Hemozoin: a waste product after heme detoxification?

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Jun Sun, Xixi Qin, Wenwen Si, Fei Wang, Yanna Li, Xiaoli Yan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5408190/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Mar, 2025 Read the published version in Parasites & Vectors → Version 1 posted 12 You are reading this latest preprint version Abstract Hemozoin is consider a waste byproduct of heme detoxification following hemoglobin digestion; consequently, the biological functions of hemozoin in hemozoin-producing organisms have often been overlooked. However, recent findings indicate that schistosoma hemozoin facilitates the transfer of iron from erythrocytes to eggs through its formation and degradation, thereby increasing interest in the role of malarial hemozoin. In this study, we compared the formation of schistosoma hemozoin and malaria hemozoin using transmission electron microscopy, which suggests why the trophozoite stage cannot eliminate hemozoin. Additionally, through transcriptome analysis of different stages of P. falciparum 3D7 WT and P. falciparum 3D7 C580Y - where the latter serves as a control with lower hemozoin production-we found that both exhibit similar expression patterns in genes related to DNA synthesis, iron, and heme utilization. Notably, during the trophozoite stage, expression levels of these genes in P. falciparum 3D7 WT are higher than in P. falciparum 3D7 C580Y , whereas during the schizont stage, they are lower. These results suggest that when P. falciparum 3D7 utilizes more heme and iron, it produces less hemozoin, whereas when it utilizes less heme and iron, it produces more hemozoin. Interrupting heme utilization and destructing hemozoin aggregation can result in parasite death. Additionally, the hemozoin released by schizonts can impair macrophage functions, or it is carried by gametocytes into the next host without being discharged as waste, suggesting that the release of malaria hemozoin protects merozoites from phagocytosis, and its transfer to the next host may fulfill the requirements for iron and heme during their development in mosquitoes. Hemozoin malarial parasites transmission electron microscopy iron heme Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Hemozoin is a critical byproduct that forms during the unique detoxification process in malaria parasites. During the blood stages of the Plasmodium species, hemozoin is synthesized as the parasite digests host erythrocyte hemoglobin to obtain its amino acids. The liberated toxic heme, a compound containing an iron atom within a porphyrin ring, is converted into an insoluble and chemically inert form to prevent cellular damage caused by its pro-oxidant properties. This substance, composed of heme polymers bonded via iron-carboxylate links, accumulates in the parasite’s food vacuole and is later released into circulation when infected red blood cells rupture. The impact of heme on parasites is a subject of interest in the scientific community. Several researchers have suggested that heme can inhibit various Plasmodium enzymes, including plasmepsins, falcipains, glycolytic glyceraldehyde-3-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase [ 1 ] [ 2 , 3 ]. Additionally, the integration of lipophilic heme into biological membranes reduces the deformability of erythrocytes and induces hemolysis. Heme in the membrane also weakens the lipid bilayer, making it more susceptible to H 2 O 2 -mediated lysis. This integration disrupts the normal dynamic interaction between the RBC membrane and its underlying cytoskeletal proteins[ 4 ]. High concentrations of heme lead to increased oxidative stress, and the presence of peroxidation products is associated with decreased RBC membrane fluidity, likely increasing cellular rigidity in parasitized red cells[ 5 ]. The catabolism of hemoglobin, which releases reactive heme and iron, is associated with the generation of redox-reactive substances such as H 2 O 2 , superoxide radicals, and the hydroxyl radical, which directly mediates lipid peroxidation[ 6 ]. The interaction between heme, intracellular hydrogen peroxide, and lipids can result in lipid peroxidation[ 7 ]. These harmful effects are directly and indirectly attributable to free heme[ 8 ], suggesting that hemozoin formation is a detoxification mechanism. Notably, the malaria parasite maintains a heme pool at a consistent level of approximately 1.6 µM throughout its development within red blood cells [ 9 ], indicating the crucial role of high concentrations of heme for the parasite. The parasite digests up to 65% of the host cell’s hemoglobin but only utilizes up to about 16% of the amino acids derived from hemoglobin digestion, raising the question of whether hemoglobin digestion serves solely for amino acids acquisition [ 10 ]. The effectiveness of iron chelators in killing malarial parasites [ 11 , 12 , 13 , 14 , 15 ] strongly suggests the essentiality of iron for these parasites. Especially, in malaria parasites, iron is released during heme degradation facilitated by glutathione (GSH) [ 16 ], suggesting that the accumulation of a substantial amount of heme likely corresponds to a significant supply and demand for iron. Notably, the considerable quantity of hemozoin formed in gametocytes and subsequently transferred to mosquitoes suggests that hemozoin plays a crucial role in the storage and utilization of heme and iron, particularly as malaria parasites initiate heme synthesis exclusively in mosquitoes[ 17 , 18 ]. Additionally, lipids play a significant role in Plasmodium , and distinct changes in lipid composition occur during different stages of development [ 19 ]. Interestingly, the parasite lacks the ability to synthesize cholesterol de novo and has limited capacity for fatty acid synthesis[ 20 , 21 ]. As a result, the parasites have to acquire and utilize host lipids. In the digestive vacuoles of Plasmodium , hemozoin formation is enhanced by host unsaturated fatty acids, whereas the electron released from heme polymerization likely oxidizes the unsaturated fatty acids of the host. The remodeled fatty acids or lipids are likely detoxified and made suitable for use by Plasmodium [ 22 , 23 ]. Furthermore, hemozoin-producing schistosomes have been found to use hemozoin formation and degradation to transfer iron and lipids to the vitelline gland and eggs [ 24 , 25 , 26 ]. These pieces of evidence suggest that malarial hemozoin formation is not merely a waste product of heme detoxification but likely plays a crucial role in the life cycle of parasites. To further investigate the role of hemozoin, we employed transmission electron microscopy (TEM) to compare the formation of hemozoin in Schistosoma and malaria. Notably, malaria hemozoin was directly observed without the conventional electron microscopy sampling process, which aimed to preserve the authentic structure of the sample and examine its association with the parasites. Additionally, gene expression analysis was conducted to investigate the relationship between iron or heme utilization and hemozoin production. Methods Ethics statement This study strictly adhered to the recommendations of the Regulations for the Administration of Affairs Concerning Experimental Animals of the State Science and Technology Commission. The protocol was approved by the Internal Review Board of Tongji University School of Medicine (TJLAC-014-017). Parasites and isolation of schistosome hemozoin granules ( SHGs ) Female mice of the Kunming strain weighting 20 to 22 g were obtained from SLRC Laboratory Animal Co., Ltd. In Shanghai, China. Cercariae freshly shed by the snails were used to infect mice percutaneously with 20 cercariae each. Adult female schistosomes were collected 42 to 45 d post-infection and washed with sterile 0.15mol/L NaCl solution (normal saline). The female schistosomes were cut into small sections in sterile normal saline solution in eppendorf tubes, and the dark suspensions were collected, following a previous study[ 25 ]. Clumps were removed, and individual hemozoin granules were retained through low-speed centrifugation. The hemozoin granules were then washed with sterile normal saline solution and utilized for a macrophage phagocytosis experiment. An intact female adult worm was cut into three sections, and the gut contents from each section were collected and treated using a previously described method [ 24 , 25 ]. The treated samples were observed using a light microscopy (LM, Nikon 50i) and a JEOL EW-1230 scanning electron microscopy with an accelerating voltage of 80 kV. Images were acquired using a digital photo-documentation system (Gatan Bioscan Camera, model 792). Malaria parasites and hemozoin analysis with LM and TEM The P. falciparum parasites (3D7 strain) were provided from the national institute of parasitic diseases of Chinese center for disease control and prevention. The P. falciparum 3D7 WT and P. falciparum 3D7 C580Y were cultured in RPMI 1640 medium with 25 mmol/L HEPEPS, 0.5% AlbuMAX, 0.2% sodium bicarbonate, 0.2 mmol/L htpoxanthine, and 20 µg/mL gentamicin sulfate at 37 ℃ incubator with 5% CO 2 and 5% O 2 . Fresh O + human RBCs from healthy human donors were used to maintain parasitemia, and parasites were maintained at 2% hematocrit based on a previous report [ 27 ]. Blood smears were stained with Giemsa in accordance with the standard staining protocol and then observed the smears with a LM (Nikon 50i). The erythrocytes infected by malaria parasites were treated with saponin at a final concentration of 0.15% and then centrifuged at 10 000 rpm for 5 min. The black deposit was collected. Then, the deposits were fixed with 2.5% glutaraldehyde–phosphate-buffered saline (PBS) (pH 7.2) buffer and then dried at room temperature, and directly observed using a JEOL EW-1230 TEM (Japan) at an accelerating voltage of 80 kV. Schitosoma samples were fixed in glutaraldehyde overnight under 4℃. Then, they were washed with PBS, fixed in 1% osmium tetroxide, dehydrated in acetone and embedded in Epon812. Then, 60 nm- thick sections were cut by using ultramicrotome (EM UC6, Leica, German) and mounted on copper slot grids coated with Formvar and stained with uranyl acetate and lead citrate for examination. Samples were observed by using a JEOL EW-1230 TEM (Japan) and the images were acquired by using a digital photodocumentation system (Gatan Bioscan Camera, Model 792). Energy dispersive spectroscopy (EDS) TEM sections of malarial hemozoin, including heme aggregation sphere (HAS) and crystal-like hemozoin, were analyzed by using energy dispersive (X-ray) spectroscopy (Oxford INCA, Lyford, OX, UK) under a JEM-2010 transmission electron microscope at an accelerating voltage of 200 kV as previously described[ 24 ]. The Oxford INCA software package was used to carry out the X-ray analysis to determine the elemental composition of the sample and generate its characteristic spectrum. Analysis of the effect of artemether on malarial parasites using light microscope BALb/c mice or ICR mice were infected by intraperitoneal injections with P. yoelii 17XNL. When the infection rate exceeded 20%, the mice were administered artemether at a dosage of 100 mg/kg. Blood samples were collected after 4 and 16 hours of treatment, respectively. Blood smears were prepared and stained with Giemsa according to standard staining protocols, and the smears were subsequently examined using a light microscope (Nikon 50i). Comparison of hemozoin content and merozoite number Plasmodium falciparum 3D7 WT and P. falciparum 3D7 C580Y were purified using a 40–70% Percoll gradient and treated with 5% sorbitol to obtain 6-h ring stages. Then, they were cultured for 36–42 h with parasitaemia adjusted to 0.5–1%. Blood smears were stained with Giemsa in accordance with the standard staining protocol and then compared the number of their merozoites with a LM (Motic, PA53 FS6). Approximately 10 ml of the culture medium was treated with 0.15% saponin solution on ice for 10 min to lyse the cells. After centrifugation at 10,000 g for 15 min, the sediment was washed with 25 mmol/L Tris (pH 7.8) containing 2.5% sodium dodecyl sulphate until the supernatant was clear. The sediment was dissolved in 250 µL of 2.5% sodium dodecyl sulphate bufer and 20 µL of 2.5 mol/L NaOH. The Nanodrop 2000 spectrophotometer was used to measure the absorbance of hemozoin at 400 nm for content quantification, as described in a previous paper [ 28 ]. Statistical analysis was conducted using T-test (unpaired) with Graphpad Prism 8.0.2 software for Windows. A p -value less than 0.05 was considered statistically significant. Transcriptome analysis of P. falciparum 3D7 The P. falciparum 3D7 were cultured in RPMI 1640 medium according to established protocols [ 27 , 28 ]. The parasites were synchronized using 5% sorbitol. Subsequently, mature schizonts were purified on a 40%/70% percoll gradient. Cultures were then treated with 5% sorbitol to obtain 0–3 h ring stages. Parasites were collected within 0–3 hours after invasion, and samples were harvested at 0, 6, 12, 18, 24, 30, 36, and 42 hours post-infection (hpi) for transcriptome analysis. Samples at different stages were collected and preserved in TRIzol (Invitrogen, USA). RNA extraction was performed using phenol and isopropanol precipitation. RNA quantification and quality assessment were carried out using a Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). RNA integrity was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Libraries were prepared using a TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, CA, USA). Transcriptome sequencing and analysis were performed by LC Sciences (Hangzhou, Zhejiang, China). RESULTS The hemozoin granules were degraded and utilized in Schistosoma japonicum gut The anterior portion of the intestinal tract of Schistosoma japonicum contains globe- and comma-shaped hemozoin granules, along with freshly-ingested host red blood cells (Fig. 1 A and 1 B). Examination using light microscopy (LM) and transmission electron microscopy (TEM) reveals that the red blood cells are surrounded by hemozoin granules, forming clusters or aggregates. Our previous studies reveal that upon attachment to erythrocytes, Schistosoma hemozoin granules can exploit these cells for the self-assembly of new hemozoin granules[ 25 ]. Under TEM, hemozoin granules are observed to be closely in contact with the matrix derived from degraded erythrocytes (Fig. 1 B). Notably, in the posterior portion of the intestine, the hemozoin granules are degraded near the intestinal microvilli (Fig. 1 C and 1 D). Initially, the heme polymer in the outer layer of the hemozoin granules decomposes, followed by subsequent decomposition of the lipid components within the granules (Fig. 1 C). Particularly, after hemozoin degradation, the transport of intestinal contents across the intestinal wall to vitelline gland cells becomes clearly visible under TEM. During this process, the intestinal contents do not appear to be processed by the cells of the intestinal wall, but instead pass directly through the gut wall via the intercellular space. Simultaneously, accumulation of lipids is observed within the vitelline gland cells (Fig. 1 E). Based on our observations, we proposed that hemozoin granules in schistosomes facilitate the transport of heme and lipids by forming and subsequently degrading (Fig. 1 F). Apparently, Schistosoma hemozoin granules play a crucial role in storing and transporting heme, iron and lipids during its development and reproduction. Morphology of malarial hemozoin differs from that of schistosoma hemozoin granules Under LM, these dark-brown malaria hemozoin appear globular or irregular in shape (Fig. 2 A and 2 B). Under TEM, we observe various morphologies of malarial hemozoin (Fig. 2 C- 2 E). The energy disperse spectroscopy can detect iron inside the hemozoin (Fig. 2 F). Notably, hemozoin is a mixture or combination of heme polymer and lipids when observed under TEM. The free heme dissolves in the lipids which facilitate the polymerization of heme into crystal-like structure. Some researchers only consider the crystal-like structure as hemozoin. The crystal-like hemozoin develops from heme aggregations in its lipid matrix[ 29 , 30 , 31 , 32 ]. Observing the fresh samples with TEM, we found malaria hemozoin is a combination within the parasites, consisting of heme aggregation or crystal-like heme polymer, and lipids. The lipid layer is external while the heme aggregation or crystal-like polymer is internal (Fig. 2 C- 2 E). It is evident that the morphology and structure of malarial hemozoin differ from those of schistosoma hemozoin granules. In schistosoma hemozoin granule, the heme layer is on the outside and is first degraded near the intestine microvilli. In contrast, the heme polymer in malarial hemozoin is located on the interior, indicating that heme is stored rather than completely degraded. This difference may be attributed to their different functions in worms and protozoa or the way they perform their functions (Fig. 2 G and 2 H). In the anterior part of the schistosoma gut, hemozoin granules can integrate with lipids in erythrocytes and absorb heme to form new hemozoin granules. In the posterior part, the hemozoin granules are degraded again to release iron and lipids near the intestinal microvilli (Fig. 2 G), as previously described[ 24 ]. By contrast, the degradation of hemozoin in malaria parasites is not observed, unlike in schistosoma hemozoin granules. It is perplexing that, if it is indeed a waste product, it should be excreted from the cell upon formation; however, we have never observed such discharge. Alternatively, hemozoin may fulfil an unidentified function. The association between malaria hemozoin formation and parasites Under light microscopy (LM), malaria hemozoin at various developmental stages can be distinctly observed. Notably, during the Plasmodium schizont stage, hemozoin appears larger and more prominent compared to other stages (Fig. 3 A). Additionally, under transmission electron microscopy (TEM), hemozoin within a schizont is observed as a combination of crystal-like structures and lipid spheres, distributed near merozoites (Fig. 3 B). When fresh samples are examined using TEM, a mass of black, cotton-like structures is frequently identified (Fig. 3 C and 3 G). Occasionally, a crystal-like structure can be detected within the “black cotton” of a parasite (Fig. 3 C). Energy spectrum analysis reveals that this “black cotton” contains iron, while the merozoites do not. Importantly, within hemozoin, the combination of crystal-like structures and lipids indicates that the crystal structures form from the lipid matrix (Fig. 3 E). These two components are interdependent. Upon treatment with sodium dodecyl sulfonate (SDS), only the crystal-like structures remain detectable (Fig. 3 F). The coexistence of crystal-like structures and lipids represents the form of hemozoin within the parasite. In the digestive vacuole, the ingested host lipids contribute to the accumulation of heme derived from hemoglobin decomposition. Furthermore, the polymerization of heme necessarily involves electron transfer between heme polymers and lipids, resulting in lipid remodeling. Given that the modified host lipids are crucial for parasites and are closely associated with heme polymers, this offers insight into why hemozoin is not expelled from parasites. Relationship between heme or iron utilization and hemozoin formation during erythrocytic stage The formation of hemozoin is observed early, with faintly visible dark brown particles detected in parasites at 12 hours post-infection. By 30 hours post-infection, noticeable hemozoin appears in the parasite (Fig. 4 A). Notably, at 42 hours post-infection, in the mature schizont, hemozoin is more prominent and larger (Fig. 4 A). It is intriguing to understand why such a significant amount of hemozoin is produced in the mature schizont. Since the formation of hemozoin is associated with hemoglobin digestion, heme release, and iron storage, we analyzed the expression of related genes to elucidate the relationship between hemozoin formation and the utilization of heme or iron. Our analysis of gene expression at different developmental stages revealed that genes related to iron, heme, DNA synthesis, and GSH exhibited predominant expression at 30 hours post-infection. Concurrently, hemozoin accumulation increased alongside cytoplasm content at this time (Fig. 4 A and 4 C). This suggests that as DNA begins to replicate, the demand for iron rises, thereby enhancing the GSH cycle to facilitate heme degradation[ 33 ]. Notably, the expression of these genes was not upregulated at 42 hours post-infection (Fig. 4 C), indicating a reduced function related to DNA synthesis and heme and iron utilization at this stage. In contrast, genes associated with reproduction and lipid utilization were upregulated at this time (Fig. 4 D), suggesting that at 42 hours post-infection, the schizont prioritizes the production of merozoites over the utilization of heme or iron, as well as the continuation of DNA synthesis. To investigate the effect of decreasing hemoglobin digestion and heme release on hemozoin formation, we compared the difference of genes expression and hemozon formation between Plasmodium falciparum 3D7 WT and P. falciparum 3D7 C580Y . Given that Plasmodium falciparum Kelch 13 protein ( Pf K13) mutations dampen haemoglobin endocytosis [ 34 , 35 ], parasites with mutations inevitably reduce iron and heme utilization. We found that the merozoite number and the total hemozoin content of P. falciparum 3D7 C580Y were lower than that in P. falciparum 3D7 WT (Fig. 4 B and 4 F), suggeseting that decreaseing hemoglobin endocytosis and heme release reduce the hemozoin production and its reproduction. However, no significant changes occur in the chronological order of genes expression related to DNA synthesis, GSH, reproduction, lipid metabolism, heme and iron utilization (Fig. 4 C). Moreover, in the schizonts of P. falciparum 3D7 C580Y , the bigger and prominent hemozoin is still observed (Fig. 4 A). Notably, both of Plasmodium falciparum 3D7 WT and P. falciparum 3D7 C580Y down-regulated gene expression related to DNA synthesis, glutathione (GSH), heme, and iron utilization at 42 hours post-infection stage (Fig. 4 C ), indicating a reduction in heme and iron utilization at this time point. Moreover, their expression level in P. falciparum 3D7 WT are lower than those in P. falciparum 3D7 C580Y (Fig. 4 E), suggesting that Plasmodium falciparum 3D7 WT , with normal iron and heme supply, decreases their utilization to a greater extent. The decreased heme utilization may serve to accumulate heme for hemozoin formation (Fig. 4 G), as lipid metabolism and reproduction-related genes were upregulated at this stage. Increased remodeled host lipids are required for merozoite production, while hemozoin formation provides the necessary remodeled lipids. Consequently, a greater accumulation of hemozoin can supply more remodeled lipids, thereby facilitating merozoite production (Fig. 4 G). What happens when hemozoin is released from parasites? When a schizont ruptures, hemozoin and merozoites are released into the host’s circulation. It has been consistently observed that mononuclear macrophages engulf significant amounts of hemozoin (Fig. 5 A), as has been observed by many researchers[ 36 ]. Furthermore, in vitro experiment have demonstrated that when a macrophage engulfs large amounts of hemozoin, it may become deformed or even rupture (Fig. 5 B and 5 C). Clearly, when a significant quantity of hemozoin is released into the circulation, the functions of macrophages are likely to be disrupted, creating conditions that allow more schizonts to survive and infect red blood cells (Figs. 5 D- 5 F). Artemether’s disruption of heme utilization leading to parasite death underscores the significance of heme and hemozoin Recent research suggests that artemisinin can form adducts with heme, thereby disrupting heme utilization and resulting in parasite death[ 37 ]. Clearly, the sequestration of heme affects the formation of hemozoin. To investigate how artemisinin interacts with hemozoin, we administered artemether to mice infected with P. yoelii 17XNL at a dosage of 100 mg/kg. At 4 to 6 hours post-treatment with artemether, cytoplasmic vacuolation was observed. After 16 to 18 hours of treatment, a significant accumulation of abnormal hemozoin was noted, accompanied by the destruction of normal cell structures (Fig. 5 G and 5 I). Notably, according to recent studies, parasite death results from heme depletion and hemozoin damage induced by artemether[ 37 ]. This suggests that heme and hemozoin formation are vital for parasite survival; if heme utilization or the interplay between heme and hemozoin is disrupted, the parasite will die (Fig. 5 J). It is likely that the equilibrium between heme and hemozoin is vital for maintaining the necessary heme levels within parasites. Additionally, the established role of schistosoma hemozoin reinforces this hypothesis[ 24 , 25 ]. Discussion Hemozoin is generally regarded as a waste product resulting from the detoxification of free heme. However, in Schistosoma, hemozoin granules are formed in the anterior portion of the gut and subsequently degraded in the posterior section[ 24 ]. During this process, iron is observed to transfer from erythrocytes to hemozoin granules, and then to vitelline gland cells through the intestinal wall, ultimately reaching the eggs [ 24 , 25 ]. Concurrently, lipids, another component of hemozoin granules, are also degraded and accumulate in vitelline gland cells. These findings indicate that the formation and degradation of hemozoin granules facilitate the transfer of heme, iron, and lipids from erythrocytes to vitelline gland cells and eggs in Schistosoma. Thus, the function of hemozoin extends beyond mere waste disposal; it serves as a crucial medium for iron storage and transport, playing a pivotal role in Schistosoma reproduction. Unlike multicellular organisms, malaria parasites, which belong to the class Protozoa, exhibit distinct characteristics. The direct observation of the relationship between hemozoin and the transport of iron, heme, or lipids within a cell is not feasible. However, various phenomena provide valuable insights, indicating that malarial hemozoin is not merely a waste product. For instance, hemozoin is exclusively released from the parasite during schizont rupture, rather than at any stage of its erythrocytic cycle. Additionally, hemozoin is transmitted to the next host along with gametocytes. The development and survival of Plasmodium parasites heavily depend on a substantial amount of iron, as evidenced by their sensitivity to iron chelators. Paradoxically, despite their reliance on a stable and high level of heme throughout their development within red blood cells[ 9 ], malarial parasites lack the proteins or mechanisms necessary to store iron or heme [ 38 , 39 ]. Therefore, establishing a mechanism to store sufficient iron and heme to meet the demands of the parasites is crucial. Hemozoin, as a polymer of iron and heme, may fulfill this demand. In fact, when malaria parasites reduce their iron and heme requirements, their hemozoin content also decreases correspondingly, suggesting a close association between heme utilization and hemozoin formation. Through the analysis of gene expression, we determined that genes associated with heme and iron utilization are predominantly expressed 30 hours post-infection. Glutathione (GSH) degrades heme to release iron in Plasmodium [ 40 , 41 , 42 , 43 ]. Genes involved in GSH synthesis and recycling are primarily expressed during this time. Furthermore, genes related to DNA synthesis are also expressed at this stage, indicating a significant release and utilization of iron that facilitates DNA synthesis [ 44 , 45 , 46 , 47 ]. Notably, hemozoin accumulation increases at this stage, although its content remains lower than that observed in later stages. At 42 hours post-infection, genes related to iron and heme utilization are down-regulated, suggesting a decreased use of these elements. This likely leads to the accumulation and formation of substantial amounts of hemozoin, the significance of which for the parasites remains unclear. Recent studies propose that artemisinin exerts its antiparasitic effects by disrupting heme utilization and altering hemozoin structure [ 37 ]. We treated mice infected with P. yoelii 17XNL using artemether and observed dead parasites exhibiting abnormal hemozoin accumulation. Previous studies also indicate that artemisinin interacts with heme to inhibit hemozoin crystallization and heme detoxification[ 48 ]. However, if hemozoin were merely a waste product of detoxification, it would be promptly expelled rather than continuously stored within the cell. Therefore, it is evident that hemozoin likely plays an unknown yet crucial role in these parasites. At 36 and 42 hours post-infection, there is a significant upregulation of genes associated with reproduction and lipid utilization. This observation aligns with the fact that the production and development of merozoites require substantial amounts of lipids. Plasmodium parasites rely on lipids obtained from their hosts [ 49 ], which have been exclusively observed in the acidic digestive vacuole of the parasites [ 50 , 51 , 52 , 53 ]. Host lipids within the digestive vacuole cannot be utilized directly by the parasites; they must undergo processing and remodeling before being utilized [ 54 , 55 , 56 ]. Notably, hemozoin formation and lipid remodeling are interconnected processes within the digestive vacuoles of malarial parasites. Specifically, both heme polymerization and hemozoin formation depend on lipids [ 57 ], while the completion of lipid oxidation and remodeling also necessitates hemozoin formation[ 58 , 59 ]. Therefore, hemozoin formation serves not only as a detoxification process but also plays a crucial role in the utilization of host lipids, especially considering the limited ability of malaria parasites to synthesize fatty acids or lipids. Previous studies have proposed that hemozoin nonenzymatically generates a significant amounts of hydroxy fatty acids [ 59 ], like hydroxyeicosatetraenoic acids [ 58 ]. Although these hydroxy fatty acids are known to contribute to hemozoin toxicity, they also play a beneficial role in the formation of the parasite’s membrane. In particular, hydroxyeicosatetraenoic acids are components of schistosoma hemozoin[ 60 ]. Furthermore, schistosoma hemozoin has been observed to transfer lipids from erythrocytes to vitelline gland cells. During the schizont stage, when numerous merozoites develop and mature, hemozoin crystals are abundantly formed. Importantly, unsaturated fatty acids can promote hemozoin formation [ 22 ]. As unsaturated fatty acids are converted into saturated fatty acids through hemozoin formation, the host-derived fatty acids or lipids undergo remodeling. The substantial formation of hemozoin during the schizont stage suggests that extensive remodeling and utilization of lipids contribute to the development of merozoites and their membranes. This observation is consistent with previous findings indicating that the schizont stage is characterized by an increase in saturated fatty acids [ 61 ]. Furthermore, the high expression of lipid-related genes at this stage further supports these observed changes and trends. Additionally, transmission electron microscopy (TEM) revealed that hemozoin consists of a lipid matrix combined with a crystal-like structure. Although hemozoin is generally considered a waste product, parasites cannot discharge it, likely due to the role of lipids at the trophozoite stage. It is noteworthy that in addition to its role in supporting parasite development and reproduction, the released hemozoin are also likely to contribute significantly to parasite protection. When a substantial amount of hemozoin is released into circulation and ingested by mononuclear macrophages or macrophages, it impairs the phagocytosis performed by these phagocytes in the host [ 59 , 62 ]. Additionally, hemozoin has been found to inhibit the differentiation and maturation of human monocyte-derived dendritic cells [ 63 ], and to impair the chemotactic motility and transendothelial migration of monocytes[ 64 ]. Consequently, hemozoin impairs the host’s immunity response, providing protection for parasites and their merozoites. In addition, a large amount of hemozoin were carried to mosquito host by gametocytes (Fig. 5 G and 5 H). Furthermore, malaria parasites initiate heme synthesis exclusively in mosquitoes[ 17 , 18 ]. The phenomenon suggests that hemozoin acts as a heme carrier to fulfill the heme requirement during the transition between the two hosts. In essence, hemozoin may play a crucial role throughout the entire life cycle of Plasmodium , encompassing growth, development, reproduction, and potentially providing protection for parasites and merozoites against elimination by macrophages. Therefore, considering the roles of hemozoin in both malaria and schistosomiasis, we propose that hemozoin should not only be regarded as a waste product of heme detoxification. Declarations Ethics approval and consent to participate This study was carried out in strict accordance with the recommendations of the Regulations for the Administration of Affairs Concerning Experimental Animals of the State Science and Technology Commission. The protocol was approved by the Internal Review Board of Tongji University School of Medicine. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Funding This research was supported by Innovation Program of Shanghai Municipal Education Commission. Author Contribution J.S. designed and conducted all experiments and wrote the main manuscript text; X.X.Q and W.W.S prepared figures 4-5; F.W., Y.N.L. and X.L.Y. prepared figures 1-3. All authors reviewed the manuscript. Acknowledgement We thank LC Sciences (Hangzhou, Zhejiang, China) for assisting with Plasmodium transcriptome and corresponding bioinformatics analysis. This research was supported by Innovation Program of Shanghai Municipal Education Commission (201901070007E00017). Availability of data and materials Data supporting the conclusions of this article are included within the article and its additional files. The raw data are provided in supplementary files. References Gluzman IY, Francis SE, Oksman A, Smith CE, Duffin KL, Goldberg DE. Order and specificity of the Plasmodium falciparum hemoglobin degradation pathway. J Clin Invest. 1994;93 4:1602–8; doi: 10.1172/JCI117140 . https://www.ncbi.nlm.nih.gov/pubmed/8163662 . Campanale N, Nickel C, Daubenberger CA, Wehlan DA, Gorman JJ, Klonis N, et al. Identification and characterization of heme-interacting proteins in the malaria parasite, Plasmodium falciparum. 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Supplementary Files supplementarydata1DNAsynthesisrelatedgenes20241020.xlsx supplementarydata2hemerelatedgenes20241020.xlsx supplementarydata3Glutathionerelatedgenes20241020.xlsx supplementarydata4ironrelatedgenes20241020.xlsx supplementarydata5reproductionrelatedgenes20241020.xlsx supplementarydata6lipidrelatedgenes20241020.xlsx Differentialgeneexpressionanalysis.zip Cite Share Download PDF Status: Published Journal Publication published 04 Mar, 2025 Read the published version in Parasites & Vectors → Version 1 posted Editorial decision: Revision requested 10 Dec, 2024 Reviews received at journal 08 Dec, 2024 Reviews received at journal 05 Dec, 2024 Reviews received at journal 02 Dec, 2024 Reviewers agreed at journal 27 Nov, 2024 Reviewers agreed at journal 25 Nov, 2024 Reviewers agreed at journal 17 Nov, 2024 Reviewers agreed at journal 15 Nov, 2024 Reviewers invited by journal 15 Nov, 2024 Editor assigned by journal 07 Nov, 2024 Submission checks completed at journal 07 Nov, 2024 First submitted to journal 07 Nov, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5408190","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":379513007,"identity":"5d40c60b-cac9-412e-8575-821211035873","order_by":0,"name":"Jun Sun","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYFACNgYGiQowK+EAAwMzsVrOGJCqhbHNAMYjQov8jLTkD5bz/siZ8y94eIChwjqxgf3sAbxaDG6kHZOQ3GZgbDnjAdBhZ9ITG3jyEvBrkU5vYwBqSdxw40DCAca2w4kNEjwGeLXIz05v/iA5B6blHxFaGG6nHZCQbABqOd8A1NJAhBaD+8/SJCSOGRsb3AAGcsKxdOM2nhwCDus5ZvxZokZOzuD8meQPH2qsZfvZzxBwGBAwS4BIiZwEhgQGcDwRBowfQCT/8QPEKB4Fo2AUjIIRCAAfBUorIOAl+wAAAABJRU5ErkJggg==","orcid":"","institution":"Tongji University","correspondingAuthor":true,"prefix":"","firstName":"Jun","middleName":"","lastName":"Sun","suffix":""},{"id":379513008,"identity":"e5e314d4-0a20-4c75-90ec-01363cbbd33b","order_by":1,"name":"Xixi Qin","email":"","orcid":"","institution":"Tongji University","correspondingAuthor":false,"prefix":"","firstName":"Xixi","middleName":"","lastName":"Qin","suffix":""},{"id":379513009,"identity":"9298b5c0-e3c7-4f6f-be06-73d725726ff7","order_by":2,"name":"Wenwen Si","email":"","orcid":"","institution":"Tongji University","correspondingAuthor":false,"prefix":"","firstName":"Wenwen","middleName":"","lastName":"Si","suffix":""},{"id":379513010,"identity":"5c9bf4de-d1d8-430d-9539-8981cbd63481","order_by":3,"name":"Fei Wang","email":"","orcid":"","institution":"Tongji University","correspondingAuthor":false,"prefix":"","firstName":"Fei","middleName":"","lastName":"Wang","suffix":""},{"id":379513011,"identity":"64bd06fd-cff2-46d1-990a-9741ebac3e18","order_by":4,"name":"Yanna Li","email":"","orcid":"","institution":"Tongji University","correspondingAuthor":false,"prefix":"","firstName":"Yanna","middleName":"","lastName":"Li","suffix":""},{"id":379513012,"identity":"4979e44f-aa9a-47bc-a17f-ec894d5b1ef1","order_by":5,"name":"Xiaoli Yan","email":"","orcid":"","institution":"Tongji University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoli","middleName":"","lastName":"Yan","suffix":""}],"badges":[],"createdAt":"2024-11-07 08:38:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5408190/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5408190/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13071-025-06699-x","type":"published","date":"2025-03-04T15:57:55+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":69843751,"identity":"44db35c8-dbb2-4535-864f-d2da340a8932","added_by":"auto","created_at":"2024-11-25 18:50:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":888184,"visible":true,"origin":"","legend":"\u003cp\u003eMorphology and degradation of schistosoma hemozoin granules. (A) A schistosoma indicating the observation position. (B) Under light microscopy (LM), the anterior part of the intestinal tract of the schistosoma is filled with dark brown hemozoin granules, which surround newly ingested red blood cells. When examined under transmission electron microscopy (TEM), the hemozoin granules appear spherical or comma-shaped, with some still connected to the matrix. (C) The degradation of hemozoin granules occurs near the microvilli of the intestinal wall in the posterior region of the schistosoma’s gut. The white arrow indicates the heme polymer layer on the outer layer of the hemozoin granules, while the black arrow indicates the lipid layer inside the hemozoin granules. A pentagram denotes a hemozoin granule whose outer layer is undergoing breakdown. (D) The hemozoin granules located near the microvilli of the intestinal wall in the posterior region of the schistosoma’s gut. (E) Intestinal contents are transmurally transported to the vitelline gland cells through the intercellular space of the intestinal wall. The black arrow indicates the location of transport across the wall, whereas the white arrows indicate the lipid drops in the vitelline gland cell. (F) The schematic diagram illustrates a process by which a hemozoin granule attaches to an erythrocyte, subsequently utilizing the erythrocyte to form additional hemozoin granules, which are then degraded near the intestinal microvilli of the Schistosoma, accompanied by the transport of iron and lipids across the intestinal wall into the vitelline gland cell. Vit, vitelline gland cells; Int.wall, intestinal wall; Li, lipids. All scale bars indicate 1 μm.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5408190/v1/8ba091128b366ecfabfa7220.png"},{"id":69843759,"identity":"13043ffd-1668-4985-a80e-2ac02b6016b2","added_by":"auto","created_at":"2024-11-25 18:50:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1354691,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of malaria hemozoin and schistosoma hemozoin. (A) Under a light microscopy (LM), the malaria hemozoin is observed in a schizont. Hz, hemozoin. (B) Freshly collected malaria hemozoin as seen under LM. (C-E) Collected malaria hemozoin observed using transmission electron microscopy (TEM). He, heme polymer or heme aggregation; Li, lipids. (F) Energy spectrum analysis reveals the presence of iron in malaria hemozoin. (G) This schematic diagram illustrates that in the anterior section of the Schistosoma intestine, free heme accumulates in lipid droplets, forming hemozoin granules. Subsequently, in the posterior region of the gut, these granules undergo degradation, beginning with the heme polymer layer and followed by the lipid layer. (H) In malaria parasites, free heme dissolves in lipid droplets, initially forming aggregates or polymers which can develop into crystal-like structures. The combination of heme polymers and lipids constitutes hemozoin, which may exist in a dynamic equilibrium with the cytoplasm, absorbing and releasing heme and lipids. All scale bars indicate 1 μm.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5408190/v1/33506242a60be9f6be4db6d6.png"},{"id":69844097,"identity":"b3030f0e-7aed-4d2f-874c-e6edd32365b4","added_by":"auto","created_at":"2024-11-25 18:58:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2628303,"visible":true,"origin":"","legend":"\u003cp\u003eMorphology of hemozoin in schizonts. (A) Malaria hemozoin (indicated by the red circle) in schizonts observed under light microscopy ( LM). The white arrow points to the merozoite. (B) Malaria hemozoin (white circle) in schizonts under transmission electron microscopy (TEM). The black arrow indicates the merozoite. (C) Under TEM, hemozoin in schizonts appears as black cotton, with occasional detection of crystal-like structure. (D) \u0026nbsp;Energy spectrum analysis reveals that this black cotton contains iron, while merozoites (indicated by white arrows) do not. (E) Under TEM, hemozoin is observed as a combination of heme polymers and lipids, where the crystal-like heme polymer is formed from the lipid matrix. (F) Upon treatment with sodium dodecyl sulfate (SDS), only the crystal-like structure remains. (G) During hemozoin formation, heme initially accumulates within a lipid droplet, resulting in the formation of a heme aggregation sphere, which appears as a black cotton-like structure in schizonts. Hz, hemozoin; Li, lipid; hem, crystal-like heme polymer; he, heme aggregation sphere. All scale bars indicate 1 μm.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5408190/v1/246129376f61ed881f6f5806.png"},{"id":69844099,"identity":"736dcee8-8cea-43f5-a842-7f58d5103f0a","added_by":"auto","created_at":"2024-11-25 18:58:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2561297,"visible":true,"origin":"","legend":"\u003cp\u003eRelationship between heme or iron utilization and hemozoin formation. (A) Formation of malaria hemozoin at 6, 12, 18, 24, 30, 36, and 42 hours post-infection with P. falciparum. (B) Comparison of merozoite numbers between \u003cem\u003eP. falciparum\u003c/em\u003e 3D7\u003csup\u003eWT\u003c/sup\u003e and \u003cem\u003eP. falciparum\u003c/em\u003e 3D7\u003csup\u003eC580Y\u003c/sup\u003e, indicating that \u003cem\u003eP. falciparum\u003c/em\u003e 3D7\u003csup\u003eC580Y\u003c/sup\u003e produces fewer merozoite compared to \u003cem\u003eP. falciparum\u003c/em\u003e 3D7\u003csup\u003eWT\u003c/sup\u003e. (C) Gene expression heat maps display expression patterns across various developmental stages between \u003cem\u003eP. falciparum\u003c/em\u003e 3D7\u003csup\u003eWT\u003c/sup\u003e and \u003cem\u003eP. falciparum\u003c/em\u003e 3D7\u003csup\u003eC580Y\u003c/sup\u003e. Supplementary data 1 - 4 provide detailed information on gene expression. (D) Heat maps of gene expression indicate the expression patterns of reproduction- and lipid-related genes (see supplementary data 5 - 6). (E) Differentiation expression analysis of genes related to DNA synthesis, iron and heme utilization (see supplementary files) (F) Comparison of hemozoin content between \u003cem\u003eP. falciparum\u003c/em\u003e 3D7\u003csup\u003eWT\u003c/sup\u003e and \u003cem\u003eP. falciparum\u003c/em\u003e 3D7\u003csup\u003eC580Y\u003c/sup\u003e, indicating that \u003cem\u003eP. falciparum\u003c/em\u003e 3D7\u003csup\u003eC580Y\u003c/sup\u003e produces less hemozoin than \u003cem\u003eP. falciparum\u003c/em\u003e 3D7\u003csup\u003eWT\u003c/sup\u003e. (G) The schematic diagram demonstrates that during the trophozoite stage, parasites consume more heme and iron, resulting in reduced heme accumulation and consequently less hemozoin formation. Conversely, in the schizont stage, parasites consume lower amounts of heme and iron, leading to increased heme accumulation and greater production of hemozoin. Furthermore, due to the interaction between hemozoin formation and host lipids, increased hemozoin production can supply more processed lipids for parasite utilization. Hz, hemozoin; All scale bars indicate 1 μm. Asterisks denote significance. **, P \u0026lt; 0.01; ***, P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5408190/v1/89944f5d3f33a575a870b1f3.png"},{"id":69843760,"identity":"f5d7b9cc-ca31-42d0-964c-933ff19df618","added_by":"auto","created_at":"2024-11-25 18:50:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":5425494,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of hemozoin on macrophages and \u003cem\u003ePlasmodium\u003c/em\u003e. (A) In infected mice, mononuclear macrophages can uptake hemozoin. (B-C) The macrophages may sustain damage when they phagocytose a significant amount of hemozoin in vitro (C), in comparison to the normal group (B). (D-F) This schematic diagram illustrates that when schizonts release merozoites, host macrophages can ingest and eliminate the merozoites (E); However, when macrophages ingest hemozoin, they are unable to eliminate merozoites due to damage or death (F). (G-I) Effect of artemether on\u003cem\u003e P. yoelii \u003c/em\u003e17XNL. Compared to the parasites observed at 0 hours post-artemether treatment, vacuoles appear in the cytoplasm of the parasites at 4 to 6 hours post-treatment. In contrast, at 16 to 18 hours post-treatment, the cells become disrupted, and hemozoin aggregates abnormally. (K) A substantial amount of hemozoin (indicated by black arrows) can be detected in gametocytes under LM. (L) This schematic diagram raise the question of whether hemozoin, as a waste product, should be discharged by gametocytes before entering the mosquito; however, they actually transport hemozoin into the mosquito. Hz, hemozoin; Mz, merozoite; All scale bars indicate 5 μm.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5408190/v1/9e61954c950c2316a3487f85.png"},{"id":78191377,"identity":"e15a2611-65c2-4d70-b570-d1a61d0ab51b","added_by":"auto","created_at":"2025-03-10 19:58:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13008168,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5408190/v1/5661e570-6835-49af-9836-fb77473fa47b.pdf"},{"id":69843756,"identity":"dac088c3-80da-4906-80f8-ceb57b840f7a","added_by":"auto","created_at":"2024-11-25 18:50:14","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":21218,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarydata1DNAsynthesisrelatedgenes20241020.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5408190/v1/e0a018a3d62ecb8f198c4219.xlsx"},{"id":69843753,"identity":"e4251161-8157-4e6f-9c14-909f7064167f","added_by":"auto","created_at":"2024-11-25 18:50:14","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":13647,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarydata2hemerelatedgenes20241020.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5408190/v1/13dd57da9fd18dc388dcdd90.xlsx"},{"id":69843752,"identity":"bceecb25-1689-42bb-b96c-9394fb1e0fd1","added_by":"auto","created_at":"2024-11-25 18:50:14","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":14472,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarydata3Glutathionerelatedgenes20241020.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5408190/v1/5ac7a15868dc40e42b88a0f5.xlsx"},{"id":69843757,"identity":"a75d0f5b-25c8-44fc-8ef2-f59832a406d6","added_by":"auto","created_at":"2024-11-25 18:50:14","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":17797,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarydata4ironrelatedgenes20241020.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5408190/v1/68e589c94e262d97ea22aac0.xlsx"},{"id":69844100,"identity":"a5a17ee4-ba97-4d24-b44b-7ceffc64fde3","added_by":"auto","created_at":"2024-11-25 18:58:15","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":16841,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarydata5reproductionrelatedgenes20241020.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5408190/v1/df0356cee1f68b9ff4c9590b.xlsx"},{"id":69844098,"identity":"e2bd7f25-bb9a-441c-811f-ba3678f4bd84","added_by":"auto","created_at":"2024-11-25 18:58:15","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":13387,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarydata6lipidrelatedgenes20241020.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5408190/v1/7bef17af712c6ad4d4558d4c.xlsx"},{"id":69843763,"identity":"9771fff8-071a-44c7-b387-eb7aa5265fc0","added_by":"auto","created_at":"2024-11-25 18:50:15","extension":"zip","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":7246522,"visible":true,"origin":"","legend":"","description":"","filename":"Differentialgeneexpressionanalysis.zip","url":"https://assets-eu.researchsquare.com/files/rs-5408190/v1/1a49d1f1d74582418033fcfa.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Hemozoin: a waste product after heme detoxification?","fulltext":[{"header":"Background","content":"\u003cp\u003eHemozoin is a critical byproduct that forms during the unique detoxification process in malaria parasites. During the blood stages of the \u003cem\u003ePlasmodium\u003c/em\u003e species, hemozoin is synthesized as the parasite digests host erythrocyte hemoglobin to obtain its amino acids. The liberated toxic heme, a compound containing an iron atom within a porphyrin ring, is converted into an insoluble and chemically inert form to prevent cellular damage caused by its pro-oxidant properties. This substance, composed of heme polymers bonded via iron-carboxylate links, accumulates in the parasite\u0026rsquo;s food vacuole and is later released into circulation when infected red blood cells rupture.\u003c/p\u003e \u003cp\u003eThe impact of heme on parasites is a subject of interest in the scientific community. Several researchers have suggested that heme can inhibit various \u003cem\u003ePlasmodium\u003c/em\u003e enzymes, including plasmepsins, falcipains, glycolytic glyceraldehyde-3-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Additionally, the integration of lipophilic heme into biological membranes reduces the deformability of erythrocytes and induces hemolysis. Heme in the membrane also weakens the lipid bilayer, making it more susceptible to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-mediated lysis. This integration disrupts the normal dynamic interaction between the RBC membrane and its underlying cytoskeletal proteins[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. High concentrations of heme lead to increased oxidative stress, and the presence of peroxidation products is associated with decreased RBC membrane fluidity, likely increasing cellular rigidity in parasitized red cells[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The catabolism of hemoglobin, which releases reactive heme and iron, is associated with the generation of redox-reactive substances such as H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, superoxide radicals, and the hydroxyl radical, which directly mediates lipid peroxidation[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The interaction between heme, intracellular hydrogen peroxide, and lipids can result in lipid peroxidation[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. These harmful effects are directly and indirectly attributable to free heme[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], suggesting that hemozoin formation is a detoxification mechanism.\u003c/p\u003e \u003cp\u003eNotably, the malaria parasite maintains a heme pool at a consistent level of approximately 1.6 \u0026micro;M throughout its development within red blood cells [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], indicating the crucial role of high concentrations of heme for the parasite. The parasite digests up to 65% of the host cell\u0026rsquo;s hemoglobin but only utilizes up to about 16% of the amino acids derived from hemoglobin digestion, raising the question of whether hemoglobin digestion serves solely for amino acids acquisition [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The effectiveness of iron chelators in killing malarial parasites [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] strongly suggests the essentiality of iron for these parasites. Especially, in malaria parasites, iron is released during heme degradation facilitated by glutathione (GSH) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], suggesting that the accumulation of a substantial amount of heme likely corresponds to a significant supply and demand for iron. Notably, the considerable quantity of hemozoin formed in gametocytes and subsequently transferred to mosquitoes suggests that hemozoin plays a crucial role in the storage and utilization of heme and iron, particularly as malaria parasites initiate heme synthesis exclusively in mosquitoes[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAdditionally, lipids play a significant role in \u003cem\u003ePlasmodium\u003c/em\u003e, and distinct changes in lipid composition occur during different stages of development [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Interestingly, the parasite lacks the ability to synthesize cholesterol de novo and has limited capacity for fatty acid synthesis[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. As a result, the parasites have to acquire and utilize host lipids. In the digestive vacuoles of \u003cem\u003ePlasmodium\u003c/em\u003e, hemozoin formation is enhanced by host unsaturated fatty acids, whereas the electron released from heme polymerization likely oxidizes the unsaturated fatty acids of the host. The remodeled fatty acids or lipids are likely detoxified and made suitable for use by \u003cem\u003ePlasmodium\u003c/em\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Furthermore, hemozoin-producing schistosomes have been found to use hemozoin formation and degradation to transfer iron and lipids to the vitelline gland and eggs [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. These pieces of evidence suggest that malarial hemozoin formation is not merely a waste product of heme detoxification but likely plays a crucial role in the life cycle of parasites. To further investigate the role of hemozoin, we employed transmission electron microscopy (TEM) to compare the formation of hemozoin in Schistosoma and malaria. Notably, malaria hemozoin was directly observed without the conventional electron microscopy sampling process, which aimed to preserve the authentic structure of the sample and examine its association with the parasites. Additionally, gene expression analysis was conducted to investigate the relationship between iron or heme utilization and hemozoin production.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEthics statement\u003c/h2\u003e \u003cp\u003eThis study strictly adhered to the recommendations of the Regulations for the Administration of Affairs Concerning Experimental Animals of the State Science and Technology Commission. The protocol was approved by the Internal Review Board of Tongji University School of Medicine (TJLAC-014-017).\u003c/p\u003e \u003cp\u003e \u003cb\u003eParasites and isolation of schistosome hemozoin granules (\u003c/b\u003eSHGs\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFemale mice of the Kunming strain weighting 20 to 22 g were obtained from SLRC Laboratory Animal Co., Ltd. In Shanghai, China. Cercariae freshly shed by the snails were used to infect mice percutaneously with 20 cercariae each. Adult female schistosomes were collected 42 to 45 d post-infection and washed with sterile 0.15mol/L NaCl solution (normal saline). The female schistosomes were cut into small sections in sterile normal saline solution in eppendorf tubes, and the dark suspensions were collected, following a previous study[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Clumps were removed, and individual hemozoin granules were retained through low-speed centrifugation. The hemozoin granules were then washed with sterile normal saline solution and utilized for a macrophage phagocytosis experiment.\u003c/p\u003e \u003cp\u003eAn intact female adult worm was cut into three sections, and the gut contents from each section were collected and treated using a previously described method [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The treated samples were observed using a light microscopy (LM, Nikon 50i) and a JEOL EW-1230 scanning electron microscopy with an accelerating voltage of 80 kV. Images were acquired using a digital photo-documentation system (Gatan Bioscan Camera, model 792).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMalaria parasites and hemozoin analysis with LM and TEM\u003c/h3\u003e\n\u003cp\u003eThe \u003cem\u003eP. falciparum\u003c/em\u003e parasites (3D7 strain) were provided from the national institute of parasitic diseases of Chinese center for disease control and prevention. The \u003cem\u003eP. falciparum\u003c/em\u003e 3D7\u003csup\u003eWT\u003c/sup\u003e and \u003cem\u003eP. falciparum\u003c/em\u003e 3D7\u003csup\u003eC580Y\u003c/sup\u003e were cultured in RPMI 1640 medium with 25 mmol/L HEPEPS, 0.5% AlbuMAX, 0.2% sodium bicarbonate, 0.2 mmol/L htpoxanthine, and 20 \u0026micro;g/mL gentamicin sulfate at 37 ℃ incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e and 5% O\u003csub\u003e2\u003c/sub\u003e. Fresh O\u003csup\u003e+\u003c/sup\u003e human RBCs from healthy human donors were used to maintain parasitemia, and parasites were maintained at 2% hematocrit based on a previous report [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Blood smears were stained with Giemsa in accordance with the standard staining protocol and then observed the smears with a LM (Nikon 50i).\u003c/p\u003e \u003cp\u003eThe erythrocytes infected by malaria parasites were treated with saponin at a final concentration of 0.15% and then centrifuged at 10 000 rpm for 5 min. The black deposit was collected. Then, the deposits were fixed with 2.5% glutaraldehyde\u0026ndash;phosphate-buffered saline (PBS) (pH 7.2) buffer and then dried at room temperature, and directly observed using a JEOL EW-1230 TEM (Japan) at an accelerating voltage of 80 kV. Schitosoma samples were fixed in glutaraldehyde overnight under 4℃. Then, they were washed with PBS, fixed in 1% osmium tetroxide, dehydrated in acetone and embedded in Epon812. Then, 60 nm- thick sections were cut by using ultramicrotome (EM UC6, Leica, German) and mounted on copper slot grids coated with Formvar and stained with uranyl acetate and lead citrate for examination. Samples were observed by using a JEOL EW-1230 TEM (Japan) and the images were acquired by using a digital photodocumentation system (Gatan Bioscan Camera, Model 792).\u003c/p\u003e\n\u003ch3\u003eEnergy dispersive spectroscopy (EDS)\u003c/h3\u003e\n\u003cp\u003eTEM sections of malarial hemozoin, including heme aggregation sphere (HAS) and crystal-like hemozoin, were analyzed by using energy dispersive (X-ray) spectroscopy (Oxford INCA, Lyford, OX, UK) under a JEM-2010 transmission electron microscope at an accelerating voltage of 200 kV as previously described[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The Oxford INCA software package was used to carry out the X-ray analysis to determine the elemental composition of the sample and generate its characteristic spectrum.\u003c/p\u003e\n\u003ch3\u003eAnalysis of the effect of artemether on malarial parasites using light microscope\u003c/h3\u003e\n\u003cp\u003eBALb/c mice or ICR mice were infected by intraperitoneal injections with \u003cem\u003eP. yoelii\u003c/em\u003e 17XNL. When the infection rate exceeded 20%, the mice were administered artemether at a dosage of 100 mg/kg. Blood samples were collected after 4 and 16 hours of treatment, respectively. Blood smears were prepared and stained with Giemsa according to standard staining protocols, and the smears were subsequently examined using a light microscope (Nikon 50i).\u003c/p\u003e\n\u003ch3\u003eComparison of hemozoin content and merozoite number\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003ePlasmodium falciparum\u003c/em\u003e 3D7\u003csup\u003eWT\u003c/sup\u003e and \u003cem\u003eP. falciparum\u003c/em\u003e 3D7\u003csup\u003eC580Y\u003c/sup\u003e were purified using a 40\u0026ndash;70% Percoll gradient and treated with 5% sorbitol to obtain 6-h ring stages. Then, they were cultured for 36\u0026ndash;42 h with parasitaemia adjusted to 0.5\u0026ndash;1%. Blood smears were stained with Giemsa in accordance with the standard staining protocol and then compared the number of their merozoites with a LM (Motic, PA53 FS6). Approximately 10 ml of the culture medium was treated with 0.15% saponin solution on ice for 10 min to lyse the cells. After centrifugation at 10,000 g for 15 min, the sediment was washed with 25 mmol/L Tris (pH 7.8) containing 2.5% sodium dodecyl sulphate until the supernatant was clear. The sediment was dissolved in 250 \u0026micro;L of 2.5% sodium dodecyl sulphate bufer and 20 \u0026micro;L of 2.5 mol/L NaOH. The Nanodrop 2000 spectrophotometer was used to measure the absorbance of hemozoin at 400 nm for content quantification, as described in a previous paper [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Statistical analysis was conducted using T-test (unpaired) with Graphpad Prism 8.0.2 software for Windows. A \u003cem\u003ep\u003c/em\u003e-value less than 0.05 was considered statistically significant.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTranscriptome analysis of\u003c/b\u003e \u003cb\u003eP. falciparum\u003c/b\u003e \u003cb\u003e3D7\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eP. falciparum\u003c/em\u003e 3D7 were cultured in RPMI 1640 medium according to established protocols [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The parasites were synchronized using 5% sorbitol. Subsequently, mature schizonts were purified on a 40%/70% percoll gradient. Cultures were then treated with 5% sorbitol to obtain 0\u0026ndash;3 h ring stages. Parasites were collected within 0\u0026ndash;3 hours after invasion, and samples were harvested at 0, 6, 12, 18, 24, 30, 36, and 42 hours post-infection (hpi) for transcriptome analysis. Samples at different stages were collected and preserved in TRIzol (Invitrogen, USA). RNA extraction was performed using phenol and isopropanol precipitation. RNA quantification and quality assessment were carried out using a Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). RNA integrity was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Libraries were prepared using a TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, CA, USA). Transcriptome sequencing and analysis were performed by LC Sciences (Hangzhou, Zhejiang, China).\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eThe hemozoin granules were degraded and utilized in\u003c/b\u003e \u003cb\u003eSchistosoma japonicum\u003c/b\u003e \u003cb\u003egut\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe anterior portion of the intestinal tract of \u003cem\u003eSchistosoma japonicum\u003c/em\u003e contains globe- and comma-shaped hemozoin granules, along with freshly-ingested host red blood cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Examination using light microscopy (LM) and transmission electron microscopy (TEM) reveals that the red blood cells are surrounded by hemozoin granules, forming clusters or aggregates. Our previous studies reveal that upon attachment to erythrocytes, \u003cem\u003eSchistosoma\u003c/em\u003e hemozoin granules can exploit these cells for the self-assembly of new hemozoin granules[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Under TEM, hemozoin granules are observed to be closely in contact with the matrix derived from degraded erythrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Notably, in the posterior portion of the intestine, the hemozoin granules are degraded near the intestinal microvilli (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Initially, the heme polymer in the outer layer of the hemozoin granules decomposes, followed by subsequent decomposition of the lipid components within the granules (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Particularly, after hemozoin degradation, the transport of intestinal contents across the intestinal wall to vitelline gland cells becomes clearly visible under TEM. During this process, the intestinal contents do not appear to be processed by the cells of the intestinal wall, but instead pass directly through the gut wall via the intercellular space. Simultaneously, accumulation of lipids is observed within the vitelline gland cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Based on our observations, we proposed that hemozoin granules in schistosomes facilitate the transport of heme and lipids by forming and subsequently degrading (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Apparently, \u003cem\u003eSchistosoma\u003c/em\u003e hemozoin granules play a crucial role in storing and transporting heme, iron and lipids during its development and reproduction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eMorphology of malarial hemozoin differs from that of schistosoma hemozoin granules\u003c/h3\u003e\n\u003cp\u003eUnder LM, these dark-brown malaria hemozoin appear globular or irregular in shape (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Under TEM, we observe various morphologies of malarial hemozoin (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). The energy disperse spectroscopy can detect iron inside the hemozoin (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Notably, hemozoin is a mixture or combination of heme polymer and lipids when observed under TEM. The free heme dissolves in the lipids which facilitate the polymerization of heme into crystal-like structure. Some researchers only consider the crystal-like structure as hemozoin. The crystal-like hemozoin develops from heme aggregations in its lipid matrix[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Observing the fresh samples with TEM, we found malaria hemozoin is a combination within the parasites, consisting of heme aggregation or crystal-like heme polymer, and lipids. The lipid layer is external while the heme aggregation or crystal-like polymer is internal (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). It is evident that the morphology and structure of malarial hemozoin differ from those of schistosoma hemozoin granules. In schistosoma hemozoin granule, the heme layer is on the outside and is first degraded near the intestine microvilli. In contrast, the heme polymer in malarial hemozoin is located on the interior, indicating that heme is stored rather than completely degraded. This difference may be attributed to their different functions in worms and protozoa or the way they perform their functions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the anterior part of the schistosoma gut, hemozoin granules can integrate with lipids in erythrocytes and absorb heme to form new hemozoin granules. In the posterior part, the hemozoin granules are degraded again to release iron and lipids near the intestinal microvilli (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG), as previously described[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. By contrast, the degradation of hemozoin in malaria parasites is not observed, unlike in schistosoma hemozoin granules. It is perplexing that, if it is indeed a waste product, it should be excreted from the cell upon formation; however, we have never observed such discharge. Alternatively, hemozoin may fulfil an unidentified function.\u003c/p\u003e\n\u003ch3\u003eThe association between malaria hemozoin formation and parasites\u003c/h3\u003e\n\u003cp\u003eUnder light microscopy (LM), malaria hemozoin at various developmental stages can be distinctly observed. Notably, during the \u003cem\u003ePlasmodium\u003c/em\u003e schizont stage, hemozoin appears larger and more prominent compared to other stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Additionally, under transmission electron microscopy (TEM), hemozoin within a schizont is observed as a combination of crystal-like structures and lipid spheres, distributed near merozoites (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). When fresh samples are examined using TEM, a mass of black, cotton-like structures is frequently identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Occasionally, a crystal-like structure can be detected within the \u0026ldquo;black cotton\u0026rdquo; of a parasite (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Energy spectrum analysis reveals that this \u0026ldquo;black cotton\u0026rdquo; contains iron, while the merozoites do not. Importantly, within hemozoin, the combination of crystal-like structures and lipids indicates that the crystal structures form from the lipid matrix (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). These two components are interdependent. Upon treatment with sodium dodecyl sulfonate (SDS), only the crystal-like structures remain detectable (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). The coexistence of crystal-like structures and lipids represents the form of hemozoin within the parasite. In the digestive vacuole, the ingested host lipids contribute to the accumulation of heme derived from hemoglobin decomposition. Furthermore, the polymerization of heme necessarily involves electron transfer between heme polymers and lipids, resulting in lipid remodeling. Given that the modified host lipids are crucial for parasites and are closely associated with heme polymers, this offers insight into why hemozoin is not expelled from parasites.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eRelationship between heme or iron utilization and hemozoin formation during erythrocytic stage\u003c/h2\u003e \u003cp\u003eThe formation of hemozoin is observed early, with faintly visible dark brown particles detected in parasites at 12 hours post-infection. By 30 hours post-infection, noticeable hemozoin appears in the parasite (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Notably, at 42 hours post-infection, in the mature schizont, hemozoin is more prominent and larger (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). It is intriguing to understand why such a significant amount of hemozoin is produced in the mature schizont.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSince the formation of hemozoin is associated with hemoglobin digestion, heme release, and iron storage, we analyzed the expression of related genes to elucidate the relationship between hemozoin formation and the utilization of heme or iron. Our analysis of gene expression at different developmental stages revealed that genes related to iron, heme, DNA synthesis, and GSH exhibited predominant expression at 30 hours post-infection. Concurrently, hemozoin accumulation increased alongside cytoplasm content at this time (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). This suggests that as DNA begins to replicate, the demand for iron rises, thereby enhancing the GSH cycle to facilitate heme degradation[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Notably, the expression of these genes was not upregulated at 42 hours post-infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), indicating a reduced function related to DNA synthesis and heme and iron utilization at this stage. In contrast, genes associated with reproduction and lipid utilization were upregulated at this time (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), suggesting that at 42 hours post-infection, the schizont prioritizes the production of merozoites over the utilization of heme or iron, as well as the continuation of DNA synthesis.\u003c/p\u003e \u003cp\u003eTo investigate the effect of decreasing hemoglobin digestion and heme release on hemozoin formation, we compared the difference of genes expression and hemozon formation between \u003cem\u003ePlasmodium falciparum\u003c/em\u003e 3D7\u003csup\u003eWT\u003c/sup\u003e and \u003cem\u003eP. falciparum\u003c/em\u003e 3D7\u003csup\u003eC580Y\u003c/sup\u003e. Given that \u003cem\u003ePlasmodium falciparum\u003c/em\u003e Kelch 13 protein (\u003cem\u003ePf\u003c/em\u003eK13) mutations dampen haemoglobin endocytosis [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], parasites with mutations inevitably reduce iron and heme utilization. We found that the merozoite number and the total hemozoin content of \u003cem\u003eP. falciparum\u003c/em\u003e 3D7\u003csup\u003eC580Y\u003c/sup\u003e were lower than that in \u003cem\u003eP. falciparum\u003c/em\u003e 3D7\u003csup\u003eWT\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF), suggeseting that decreaseing hemoglobin endocytosis and heme release reduce the hemozoin production and its reproduction. However, no significant changes occur in the chronological order of genes expression related to DNA synthesis, GSH, reproduction, lipid metabolism, heme and iron utilization (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Moreover, in the schizonts of \u003cem\u003eP. falciparum\u003c/em\u003e 3D7\u003csup\u003eC580Y\u003c/sup\u003e, the bigger and prominent hemozoin is still observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eNotably, both of \u003cem\u003ePlasmodium falciparum\u003c/em\u003e 3D7\u003csup\u003eWT\u003c/sup\u003e and \u003cem\u003eP. falciparum\u003c/em\u003e 3D7\u003csup\u003eC580Y\u003c/sup\u003e down-regulated gene expression related to DNA synthesis, glutathione (GSH), heme, and iron utilization at 42 hours post-infection stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC ), indicating a reduction in heme and iron utilization at this time point. Moreover, their expression level in \u003cem\u003eP. falciparum\u003c/em\u003e 3D7\u003csup\u003eWT\u003c/sup\u003e are lower than those in \u003cem\u003eP. falciparum\u003c/em\u003e 3D7\u003csup\u003eC580Y\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), suggesting that \u003cem\u003ePlasmodium falciparum\u003c/em\u003e 3D7\u003csup\u003eWT\u003c/sup\u003e, with normal iron and heme supply, decreases their utilization to a greater extent. The decreased heme utilization may serve to accumulate heme for hemozoin formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG), as lipid metabolism and reproduction-related genes were upregulated at this stage. Increased remodeled host lipids are required for merozoite production, while hemozoin formation provides the necessary remodeled lipids. Consequently, a greater accumulation of hemozoin can supply more remodeled lipids, thereby facilitating merozoite production (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eWhat happens when hemozoin is released from parasites?\u003c/h2\u003e \u003cp\u003eWhen a schizont ruptures, hemozoin and merozoites are released into the host\u0026rsquo;s circulation. It has been consistently observed that mononuclear macrophages engulf significant amounts of hemozoin (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), as has been observed by many researchers[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Furthermore, in vitro experiment have demonstrated that when a macrophage engulfs large amounts of hemozoin, it may become deformed or even rupture (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Clearly, when a significant quantity of hemozoin is released into the circulation, the functions of macrophages are likely to be disrupted, creating conditions that allow more schizonts to survive and infect red blood cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eArtemether\u0026rsquo;s disruption of heme utilization leading to parasite death underscores the significance of heme and hemozoin\u003c/b\u003e \u003c/p\u003e \u003cp\u003eRecent research suggests that artemisinin can form adducts with heme, thereby disrupting heme utilization and resulting in parasite death[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Clearly, the sequestration of heme affects the formation of hemozoin. To investigate how artemisinin interacts with hemozoin, we administered artemether to mice infected with \u003cem\u003eP. yoelii\u003c/em\u003e 17XNL at a dosage of 100 mg/kg. At 4 to 6 hours post-treatment with artemether, cytoplasmic vacuolation was observed. After 16 to 18 hours of treatment, a significant accumulation of abnormal hemozoin was noted, accompanied by the destruction of normal cell structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). Notably, according to recent studies, parasite death results from heme depletion and hemozoin damage induced by artemether[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. This suggests that heme and hemozoin formation are vital for parasite survival; if heme utilization or the interplay between heme and hemozoin is disrupted, the parasite will die (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ). It is likely that the equilibrium between heme and hemozoin is vital for maintaining the necessary heme levels within parasites. Additionally, the established role of schistosoma hemozoin reinforces this hypothesis[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eHemozoin is generally regarded as a waste product resulting from the detoxification of free heme. However, in Schistosoma, hemozoin granules are formed in the anterior portion of the gut and subsequently degraded in the posterior section[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. During this process, iron is observed to transfer from erythrocytes to hemozoin granules, and then to vitelline gland cells through the intestinal wall, ultimately reaching the eggs [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Concurrently, lipids, another component of hemozoin granules, are also degraded and accumulate in vitelline gland cells. These findings indicate that the formation and degradation of hemozoin granules facilitate the transfer of heme, iron, and lipids from erythrocytes to vitelline gland cells and eggs in Schistosoma. Thus, the function of hemozoin extends beyond mere waste disposal; it serves as a crucial medium for iron storage and transport, playing a pivotal role in Schistosoma reproduction.\u003c/p\u003e \u003cp\u003eUnlike multicellular organisms, malaria parasites, which belong to the class Protozoa, exhibit distinct characteristics. The direct observation of the relationship between hemozoin and the transport of iron, heme, or lipids within a cell is not feasible. However, various phenomena provide valuable insights, indicating that malarial hemozoin is not merely a waste product. For instance, hemozoin is exclusively released from the parasite during schizont rupture, rather than at any stage of its erythrocytic cycle. Additionally, hemozoin is transmitted to the next host along with gametocytes. The development and survival of \u003cem\u003ePlasmodium\u003c/em\u003e parasites heavily depend on a substantial amount of iron, as evidenced by their sensitivity to iron chelators. Paradoxically, despite their reliance on a stable and high level of heme throughout their development within red blood cells[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], malarial parasites lack the proteins or mechanisms necessary to store iron or heme [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Therefore, establishing a mechanism to store sufficient iron and heme to meet the demands of the parasites is crucial. Hemozoin, as a polymer of iron and heme, may fulfill this demand. In fact, when malaria parasites reduce their iron and heme requirements, their hemozoin content also decreases correspondingly, suggesting a close association between heme utilization and hemozoin formation.\u003c/p\u003e \u003cp\u003eThrough the analysis of gene expression, we determined that genes associated with heme and iron utilization are predominantly expressed 30 hours post-infection. Glutathione (GSH) degrades heme to release iron in Plasmodium [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Genes involved in GSH synthesis and recycling are primarily expressed during this time. Furthermore, genes related to DNA synthesis are also expressed at this stage, indicating a significant release and utilization of iron that facilitates DNA synthesis [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Notably, hemozoin accumulation increases at this stage, although its content remains lower than that observed in later stages. At 42 hours post-infection, genes related to iron and heme utilization are down-regulated, suggesting a decreased use of these elements. This likely leads to the accumulation and formation of substantial amounts of hemozoin, the significance of which for the parasites remains unclear. Recent studies propose that artemisinin exerts its antiparasitic effects by disrupting heme utilization and altering hemozoin structure [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. We treated mice infected with \u003cem\u003eP. yoelii\u003c/em\u003e 17XNL using artemether and observed dead parasites exhibiting abnormal hemozoin accumulation. Previous studies also indicate that artemisinin interacts with heme to inhibit hemozoin crystallization and heme detoxification[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. However, if hemozoin were merely a waste product of detoxification, it would be promptly expelled rather than continuously stored within the cell. Therefore, it is evident that hemozoin likely plays an unknown yet crucial role in these parasites.\u003c/p\u003e \u003cp\u003eAt 36 and 42 hours post-infection, there is a significant upregulation of genes associated with reproduction and lipid utilization. This observation aligns with the fact that the production and development of merozoites require substantial amounts of lipids. Plasmodium parasites rely on lipids obtained from their hosts [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], which have been exclusively observed in the acidic digestive vacuole of the parasites [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Host lipids within the digestive vacuole cannot be utilized directly by the parasites; they must undergo processing and remodeling before being utilized [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Notably, hemozoin formation and lipid remodeling are interconnected processes within the digestive vacuoles of malarial parasites. Specifically, both heme polymerization and hemozoin formation depend on lipids [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], while the completion of lipid oxidation and remodeling also necessitates hemozoin formation[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Therefore, hemozoin formation serves not only as a detoxification process but also plays a crucial role in the utilization of host lipids, especially considering the limited ability of malaria parasites to synthesize fatty acids or lipids. Previous studies have proposed that hemozoin nonenzymatically generates a significant amounts of hydroxy fatty acids [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e], like hydroxyeicosatetraenoic acids [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Although these hydroxy fatty acids are known to contribute to hemozoin toxicity, they also play a beneficial role in the formation of the parasite\u0026rsquo;s membrane. In particular, hydroxyeicosatetraenoic acids are components of schistosoma hemozoin[\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Furthermore, schistosoma hemozoin has been observed to transfer lipids from erythrocytes to vitelline gland cells. During the schizont stage, when numerous merozoites develop and mature, hemozoin crystals are abundantly formed. Importantly, unsaturated fatty acids can promote hemozoin formation [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. As unsaturated fatty acids are converted into saturated fatty acids through hemozoin formation, the host-derived fatty acids or lipids undergo remodeling. The substantial formation of hemozoin during the schizont stage suggests that extensive remodeling and utilization of lipids contribute to the development of merozoites and their membranes. This observation is consistent with previous findings indicating that the schizont stage is characterized by an increase in saturated fatty acids [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Furthermore, the high expression of lipid-related genes at this stage further supports these observed changes and trends. Additionally, transmission electron microscopy (TEM) revealed that hemozoin consists of a lipid matrix combined with a crystal-like structure. Although hemozoin is generally considered a waste product, parasites cannot discharge it, likely due to the role of lipids at the trophozoite stage.\u003c/p\u003e \u003cp\u003eIt is noteworthy that in addition to its role in supporting parasite development and reproduction, the released hemozoin are also likely to contribute significantly to parasite protection. When a substantial amount of hemozoin is released into circulation and ingested by mononuclear macrophages or macrophages, it impairs the phagocytosis performed by these phagocytes in the host [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Additionally, hemozoin has been found to inhibit the differentiation and maturation of human monocyte-derived dendritic cells [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e], and to impair the chemotactic motility and transendothelial migration of monocytes[\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Consequently, hemozoin impairs the host\u0026rsquo;s immunity response, providing protection for parasites and their merozoites. In addition, a large amount of hemozoin were carried to mosquito host by gametocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). Furthermore, malaria parasites initiate heme synthesis exclusively in mosquitoes[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The phenomenon suggests that hemozoin acts as a heme carrier to fulfill the heme requirement during the transition between the two hosts.\u003c/p\u003e \u003cp\u003eIn essence, hemozoin may play a crucial role throughout the entire life cycle of \u003cem\u003ePlasmodium\u003c/em\u003e, encompassing growth, development, reproduction, and potentially providing protection for parasites and merozoites against elimination by macrophages. Therefore, considering the roles of hemozoin in both malaria and schistosomiasis, we propose that hemozoin should not only be regarded as a waste product of heme detoxification.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e \u003cp\u003e This study was carried out in strict accordance with the recommendations of the Regulations for the Administration of Affairs Concerning Experimental Animals of the State Science and Technology Commission. The protocol was approved by the Internal Review Board of Tongji University School of Medicine.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was supported by Innovation Program of Shanghai Municipal Education Commission.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJ.S. designed and conducted all experiments and wrote the main manuscript text; X.X.Q and W.W.S prepared figures 4-5; F.W., Y.N.L. and X.L.Y. prepared figures 1-3. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank LC Sciences (Hangzhou, Zhejiang, China) for assisting with Plasmodium transcriptome and corresponding bioinformatics analysis. This research was supported by Innovation Program of Shanghai Municipal Education Commission (201901070007E00017).\u003c/p\u003e\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e \u003cp\u003eData supporting the conclusions of this article are included within the article and its additional files. The raw data are provided in supplementary files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGluzman IY, Francis SE, Oksman A, Smith CE, Duffin KL, Goldberg DE. Order and specificity of the Plasmodium falciparum hemoglobin degradation pathway. 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Free Radic Biol Med. 2014;75:210\u0026ndash;21; doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.freeradbiomed.2014.07.004\u003c/span\u003e\u003cspan address=\"10.1016/j.freeradbiomed.2014.07.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/pubmed/25017964\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/pubmed/25017964\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Hemozoin, malarial parasites, transmission electron microscopy, iron, heme","lastPublishedDoi":"10.21203/rs.3.rs-5408190/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5408190/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHemozoin is consider a waste byproduct of heme detoxification following hemoglobin digestion; consequently, the biological functions of hemozoin in hemozoin-producing organisms have often been overlooked. However, recent findings indicate that schistosoma hemozoin facilitates the transfer of iron from erythrocytes to eggs through its formation and degradation, thereby increasing interest in the role of malarial hemozoin. In this study, we compared the formation of schistosoma hemozoin and malaria hemozoin using transmission electron microscopy, which suggests why the trophozoite stage cannot eliminate hemozoin. Additionally, through transcriptome analysis of different stages of \u003cem\u003eP. falciparum\u003c/em\u003e 3D7\u003csup\u003eWT\u003c/sup\u003e and \u003cem\u003eP. falciparum\u003c/em\u003e 3D7\u003csup\u003eC580Y\u003c/sup\u003e- where the latter serves as a control with lower hemozoin production-we found that both exhibit similar expression patterns in genes related to DNA synthesis, iron, and heme utilization. Notably, during the trophozoite stage, expression levels of these genes in \u003cem\u003eP. falciparum\u003c/em\u003e 3D7\u003csup\u003eWT\u003c/sup\u003e are higher than in \u003cem\u003eP. falciparum\u003c/em\u003e 3D7\u003csup\u003eC580Y\u003c/sup\u003e, whereas during the schizont stage, they are lower. These results suggest that when \u003cem\u003eP. falciparum\u003c/em\u003e 3D7 utilizes more heme and iron, it produces less hemozoin, whereas when it utilizes less heme and iron, it produces more hemozoin. Interrupting heme utilization and destructing hemozoin aggregation can result in parasite death. Additionally, the hemozoin released by schizonts can impair macrophage functions, or it is carried by gametocytes into the next host without being discharged as waste, suggesting that the release of malaria hemozoin protects merozoites from phagocytosis, and its transfer to the next host may fulfill the requirements for iron and heme during their development in mosquitoes.\u003c/p\u003e","manuscriptTitle":"Hemozoin: a waste product after heme detoxification?","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-25 18:50:09","doi":"10.21203/rs.3.rs-5408190/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-12-10T20:20:06+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-09T04:05:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-05T20:24:18+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-02T17:00:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"265022888654317375554476796059694707243","date":"2024-11-27T17:04:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"169489180439025670291704347657744039249","date":"2024-11-25T17:49:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"235171761092552341579184296462999173223","date":"2024-11-17T16:52:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"19125768323799434170566715972232895180","date":"2024-11-15T21:53:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-15T16:50:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-07T14:38:08+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-07T14:32:29+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasites \u0026 Vectors","date":"2024-11-07T08:31:11+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8da66c8a-fe63-4ed6-93e6-b7ce8a6e31e1","owner":[],"postedDate":"November 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-03-10T19:58:21+00:00","versionOfRecord":{"articleIdentity":"rs-5408190","link":"https://doi.org/10.1186/s13071-025-06699-x","journal":{"identity":"parasites-and-vectors","isVorOnly":false,"title":"Parasites \u0026 Vectors"},"publishedOn":"2025-03-04 15:57:55","publishedOnDateReadable":"March 4th, 2025"},"versionCreatedAt":"2024-11-25 18:50:09","video":"","vorDoi":"10.1186/s13071-025-06699-x","vorDoiUrl":"https://doi.org/10.1186/s13071-025-06699-x","workflowStages":[]},"version":"v1","identity":"rs-5408190","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5408190","identity":"rs-5408190","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}