Fig 1.
Ts-Asp2 liberates heme from host myoglobin, and hemin supplementation drives molting in vitro.
(A) Colorimetric detection of ALAD activity. Reaction mixtures containing δ-aminolevulinic acid (ALA, 2.5–50 μg/μL) were incubated with T. spiralis (Ts) lysates, E. coli lysates (positive control), or PBS (negative control). Porphobilinogen (PBG) was detected with Ehrlich’s reagent. A pink-to-red colorimetric signal (indicative of PBG formation) was observed in E. coli lysates in an ALA concentration-dependent manner. No visible color change was detected in Ts lysates or the PBS control. (B) Absorbance quantification at 555 nm. E. coli lysates exhibited dose-dependent PBG production (p < 0.0001 vs. Ts/PBS). Ts lysates and PBS showed negligible absorbance across all ALA concentrations, with no significant difference between Ts and PBS. Statistical analysis was performed using two-way ANOVA (n = 3). (C) TMB-based proteolytic assay. Mb (1 μg/μL) was incubated with rTs-Asp2 (0.5 μg/μL) ± Pepstatin A (10 μM, aspartic protease inhibitor) at 37°C for 4 h. Mb alone exhibited the strongest blue coloration (oxidized TMB), followed by rTs-Asp2 + Mb + PepA, whereas rTs-Asp2 + Mb showed minimal coloration. No signal was detected in the rTs-Asp2-alone group. (D) Absorbance spectra of the TMB oxidation products. Mb alone had the highest peaks at 370 and 652 nm (TMB oxidation markers). rTs-Asp2 + Mb exhibited significantly reduced signals, whereas rTs-Asp2 + Mb + PepA displayed intermediate peaks. No peaks were detected for rTs-Asp2 alone. Statistical analysis was performed using two-way ANOVA (n = 3). (E) SDS‒PAGE analysis of Mb integrity. Mb alone and rTs-Asp2 + Mb + PepA had intact 15 kDa bands, whereas rTs-Asp2 + Mb had a significantly fainter band. (F) Hemin-dependent molting rate. Larvae cultured with hemin (0–100 μM) for 24 h exhibited dose-dependent molting (0 μM vs. 100 μM, p < 0.0001; n = 3 biological replicates, one-way ANOVA). (G) Microscopic visualization of molting. Shed cuticles (translucent sheaths, arrows) were rare at 0 μM but increased with increasing hemin concentration. Scale bars: 200 μm (main panels), 100 μm (insets). (H) ZnMP fluorescence imaging. Larvae cultured in PBS, 25 μM hemin, 6.25 μM hemoglobin (Hb), or 25 μM myoglobin (Mb) presented the strongest anterior-localized granular fluorescence, with diminished signals in the Hemin/Hb/Mb groups (excitation/emission: 561/595 nm). (I) ZnMP fluorescence intensity in larvae (one-way ANOVA, n = 3).
Fig 2.
Ts-HRG-1 has a phylogenetically conserved transmembrane architecture with intact heme-binding motifs.
(A) A maximum-likelihood phylogenetic tree of HRG-1 homologs (MEGA) grouped T. spiralis (Ts) HRG-1 within the animal parasitic nematode clade, which is distinct from that of plant parasitic nematodes, non-parasitic nematodes, and non-nematodes. (B) Sequence alignment (MEGA) of Ts-HRG-1 with homologs from Hc, Ce, mouse, and zebrafish revealed four conserved transmembrane domains (TMD1-4, purple boxes) and an intact heme-binding Y-x-R-x-R motif (yellow box). (C) Homology models (ChimeraX) depicting HRG-1 structures across species, color-coded to show extracellular loops (pink), transmembrane helices (green), and the surrounding phospholipid bilayer (red: outer leaflet; blue: inner leaflet). TMDs are spatially conserved, which is consistent with a shared transmembrane architecture.
Fig 3.
rTs-HRG-1 binds hemin at conserved residues and induces a hypsochromic spectral shift.
(A) Molecular docking (AutoDock) identified Arg33, Lys145, and Tyr153 as key residues that form hydrogen bonds and hydrophobic interactions with hemin. Electrostatic surface models highlight the hemin-binding pocket. (B) Binding affinity analysis (Discovery Studio) confirmed the hemin-Ts-HRG-1 interaction, with schematic diagrams depicting binding poses (see in-fig legends). (C) Nondenaturing PAGE of rTs-HRG-1 incubated with hemin (37°C, 2 h). TMB staining revealed a distinct band in the rTs-HRG-1 + hemin lane (hemin-bound complex), which was absent in the rTs-HRG-1 alone lane. Coomassie blue staining confirmed equal protein loading. Hemoglobin (positive control) showed intrinsic heme-dependent TMB reactivity. (D) Absorbance spectra (340–750 nm). Free hemin exhibited a peak at 390 nm (Soret band), which shifted to 370 nm in the rTs-HRG-1 + hemin group, indicating hemin‒protein interactions. No peak was observed for rTs-HRG-1 alone.
Fig 4.
Ts-HRG-1 restores the growth of heme-auxotrophic Δhem1 yeast by functional complementation.
(A) Schematic diagram of HEM1 knockout and complementation in S. cerevisiae BY4741. The CRISPR/Cas9 system (pCAS plasmid) with HEM1-targeting sgRNA was used to generate the Δhem1 strain, which was subsequently transformed with empty vector (pYES2-CT), Ce-HRG-4, or Ts-HRG-1 constructs. (B) Validation of Δhem1 knockout: parental and Δhem1 strains were cultured on YPD ± 250 μM δ-aminolevulinic acid (ALA). Δhem1 failed to grow without ALA, confirming auxotrophy. (C) Western blotting (with an anti-His tag) confirmed the expression of Ce-HRG-4 and Ts-HRG-1 in the transformed strains. (D) Growth validation of transformed Δhem1 strains: Empty vector-, Ce-HRG-4-, and Ts-HRG-1-expressing strains grew similarly on SD ± ALA, confirming plasmid neutrality. (E) Hemin concentration-dependent growth rescue. Δhem1 strains expressing Ts-HRG-1, Ce-HRG-4, or the empty vector were cultured in SD medium supplemented with hemin (0.25, 2.5, or 10 μM). Ts-HRG-1 restored growth at 2.5 μM hemin, with further enhancement at 10 μM, mimicking that of Ce-HRG-4. No rescue occurred at 0.25 μM or with the empty vector. (F) Growth curves of Δhem1 yeast strains expressing the empty vector (control), Ts-HRG-1, or Ce-HRG-4 in SD medium containing 2.5 μM hemin. The indicated P value (p < 0.0001) represents the comparison of OD600 values at the 22-hour time point, analyzed by one-way ANOVA (n = 3).
Fig 5.
Ts-HRG-1 shows tissue-specific localization in ML.
(A) Immunofluorescence localization of Ts-HRG-1 in ML sections using a Ts-HRG-1 polyclonal antibody (red; Alexa Fluor 555-conjugated secondary antibody and Alexa Fluor 647-conjugated secondary antibody) and DAPI (blue, nuclei). The negative control serum (preimmune serum with identical secondary antibodies) showed no specific signal. Ts-HRG-1 localized to the stichosome (arrows). Scale bars: 20–50 μm. (B) Immunofluorescence localization of Ts-HRG-1 in intact ML using a Ts-HRG-1 polyclonal antibody (red; Alexa Fluor 555-conjugated secondary antibody and Alexa Fluor 647-conjugated secondary antibody) and DAPI (blue, nuclei). The negative control serum (preimmune serum with identical secondary antibodies) showed no specific signal. Ts-HRG-1 localized to the cuticle (arrows). Scale bars: 20–50 μm. (C) Colocalization of Ts-HRG-1-GFP (green) with organelle markers: Golgi, lysosomes, and plasma membrane. Strong colocalization (yellow) was observed with the plasma membrane (arrowheads), but minimal overlap with the Golgi or lysosomes was detected. (D) Colocalization analysis. Pearson’s correlation coefficient (Rr): plasma membrane = 0.782, Golgi = 0.274, and lysosomes = 0.239. The intensity profiles show high overlap between the Ts-HRG-1-GFP and mCherry signals.
Fig 6.
Ts-HRG-1 knockdown impairs molting and heme uptake in ML.
(A) ML were cultured with 0–100 μM hemin for 24 h, and the expression of Ts-HRG-1 was analyzed by qPCR. Statistical significance was determined by one-way ANOVA (n = 3). (B) qPCR analysis of Ts-HRG-1 expression in ML treated with either NC (nontargeting control) or Ts-HRG-1 RNAi and cultured with 0 or 25 μM Hemin for 24 h. Statistical significance was determined by one-way ANOVA (n = 3). (C) Western blot (anti-Ts-HRG-1, with α-tubulin as a loading control). (D) Grayscale quantification of the WB bands. Statistical analysis was performed using one-way ANOVA (n = 3). (E) Microscopy images of ML molting. (F) Molting rate quantification. Statistical analysis was performed using one-way ANOVA (n = 3). (G) Schematic of the mouse infection assay. PBS-, NC-, or Ts-HRG-1 RNAi-treated ML (200 larvae/mouse) were orally administered to the mice, with the larvae collected on Day 6 (AD6) and Day 35 (ML stage). (H) Larval burden analysis. AD6: No difference across groups. Statistical analysis was performed using one-way ANOVA (n = 6). (I) NBL: The RNAi group presented reduced larval counts (p < 0.05 vs. the NC group). Statistical analysis was performed using one-way ANOVA (n = 12). (J) The Day 35 ML: RNAi group presented a significantly lower ML burden (p < 0.05 vs. the NC group). Statistical analysis was performed using one-way ANOVA (n = 6).
Fig 7.
Detection of a direct interaction between Ts-HRG-1 and Ts-ATP6V0C in vitro.
(A) Yeast two-hybrid assay. AD-Ts-ATP6V0C and BD-Ts-HRG-1 were cotransformed into yeast. Growth on SD-TLHA + 10 mM 3-AT confirmed the interaction, with no autoactivation in the controls (AD/BD empty vectors). (B) Coimmunoprecipitation (Co-IP). Lysates from HeLa cells coexpressing Ts-HRG-1-GFP and Ts-ATP6V0C-mCherry were immunoprecipitated with anti-GFP. Ts-ATP6V0C-mCherry was detected in the IP fraction (anti-mCherry blot), confirming the physical interaction. No signal was observed in the GFP-empty controls. (C) Colocalization analysis. Pearson’s correlation coefficient (Rr = 0.773) and distance-dependent intensity profiles confirmed the spatial proximity between Ts-HRG-1 and Ts-ATP6V0C. (D) Colocalization in HeLa cells. Ts-HRG-1-GFP (green) and Ts-ATP6V0C-mCherry (red) strongly colocalized (yellow) in the cell. Scale bar: 5 μm.
Fig 8.
BafA1 and Ts-HRG-1 knockdown suppress larval development and heme uptake in T. spiralis.
(A) ZnMP fluorescence in treated larvae. ML pretreated with Ts-HRG-1 RNAi, Ts-ATP6V0C RNAi or 2.5μg/mL BafA1 (for 24 h) and fed ZnMP displayed markedly reduced intracellular fluorescence, suggesting impaired heme uptake. (B) Quantification of ZnMP fluorescence intensity. The intensity was significantly reduced in the Ts-HRG-1 RNAi, Ts-ATP6V0C RNAi and BafA1 groups. Statistical analysis was performed using one-way ANOVA (n = 3). (C) ML molting rate analysis of the CON, Ts-HRG-1 RNAi, Ts-ATP6V0C RNAi and BafA1 (2.5 μg/mL) groups; data are presented as the means ± SEMs (one-way ANOVA, n = 3). (D) Mortality of NBL: Concentration-dependent mortality following 24-h BafA1 exposure. One-way ANOVA vs. control, n = 3. (E) Mortality of AD6: Dose‒dependent mortality after 24 h of BafA1 treatment (0–10 μg/mL). One-way ANOVA vs. control, n = 3. (F) Molting rate of ML: BafA1-induced suppression of molting after 24 h of treatment. One-way ANOVA vs. control, n = 3. (G) ML: Ts-ATP6V0C mRNA measurement following 24 h of BafA1 treatment. Data = mean ± SEM; one-way ANOVA vs. control. 18S rRNA internal control (n = 3). (H) Ts-HRG-1 qPCR analysis: CON, Ts-HRG-1 RNAi, Ts-ATP6V0C RNAi and BafA1 (2.5 μg/mL) groups with means ± SEMs; one-way ANOVA (18s rRNA reference). (I) NBL: Ts-HRG-1 mRNA quantification after 24 h of BafA1 treatment. Data = mean ± SEM; one-way ANOVA vs. control. 18S rRNA internal control, n = 3. (J) AD6: Ts-HRG-1 mRNA levels after 24 h of BafA1 treatment. Data = mean ± SEM; one-way ANOVA vs. control. 18S rRNA internal control, n = 3. (K) ML: Ts-HRG-1 mRNA measurement following 24 h of BafA1 treatment. Data = mean ± SEM; one-way ANOVA vs. control. 18S rRNA internal control, n = 3.
Fig 9.
BafA1 treatment reduces T. spiralis larval burden and pathology in mice.
(A) Experimental design. Mice (n = 6/group) were administered BafA1 (0.1, 0.5, or 1 mg/kg) via oral gavage during the intestinal phase (0–7 dpi; Int. ph) or encystment phase (21–29 dpi; Encyst. ph). ML were recovered at 35 dpi for quantification. (B) Larval burden analysis. BafA1 treatment during the intestinal phase (0–7 dpi; Int. ph) significantly reduced the ML count (p < 0.05), and treatment during the encystment phase (21–29 dpi; Encyst. ph) reduced the ML count (p < 0.05). The data were analyzed with GraphPad Prism (mean ± SEM). Statistical analysis was performed using one-way ANOVA (n = 6). (C) H&E staining of representative diaphragm sections. Treatment groups: Nondosing group (CON), BafA1-administered at 0.1, 0.5, or 1 mg/kg during the intestinal phase (Int. ph) and encystment phase (Encyst. ph). Scale bar: 200 μm.
Fig 10.
Key findings are integrated: Ts-Asp2 degrades host myoglobin (Mb) or hemoglobin (Hb) to release hemin, which is transported by Ts-HRG-1 and its interacting protein Ts-ATP6V0C.