weas utilized as control.2. All the livers were grinded to power with liquid nitrogen rapidly. Total RNAs of each animal liver tissues were extracted using Trizol reagent. After further DNase digestion, cDNAs were synthesized. Then the transcript expressional profiling of Sjp40 in each rabbits group was determined by qPCR. Total RNA of each mice group was send for high-throughput sequencing.3. Total soluble protein (TSP) was extracted. Anti Sjp40-McAb 9G7 and anti Toxoplasma gondii tSAG1-McAb Y3A8 (as a control antibody) were purified by (NH4)2SO4 salt-out method and Protein G affinity column. We detected the specific expression of Sjp40 in the livers of infected rabbit models at 29 dpi and 45 dpi by Western blot.4. We prepared the paraffin sections from each group’s rabbits’livers for further HE staining to observe the formation process of egg granulomas and for indirect immunofluorescence to show Sjp40 location in the trapped eggs and egg granulomas in the livers of infected New Zealand rabbits.5. Experimental data were analyzed using IBM SPSS 19.0. Student’s t-test was used to compare the means of Sjp40 transcript level between 29 dpi group and 45 dpi group. P<0.05 were considered statistically significant.Results1. The transcript level of Sjp40 in the eggs trapped in the infectious rabbits’livers at 45 dpi was significantly higher than that at 29 dpi (p<0.05).2. Anti Sjp40-McAb 9G7 checked the special protein-level expression of Sjp40 in the infected rabbits’livers both at 29 dpi and 45 dpi.3. Results of HE staining showed there was a few immaturate eggs deposited in 29 dpi livers and no egg granulomatous lesions was found. While there were a lot of mature egg deposited in 45 dpi livers and acute liver granulomatous lesions were found.4. Results of immunofluorescence demonstrated that Sjp40 located inside the immature eggs at 29 dpi infected rabbits’ livers, but spread from the mature eggs with miracidium inside to the surrounding egg granulomas at 45 dpi.5. Initial transcriptomics results of BALB/c mice livers 29 dpi were as follows. 105,021,590 (91.47%) mappes reads could de aligned to mouse genome (http://hgdownload.cse.ucsc.edu/downloads.html#mouse) and 43,117 (1.5%) mappes reads could be aligned to Schistosoma japonicum genome (http://lifecenter.sgst.cn/schistosoma/cn/schdownload.do).4238 knowm transcripts and 221 new transcripts were obtained. Major egg antigen p40 topped first, when transcrips whose coverage greater than 10 and rpkm value greater than 500 were ranked.Conclusion1. The transcript levels of Sjp40 were significantly increased along with the maturing and developing of eggs trapped in the rabbits’ livers. Sjp40 protein spread from eggs to surrounding egg granuloma at 45 dpi livers which is a stage of acute liver granulomatous lesions. It implicates that Sjp40 plays a key role during the pathological process of egg granulomas formation in the livers of infected New Zealand white rabbits. This work lays a foundation for the further study on the pathogenic molecular mechanism of Sjp40 in schistosomiasis japomca.2. Preliminary high-throughput sequencing results indicate that Sjp40 top first, among transcrips whose coverage greater than 10 and rpkm value greater than 500. It indicates that Sjp40 may play a key role in immaturate eggs deposited in 29 dpi livers of infected animal models. |
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