Haem transporter HRG-1 is essential in the barber’s pole worm and an intervention target candidate

Parasitic roundworms (nematodes) have lost genes involved in the de novo biosynthesis of haem, but have evolved the capacity to acquire and utilise exogenous haem from host animals. However, very little is known about the processes or mechanisms underlying haem acquisition and utilisation in parasites. Here, we reveal that HRG-1 is a conserved and unique haem transporter in a broad range of parasitic nematodes of socioeconomic importance, which enables haem uptake via intestinal cells, facilitates cellular haem utilisation through the endo-lysosomal system, and exhibits a conspicuous distribution at the basal laminae covering the alimentary tract, muscles and gonads. The broader tissue expression pattern of HRG-1 in Haemonchus contortus (barber’s pole worm) compared with its orthologues in the free-living nematode Caenorhabditis elegans indicates critical involvement of this unique haem transporter in haem homeostasis in tissues and organs of the parasitic nematode. RNAi-mediated gene knockdown of hrg-1 resulted in sick and lethal phenotypes of infective larvae of H. contortus, which could only be rescued by supplementation of exogenous haem in the early developmental stage. Notably, the RNAi-treated infective larvae could not establish infection or survive in the mammalian host, suggesting an indispensable role of this haem transporter in the survival of this parasite. This study provides new insights into the haem biology of a parasitic nematode, demonstrates that haem acquisition by HRG-1 is essential for H. contortus survival and infection, and suggests that HRG-1 could be an intervention target candidate in a range of parasitic nematodes.

Interestingly, the latter protein was first discovered in the free-living nematode, 7 Here, we modelled haem acquisition in transgenic C. elegans using zinc mesoporphyrin (ZnMP)a fluorescent 128 haem analogue (Fig 3A). In the transgenic worms, we showed that heterologous Hc-HRG-1 fused with a green 129 fluorescence protein (GFP) tag (PCe-hrg-1::Hc-HRG-1::GFP) co-localised within intestinal cells to ZnMP, in a 130 punctate manner (Fig 3B, subpanels a and b; S1 Fig), consistent with the cellular distribution of HRG-1 in C.

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elegans [18]. This result indicated transfer of haem to intracellular organelles, confirmed to be lysosome-132 related organelles based on co-localisation of ZnMP with lysosome-associated membrane protein (LMP) fused 133 to GFP (PCe-lmp-2::Ce-LMP-2::GFP) in C. elegans (Fig 3C; S1 Fig). Subsequently, we explored the subcellular 134 distribution of Hc-HRG-1, and showed in transfected HeLa cells that Hc-HRG-1::GFP was predominantly 135 distributed in the plasma membrane (Dil-co-stained), in early and late endosomes, identified independently 136 using RAB5A and RAB7A (Ras-related protein; specific marker for endosomes) fused to an mCherry tag, and 137 lysosomes (Lyso-Tracker-stained) (Fig 4). These findings are consistent with the canonical movement of haem 138 within a cell, which is usually mediated by HRG-4 (in the apical plasma membrane) and HRG-1 (in the 139 subcellular compartment) in C. elegans [18,22], suggesting biological roles of the unique HRG-1 orthologue 140 of parasitic nematodes in both haem uptake across plasma membrane and haem transport into subcellular 141 compartments.

H. contortus 145
Here, we searched for molecules that interact with Hc-HRG-1 to facilitate haem uptake and transport in H.

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contortus. Yeast-two-hybrid (Y2H), glutathione-S-transferase (GST) pull-down and co-immunoprecipitation 147 (co-IP) experiments (Fig 5A-5D) revealed a specific interaction between Hc-HRG-1 and a vacuolar H(+)-148 ATPase (V-ATPase) domain-containing protein (Hc-VHA-2) of H. contortus, with a punctate co-localisation 8 of these two proteins detected in transfected HeLa cells (Fig 5E). The sequential deletion of each of the four 150 transmembrane domains of Hc-HRG-1 showed that the third and fourth domains (representing the HRG 151 superfamily; Fig 5F) are essential for this specific interaction with Hc-VHA-2 in HEK293T cells (Fig 5G), 152 further indicating an involvement of the endo-lysosomal system in haem trafficking in the barber's pole worm 153 (see Fig 2), similar to that reported for mouse, rat and human cells [23].

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Interestingly, in adult H. contortus, while Hc-HRG-1 was not predominantly detected in the male reproductive 188 tract, it was abundant in the uterus of the female (Fig 7B and 7D), suggesting that this haem transporter is 189 integral to egg production and/or pre-embryonic development.
(P < 0.05) after four days of treatment, compared with control ( Fig 8B-8D

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contortus larvae resulted in sick (exhibiting retarded development, limited motility and viability) or lethal 199 phenotypes. Specifically, increased numbers of sick (30%; P < 0.001) and dead larvae (24%; P < 0.01) were 200 found after three days of RNAi treatment, compared untreated control worms (~12%) (Fig 8E and 8F), and the 201 percentages of sick (18%; P < 0.001) and dead larvae (38%; P < 0.01) increased significantly after four more 202 days (Fig 8G and 8H). We could partially rescue impaired haem acquisition in RNAi-treated larvae by adding 203 exogenous haem (5 µM and 10 µM) to the culture medium, achieving enhanced rescue at the higher 204 concentration (Fig 8E-8H). These findings suggest the essentiality of Hc-hrg-1 and the associated haem 205 acquisition and homeostasis in the larvae development and survival of H. contortus.

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Nematodes are a very diverse and large group of animals that inhabit almost every ecosystem, and can be 227 classified into five distinct clades (I to V), many of which are parasites of mammals [31,32]. Some species, 228 such as Haemonchus, Ancylostoma and Necator, are blood-feeding worms, with direct life cycles, and others 229 (e.g., within the spiruroid and filarioid groups) are transmitted to their definitive animal hosts via arthropod 230 vectors (e.g., black flies or mosquitoes) [32]. Not only are these parasites interesting from a biological 231 perspective, in that they maintain a very intimate relationship with the host that they infect, and rely heavily 232 on maintaining a balance in this relationship, so that neither parasite nor host is disadvantaged in a major way, 233 and with the parasites finding ways of acquiring nutrients from the host to survive and/or modulating or 234 suppressing the host immune response(s) to evade or avoid direct immune attack. Despite this intricate host-235 parasite relationship, many parasitic nematodes, such as blood-feeding nematodes, can cause devastating 12 diseases in humans and animals [33,34]; they deprive the host of blood, causing anaemia and associated 237 symptoms (e.g., impaired intellectual and physical development, and reduced economic productivity), and can 238 lead to death, particularly in young individuals. Given the socioeconomic impact that these parasites cause in 239 animal and human populations [35,36], and the challenges associated with their control, particularly relating 240 to resistance to, the inefficacy of, current treatments and lack of vaccines [37,38], there is a need to find better 241 ways of controlling parasitic nematode infections; this could be achieved through finding the Achilles heel in 242 these nematodes to disrupt or interrupt biological processes or pathways. For these reasons, our research 243 mission has been focused on understanding the biology and biochemistry of socioeconomically important 244 parasitic nematodes, with an emphasis on finding new intervention targets.

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Here, we elucidated the structure and function of the haem transporter (HRG-1) in one of the most 246 pathogenic, blood-feeding nematodes of the gastrointestinal tract of ruminants -H. contortus (barber's pole 247 worm) using various complementary experimental tools (e.g., yeast, C. elegans and mammalian cells) and 248 molecular assays (e.g., heterologous expression and RNAi). This transporter is of particular interest, because 249 many species/lineages have lost genes that encode enzymes involved specifically in endogenous haem 250 biosynthesis, such that they are completely reliant on acquiring exogenous haem from the environment within 251 or outside of their host, depending on their developmental stage, which means that this transporter is critical 252 for the survival of the parasite, thus representing an Achilles heel.

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Our findings revealed that most parasitic nematodes studied (for which molecular data sets were available) 254 only have one hrg-1 gene, and that this gene and its gene product are functionally essential for the acquisition 255 of exogenous haem and nematode survival, and that the protein (HRG-1) is structurally conserved in the 256 nematodes and selective for the parasite in that it is structurally very distinct from the orthologous host 257 molecule(s). These features, together with evidence that third-stage larvae of H. contortus whose hrg-1 gene 13 had been "knocked down" did not establish within the permissive host animal, indicating that HRG-1 is indeed 259 a promising intervention target. Given that HRG-1 is a membrane-bound protein expressed in the nematode's

Statistical analysis 443
Data were analysed using GraghPad Prism 8 and shown as means ± standard deviation (SD) or means ± 444 standard error of the mean (SEM). All statistical analyses were carried out using one-way ANOVA or Student's S2