Fig 1.
Structural conservation of HRG-1 in parasitic nematodes and distinctiveness from mammalian host orthologues.
(A) Structures of HRG-1 predicted for parasitic nematodes and mammalian hosts using AlphaFold2. Structural alignment and comparisons are performed between Hc-HRG-1 of Haemonchus contortus and orthologues of Nippostrongylus brasiliensis (Nb-HRG-1), Ancylostoma ceylanicum (Ac-HRG-1), Strongyloides stercoralis (Ss-HRG-1), Strongyloides ratti (St-HRG-1), Parastrongyloides trichosuri (Pt-HRG-1), Brugia malayi (Bm-HRG-1), Onchocerca volvulus (Ov-HRG-1), Ascaris suum (As-HRG-1), Trichinella pseudospiralis (Tp-HRG-1), Trichuris muris (Tm-HRG-1), Trichuris trichiura (Tt-HRG-1), and mammalian host animals including sheep (UniProt ID: A0A6P3YH43), goat (A0A452EBG4), cattle (E1BKJ0), mouse (Q9D8M3), rat (A0A8L2R3J2) and human (Q6P1K1). Root mean square deviation (RMSD) values are shown to indicate the structural distinctiveness between Hc-HRG-1 of H. contortus and orthologues in other parasitic nematodes and animals. (B) Structural superposition of HRG-1s in Caenorhabditis elegans (Q21642) and orthologues in parasitic nematodes. An overall RMSD value is measured at 1.065 among the HRG-1 orthologues of the free-living C. elegans and selected parasitic nematodes H. contortus, N. brasiliensis and A. ceylanicum (clade V), S. stercoralis, S. ratti and P. trichosuri (clade IV), B. malayi, O. volvulus and A. suum (clade III), T. pseudospiralis, T. muris and T. trichiura (clade I). (C) in silico docking of haem and predicted three-dimensional structure of Hc-HRG-1 using AlphaFold2 [57]. (D) Maximum absorbance for haem (at a wavelength of 386 nm) and for haem + Hc-HRG-1 (at a wavelength of 408 nm).
Fig 2.
Hc-HRG-1 mediates haem acquisition in a haem-deficient strain of yeast.
(A) The haem-deficient strain [Δhem1] of yeast is constructed by inserting δ-aminolevulinic acid synthase coding gene leu2, its promoter Pleu and loxP sites (donor fragment) into the hem1 locus of Saccharomyces cerevisiae BY4741 strain by homologous recombination. As hem1 is involved in the first step of haem biosynthesis, Δhem1 cannot synthesise endogenous haem de novo and cannot grow normally on plate unless supplemented with 5-aminolevulinic acid (ALA, an intermediate in the haem biosynthesis). (B) Haem spot growth assay of Δhem1 transformed with empty parental vector (negative control), vector expressing HRG-1 paralogue in Caenorhabditis elegans (Ce-HRG-4) or HRG-1 orthologue in Haemonchus contortus (Hc-HRG-1). 10-fold serially diluted haem-depleted cells are spotted on plates supplemented with the indicated concentrations of haem. 250 μM ALA is used as positive control. Neither ALA nor haem is used in negative control. (C) Gallium protoporphyrin IX (GaPPIX, a toxic haem analogue) spot growth assay of yeast transformed with empty parental vector and vector expressing Ce-HRG-4 and Ce-HRG-1 of C. elegans or Hc-HRG-1 of H. contortus. Transformants are spotted by 10-fold serial dilutions on plates supplemented with 0, 10 or 20 μM GaPPIX. (D) Schematic diagram showing transmembrane domains (TMD indicated in yellow) and residues essential for haem transport (indicated in red) in the haem spot growth assay. Positions of essential residues are numbered.
Fig 3.
Co-localisation of heterologous HRG-1 of Haemonchus contortus and a haem fluorescent analogue in the intestine of Caenorhabditis elegans.
(A) Structures of haem and the fluorescent zinc mesoporphyrinIX (ZnMP). (B) Heterologous expression of Haemonchus contortus hrg-1 (Hc-hrg-1) in Caenorhabditis elegans. Expression of Hc-HRG-1 fused with a green fluorescent protein (GFP) tag in transfected C. elegans is driven by the endogenous promoter of Ce-hrg-1 (PCe-hrg-1). Predominant distribution of Hc-HRG-1 is shown in the intestine of C. elegans, with punctate co-localisation of Hc-HRG-1 and ZnMP (indicated by white arrows). Details are shown in subpanels a (a1-a4) and b (b1-b4). (C) Co-localisation of ZnMP and a lysosome-associated membrane protein (Ce-LMP-2) fused with a GFP tag in the intestine of transfected C. elegans. Expression of Ce-LMP-2-GFP is driven by the endogenous promoter of Ce-lmp-2 in this nematode. Details are shown in subpanels a (a1-a4) and b (b1-b4). DIC indicates images capture using differential interference contrast technique. Scale bars, 50 μm or 10 μm.
Fig 4.
Heterologous expression and localisation of Hc-HRG-1 in mammalian HeLa cells.
Some cells are transfected with Hc-hrg-1-gfp, then stained with Lyso-tracker Red (staining lysosomes) or DiI (staining plasma) and subjected to confocal microscopy analysis. Other cells are co-transfected with Hc-hrg-1-gfp and hRab5a-mCherry (a marker for early endosomes) or hRab7a-mCherry (a marker for late endosomes) plasmids and analysed using confocal microscopy. Blue staining indicates the nucleus while the yellow signal indicates the co-localisation of Hc-HRG-1-GFP and Dil, RAB5A, RAB7A or Lyso-Tracker in transfected cells. Scale bars (5 μm) are shown in the images. A schematic diagram summarising the cellular distribution of Hc-HRG-1 and its movement from plasma membrane to early endosome, late endosome and then lysosome is shown.
Fig 5.
Identification of a V-ATPase subunit interacting with HRG-1 in Haemonchus contortus.
(A) pGBKT7 vector expressing GAL4 transcription factor binding domain (BD)-Hc-HRG-1 and a pGADT7-Rec-cDNA library expressing prey protein-GAL4 activation domain (AD) are constructed for H. contortus. Complex of GAL4 BD-Hc-HRG-1 and prey protein-GAL4 AD initiates transcription of reporter gene. (B) Hybridisation between the prey (BD-Hc-HRG-1) and bait (AD-Hc-VHA-2) transformants. Hybridisation of Y187-pGADT7-T (AD-T) and Y2H-pGBKT7-lam (BD-lam) is used as negative control, whereas AD-T and Y2H-pGBKT7-p53 (BD-53) is used as blank control. Proliferating blue dots indicate interaction and non-proliferating white dots indicate no interaction. (C) Pull-down assay. The glutathione-S-transferase (GST) tag or Hc-HRG-1-GST fusion protein absorbed beads are incubated with the whole-cell lysate (WCL) of human embryo kidney cells (HEK293T) transfected with pcDNA3.1-Hc-VHA-2 fused with a FLAG tag, and analysed by Western blot using antibodies against GST and FLAG tags. (D) Co-immunoprecipitation (Co-IP) assay. HEK293T cells transfected with plasmids expressing Hc-HRG-1-HA (YPYDVPDYA-tag) and Hc-VHA-2-FLAG is subjected to immunoprecipitation (Co-IP) with anti-FLAG antibody-conjugated magnetic beads. The Co-IP and WCLs are individually analysed by Western blot using specific antibodies indicated. Actin is used as an internal control. (E) Co-localization of Hc-HRG-1-GFP and Hc-VHA-2-mCherry in HeLa cells. Cells are co-transfected with plasmids expressing Hc-HRG-1-GFP and Hc-VHA-2-mCherry fusion proteins and analysed using confocal microscopy after 24 hours. Blue staining indicates nucleus and yellow pixels indicates co-localization of the two proteins. Scale bar, 10 μm. (F) Domain mapping of Hc-HRG-1 that interacts with Hc-VHA-2. Schematic diagrams showing the domain architecture of Hc-HRG-1 and deletion (Δ1, Δ2, Δ3 and Δ4) of individual transmembrane domains (TMD). The TMD3 and TMD4 represent a HRG superfamily domain. (G) Co-IP assay indicates the TMD3 and TMD4 of Hc-HRG-1-HA are essential for its interaction with Hc-VHA-2-FLAG. Tubulin is used as an internal control. Scale bars, 50 μm or 10 μm.
Fig 6.
Haem responsive transcription of hrg-1 and its roles in haem homeostasis in Haemonchus contortus.
(A) Relative mRNA level of hrg-1 in the egg, first- (L1s), second- (L2s), third- (L3s) and fourth-stage larvae (L4s), and adult female (Af) and adult male (Af) of H. contortus. Hc-18sRNA is used as an internal control. One-way ANOVA is used for statistical analyses. Different letter among data indicates a significant difference. (B) Relative mRNA level of hrg-1 in L1s/L2s, L3s, adult females and adult males of H. contortus when exposing to 0 μM, 20 μM and 100 μM of hemin chloride. (C) Relative mRNA levels of Ce-hrg-1 in C. elegans N2 worms fed with bacteria expressing double stranded RNA targeting cry1Ac of Bacillus thuringiensis (irrelavent control), Ce-hrg-1 of C. elegans (Ce-hrg-1RNAi) and Hc-hrg-1 of H. contortus (Hc-hrg-1RNAi). Ce-actin-1 is used as an internal control. (D) Hc-hrg-1RNAi mediated gene knockdown of Ce-hrg-1 results in accumulation of zinc mesoporphyrin IX (ZnMP; a fluorescent haem analogue) in the intestine of treated worms, compared with untreated worms (Control). DIC, differential interference contrast. Scale bar, 10 μm. (E) Quantification of ZnMP using ImageJ software. (F) Relative mRNA levels of Hc-vha-2 in H. contortus fed with bacteria expressing double stranded RNA targeting cry1Ac of Bacillus thuringiensis (Bt-cry1Ac; irrelavent control) and Hc-vha-2 (Hc-vha-2RNAi). (G) Relative mRNA levels of Hc-hrg-1 in H. contortus fed with bacteria expressing double stranded RNA targeting Bt-cry1Ac (irrelavent control) and Hc-vha-2. (H) Relative mRNA levels of Hc-vha-2 in H. contortus fed with bacteria expressing double stranded RNAs targeting Hc-hrg-1 and Hc-vha-2. (I) Relative mRNA levels of Hc-hrg-1 in H. contortus fed with bacteria expressing double stranded RNAs targeting Hc-hrg-1 and Hc-vha-2. Data are showed as mean ± standard deviation (SD), n = 10. A 2−ΔΔCT method is used for relative transcriptional data normalisation. Data are showed as mean ± standard deviation, n ≥ 3. Student t-test is performed for statistical analyses. ns: non-significant, *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 7.
Tissue expression of HRG-1 in Caenorhabditis elegans and Haemonchus contortus.
(A) Heterologous expression of H. contortus HRG-1 fused with green fluorescent protein (GFP) in the anterior, medial and posterior intestinal tract of C. elegans. (B) A schematic diagram indicating specific HRG-1 expression on the apical and basal membrane of intestine in C. elegans. No distribution of HRG-1 is found in other tissues. (C) Expression of HRG-1 at the basal laminae that covering both intestine (indicated by in), reproductive tract (indicated by re) and muscle of adult female and male H. contortus. 4’,6-diamidino-2-phenylindole (DAPI) stains nucleus in blue. Scale bar, 50 μm or 20 μm. (D) A schematic diagram indicating a broad tissue expression (particularly at basal membranes) of HRG-1 in adult worms (female and male) of H. contortus.
Fig 8.
RNA interference mediated gene knockdown of hrg-1 results in compromised haem uptake in and survival of Haemonchus contortus.
(A) Relative mRNA levels of Hc-hrg-1 in H. contortus larvae fed with bacteria expressing double stranded RNA targeting cry1Ac of Bacillus thuringiensis (irrelavent control) and Hc-hrg-1 after three (Day3) and seven days (Day7). Hc-18sRNA is used as an internal control. (B-D) RNAi-treated worms are fed with 0 μM, 5 μM and 10 μM of Ga (III) complex of the haem precursor protoporphyrin IX (GappIX; a toxic haem analogue) for 6 days, and subjected to mortality analysis every day. (E-H) Percentages of sick (low vitality, developmental retarded or deformed) and dead larvae are found in H. contortus fed with bacteria expressing double stranded RNA targeting Hc-hrg-1 at day 3 and 7, compared with that targeting irrelavent control. Supplementation of 5 μM or 10 μM of haem into culture medium completely or partilly rescue the sick and lethal phenotype of treated larvae. (I) Percentages of dead and sick (low vitality, developmental retarded or deformed) larvae after Hc-vha-2RNAi measured at day 7. (J) Percentages of dead larvae after Hc-vha-2RNAi treatment with supplementation of 0 μM, 5 μM and 10 μM hemin chloride. Treatment with Bt-cry1AcRNAi is used as irrelavant control. Hc-18sRNA is used as an internal control. A 2−ΔΔCT method is used for relative transcriptional data normalisation. Data are showed as mean ± SD, n ≥ 3. Student t-test is performed for statistical analyses. ns, no significance, *P < 0.05, **P < 0.01, ***P < 0.001. (K) A schematic diagaram indicating the roles of Hc-hrg-1 and Hc-vha-2 in haem homeostasis and utilisation in H. contortus. Low transcriptional level of Hc-hrg-1 do not affect the transcription of Hc-vha-2, whereas lower mRNA level upregulates the transcription of Hc-hrg-1.
Fig 9.
Animal infection with RNA interference treated infective larvae of Haemonchus contortus.
(A-C) Eggs (n = 10,000) of H. contortus are collected from faeces, hatched on plates and fed with faecal matter (Blank control) or bacteria expressing double stranded RNA targeting cry1Ac of Bacillus thuringiensis (Bt-cry1Ac; irrelavent control) or Hc-hrg-1 (hrg-1RNAi) for 7 days. Infective larvae of these treated worms are used to infect helminth-free sheep. Faeces are collected from the infected sheep every day from 21 days post infection to 63 days post infection to perform worm egg counting using a flotation method. (D) The numbers of eggs per gram (EPG) of faeces collected from sheep of blank control, irrelevant control and hrg-1RNAi groups are calculated and shown as mean ± standard error of the mean (n = 4) in the line graph. Images in this figure are drawn by the authors.