Animals and parasites
Schistosoma japonicum cercariae (Anhui province isolate) were provided by the Department of Vector Biology, National Institute of Parasitic Disease, Chinese Center for Disease Control and Prevention (NIPD, China CDC). C57BL/6 mice (8 weeks age), purchased from Shanghai Animal Center, Chinese Academy of Sciences (Shanghai, China), were infected with S. japonicum cercariae. Two specific pathogen-free Japanese White Rabbits were each injected with 0.25 mg recombinant SjFer0 protein 4 times every 2 weeks and then sacrificed at week 10; polyclonal antibodies were extracted from the collected blood samples by ABclonal Technology Co., Ltd (Woburn, MA, USA).
All animal experiments complied with the Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of the National Institute of Parasitic Diseases, Chinese CDC (Shanghai, China).
Bioinformatics of three S. japonicum ferritin isoforms
Information on the sequences of SjFer0, SjFer1 and SjFer2 is available on the Uniprot website (https://www.uniprot.org/) under accession numbers B3GUY2, C1LRQ1 and C1L7G5, respectively. Protein domains were analyzed by the SMART tool (http://smart.embl-heidelberg.de/) [28]. Information on the chromosomal position of the three three ferritins is derived from the V3 genome assembly S. japonicum (SRA accession number PRJNA739049).
Phylogenetic analysis of Ferritin protein
Ferritin protein sequences of multiple species were acquired from the Uniprot database. MEGA-X software was used to construct the phylogenetic tree with the maximum likelihood method, then improved by the iTOL online tool (https://itol.embl.de/index.shtml).
Examination of the expression profile of SjFer0, SjFer1 and SjFer2 at different time points after infection
C57BL/6 mice were infected with 60 cercariae percutaneously through the shaved abdomen. Mice were euthanized at two-day interval during the 14-30 days post-infection (dpi), respectively, and perfused with a 4 °C saline solution containing heparin sodium via the hepatic portal vein to obtain schistosomula or adult schistosomes. Male and female S. japonicum collected at each time point, with the exception of 14 dpi, were separated with a soft brush for total RNA extraction with RNAiso Plus (TRIzol; Takara Bio, Shiga, Japan). The expression profile of ferritin messenger RNAs (mRNAs) was examined by quantitative PCR (qPCR) with the PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time; Takara Bio) and 2× SYBR Green qPCR Master Mix (Bimake, Houston, TX, USA). The qPCR primers of SjFer0, SjFer1, SjFer2 and 26S proteasome non-ATPase regulatory subunit 4 (PSMD4) for internal reference [29] were designed with the Primer Premier 6.0 software package (Additional file 1: Table S1).
RNA interference assay in vivo
RNA interference (RNAi) was performed using the method as described previously [30]. To produce double-stranded RNA (dsRNA) for the assay, the target DNA sequences were amplified from S. japonicum complementary DNA (cDNA) by primers (Additional file 2: Table S2) containing the T7 promoter and then transcribed to dsRNA by the MEGAscriptTM T7 High Yield Transcription kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). The mice were assigned to SjFer0, SjFer1, SjFer2 and green fluorescent protein (GFP) groups, with four mice in each group, and each group was treated with 10 μg target dsRNA via intravenous injection into the tail at 1, 6, 10, 14, 18, 22 and 26 dpi. Schistosomes were recovered by perfusion [30] at 30 dpi. The worms were grouped with six pairs each, washed three times with diethyl pyrocarbonate (DEPC)-treated phosphate buffered saline (PBS), frozen in liquid nitrogen and then stored until RNA extraction.
To assess longer time of interference up to 42 days on the impact of SjFer0 on schistosome growth and development or egg production and hepatic fibrosis, 16 mice, in four groups of four mice each, respectively, were administered 10 μg SjFer0 dsRNA, or GFP dsRNA as a control, under two injection schemes: scheme A, injections on 1, 6, 10, 14, 18, 22, 26, 30, 34 and 38 dpi; scheme B, injections on 26, 30, 34 and 38 dpi. The mice were sacrificed to recover schistosomes at 42 dpi, and the subsequent treatment of the worms was the same as described for the 30 dpi assay.
Measuring schistosome body length and carmine alum staining
Schistosomes were separated and treated with AFA buffer (alcohol-formalin-acetic acid: alcohol 95%, formalin 3%, glacial acetic acid 2%). Digital images of the schistosomes were taken, and the images analyzed with ImageJ software(https://imagej.nih.gov/ij/) to measure schistosome body length.
The schistosomes were stained with Mayer's carmine alum stain after dehydration through an alcohol gradient (30, 50 and 70% ethanol). The worms were then permeabilized with methyl salicylate after decolorization with hydrochloric acid alcohol (3% hydrochloric acid, 70% ethanol) and dehydration through an alcohol gradient (70, 85 and 100% ethanol). As a final step, the parasites were mounted on a Nikon fluorescence microscope and Nikon A1-Ni laser confocal microscope (Nikon Corp., Tokyo, Japan) for morphological observation.
Hematoxylin/eosin staining of liver tissue and egg counting in liver
A portion of the mouse liver at 42 dpi was fixed with formaldehyde solution and sent to Wuhan Servicebio Co., Ltd (Wuhan, China) for staining with hematoxylin/eosin (HE). The remaining liver tissues were weighed (W, in grams) and digested with 5% NaOH (w/v) (V, in milliliters) at 37 °C for 24 h for egg counting under microscope (10 μl of digested suspension). The eggs in each sample were counted at least three times and a mean egg number (x) determined. The number of paired schistosomes was recorded (n pairs) when recovering. Consequently, the number of eggs that each pair of schistosomes produced per gram of liver tissue (N) is given by the following formula: N = 100 × x × V/(n × W).
Preparation of a rabbit polyclonal antibodies against SjFer0
The SjFer0 sequence was amplified with the forward primer 5ʹ-AATGGGTCGCGGATCCCCTATTCAAACTAACGGTGAG-3ʹ and reverse primer 5ʹ-AATGGGTCGCGGATCCCCTATTCAAACTAACGGTGAG-3ʹ, subsequently cloned into the pET-28a vector and then linearized by BamHI and XhoI enzymes (New England Biolabs [NEB], Ipswich, MA, USA) for expression in Escherichia coli BL21 (DE3) (TIANGEN BIOTECH, Beijing, China). The expressed recombinant SjFer0 was purified by Ni column chromatography and concentrated by centrifugation at 5000 g at 4 °C in an ultrafiltration tube (MilliporeSigma, Burlington, MA, USA). The purified and concentrated recombinant SjFer0 protein was used to produce rabbit polyclonal antibody against SjFer0 by ABclonal Technology Co., Ltd (check section 3 for details).
Immunofluorescence localization of SjFer0
Schistosomes recovered at 35 dpi from infected mice were cultured in Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) at 37 °C until the paired parasites separated. The worms were then killed by 0.6 M MgCl2 and fixed in 4% paraformaldehyde for 4 h at room temperature. The fixed samples were washed 10 min in PBSTx (PBS, 0.3% Triton X-100), 50% methanol (prepared with PBSTx) and 100% methanol. Samples could be stored in methanol at – 20 °C for several weeks.
The immunofluorescence assay was performed as previously described [31] with slight modifications. Briefly, on day 1, worm samples were hydrated for 10 min in 50% methanol (prepared with PBSTx) and PBSTx, then bleached for 90 min under intense light in a bleaching buffer (900 ul H2O, 50 ul formamide, 25 μl 20× SSC, 40 μl 30% H2O2), followed by two 1-min washes in PBST (PBS, 0.2% Tween20), digestion for 30 min in proteinase K (2 μg/ml) and 10 min in 4% paraformaldehyde (PFA), then 2% PFA and a wash in PBST. As a last step, the worm samples were blocked in blocking buffer (5% serum prepared with PBST) at 4 °C overnight. On day 2, three 20-min washes in PBST were carried out. Then the females were treated with 50 mM CuSO4 (pH = 5.0, regulated with 1 M NH4Ac) for 6 h and the males were treated with 0.5% Sudan black B for 6 h, followed by six 20-min washes in PBST. Next, the parasites were incubated in SjFer0 antibody or the negative rabbit-derived serum solution (1:500 dilutions in blocking buffer) at 4 °C overnight. On day 3, five 20-min washes in PBST were carried out. Then the commercialized secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor 488; ab150077; 1:1000 dilution; Abcam, Cambridge, UK) was applied, and the solution was incubated at room temperature for 3 h, followed by three 20-min washes in PBST. The 4’,6-diamidino-2-phenylindole (DAPI) dye (Thermo Fisher Scientific, Waltham, MA, USA) was applied at 1:10,000 dilution and incubated at 4 °C overnight. On the last day, the worms were washed three times in PBST (20 min each wash) and then transferred to 80% glycerin for mounting and observation under a laser confocal microscope (Nikon Corp.).
Functional expression of SjFer0 in Saccharomyces cerevisiae
The fet3fet4 double mutant Saccharomyces cerevisiae (strain DEY1453) is defective in low-affinity and high-affinity iron transport systems [32]. It shows more insufficient growth than the wild-type strain DY1450 on an iron-limited medium. These strains were kindly provided by Dr. Chen R (Chinese Academy of Agricultural Sciences, Beijing, China) with permission from Dr. David Eide (Nutritional Science Program, University of Missouri, Columbia, MO, USA).
The protocol used in the present study was a slight modification of that described by Ballesteros et al. [33]. Briefly, the SjFer0 sequence was first amplified with forward primer 5′-CCGAGCTCGGATCCATGAAAATCATGATGTTGATGAC-3′ and reverse primer 5′-GATGCATGCTCGAGTCAACGAAGTTCTTTATCCATC-3′ and then cloned into the pYES2 vector linearized by the BamHI and XhoI enzymes. Empty pYES2 vector was transformed with PEG and LiAc into wild-type strain DY1450 and fet3fet4 double mutant strain DEY1453 as a positive and negative control, respectively. The pYES2-SjFer0 vector was transformed into strain DEY1453. Culture at 30 °C for several days on uracil (URA)-selective synthetic defined (SD) media (Invitrogen, Thermo Fisher Scientific) supplemented with adenine hemisulfate and 2% glucose was used to select the pYES2-containing transformants because pYES2 expresses the URA3 gene.
A single colony was inoculated into URA-selective SD liquid culture media at 30 °C overnight. The cultures were then diluted in series (OD600 = 1.0, 0.1, 0.01 and 0.001), following which 5 μl of each dilution was spotted onto selective SD medium URA− plates supplemented with adenine hemisulfate and 2% galactose (for induced expression), 1% raffinose and 10 μM ferric chloride. The plates were incubated at 30 °C to observe colony’s growth.
RNA-sequencing after SjFer0 dsRNA interference in vivo
C57BL/6 mice infected with 60 cercariae were injected with 10 μg SjFer0 dsRNA or GFP dsRNA on 14 dpi and 18 dpi. The mice were sacrificed at 22 dpi and schistosome samples were collected. Second-generation RNA sequencing (RNA-Seq) was performed by Beijing Novogene Technology Co., Ltd. (Novogene, Beijing, China). Quality control (QC) of the raw RNA-Seq data was performed using the FASTQC program. Low-quality reads and adapter contamination were trimmed using the fastp tool [34].
The STAR tool [35] was used to map the clean reads to V3 genome assembly S. japonicum (SRA accession number: PRJNA739049). Transcript abundances were imported into the R program after further estimating with RSEM [36]. Differential expression analysis was performed with R package DEseq2, and Gene Ontology (GO) enrichment analysis was performed with the R package clusterProfile [37].
Statistical analysis
Digital experimental data were visualized by GraphPad Prism 8.0 software (GraphPad Software, San Diego, CA, USA). The results were analyzed using t-test. Results were considered to be significantly different at *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.