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. 2022 May 24;15(1):177.
doi: 10.1186/s13071-022-05247-1.

A new ferritin SjFer0 affecting the growth and development of Schistosoma japonicum

Fanyuan Zeng  1 , Cun Yi  1 , Wei Zhang  1 , Shaoyun Cheng  1 , Chengsong Sun  1 , Fang Luo  1 , Zheng Feng  2 , Wei Hu  3   4   5
Affiliations

A new ferritin SjFer0 affecting the growth and development of Schistosoma japonicum

Fanyuan Zeng et al. Parasit Vectors. .

Abstract

Background: Schistosomiasis, an acute and chronic parasitic disease, causes substantial morbidity and mortality in tropical and subtropical regions of the world. Iron is an essential constituent of numerous macromolecules involving in important cellular reactions in virtually all organisms. Trematodes of the genus Schistosoma live in iron-rich blood, feed on red blood cells and store abundant iron in vitelline cells. Ferritins are multi-meric proteins that store iron inside cells. Three ferritin isoforms in Schistosoma japonicum are known, namely SjFer0, SjFer1 and SjFer2; however, their impact on the growth and development of the parasites is still unknown. In this study we report on and characterize the ferritins in S. japonicum.

Methods: A phylogenetic tree of the SjFer0, SjFer1 and SjFer2 genes was constructed to show the evolutionary relationship among species of genus Schistosoma. RNA interference in vivo was used to investigate the impact of SjFer0 on schistosome growth and development. Immunofluorescence assay was applied to localize the expression of the ferritins. RNA-sequencing was performed to characterize the iron transport profile after RNA interference.

Results: SjFer0 was found to have low similarity with SjFer1 and SjFer2 and contain an additional signal peptide sequence. Phylogenetic analysis revealed that SjFer0 can only cluster with some ferritins of other trematodes and tapeworms, suggesting that this ferritin branch might be unique to these parasites. RNA interference in vivo showed that SjFer0 significantly affected the growth and development of schistosomula but did not affect egg production of adult female worms. SjFer1 and SjFer2 had no significant impact on growth and development. The immunofluorescence study showed that SjFer0 was widely expressed in the somatic cells and vitelline glands but not in the testicle or ovary. RNA-sequencing indicated that, in female, the ion transport process and calcium ion binding function were downregulated after SjFer0 RNA interference. Among the differentially downregulated genes, Sj-cpi-2, annexin and insulin-like growth factor-binding protein may be accounted for the suppression of schistosome growth and development.

Conclusions: The results indicate that SjFer0 affects the growth and development of schistosomula but does not affect egg production of adult female worms. SjFer0 can rescue the growth of the fet3fet4 double mutant Saccharomyces cerevisiae (strain DEY1453), suggesting being able to promote iron absorption. The RNA interference of SjFer0 inferred that the suppression of worm growth and development may via down-regulating Sj-cpi-2, annexin, and IGFBP.

Keywords: Ferritin; Growth and development; RNA interference; Schistosoma japonicum.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1
Domain and chromosome locations of the three Schistosoma japonicum ferritins (SjFer0, SjFer1 and SjFer2). a Protein domain of S. japonicum ferritins. SjFer0 contains an additional signal peptide sequence in addition to a conserved domain. b Location on chromosomes of S. japonicum ferritins. SjFer0 and SjFer2 are located at a distant from each other on chromosome 5, and SjFer1 is on chromosome 8
Fig. 2
Fig. 2
Phylogenetic analysis of ferritins. The sequence ID consists of the Uniprot entry name + species name + protein annotation. Protein sequences were divided into ten groups that are color-coded (see list to the top left of figure): Schistosome (Schistosoma japonicum [SCHJA], S. mansoni [SCHMA] and S. haematobium [SCHHA]); Other trematodes (Clonorchis sinensis ([lOSI], Fasciola hepatica [FASHE]; Cestoda (Taenia asiatica [TAEAS], Echinococcus granulosus [ECHGR]); Nematodes (Caenorhabditis elegans [CAEEL]); Mollusca (Pomacea canaliculata [POMCA], Biomphalaria glabrata [BIOGL]); Arthropods (Oncopeltus fasciatus [ONCFA], Apis mellifera [APIME] and Daphnia pulex [DAPPU]); Fish (Danio rerio [DANRE]; Amphibians (Xenopus laevis [XENLA]; Mammals (Homo sapiens ([HUMAN], Mus [MOUSE]); Bacteria (Thermodesulfobacterium Commune ([BACT])
Fig. 3
Fig. 3
Expression patterns of SjFer0, SjFer1 and SjFer2 at different time points after infection. a–f mRNA expression of SjFer0, SjFer1 and SjFer2 at 14 ~ 30 dpi (a–c) and 14 ~ 30dpi (d–f). Each sample had four duplicates. Error bars: 95% confidence intervals
Fig. 4
Fig. 4
SjFer0 dsRNA interference in vivo. a SjFer0 mRNA expression levels detected by RT-qPCR. Error bars: 95% confidence intervals, n = 4. Asterisks indicate a significant difference at *P < 0.05, ***P < 0.001 (t-test). b Worm body length measurements. Asterisks indicate a significant difference at ****P < 0.0001 (t-test, n > 30). c Schistosome stained with carmine alum was observed by fluorescence microscopy. The gonad was less stained and there was little hemozoin deposition in the SjFer0 dsRNA interference group. Scale bar: 100 um. d Schistosome stained with carmine alum observed by laser scanning confocal microscopy (LSCM). The SjFer0 dsRNA interfered worm has a fewer eggs and less yolk than the GFP group. Scale bar: 25 um. Abbreviations: F, Female; GFP, green fluorescent protein; M, male; RT–qPCR, reverse transcription–quantitative PCR
Fig. 5
Fig. 5
SjFer0 functions compared between schistosomula and adult schistosomes. a–c, g, j dsRNA injection was started on 26 dpi. d-f, h, k dsRNA injection was started on 1 dpi. a, d SjFer0 mRNA expression levels detected by RT-qPCR. Error bars: 95% confidence intervals, n = 4. Asterisks indicate significant difference at ***P < 0.001 and ****P < 0.0001 (t-test). b, e Effect of dsRNA interference on the egg production of schistosomes. The statistics are based on the egg number of each pair of schistosomes in 1 g mouse liver tissue. Error bars: 95% confidence intervals, n = 4. Asterisks indicate signifiant difference at ****P < 0.0001; ns indicates no significant difference (P > 0.05) (t-test). c, f Excised mouse liver. g, h HE staining of liver tissues. j, k Schistosome stained with carmine alum. Observation by fluorescence microscopy. Scale bar: 100 um
Fig. 6
Fig. 6
SjFer0 immunofluorescence. a SjFer0 immunofluorescence in male worms. SjFer0 protein is widely expressed in the somatic cells of adult males but not in the testicle. b SjFer0 immunofluorescence in females. SjFer0 is detected in the somatic cells and vitelline glands of adult females but not in the ovaries. Vitelline gland expression is extracellular. The experimental mice group (SjFer0) is that injected with SjFer0 polyclonal antibody, and the Control group (Ctrl) is rabbit-derived negative serum. The secondary antibody was FITC-labeled Goat Anti-Rabbit IgG H&L (Alexa Fluor 488). Abbreviations: DAPI (4’,6-diamidin-2-phenylindol): blue staining; FITC (fluorescein isothyocianate): green staining. Scale bar: 50 um
Fig. 7
Fig. 7
Functional expression of SjFer0 in Saccharomyces cerevisiae. The fet3fet4 double mutant strain DEY1453 was transformed with an empty vector pYES2 or with SjFer0. The wild-type DY1450 strain transformed with an empty vector pYES2 served as a positive control. Samples of 5 μl of a serial dilution (OD600 = 1.0, 0.1, 0.01, and 0.001) were spotted onto the induced expression plate with 10 μM FeCl3
Fig. 8
Fig. 8
RNA-sequencing after SjFer0 RNA-interference. Parasites were injected with SjFer0 and GFP dsRNA at 14 dpi and 18 dpi and collected at 22 dpi. a PCA analysis. b Heatmap of the female gene expression level. Downregulated and upregulated condition: log2FoldChange; c Gene set enrichment analysis. P value < 0.05, q value < 0.25. d Volcano map of differential genes. P < 0.05, log2-FoldChange > 1 or log2FoldChange < −1. Abbreviations: BP, Biological process; CC, cellular component; MF, molecular function; PCA, principal component analysis
Fig. 9
Fig. 9
Quantitative PCR verification of representative differential gene expression. The expression changes of our downregulated genes (Sjc_0005644, Sjc_0006159, Sjc_0007803 and Sjc_0008511) and one upregulated gene (Sjc_0002722) were tested by RT-qPCR. Error bars: 95% confidence intervals, n = 3. Asterisks indicate signficant difference at *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 (t-test)
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