Targeting Host Metabolic Niche to Kill Malaria Parasites

Targeting Host Metabolic Niche to Kill Malaria Parasites | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Targeting Host Metabolic Niche to Kill Malaria Parasites Rays Jiang, Faiza Siddiqui, Swamy Adapa, Liwang Cui This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4535885/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 12 You are reading this latest preprint version Abstract Malaria remains a global health challenge, exacerbated by artemisinin resistance. Inspired by our recent study targeting aberrant cancer heme metabolism, we propose a novel "bait-and-kill" strategy, focusing on the unique metabolic vulnerability of infected Red Blood Cells (iRBCs) to destroy host niche. We exploit three key factors: 1) mature RBCs inherently possess a truncated heme biosynthesis pathway capable of accumulating heme intermediates, i.e ., porphyrins, 2) Uninfected RBCs exhibit impermeability to the heme precursor ALA (Aminolaevulinic acid), while infected RBCs demonstrate increased permeability, and 3) heme/porphyrin mediated activation of artemisinin has been established as the primary mechanism of action for their antimalarial activity. Utilizing the heightened membrane permeability of iRBCs, we employ the heme precursor ALA as “bait”, inducing heme intermediates accumulation. This synergizes with artemisinin, acting as the 'kill' agent, to effectively eradicate parasites. Uninfected RBCs do not uptake ALA, avoiding collateral damage. We present experimental characterization of drug-drug synergy in a malaria liver stage host cell line and successful elimination of artemisinin-resistant parasites during the blood stage, particularly parasites from the Great Mekong sub-region, a hotspot for antimalarial drug resistance. Leveraging safe drugs like ALA and artemisinin, tested globally, this synergistic strategy holds promise for large-scale deployment in malaria control. Health sciences/Diseases/Infectious diseases/Malaria Biological sciences/Drug discovery Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Human red blood cells (RBCs) represent a terminally differentiated cellular entity characterized by the absence of an endomembrane system, nuclei, or mitochondria [ 1 – 4 ]. Because of their minimalist structure, RBCs possess unique metabolic properties that hold potential for the development of host-targeted therapies against malaria. Heme metabolism in RBC plays a critical role in malaria [ 5 ], serving as the foundation for artemisinin activation [ 6 ] and hemozoin formation by the parasite. In human RBCs, the metabolic profile is characterized by a significant reliance on cytoplasmic pathways, particularly glycolysis, due to the absence of metabolic processes associated with cell division and mitochondrial-based energy or metabolite production [ 3 , 4 ]. Initially, young RBCs undertake heme biosynthesis through a two-part process involving both mitochondrial and cytoplasmic steps [ 7 , 8 ], intricately coordinated during erythroid development. However, upon maturation, only the cytoplasmic steps persist [ 3 , 4 ], presenting metabolic characteristics unique to mature RBCs. Our recent studies have demonstrated the viability of a bait-and-kill strategy aimed at exploiting the metabolic vulnerabilities of cancer cells by leveraging their 'truncated' heme biosynthesis pathway [ 9 – 11 ]. Unlike normal cells, cancer cells possess this aberrant pathway, allowing for the accumulation of heme intermediates without complete conversion into the final product, heme. This strategy capitalizes on the clinical safety profile of ALA [ 12 , 13 ], a heme precursor, which bypasses the initial mitochondrial step, thereby triggering the accumulation of heme intermediates, specifically porphyrins, within cancer cells. Subsequently, these accumulated porphyrins are targeted for elimination using compounds that induce redox stress [ 9 ]. Importantly, normal human cells, characterized by balanced heme pathways, remain unaffected due to their lack of porphyrin accumulation [ 9 ]. We propose that this strategy holds promise for malaria treatment due to three key factors: Firstly, mature RBCs inherently possess a truncated heme biosynthesis pathway [ 1 , 2 , 14 ]. Secondly, healthy/uninfected RBCs are impermeable to the heme precursor ALA, while infected RBCs demonstrate increased permeability [ 14 – 18 ], resulting in porphyrin accumulation. Thirdly, heme mediated activation of artemisinin has been established as the major mechanism of action for its antimalarial activity [ 6 ]. Additionally, evidence from human ferrochelatase (last step of heme biosynthesis) deficient erythropoietic protoporphyria patients indicates resistance to malarial parasite growth, suggesting that natural human genetic variation in aberrant heme metabolism can confer malaria resistance [ 19 ]. With the emergence of artemisinin resistance [ 20 – 22 ], there is an urgent imperative to explore novel and safe antimalarial drugs. In this study, we use the latest quantitative proteomics and erythropoiesis data to elucidate the unique heme pathway of RBCs in their terminally differentiated state. Contrary to previous antimalarial work involving light as PDT therapy [ 14 ], or using human proteins targeting circulating heme [ 23 ], our approach is entirely based on clinically safe drugs. In agreement with previously published results by Sigala et al. [ 14 ], we found RBC heme metabolism is intrinsically distinct than other human cells that can be used for host-niche targeting. Importantly, we generate quantitative drug interaction data for the first time, showing the synergy between ALA and artemisinin. This synergy is leveraged to effectively target clinically procured artemisinin-resistant parasites. Results Result 1: RBC possesses a unique truncated heme biosynthesis pathway Human RBCs are terminally differentiated cells characterized by distinct proteomic and metabolic profiles [ 3 , 4 ]. This distinctiveness in heme biosynthesis enzymes and the resulting substrate production offers a promising approach for targeting malaria parasites by disrupting the unique host heme metabolic niche (Fig. 1 A). Porphyrin production in cancers, a consequence of this unique pathway [ 9 ], exhibits redox activity [ 24 ] and can be exploited to selectively eliminate pathological cells. Our rationale is that we can employ a similar approach to kill malaria-infected RBCs due to the similarity in the 'truncated' heme biosynthesis pathways present in both cancers and RBCs. First, we conducted terminal erythropoiesis analysis, based on previously published human hematopoiesis data [ 25 ] and quantitative proteomic analysis of mature RBCs [ 2 ], to elucidate the heme metabolic processes in the mature cell stage. Our aim was to better understand the metabolic vulnerability of host cells during P. falciparum infection. Utilizing human hematopoiesis gene expression data, we examined the expression of genes involved in heme metabolic processes across various erythroid progenitor stages (Fig. 1 B) (Supplemental Table S1 ). Our analysis encompassed the different temporal steps of erythropoiesis, including common myeloid progenitor (MYE_0), megakaryocyte/erythroid progenitor (MYE_1 and MYE_2), and erythroid cells (ERY1-4). We investigated various key aspects of heme metabolism in RBCs, including heme biosynthesis pathway genes, heme degradation and binding processes, mitochondrial hemoprotein genes, heme/iron trafficking genes. We also examined erythropoiesis principal regulator genes, such as EIF2K1 (heme regulated global translation initiation kinase) [ 26 , 27 ], BACH1 (heme regulated transcription activator) [ 28 ], GATA2 (early erythropoiesis regulator)[ 29 , 30 ] and FOXO1 (late erythropoiesis regulator) [ 31 , 32 ]. Consistent with established findings [ 25 ], we observed distinct gene expression patterns in the heme biosynthesis pathway during erythrocyte maturation. We noted a progressive upregulation of the heme biosynthesis pathway, reaching its peak in the later stages of maturation with high levels of expression of all eight steps of genes. Particularly noteworthy were the robust expression levels of key mitochondrial hemoprotein encoding genes, such as UQCRH and CYC1 , which play crucial roles in powering cellular respiration, during erythroid differentiation. This highlights the significance of mitochondrial metabolism and electron transport chain function in erythroid development and differentiation. Furthermore, our analysis revealed a coordinated upregulation of iron import gene TFRC , indicative of fueling hemoglobin synthesis, and iron export gene SLC40A1, to maintain heme homeostasis. This was accompanied by a downregulation of heme breakdown pathways, BLVRA and BLVRB , indicative of active heme production and its incorporation into cellular components during erythroid differentiation. Subsequent to RBC maturation, the expulsion of DNA regulatory machinery and elimination of subcellular organelles predominates. Examination of recent quantitative RBC proteomic data [ 1 , 2 ] delineates the transition from late differentiated erythroid to fully mature RBCs (Fig. 1 C) (Supplemental Table S2). Mature RBCs exhibit minimal expression levels of mitochondrial hemoproteins (UQCRH and CYC1) and master regulators of heme metabolism (EIF2K1 and BACH1) during differentiation, indicating a cessation of both nuclear and mitochondrial metabolism and heme-regulated developmental processes in mature proteome. Notably, significant reductions are observed in both the initial (ALAS2) and final steps (CPOX, PPOX, and FECH) of heme biosynthesis occurring in mitochondria, with levels low or undetectable per cell. In contrast, mid-step cytosolic enzymes (ALAD, HMBS, UROS, and UROD ) persist at considerable levels (7000 to 800,000 copies protein per cell) in two independently generated proteome datasets, suggesting that only the mid-steps of heme biosynthesis enzymes constitute part of the canonical proteome within mature RBCs. This indicates an intrinsically truncated heme biosynthesis pathway akin to our findings in cancer cells, predisposing the pathway to the accumulation of intermediates. Additionally, the presence of low levels of iron import proteins (TFRC) and high levels of iron export (SLC40A1) and heme degradation proteins (BLVRA and BLVRB) further supports the absence of heme biosynthesis but the maintenance of heme breakdown during the mature RBC lifespan. Furthermore, RBC hemoproteins responsible for antioxidant responses [ 33 – 35 ], such as catalase (CAT), cytochrome b5 reductase A (CYB5A), and cytochrome b5 reductase D (CYRBD1), are prominently expressed in the mature proteome, indicating their crucial role in detoxifying radical species and maintaining RBC integrity and function against the onslaught of redox stress during RBC circulation. Result 2: iRBC in dihydroartemisinin (DHA) resistance stage are remodeled for host cell permeability change The emergence of artemisinin resistance in malaria parasites has raised significant concerns in recent years. Notably, key genes associated with artemisinin resistance, such as Kelch 13 [ 20 , 36 ], ubiquitin hydrolase ( UBP1 ) [ 37 ], AP2 adaptor complex µ-subunit ( AP2-MU ) [ 38 , 39 ], Kelch13 interaction candidate ( KIC5 ) [ 40 ], and Kelch13 interaction candidate ( KIC7 ) [ 41 ], exhibit enrichment of expression in the ring stage of infection [ 20 , 36 ], suggesting their potential involvement in resistance mechanisms during the early stages of parasite colonization. To combat drug resistance effectively, targeting infected host cells during this critical stage becomes imperative. We hypothesize that infected RBCs provide an optimal environment for inducing porphyrin accumulation due to their increased permeability, the precursor of heme intermediates, facilitated by parasite-induced changes in host cell permeability [ 42 , 43 ]. We investigated the gene expression and proteomic evidence of P. falciparum parasites inducing changes in host cell permeability during the erythrocytic stage, with a specific focus on alterations in infected RBCs (iRBCs) associated with DHA resistance (Fig. 2 A) (Supplemental Table S3). Initially, we obtained a predicted set of 386 parasite-exported proteins from PlasmoDB v68, which revealed a distinct gene expression pattern of protein export during blood stage growth. Notably, we observed a peak in gene expression occurring at the ring and early trophozoite stages in the blood stage expression dataset [ 44 ]. Intriguingly, this timeframe coincided with the expression of genes associated with artemisinin resistance. This synchrony suggests a potential opportunity to leverage enhanced host permeability as a strategy for targeting drug resistance. To evaluate the essentiality of the export process, we examined P. falciparum exported protein-encoding genes in the context of saturation mutagenesis through transposon tagging (Fig. 2 B). The mutagenesis index, reflecting the essentiality of in vitro blood stage parasite survival, indicated that lower values correspond to greater essentiality [ 45 ]. Our analysis unveiled a bimodal forward genetic gene essentiality pattern, with approximately 60 exported protein genes identified as essential for blood stage parasite survival. This suggests their critical role in host remodeling and export mechanisms. The identification of these essential genes further supports the rationale for targeting this process as a potential antimalarial strategy. Moreover, we investigated parasite-encoded genes implicated in modulating host RBC permeability, such as CLAG3.1 [ 46 ], CuTP [ 47 ], RhopH2 [ 48 ], and RhopH3 [ 49 ], alongside CTR1 [ 50 ] and HlyIII [ 51 ], to understand their expression patterns and genetic essentiality. Our analysis revealed that during the ring stage of infection, four of these genes were upregulated, as evidenced by the high protein expression levels of CLAG3.1, CuTP, RhopH2 , and RhopH3 (Fig. 2 C). Additionally, a subset of these genes, including RhopH2, RhopH3, CTR1 , and HlyIII , demonstrated forward genetic growth essentiality, indicating the critical role of host cell remodeling for parasite survival within RBCs (Fig. 2 D). Taken together, these findings highlight the potential to leverage host permeability changes induced by infection as a strategy for targeting the host heme metabolic niche. Result 3: Normal RBCs do not accumulate porphyrin, while cancerous blood lineage cells show accumulation. Our recent study targeting the truncated heme biosynthesis pathway in cancer cells has prompted us to investigate a similar pathway in normal human RBCs. Our anti-cancer strategy relies on the premise that normal cells do not accumulate porphyrins, even under ALA induction, providing a therapeutic window for selective targeting of cancer cells. Similarly, to target malaria-infected cells, we conducted further experimentation to demonstrate that normal human RBCs cannot be induced to accumulate porphyrins due to the lack of permeability changes induced by parasites, preventing ALA entry and subsequent PPIX accumulation. First, we investigated the accumulation of PPIX in blood cancer cell lines, expanding upon our previous findings that leukemic cell lines exhibit an aberrant heme biosynthesis pathway, resulting in the buildup of intermediates. To validate this observation, we analyzed in vitro CRISPR KO essentiality data on leukemic cell lines, leveraging the Cancer Dependency Map (DepMap) dataset (23Q4). This dataset offers data of genetic dependence inferred from cell survival upon specific gene knockout, allowing us to assess the essentiality of heme biosynthesis pathway genes in blood cancer cells. Our analysis unveiled an aberrant heme biosynthesis pathway in blood cancer cell lines, characterized by varying essentiality among different pathway genes (Fig. 3 A). Mid-step heme biosynthesis genes, particularly UROD , emerged as crucial for blood cancer cell survival, indicating their significance in sustaining the dysregulated heme biosynthesis process. In contrast, the initial steps or the last step of the pathway displayed low essentiality, suggesting an enzymatic imbalance that leads to inefficiency in heme production and the accumulation of intermediates, namely porphyrins. These analysis confirm the dysregulation of heme biosynthesis in blood cancer cells, highlighting potential vulnerabilities that parallel those observed in malaria-infected cells, providing rationales for targeted therapeutic interventions. Next, we conducted ALA induction experiments on both normal human PBMCs and a selection of four blood cancer cell lines to further elucidate heme biosynthesis dynamics (Fig. 3 B). Normal PBMCs exhibited no accumulation of PPIX, irrespective of ALA presence, indicating stringent heme biosynthesis control and the absence of intermediate buildup. In contrast, blood cancer cell lines robustly accumulated porphyrins upon ALA addition, indicating the aberrant accumulation of heme intermediates in these cells. Flow cytometry data shows a substantial amount PPIX accumulation in > 99% the blood cancer K562 cells following ALA addition (Fig. 3 C). These findings show the distinction between normal human RBCs, which do not accumulate PPIX, and blood cancer cells, which possess a truncated pathway leading to PPIX accumulation. This evidence guides our next investigation into the analogous heme biosynthesis scenario of malaria-infected RBCs for host niche targeting. Result 4: iRBCs Specifically Accumulate Porphyrin To demonstrate the accumulation of Protoporphyrin IX (PPIX) within infected red blood cells (iRBCs) during malaria infection, we used two strains of malaria parasites to infect human RBCs (Fig. 4 ). We specifically tracked the parasites in the trophozoite stage, characterized by increased parasite biomass and clearer marker visualization. Two strains of the malaria parasite, P. falciparum 3D7 and F09A44, were tested for PPIX accumulation. PPIX fluorescence was detected using excitation at 405 nm and emission in the red channel at 633 nm. Additionally, SYBR Green dye, with excitation and emission at 498/522 nm, labeled nucleic acids of the parasites. Normal RBCs (> 99.9%) did not exhibit PPIX accumulation regardless of ALA presence. For 3D7-infected RBCs with lower parasitemia, PPIX accumulation was absent without ALA but increased upon ALA addition (Fig. 4 D & E). Similarly, F09A44-infected RBCs with higher parasitemia showed no PPIX accumulation without ALA, but levels rose after ALA addition (Fig. 4 D & E). Importantly, the majority of RBCs, both in the total normal population and uninfected subpopulations, did not accumulate significant amounts of PPIX. Unlike all nucleated healthy human cell types we tested [ 9 – 11 ], a small increase in some uninfected RBCs co-cultured with infected RBCs was observed, possibly due to parasite-released micro-vesicles [ 52 ]. However, no hemolysis was observed in uninfected cells, consistent with decades of ALA human clinical safe use without reported hematological effects. Taken together, these findings suggest that active malaria infection facilitates ALA entry and subsequent heme precursor conversion, bypassing the initial mitochondrial step and leading to porphyrin accumulation in infected RBCs. Result 5: DHA and ALA Synergize in Killing HC-04 Cancer Cells To explore the potential synergy between DHA and ALA in targeting porphyrin accumulation, we conducted a series of experiments using cell viability assays. As RBCs are terminally differentiated and unsuitable for quantitative drug synergy assays, we utilized the HC-04 liver cancer cell line [ 53 – 55 ]. This cell line can serve as a host cell for in vitro malaria liver stage development experiments and exhibits onco-transformation properties. First, employing CRISPR KO in vitro gene essentiality analysis in a set of liver cancer cell lines, we assessed heme biosynthesis pathway gene essentiality (Fig. 5 A). Our results indicate that liver cancer cell lines exhibit aberrant heme biosynthesis patterns, with only the mid-steps of the pathway (UROS and UROD) being essential for in vitro cell survival. This suggests that while liver cancer cells do not require endogenous heme synthesis, they are capable of producing intermediate porphyrins due to the essentiality of mid-steps, a scenario similar to cancer blood cell lines and RBCs. Next, we investigated the accumulation of Protoporphyrin IX (PPIX) upon ALA incubation in normal primary human hepatocytes and three liver cancer cell lines, including HC-04 (Fig. 5 B). Consistent with our recent findings [ 9 ], normal primary human hepatocytes did not exhibit PPIX accumulation upon ALA treatment, indicating tightly controlled heme biosynthesis without intermediate accumulation to support liver function. In contrast, all three liver cancer cell lines, including HC-04, showed high levels of PPIX accumulation after 72 hours of ALA treatment, suggesting incomplete heme biosynthesis and the accumulation of intermediates in liver cancer cells. Further, we conduct experiments to quantify the cytotoxic effects of ALA and DHA, both individually and in combination, across a broad range of drug concentrations (Fig. 5 C). We assessed a wide spectrum of drug concentrations, ranging from nanomolar (nM) to 133 micromolar (µM) concentrations of DHA and from nanomolar (nM) to millimolar (mM) concentrations of ALA. This drug-drug interaction study is performed in experimental triplicates (Supplemental Table S4). Our findings revealed that while treatment with either ALA or DHA alone demonstrated minimal toxicity, their combined administration exhibited a robust synergy in killing cancer cells. Remarkably, this synergistic effect was evident even at low concentrations of DHA in the nanomolar (nM) range. This observation suggests that ALA sensitizes DHA, likely through the accumulation of PPIX, thereby enhancing its efficacy in targeting and eliminating cancer cells. Notably, nanomolar concentrations of DHA proved sufficient for inducing cell death following sensitization by ALA, indicating the potential of this strategy to overcome malaria artemisinin resistance. Result 6: DHA and ALA Kill DHA-Resistant Parasites Based on our investigation into RBC heme metabolism and DHA sensitization, we propose a Bait-and-Kill Strategy to combat artemisinin-resistant malaria, utilizing ALA as the “bait” and artemisinin as the ‘kill’ agent. Given the global impact of malaria, the success of this strategy hinges on the safety profile of ALA (Table 1 ). Notably, ALA has been employed as an FDA-approved cancer imaging agent since 2007, with extensive use in Europe preceding its adoption in the United States. Over 58,000 individuals worldwide have used ALA with no discernible adverse effects. ALA is routinely utilized in mouse models for both imaging and treatment, with a good safety profile[ 12 ]. Studies in Japan have examined its long-term use for up to three months [ 13 ] and large dose administration of up to 2250 mg/day without reported toxicity [ 56 ]. Presently, ALA is marketed as a nutritional supplement in Japan (SB Pharma). Human pharmacodynamic studies [ 57 , 58 ] have directly monitored ALA metabolism, revealing no toxic effects. PPIX, metabolized from exogenous ALA, can persist in human sera for several hours before complete elimination, typically occurring within 35–48 hours. Detailed information regarding ALA's drug use, safety profile, and human pharmacodynamics studies is provided in Supplementary Table S5. Collectively, ALA's widespread use across diverse populations suggests its safety for use as a public health intervention against infectious diseases. Table 1 Summary of ALA dosage for safety and efficacy in human clinical studies. (An extensive set of results are provided in Supplemental Table S1 ) Experimental system ALA dosage Disease model ALA effect References Mouse model 40–600 mg/kg Imaging, Cancer treatment, Malaria treatment No toxicity. Unpublished data Predina et al [ 78 ] Stowers et al [ 79 ] Suzuki et al [ 80 ] Human Imaging (FDA approved) 20 mg/kg Brain cancer surgery imaging No toxicity. FDA Gleolan report NDA 208630 Human High dose up to 2250 mg/day for 3–7 days COVID treatment in Japan No toxicity. Kaketani and Nakajima [ 56 ] Human Long term 200 mg/day for 3 months Type 2 diabetes treatment No toxicity. Al-Saber et al [ 13 ] Human Pharmacodynamics 40–60 mg/kg ALA pharmacodynamics study No toxicity. Webber et al [ 57 ] Rick et al [ 58 ] Abbreviations: ALA, 5-aminolevulinate; DHA, dihydroartemisinin; PPIX, protoporphyrin IX To evaluate the Bait-and-Kill Strategy against artemisinin-resistant clinical isolates, we utilized the clinical isolate F09A44 (a Southeast Asian clinical isolate) carrying the K13 (C469Y) mutation associated with drug resistance. Characterized as artemisinin-resistant, the Ring Survival Assay (RSA) revealed that 700 nM DHA alone resulted in approximately 10% parasite survival (Fig. 6 A). Subsequently, we applied a combination drug therapy comprising ALA and DHA. Initially, 1 mM ALA was administered to facilitate PPIX accumulation, followed by the addition of 700 nM DHA for parasite eradication. ALA was replenished at only 6 hours (wash out time of DHA), or two days or all three days until 72 hours, the readout time for RSA. Our results demonstrated that the combination of ALA and DHA when ALA was replenished everyday resulted in complete parasite elimination by day 3, with no parasite recurrence observed even after 3 weeks of culture. The experiment was conducted in biological replicates (n = 3), providing evidence that this strategy effectively eradicates resistant parasites without recrudescence from parasite dormancy. In summary, our proposed Bait-and-Kill Strategy targets artemisinin-resistant parasites (Fig. 6 B). We show that uninfected red blood cells (RBCs) do not uptake ALA and therefore do not produce PPIX, remaining unharmed by the drug combination. In contrast, infected RBCs uptake ALA, leading to PPIX production, sensitizing DHA to kill the parasite. This approach selectively targets malaria-infected red blood cells, as ALA is exclusively taken up by these cells and cannot enter normal RBCs. Conclusion Amidst the persistent threat of malaria and the rise of artemisinin resistance, urgent and innovative solutions are imperative. Drawing from our recent discoveries concerning cancer heme metabolic vulnerability, we have introduced a novel therapeutic strategy termed the "bait-and-kill" approach. This strategy targets analogous metabolic susceptibilities found in both malaria-infected red blood cells (iRBCs) and cancer cells. By exploiting the truncated heme biosynthesis pathway in mature RBCs and the heightened permeability of infected cells to the heme precursor ALA, we have effectively demonstrated the potent synergy between ALA and artemisinin in eradicating artemisinin-resistant Southeast Asian clinical isolates. Significantly, our strategy provides a targeted intervention that selectively eliminates infected RBCs while preserving uninfected cells, thereby minimizing collateral damage. These findings offer promise for the development of large-scale public health interventions leveraging clinically safe drugs. Discussion In our study, we show the importance of considering human host metabolism as a pivotal battleground in the fight against malaria infection, especially given the emerging resistance to artemisinin [ 20 – 22 ], the primary treatment for malaria. Malaria parasites, as obligate intracellular organisms, heavily rely on reshaping host cells to ensure their survival. Notably, the occurrence of metabolic polymorphisms in malaria-endemic regions, such as G6PD deficiency [ 59 ], highlights how natural selection has historically provided solutions to combat malaria infection through adjustments in host metabolism [ 5 , 60 ]. RBCs are functionally robust cells, constituting the most abundant yet metabolically minimalist cell type in the human body [ 4 , 34 ]. Upon release from bone marrow development, RBCs lack nuclei and organelles for most cellular repair processes, yet circulate for approximately three months [ 34 , 35 ], equipped with a robust antioxidant system, including high levels of hemoproteins like catalase and cytochrome b5 reductase [ 33 , 34 ]. In the context of malaria infections, natural selection has favored mechanisms that increase redox stress to kill parasites while preserving normal RBC function, as evidenced by the worldwide high prevalence of hematological diseases like G6PD deficiency and sickle cell anemia [ 61 – 63 ]. Our aim is to exploit this redox stress using bait-and-kill approaches. Our unique approach selectively enhances host redox stress in infected cells. To further elucidate the mechanisms, we are investigating a range of human malaria-related hematological diseases to understand the role of inherent host metabolic polymorphisms in determining infection outcomes. Here, we leverage the distinctive metabolic characteristics of human mature red blood cells (RBCs), which possess a truncated heme biosynthesis pathway. Drawing inspiration from recent discoveries targeting similar pathways in cancer cells [ 9 – 11 ], we harness the resulting accumulation of redox-active heme intermediates [ 24 , 64 ] to effectively induce cell death. Moreover, there is potential for further exploration into strategies that exploit host redox stress during infection, offering novel ways in treating other types of intracellular pathogens. The precise mode of action underlying the synergy between ALA and DHA in parasite killing remains to be elucidated in future research. Artemisinin's mechanism of action relies on its activation by heme present within infected cells, triggering redox damage and ultimately killing the parasite [ 6 ]. Recent advances have demonstrated that porphyrins, similar to heme, can activate artemisinin [ 65 ](unpublished). By inducing infected cells to accumulate porphyrins, we sensitize artemisinin, providing a novel approach to combat artemisinin-resistant parasites. Furthermore, this combinatorial approach could potentially extend to testing artemisinin in combination therapy with other frontline antimalarials [ 21 ], thereby revitalizing existing drugs that have encountered resistance. The quantification of synergy conducted in HC-04 cells, a liver cancer cell line utilized as a host for liver stage malaria assays [ 54 , 55 ], demonstrates the potential of targeting aberrant heme biosynthesis in malaria-infected liver cells. Given that malaria can infect diverse host cells, including liver [ 66 – 68 ] and bone marrow cells [ 69 , 70 ], which undergo parasite-induced remodeling, the bait-and-kill strategy holds promise for application across various stages of malaria infection. Evidence indicates that the heme biosynthesis pathway is dysregulated in infected liver and marrow niches, presenting opportunities to develop interventions that target multiple life stages of malaria beyond red blood cell infection. Further investigation could explore the efficacy of the bait-and-kill strategy other phases of malaria infection, thereby taking initial steps for therapeutic interventions against malaria by targeting different host niches. Materials and Methods Analysis of Cancer Gene Essentiality in Heme Biosynthesis Using CRISPR KO data The assessment of cancer gene essentiality via CRISPR/Cas9 was conducted by leveraging data available on the DepMap Portal, using established methodologies [ 71 – 73 ], similar to methods we previously used [ 9 ]. Whole-genome CRISPR/Cas9 datasets were utilized to identify notable reductions in the growth of mutant cells following targeted gene knockouts in pooled experiments. Gene essentiality was inferred based on the dependency of a particular gene, determined through CRISPR/Cas9 gRNA-mediated gene knockout. Essential scores were employed to evaluate cell growth fitness, with lower scores indicating a more significant impact on cell viability upon gene loss. Specifically, scores of 0, 0 represented no change in fitness, loss of fitness, and gain in fitness (suggesting potential growth advantage for the cell line) under the assay conditions. Identification of commonly essential genes was based on their significance for the fitness of most cell lines across various cancer types [ 74 , 75 ]. Analysis of human mature red blood cell (RBC) heme biosynthesis and malaria parasite host remodeling data Hemopoiesis gene expression data were sourced from previously published datasets [ 25 ], focusing on various stages of erythropoiesis, such as common myeloid progenitor (MYE_0), megakaryocyte/erythroid progenitor (MYE_1 and MYE_2), and erythroid cells (ERY1-4). Quantitative proteomic data of human mature red blood cells (RBCs) were obtained from comprehensive datasets [ 1 , 2 ] comprising a total of 18,581 proteins present in RBCs. Within this dataset, approximately 1200 proteins constitute the canonical RBC proteome, including mid-steps of heme biosynthesis enzymes ALAD, HMBS, UROS and UROD. The full sets of RBC proteome exhibit a broad range of protein abundances, from high to low, with the lowest abundance represented by a single peptide, for example, the mitochondrial hemoprotein COX10. Proteins considered abundantly present were those ranking above the 0.75 percentile in peptide counts within RBC proteomes. Differential expression levels between early and late stages were assessed using the Wilcoxon test, with P values adjusted using the Benjamini-Hochberg method for multiple hypothesis testing. For malaria host modeling protein analysis, the set of host-targeted proteins were retrieved from PlasmoDB v68 [ 76 ] by combining searches for the 'PEXEL' motif and 'Host-Targeted' motif in the reference P. falciparum 3D7 genome [ 15 , 16 ]. Gene essentiality data, represented by the Mutagenesis Index score, were obtained based on saturation transposon mutagenesis phenotypic data of parasite in vitro survival in blood stages. Host permeability proteins were identified based on the Malaria Parasite Metabolic Pathway (MPMP) v2023 ( http://mpmp.huji.ac.il ), specifically focusing on the permeability of the membrane of infected RBCs. Malaria gene expression data were retrieved from transcriptomes of seven sexual and asexual life stages [ 44 ], including two gametocyte stages (II and V), ookinete, and four time points of erythrocytic stages (ring, early trophozoite, late trophozoite, and schizont). Bimodal distribution estimation was performed using histograms displaying two prominent peaks, and Kernel Density Estimation (KDE) was employed to estimate the probability density function of the data, providing a smoothed estimate of the underlying distribution and identifying multiple modes. Primary Liver Cell and Hepatoma Cell Line Preparation In vitro culture of primary human hepatocytes began by sterilizing 384-well plates (Greiner, Cat No. 781091) in a class II biosafety cabinet and placing them in a secondary container to prevent evaporation. The wells were coated with 40 µL of 15 µg µL-1 rat tail collagen I (Corning, Cat No. 354236) in sterile filtered 0.02 M acetic acid (Thermo Fisher Scientific) and kept at 37°C overnight. Before seeding, the wells were washed thrice with sterile phosphate-buffered saline (PBS) and filled with 20 µL of in vitro GRO® CP plate medium (BioIVT, Cat No. Z99029), supplemented with 1x Pen-Strep-Neo solution (100x, Fisher, Cat No. 15640-055) and 20 µM gentamicin (1000x, Fisher, Cat No. 15-710-072). Cryopreserved primary human hepatocytes (BioIVT, Cat No. M00995-P) were thawed by immersion in a 37°C water bath for 2 minutes, sterilized with 70% ethanol in a sterile field, and added directly to 4 mL plate medium. Live and dead cells were quantified using trypan blue exclusion on a Neubauer improved hemocytometer. The hepatocyte density was adjusted to 1 × 103 live cells µL-1, and 18 µL of the cell suspension was added to each well. Medium exchange with the GRO® CP plating medium occurred thrice weekly. Three hepatoma cell lines, namely HC-04, HepG2, and SNU449, were used as liver cancer cell lines for comparative analysis. These cryopreserved cell lines were thawed, suspended in a hepatocyte culture medium previously prepared, and transferred to T75 flasks coated with collagen (Corning, Cat No. 354236) at a density of 5 µg/cm². The culture medium consisted of a 1:1 (v/v) mixture of F12 base medium (Invitrogen, Cat No. 11765-054) and MEM base medium (Invitrogen, Cat No. A10490-01), supplemented with 10% FBS (Hyclone, Cat No. SH30070), 1.0 M HEPES (Invitrogen, Cat No. 15630-080), and 200 mM glutamine (Invitrogen, Cat No. 25030-081). The cells were cultured until reaching 70% confluence, with the medium being changed every other day. Once the desired confluence was attained, the cells were trypsinized using TrypLE™ Express Enzyme (1X) (Gibco, Cat No. 12605028), washed with hepatocyte culture medium, and then seeded at a density of 6000 cells/well in 384-well plates (Greiner, Cat No. 781091), with each well receiving 20 µL of the aforementioned medium. Cells were then incubated either in the absence or presence of 1.0 mM ALA at 37°C for 4 hours under very low light conditions. During the last 45 minutes of incubation, a staining solution diluted in phenol-free, serum-free RPMI (Gibco, Cat No. 11835055), containing Hoechst 33342 (Life Technologies, Cat No. H3570) at a final concentration of 10 µM, was added to the cells. Cellular PPIX Quantification Quantification of intracellular protoporphyrin IX (PPIX) accumulation was performed using fluorescence-activated cell sorting (FACS), following established protocols [ 77 ]. Cells were cultured in medium as described in the Cell Culture section, supplemented with 1.0 mM 5-aminolevulinic acid (ALA), and maintained at 37°C for 4 hours under low light conditions. After incubation, cells underwent triple washing with Dulbecco's phosphate-buffered saline (DPBS, 1X, Ca2+- and Mg2+-free; Corning, Cat No. 21-031-CV) and were resuspended in 250 µL of 1x DPBS. Subsequently, cells were washed once with serum-free medium (Gibco, Cat No. 11835055) and seeded in 6-well plates with the designated medium (as described in the Cell Culture section), with or without the addition of ALA, followed by incubation at 37°C. Intracellular PPIX concentration was evaluated 18 hours later using FACS. FACS analyses were conducted using a BD LSR II Analyzer (Becton, Dickinson, and Company) equipped with FACSDiva Version 6.1.3 software. To minimize background red fluorescence, the 633 nm-red laser was deactivated during PPIX emission data collection. PPIX emission within the 619 nm and 641 nm range (630/22BP filter) was measured following excitation with the 405 nm laser. Forward-scatter (FSC) versus side-scatter (SSC) dot plots were utilized to gate the entire cell population while excluding cell debris. A minimum of 10,000 cells within the gated region were then represented in dot plots of SSC vs. PPIX fluorescence, with the gate defined using cells lacking perturbation as negative controls. Malaria Parasite Culture For the cultivation of asexual blood-stage parasites, O + human red blood cells (RBCs) were utilized and maintained in a controlled environment at 37°C with 5% CO 2 humidity. The parasite culture medium consisted of RPMI 1640 supplemented with 25 mM NaHCO 3 , 11 mM glucose, 25 mM HEPES (pH 7.4), 0.367 mM hypoxanthine, and 5 µg/liter gentamicin. Additionally, 0.5% AlbuMAX II lipid-rich bovine serum albumin from Thermo Fisher Scientific, MA, was added to enhance the lipid content. To synchronize the parasites at the ring stage, a 5% D-sorbitol treatment method was employed. Malaria Ring-Stage Survival Assays The Ring-Stage Survival Assay (RSA) was conducted following established protocols. Initially, schizonts were purified from tightly synchronized cultures using a 75% Percoll gradient (Sigma-Aldrich). After purification, the schizonts were washed once in RPMI 1640 incomplete medium and allowed to rupture, invading fresh red blood cells (RBCs) for a duration of 3 hours. Following invasion, the cultures underwent another synchronization process using sorbitol to select for early rings and eliminate any remaining schizonts. For the RSA 0 –3 h assay, ring-stage parasites (0 to 3 hours post-invasion) at a parasitemia of 1% and hematocrit of 1% were exposed to 700 nM Dihydroartemisinin (DHA) for a period of 6 hours, followed by a single wash. Subsequently, the cultures were allowed to incubate for 66 hours, after which approximately 10,000 RBCs were blindly counted on thin blood smears to determine the number of viable parasites. For ALA combination assays, 1 mM ALA was added along with 700 nM DHA to the 0–3 hours rings and aliquoted in four distinct wells. 1mM ALA was either replenished after DHA washout on day 1 or day 1 & 2 or all 3 days until readout. Smears were prepared at 72 hours, and further parasite growth was monitored through Geimsa stained smears for over 3 weeks. For all assays, parallel dimethyl sulfoxide (DMSO)-treated controls (0.1% concentration) were included, and survival rates were expressed as ratios of viable parasites in DHA-exposed or DHA + ALA exposed Vs DMSO-exposed samples. All assays were performed in 3 biological replicates. Declarations Data Availability Supplemental files containing the datasets necessary to interpret, replicate, and build upon the methods or findings are available. The raw data of plate readouts from the drug synergy studies, conducted in experimental triplicates, are provided in the supplemental materials. Author Contributions RJ contributed to project design, data analysis, writing, and fund acquisition and management. FS and SA conducted the experiments, performed analysis, and contributed to the writing. LC contributed to the discussion and project management. Competing Interests The authors declare no competing interests. Acknowledgements We gratefully acknowledge the College of Public Health, University of South Florida, for providing research funding for this project. Additionally, RJ received funding from Women’s Philanthropy Leadership (WLP) FY23-25 and FLDOH 9BC14 to support research related to the manuscript. References Bryk AH, Wiśniewski JR: Quantitative Analysis of Human Red Blood Cell Proteome. J Proteome Res 2017, 16(8):2752–2761. Sae-Lee W, McCafferty CL, Verbeke EJ, Havugimana PC, Papoulas O, McWhite CD, Houser JR, Vanuytsel K, Murphy GJ, Drew K et al : The protein organization of a red blood cell. Cell Rep 2022, 40(3):111103. 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Additional Declarations (Not answered) Supplementary Files SupplementalTableS1.xlsx Supp Table S1 SupplementalTableS2.xlsx Supp Table S2 SupplementalTableS3.xlsx Supp Table S3 SupplementalTableS4.xlsx Supp Table S4 SupplementalTableS5.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: withdrawn 23 Mar, 2025 Editorial decision: revise 30 Jul, 2024 Review # 3 received at journal 26 Jul, 2024 Review # 1 received at journal 09 Jul, 2024 Reviewer # 3 agreed at journal 07 Jul, 2024 Reviewer # 2 agreed at journal 22 Jun, 2024 Reviewer # 1 agreed at journal 22 Jun, 2024 Reviewers invited by journal 19 Jun, 2024 Submission checks completed at journal 13 Jun, 2024 First submitted to journal 10 Jun, 2024 Unknown event 06 Jun, 2024 Editor assigned by journal 05 Jun, 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-4535885","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":316353322,"identity":"5d99f242-4cc7-4577-bb50-6f81315b3ac4","order_by":0,"name":"Rays Jiang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuUlEQVRIiWNgGAWjYBACxmYQWWHDwMB8gIGBh3gtZ9IYGNgSiNQC0dd2mAQtzO3Mzx5+YTufOL+NgfHB2zaiHMZmbizDcztxwzEGZsO5xGlhMJOWkABqkW9gk+YlTgv7N2kJg3Mgh7H/JlILj5nkh4QDiQ3HGNiYidVSJs1wINl4wzHGZsk554jQYth/fJvkz392svPbmA9+eFNGjJYGYEBDooOxgQj1QCAPUvuDOLWjYBSMglEwUgEA2Uoze/Z4IawAAAAASUVORK5CYII=","orcid":"","institution":"University of South Florida","correspondingAuthor":true,"prefix":"","firstName":"Rays","middleName":"","lastName":"Jiang","suffix":""},{"id":316353323,"identity":"6a2353e7-073d-4672-86a1-e85e12204ac2","order_by":1,"name":"Faiza Siddiqui","email":"","orcid":"","institution":"university of South Florida","correspondingAuthor":false,"prefix":"","firstName":"Faiza","middleName":"","lastName":"Siddiqui","suffix":""},{"id":316353324,"identity":"589930ba-d3b6-4e2a-95be-7d7cd4ec8986","order_by":2,"name":"Swamy Adapa","email":"","orcid":"","institution":"University of South Florida","correspondingAuthor":false,"prefix":"","firstName":"Swamy","middleName":"","lastName":"Adapa","suffix":""},{"id":316353325,"identity":"51108698-e51b-441d-a876-aec1b9bc83e0","order_by":3,"name":"Liwang Cui","email":"","orcid":"https://orcid.org/0000-0002-8338-1974","institution":"University of South Florida","correspondingAuthor":false,"prefix":"","firstName":"Liwang","middleName":"","lastName":"Cui","suffix":""}],"badges":[],"createdAt":"2024-06-05 18:55:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4535885/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4535885/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60172601,"identity":"1fc452cf-f0cd-40d1-9368-555124b660ce","added_by":"auto","created_at":"2024-07-12 15:17:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":375105,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eErythropoiesis Gene Expression and Mature RBC Proteome Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Schematic representation illustrating the heme biosynthesis pathway in developing versus terminally differentiated stages. Upon erythroid maturation, the loss of cell organelles including mitochondria results in the diminished presence of mitochondrial heme biosynthesis enzymes. Evidence of residual functions of the last steps has been found in in-depth analysis of the proteome of mature human RBCs, as well as previously demonstrated (Sigala et al.). Right panel: Malaria-infected RBCs can uptake the heme precursor ALA and accumulate porphyrins.\u003c/p\u003e\n\u003cp\u003eB. Human erythropoiesis gene expression analysis reveals upregulation of the entire heme biosynthesis pathway into the last stages before maturation. The erythropoiesis stages are: common myeloid progenitor (MYE_0), megakaryocyte/erythroid progenitor (MYE_1 and MYE_2), and Erythroid cells (ERY1-4). Mitochondrial heme protein genes, such as \u003cem\u003eUQCRH\u003c/em\u003e and \u003cem\u003eCYC1\u003c/em\u003e, are robustly expressed. For heme synthesis related trafficking, there is concurrent upregulation of iron import (\u003cem\u003eTFRC\u003c/em\u003e) and export (\u003cem\u003eSLC40A1\u003c/em\u003e) genes. Heme breakdown is downregulated. On the right panel, by contrast, the fully mature RBC proteome shows abundant cytoplasmic heme biosynthesis enzymes, including ALAD, HMBS, UROS, and UROD, while mitochondrial enzymes are depleted. Iron export (SLC40A1) and heme degradation proteins (BLVRB, BLVRA) are present at high levels in mature RBCs, whereas iron import and mitochondrial located heme biosynthesis proteins (ALAS1, ALAS2, CPOX, PPOX, and FECH) are greatly reduced.\u003c/p\u003e\n\u003cp\u003eC. Mid-step heme biosynthesis genes and heme degradation genes are among the highest-level proteins in mature RBCs, while first or last heme biosynthesis genes are present at low levels or are undetectable, indicating an intrinsic truncated pathway present in normal human mature RBCs. RBC hemoproteins responsible for antioxidant responses, such as catalase (CAT), cytochrome b5 reductase A (CYB5A), and cytochrome b5 reductase D (CYRBD1), are prominently expressed in the mature proteome, indicating the importance in detoxifying radical species. Heme degradation and iron export proteins are present, suggesting breakdown during the RBC lifespan during the halting of heme biosynthesis. Mitochondrial respiratory hemoproteins and heme regulators during development are present at very low levels, confirming extrusion of mitochondria and cessation of key erythroid developmental processes.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-4535885/v1/7a5eba39076e418a23bb6f79.png"},{"id":60171203,"identity":"eff2e311-d71a-47a7-860d-b84c8ee7492d","added_by":"auto","created_at":"2024-07-12 15:09:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":174507,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGene Expression and Proteomic Evidence of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. falciparum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e Parasite Inducing Host Cell Permeability Changes During Erythrocytic Stage\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Predicted exported proteins (\u0026gt;400) by \u003cem\u003eP. falciparum\u003c/em\u003e during the blood stage growth exhibit a peak gene expression pattern at the ring and early trophozoite stages. In contrast, non-exported protein-encoding genes show a peak expression pattern in the late trophozoite and schizont stages. Several previously reported key players of artemisinin resistance-related genes, such as \u003cem\u003eK13, UBP1, AP2-MU, KIC5,\u003c/em\u003e and \u003cem\u003eKIC7\u003c/em\u003e, are enriched in the ring stage of infection.\u003c/p\u003e\n\u003cp\u003eB. \u003cem\u003eP. falciparum\u003c/em\u003e exported protein-encoding genes display a bimodal forward genetic gene essentiality pattern, as evidenced by transposon mutagenesis phenotype survival data. While the majority of exported protein genes are classified as non-essential during in vitro blood stage culture, a subset of 60 exported protein genes are phenotyped as essential for blood stage parasite survival, indicating their importance in the export and host remodeling process.\u003c/p\u003e\n\u003cp\u003eC. A set of previously published parasite-encoded genes that modulate host RBC permeability are upregulated in the ring stage. Whole proteome protein abundance data reveal that \u003cem\u003eCLAG3.1, CuTP, RhopH2\u003c/em\u003e, and \u003cem\u003eRhopH3\u003c/em\u003eproteins are highly expressed in the ring stage.\u003c/p\u003e\n\u003cp\u003eD. A subset of parasite-host permeability-changing genes, including \u003cem\u003eRhopH2, RhopH3, CTR1\u003c/em\u003e, and \u003cem\u003eHlyIII\u003c/em\u003e, demonstrate forward genetic growth essentiality during random transposon mutagenesis under standard \u003cem\u003ein vitro\u003c/em\u003eblood stage culture conditions, indicating the significance of host cell remodeling for parasite survival within RBCs.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-4535885/v1/2e61ed36fc4f75012e443f45.png"},{"id":60171207,"identity":"d81f82fc-b7c3-4ac6-b06e-7ce3d12a8cf2","added_by":"auto","created_at":"2024-07-12 15:09:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":99785,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAccumulation of Protoporphyrin IX (PPIX) in Blood Cancer Cell Lines but Absent in Normal Human Peripheral Blood Mononuclear Cells (PBMCs)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Blood cancer cell lines exhibit an aberrant heme biosynthesis pathway, as demonstrated by in vitro CRISPR KO gene essentiality analysis. Partial heme biosynthesis pathway genes, particularly UROD, are crucial for blood cancer in vitro survival. Conversely, the first steps of the pathway show low essentiality, suggesting an imbalanced heme biosynthesis in blood cancer cells prone to intermediate accumulation, \u003cem\u003ei.e.,\u003c/em\u003e Protoporphyrin.\u003c/p\u003e\n\u003cp\u003eB. Normal human peripheral blood mononuclear cells (PBMCs) do not accumulate PPIX, with or without ALA, indicating strict heme biosynthesis control and absence of intermediate buildup. ALA is either efficiently converted to the final heme product or degraded in normal human PBMCs. In contrast, the blood cancer cell lines HEL, HEL-ALAS2KO, K562, and BAF3_Jak2mt, all robustly accumulate porphyrins to thousands-fold higher levels after adding ALA.\u003c/p\u003e\n\u003cp\u003eC. Flow cytometry data reveal that untreated blood cancer cell lines exhibit no PPIX accumulation. However, upon adding PPIX for 48 hours, more than 99% of blood cancer cells become PPIX positive, indicating massive heme intermediate accumulation without conversion to the final heme product.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-4535885/v1/395fea3a09b973769bd0d10f.png"},{"id":60173697,"identity":"8f071481-25ed-443b-b422-3d6736f3cf2b","added_by":"auto","created_at":"2024-07-12 15:25:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":213071,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eP. falciparum-Infected RBCs Accumulate Porphyrins upon Adding ALA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwo parasite strains, \u003cem\u003eP. falciparum\u003c/em\u003e 3D7 and A44 (A44 refers to the clinical strain F09A44), were tested for PPIX accumulation during RBC infection \u003cem\u003ein vitro\u003c/em\u003e. \u003cstrong\u003eA:\u003c/strong\u003e Flow cytometry scatterplot separating infected RBCs from uninfected RBCs using SYBR Green (SRBG) nucleic acid signal. The Y-axis represents flow cytometry Side Scatter (SSC), and the X-axis represents SYBR levels. \u003cstrong\u003eB:\u003c/strong\u003e SRBG staining reveals varying levels of parasitemia with 3D7 and A44 in vitro infections. \u003cstrong\u003eC:\u003c/strong\u003e Upon adding ALA, similar percentages of SRBG-positive (infected) RBCs were found\u003cstrong\u003e. D:\u003c/strong\u003e Without adding ALA, no PPIX-positive cell populations were detected in infected RBCs or control normal RBCs. \u003cstrong\u003eE:\u003c/strong\u003e Both 3D7 and A44 infected cells accumulated PPIX upon ALA addition. \u003cstrong\u003eF:\u003c/strong\u003e Without ALA, most RBCs are negative for PPIX and SYBR, with a slight reduction in PPIX and SYBR negativity observed in infected RBCs due to parasite nucleic acid content. \u003cstrong\u003eG: \u003c/strong\u003eUpon ALA addition, most RBCs are negative for PPIX and SYBR. PPIX signal was visualized using excitation at 405 nm and emission in the red channel at 633 nm. SYBR Green dye was used to label nucleic acids, with excitation and emission at 498/522 nm.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-4535885/v1/c06d93ac44b71034e0d06e80.png"},{"id":60171210,"identity":"0c5ec6c3-6499-46f6-abff-921f159c943a","added_by":"auto","created_at":"2024-07-12 15:09:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":95702,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTargeting Porphyrin Accumulation with ALA and Artemisinin Synergy in Human Liver Cancer Cell Lines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. CRISPR KO in vitro gene essentiality data demonstrate that liver cancer cell lines exhibit aberrant heme biosynthesis. Only the mid steps (UROS and UROD) are required for in vitro cell survival, while the first and last steps are dispensable. This indicates that liver cancer cells do not need to synthesize heme endogenously in vitro because the complete pathway is not required, but they are capable of producing intermediate porphyrins due to the essentiality of mid-steps.\u003c/p\u003e\n\u003cp\u003eB. Normal primary human hepatocytes do not show Protoporphyrin IX (PPIX) accumulation upon ALA incubation, indicating tightly controlled heme biosynthesis without intermediate accumulations. In contrast, the three liver cancer cell lines, HC-04, HepG2, and SNU449, all exhibit high levels of PPIX in more than 99% of cells upon adding ALA for 72 hours, suggesting incomplete heme biosynthesis and accumulation of intermediates in cancer cells.\u003c/p\u003e\n\u003cp\u003eC. Drug synergy between ALA and DHA in HC-04 cells shows potent synergy in killing cancer cells. While each ALA or DHA alone shows no toxicity, even at the highest tested dose against HC-04, their combination effectively kills cancer cells even at low concentrations with DHA in the nanomolar (nM) ranges. (DHA: dihydroartemisinin, the active metabolite of ART)\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-4535885/v1/76e445c134fd3ba84f917259.png"},{"id":60171211,"identity":"37504862-e827-4cd5-8534-b1d121f72a69","added_by":"auto","created_at":"2024-07-12 15:09:29","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":168336,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKilling Artemisinin-Resistant Malaria with Bait-and-Kill Strategy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Artemisinin-resistant clinical isolates were effectively targeted using the bait-and-kill strategy, which synergizes ALA and DHA. The experiment utilized the clinical artemisinin-resistant parasite F09A44. Results from the Ring Survival Assay (RSA) showed that DHA alone led to approximately 10% parasite survival, while the combination of ALA+DHA reduced parasite survival to around 1% by day 3. Long-term culture confirmed complete parasite elimination, with no parasite reoccurrence observed after 3 weeks of culture.\u003c/p\u003e\n\u003cp\u003eB. Schematic representation of the proposed bait-and-kill strategy for targeting artemisinin-resistant parasites. Uninfected red blood cells (RBCs) do not uptake ALA and thus do not produce Protoporphyrin IX (PPIX), depicted in red. In contrast, infected RBCs uptake ALA, leading to PPIX production. This approach selectively targets malaria-infected red blood cells, as ALA is taken up by these cells and most cannot enter normal RBCs. The metabolism of human RBCs results in the production of porphyrins (PPIX) only in infected cells. Artemisinin synergizes with porphyrin to effectively kill malaria parasites. Bottom Right: In the experiments in panel A, uninfected RBCs are not observed to be affected after 3 days of culture with a high concentration of 1 mM ALA.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-4535885/v1/cb7df2c9ebdba0b7fc2c52ef.png"},{"id":60174630,"identity":"46ea7afc-c376-4cea-a59d-55d64f0652bb","added_by":"auto","created_at":"2024-07-12 15:33:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1533243,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4535885/v1/463e84e1-d3d7-4fa9-97e9-e2a5d2f985b4.pdf"},{"id":60171201,"identity":"499400bd-171a-4954-a186-0be6607aac64","added_by":"auto","created_at":"2024-07-12 15:09:28","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14424,"visible":true,"origin":"","legend":"Supp Table S1","description":"","filename":"SupplementalTableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4535885/v1/0b2327312b923c745ffecf75.xlsx"},{"id":60172605,"identity":"3ad0fe10-eabe-4390-a37c-6b7adc74c3e7","added_by":"auto","created_at":"2024-07-12 15:17:28","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":14962,"visible":true,"origin":"","legend":"Supp Table S2","description":"","filename":"SupplementalTableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4535885/v1/fb76054ee0712b281370e353.xlsx"},{"id":60171205,"identity":"490a3a7a-623c-4f1b-9a0d-4a019e59e841","added_by":"auto","created_at":"2024-07-12 15:09:28","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":60732,"visible":true,"origin":"","legend":"Supp Table S3","description":"","filename":"SupplementalTableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4535885/v1/cecc53aba8612ca3d4d0bb25.xlsx"},{"id":60172604,"identity":"9a77283c-ec64-4fbf-a0e1-095f52d94876","added_by":"auto","created_at":"2024-07-12 15:17:28","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":16929,"visible":true,"origin":"","legend":"Supp Table S4","description":"","filename":"SupplementalTableS4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4535885/v1/70a6f179f1a700d2420d7b87.xlsx"},{"id":60171212,"identity":"d545600f-0f94-4a82-83a0-1caee339ba76","added_by":"auto","created_at":"2024-07-12 15:09:29","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":29432,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalTableS5.docx","url":"https://assets-eu.researchsquare.com/files/rs-4535885/v1/0a996ec7b3aa526ddc241eac.docx"}],"financialInterests":"(Not answered)","formattedTitle":"Targeting Host Metabolic Niche to Kill Malaria Parasites","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHuman red blood cells (RBCs) represent a terminally differentiated cellular entity characterized by the absence of an endomembrane system, nuclei, or mitochondria [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Because of their minimalist structure, RBCs possess unique metabolic properties that hold potential for the development of host-targeted therapies against malaria.\u003c/p\u003e \u003cp\u003eHeme metabolism in RBC plays a critical role in malaria [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], serving as the foundation for artemisinin activation [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and hemozoin formation by the parasite. In human RBCs, the metabolic profile is characterized by a significant reliance on cytoplasmic pathways, particularly glycolysis, due to the absence of metabolic processes associated with cell division and mitochondrial-based energy or metabolite production [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Initially, young RBCs undertake heme biosynthesis through a two-part process involving both mitochondrial and cytoplasmic steps [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], intricately coordinated during erythroid development. However, upon maturation, only the cytoplasmic steps persist [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], presenting metabolic characteristics unique to mature RBCs.\u003c/p\u003e \u003cp\u003eOur recent studies have demonstrated the viability of a bait-and-kill strategy aimed at exploiting the metabolic vulnerabilities of cancer cells by leveraging their 'truncated' heme biosynthesis pathway [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Unlike normal cells, cancer cells possess this aberrant pathway, allowing for the accumulation of heme intermediates without complete conversion into the final product, heme. This strategy capitalizes on the clinical safety profile of ALA [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], a heme precursor, which bypasses the initial mitochondrial step, thereby triggering the accumulation of heme intermediates, specifically porphyrins, within cancer cells. Subsequently, these accumulated porphyrins are targeted for elimination using compounds that induce redox stress [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Importantly, normal human cells, characterized by balanced heme pathways, remain unaffected due to their lack of porphyrin accumulation [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe propose that this strategy holds promise for malaria treatment due to three key factors: Firstly, mature RBCs inherently possess a truncated heme biosynthesis pathway [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Secondly, healthy/uninfected RBCs are impermeable to the heme precursor ALA, while infected RBCs demonstrate increased permeability [\u003cspan additionalcitationids=\"CR15 CR16 CR17\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], resulting in porphyrin accumulation. Thirdly, heme mediated activation of artemisinin has been established as the major mechanism of action for its antimalarial activity [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Additionally, evidence from human ferrochelatase (last step of heme biosynthesis) deficient erythropoietic protoporphyria patients indicates resistance to malarial parasite growth, suggesting that natural human genetic variation in aberrant heme metabolism can confer malaria resistance [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWith the emergence of artemisinin resistance [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], there is an urgent imperative to explore novel and safe antimalarial drugs. In this study, we use the latest quantitative proteomics and erythropoiesis data to elucidate the unique heme pathway of RBCs in their terminally differentiated state. Contrary to previous antimalarial work involving light as PDT therapy [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], or using human proteins targeting circulating heme [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], our approach is entirely based on clinically safe drugs. In agreement with previously published results by Sigala et al. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], we found RBC heme metabolism is intrinsically distinct than other human cells that can be used for host-niche targeting. Importantly, we generate quantitative drug interaction data for the first time, showing the synergy between ALA and artemisinin. This synergy is leveraged to effectively target clinically procured artemisinin-resistant parasites.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eResult 1: RBC possesses a unique truncated heme biosynthesis pathway\u003c/h2\u003e\n\u003cp\u003eHuman RBCs are terminally differentiated cells characterized by distinct proteomic and metabolic profiles [\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e]. This distinctiveness in heme biosynthesis enzymes and the resulting substrate production offers a promising approach for targeting malaria parasites by disrupting the unique host heme metabolic niche (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). Porphyrin production in cancers, a consequence of this unique pathway [\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e], exhibits redox activity [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e] and can be exploited to selectively eliminate pathological cells. Our rationale is that we can employ a similar approach to kill malaria-infected RBCs due to the similarity in the 'truncated' heme biosynthesis pathways present in both cancers and RBCs.\u003c/p\u003e\n\u003cp\u003eFirst, we conducted terminal erythropoiesis analysis, based on previously published human hematopoiesis data [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e] and quantitative proteomic analysis of mature RBCs [\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e], to elucidate the heme metabolic processes in the mature cell stage. Our aim was to better understand the metabolic vulnerability of host cells during \u003cem\u003eP. falciparum\u003c/em\u003e infection. Utilizing human hematopoiesis gene expression data, we examined the expression of genes involved in heme metabolic processes across various erythroid progenitor stages (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB) (Supplemental Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). Our analysis encompassed the different temporal steps of erythropoiesis, including common myeloid progenitor (MYE_0), megakaryocyte/erythroid progenitor (MYE_1 and MYE_2), and erythroid cells (ERY1-4). We investigated various key aspects of heme metabolism in RBCs, including heme biosynthesis pathway genes, heme degradation and binding processes, mitochondrial hemoprotein genes, heme/iron trafficking genes. We also examined erythropoiesis principal regulator genes, such as \u003cem\u003eEIF2K1\u003c/em\u003e (heme regulated global translation initiation kinase) [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e], \u003cem\u003eBACH1\u003c/em\u003e (heme regulated transcription activator) [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e], \u003cem\u003eGATA2\u003c/em\u003e (early erythropoiesis regulator)[\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e] and \u003cem\u003eFOXO1\u003c/em\u003e (late erythropoiesis regulator) [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e]. Consistent with established findings [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e], we observed distinct gene expression patterns in the heme biosynthesis pathway during erythrocyte maturation. We noted a progressive upregulation of the heme biosynthesis pathway, reaching its peak in the later stages of maturation with high levels of expression of all eight steps of genes. Particularly noteworthy were the robust expression levels of key mitochondrial hemoprotein encoding genes, such as \u003cem\u003eUQCRH\u003c/em\u003e and \u003cem\u003eCYC1\u003c/em\u003e, which play crucial roles in powering cellular respiration, during erythroid differentiation. This highlights the significance of mitochondrial metabolism and electron transport chain function in erythroid development and differentiation. Furthermore, our analysis revealed a coordinated upregulation of iron import gene \u003cem\u003eTFRC\u003c/em\u003e, indicative of fueling hemoglobin synthesis, and iron export gene SLC40A1, to maintain heme homeostasis. This was accompanied by a downregulation of heme breakdown pathways, \u003cem\u003eBLVRA\u003c/em\u003e and \u003cem\u003eBLVRB\u003c/em\u003e, indicative of active heme production and its incorporation into cellular components during erythroid differentiation.\u003c/p\u003e\n\u003cp\u003eSubsequent to RBC maturation, the expulsion of DNA regulatory machinery and elimination of subcellular organelles predominates. Examination of recent quantitative RBC proteomic data [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e] delineates the transition from late differentiated erythroid to fully mature RBCs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC) (Supplemental Table S2). Mature RBCs exhibit minimal expression levels of mitochondrial hemoproteins (UQCRH and CYC1) and master regulators of heme metabolism (EIF2K1 and BACH1) during differentiation, indicating a cessation of both nuclear and mitochondrial metabolism and heme-regulated developmental processes in mature proteome. Notably, significant reductions are observed in both the initial (ALAS2) and final steps (CPOX, PPOX, and FECH) of heme biosynthesis occurring in mitochondria, with levels low or undetectable per cell. In contrast, mid-step cytosolic enzymes (ALAD, HMBS, UROS, and UROD ) persist at considerable levels (7000 to 800,000 copies protein per cell) in two independently generated proteome datasets, suggesting that only the mid-steps of heme biosynthesis enzymes constitute part of the canonical proteome within mature RBCs. This indicates an intrinsically truncated heme biosynthesis pathway akin to our findings in cancer cells, predisposing the pathway to the accumulation of intermediates. Additionally, the presence of low levels of iron import proteins (TFRC) and high levels of iron export (SLC40A1) and heme degradation proteins (BLVRA and BLVRB) further supports the absence of heme biosynthesis but the maintenance of heme breakdown during the mature RBC lifespan. Furthermore, RBC hemoproteins responsible for antioxidant responses [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e], such as catalase (CAT), cytochrome b5 reductase A (CYB5A), and cytochrome b5 reductase D (CYRBD1), are prominently expressed in the mature proteome, indicating their crucial role in detoxifying radical species and maintaining RBC integrity and function against the onslaught of redox stress during RBC circulation.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003eResult 2: iRBC in dihydroartemisinin (DHA) resistance stage are remodeled for host cell permeability change\u003c/h2\u003e\n\u003cp\u003eThe emergence of artemisinin resistance in malaria parasites has raised significant concerns in recent years. Notably, key genes associated with artemisinin resistance, such as \u003cem\u003eKelch 13\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e], ubiquitin hydrolase (\u003cem\u003eUBP1\u003c/em\u003e) [\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e], AP2 adaptor complex \u0026micro;-subunit (\u003cem\u003eAP2-MU\u003c/em\u003e) [\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e], Kelch13 interaction candidate (\u003cem\u003eKIC5\u003c/em\u003e) [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e], and Kelch13 interaction candidate (\u003cem\u003eKIC7\u003c/em\u003e) [\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e], exhibit enrichment of expression in the ring stage of infection [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e], suggesting their potential involvement in resistance mechanisms during the early stages of parasite colonization. To combat drug resistance effectively, targeting infected host cells during this critical stage becomes imperative. We hypothesize that infected RBCs provide an optimal environment for inducing porphyrin accumulation due to their increased permeability, the precursor of heme intermediates, facilitated by parasite-induced changes in host cell permeability [\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eWe investigated the gene expression and proteomic evidence of \u003cem\u003eP. falciparum\u003c/em\u003e parasites inducing changes in host cell permeability during the erythrocytic stage, with a specific focus on alterations in infected RBCs (iRBCs) associated with DHA resistance (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA) (Supplemental Table S3). Initially, we obtained a predicted set of 386 parasite-exported proteins from PlasmoDB v68, which revealed a distinct gene expression pattern of protein export during blood stage growth. Notably, we observed a peak in gene expression occurring at the ring and early trophozoite stages in the blood stage expression dataset [\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e]. Intriguingly, this timeframe coincided with the expression of genes associated with artemisinin resistance. This synchrony suggests a potential opportunity to leverage enhanced host permeability as a strategy for targeting drug resistance.\u003c/p\u003e\n\u003cp\u003eTo evaluate the essentiality of the export process, we examined \u003cem\u003eP. falciparum\u003c/em\u003e exported protein-encoding genes in the context of saturation mutagenesis through transposon tagging (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). The mutagenesis index, reflecting the essentiality of \u003cem\u003ein vitro\u003c/em\u003e blood stage parasite survival, indicated that lower values correspond to greater essentiality [\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e]. Our analysis unveiled a bimodal forward genetic gene essentiality pattern, with approximately 60 exported protein genes identified as essential for blood stage parasite survival. This suggests their critical role in host remodeling and export mechanisms. The identification of these essential genes further supports the rationale for targeting this process as a potential antimalarial strategy.\u003c/p\u003e\n\u003cp\u003eMoreover, we investigated parasite-encoded genes implicated in modulating host RBC permeability, such as \u003cem\u003eCLAG3.1\u003c/em\u003e[\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e], \u003cem\u003eCuTP\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e], \u003cem\u003eRhopH2\u003c/em\u003e[\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e], and \u003cem\u003eRhopH3\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e], alongside \u003cem\u003eCTR1\u003c/em\u003e[\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e] and \u003cem\u003eHlyIII\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e], to understand their expression patterns and genetic essentiality. Our analysis revealed that during the ring stage of infection, four of these genes were upregulated, as evidenced by the high protein expression levels of \u003cem\u003eCLAG3.1, CuTP, RhopH2\u003c/em\u003e, and \u003cem\u003eRhopH3\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC). Additionally, a subset of these genes, including \u003cem\u003eRhopH2, RhopH3, CTR1\u003c/em\u003e, and \u003cem\u003eHlyIII\u003c/em\u003e, demonstrated forward genetic growth essentiality, indicating the critical role of host cell remodeling for parasite survival within RBCs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD). Taken together, these findings highlight the potential to leverage host permeability changes induced by infection as a strategy for targeting the host heme metabolic niche.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResult 3: Normal RBCs do not accumulate porphyrin, while cancerous blood lineage cells show accumulation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur recent study targeting the truncated heme biosynthesis pathway in cancer cells has prompted us to investigate a similar pathway in normal human RBCs. Our anti-cancer strategy relies on the premise that normal cells do not accumulate porphyrins, even under ALA induction, providing a therapeutic window for selective targeting of cancer cells. Similarly, to target malaria-infected cells, we conducted further experimentation to demonstrate that normal human RBCs cannot be induced to accumulate porphyrins due to the lack of permeability changes induced by parasites, preventing ALA entry and subsequent PPIX accumulation.\u003c/p\u003e\n\u003cp\u003eFirst, we investigated the accumulation of PPIX in blood cancer cell lines, expanding upon our previous findings that leukemic cell lines exhibit an aberrant heme biosynthesis pathway, resulting in the buildup of intermediates. To validate this observation, we analyzed \u003cem\u003ein vitro\u003c/em\u003e CRISPR KO essentiality data on leukemic cell lines, leveraging the Cancer Dependency Map (DepMap) dataset (23Q4). This dataset offers data of genetic dependence inferred from cell survival upon specific gene knockout, allowing us to assess the essentiality of heme biosynthesis pathway genes in blood cancer cells. Our analysis unveiled an aberrant heme biosynthesis pathway in blood cancer cell lines, characterized by varying essentiality among different pathway genes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). Mid-step heme biosynthesis genes, particularly \u003cem\u003eUROD\u003c/em\u003e, emerged as crucial for blood cancer cell survival, indicating their significance in sustaining the dysregulated heme biosynthesis process. In contrast, the initial steps or the last step of the pathway displayed low essentiality, suggesting an enzymatic imbalance that leads to inefficiency in heme production and the accumulation of intermediates, namely porphyrins. These analysis confirm the dysregulation of heme biosynthesis in blood cancer cells, highlighting potential vulnerabilities that parallel those observed in malaria-infected cells, providing rationales for targeted therapeutic interventions.\u003c/p\u003e\n\u003cp\u003eNext, we conducted ALA induction experiments on both normal human PBMCs and a selection of four blood cancer cell lines to further elucidate heme biosynthesis dynamics (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). Normal PBMCs exhibited no accumulation of PPIX, irrespective of ALA presence, indicating stringent heme biosynthesis control and the absence of intermediate buildup. In contrast, blood cancer cell lines robustly accumulated porphyrins upon ALA addition, indicating the aberrant accumulation of heme intermediates in these cells. Flow cytometry data shows a substantial amount PPIX accumulation in \u0026gt;\u0026thinsp;99% the blood cancer K562 cells following ALA addition (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC). These findings show the distinction between normal human RBCs, which do not accumulate PPIX, and blood cancer cells, which possess a truncated pathway leading to PPIX accumulation. This evidence guides our next investigation into the analogous heme biosynthesis scenario of malaria-infected RBCs for host niche targeting.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003eResult 4: iRBCs Specifically Accumulate Porphyrin\u003c/h2\u003e\n\u003cp\u003eTo demonstrate the accumulation of Protoporphyrin IX (PPIX) within infected red blood cells (iRBCs) during malaria infection, we used two strains of malaria parasites to infect human RBCs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). We specifically tracked the parasites in the trophozoite stage, characterized by increased parasite biomass and clearer marker visualization. Two strains of the malaria parasite, P. falciparum 3D7 and F09A44, were tested for PPIX accumulation. PPIX fluorescence was detected using excitation at 405 nm and emission in the red channel at 633 nm. Additionally, SYBR Green dye, with excitation and emission at 498/522 nm, labeled nucleic acids of the parasites. Normal RBCs (\u0026gt;\u0026thinsp;99.9%) did not exhibit PPIX accumulation regardless of ALA presence. For 3D7-infected RBCs with lower parasitemia, PPIX accumulation was absent without ALA but increased upon ALA addition (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD \u0026amp; E). Similarly, F09A44-infected RBCs with higher parasitemia showed no PPIX accumulation without ALA, but levels rose after ALA addition (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD \u0026amp; E). Importantly, the majority of RBCs, both in the total normal population and uninfected subpopulations, did not accumulate significant amounts of PPIX. Unlike all nucleated healthy human cell types we tested [\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e], a small increase in some uninfected RBCs co-cultured with infected RBCs was observed, possibly due to parasite-released micro-vesicles [\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e]. However, no hemolysis was observed in uninfected cells, consistent with decades of ALA human clinical safe use without reported hematological effects. Taken together, these findings suggest that active malaria infection facilitates ALA entry and subsequent heme precursor conversion, bypassing the initial mitochondrial step and leading to porphyrin accumulation in infected RBCs.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003ch2\u003eResult 5: DHA and ALA Synergize in Killing HC-04 Cancer Cells\u003c/h2\u003e\n\u003cp\u003eTo explore the potential synergy between DHA and ALA in targeting porphyrin accumulation, we conducted a series of experiments using cell viability assays. As RBCs are terminally differentiated and unsuitable for quantitative drug synergy assays, we utilized the HC-04 liver cancer cell line [\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e]. This cell line can serve as a host cell for \u003cem\u003ein vitro\u003c/em\u003e malaria liver stage development experiments and exhibits onco-transformation properties.\u003c/p\u003e\n\u003cp\u003eFirst, employing CRISPR KO in vitro gene essentiality analysis in a set of liver cancer cell lines, we assessed heme biosynthesis pathway gene essentiality (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). Our results indicate that liver cancer cell lines exhibit aberrant heme biosynthesis patterns, with only the mid-steps of the pathway (UROS and UROD) being essential for \u003cem\u003ein vitro\u003c/em\u003e cell survival. This suggests that while liver cancer cells do not require endogenous heme synthesis, they are capable of producing intermediate porphyrins due to the essentiality of mid-steps, a scenario similar to cancer blood cell lines and RBCs.\u003c/p\u003e\n\u003cp\u003eNext, we investigated the accumulation of Protoporphyrin IX (PPIX) upon ALA incubation in normal primary human hepatocytes and three liver cancer cell lines, including HC-04 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). Consistent with our recent findings [\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e], normal primary human hepatocytes did not exhibit PPIX accumulation upon ALA treatment, indicating tightly controlled heme biosynthesis without intermediate accumulation to support liver function. In contrast, all three liver cancer cell lines, including HC-04, showed high levels of PPIX accumulation after 72 hours of ALA treatment, suggesting incomplete heme biosynthesis and the accumulation of intermediates in liver cancer cells.\u003c/p\u003e\n\u003cp\u003eFurther, we conduct experiments to quantify the cytotoxic effects of ALA and DHA, both individually and in combination, across a broad range of drug concentrations (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC). We assessed a wide spectrum of drug concentrations, ranging from nanomolar (nM) to 133 micromolar (\u0026micro;M) concentrations of DHA and from nanomolar (nM) to millimolar (mM) concentrations of ALA. This drug-drug interaction study is performed in experimental triplicates (Supplemental Table S4). Our findings revealed that while treatment with either ALA or DHA alone demonstrated minimal toxicity, their combined administration exhibited a robust synergy in killing cancer cells. Remarkably, this synergistic effect was evident even at low concentrations of DHA in the nanomolar (nM) range. This observation suggests that ALA sensitizes DHA, likely through the accumulation of PPIX, thereby enhancing its efficacy in targeting and eliminating cancer cells. Notably, nanomolar concentrations of DHA proved sufficient for inducing cell death following sensitization by ALA, indicating the potential of this strategy to overcome malaria artemisinin resistance.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n\u003ch2\u003eResult 6: DHA and ALA Kill DHA-Resistant Parasites\u003c/h2\u003e\n\u003cp\u003eBased on our investigation into RBC heme metabolism and DHA sensitization, we propose a Bait-and-Kill Strategy to combat artemisinin-resistant malaria, utilizing ALA as the \u0026ldquo;bait\u0026rdquo; and artemisinin as the \u0026lsquo;kill\u0026rsquo; agent. Given the global impact of malaria, the success of this strategy hinges on the safety profile of ALA (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Notably, ALA has been employed as an FDA-approved cancer imaging agent since 2007, with extensive use in Europe preceding its adoption in the United States. Over 58,000 individuals worldwide have used ALA with no discernible adverse effects. ALA is routinely utilized in mouse models for both imaging and treatment, with a good safety profile[\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e]. Studies in Japan have examined its long-term use for up to three months [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e] and large dose administration of up to 2250 mg/day without reported toxicity [\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e]. Presently, ALA is marketed as a nutritional supplement in Japan (SB Pharma). Human pharmacodynamic studies [\u003cspan class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e] have directly monitored ALA metabolism, revealing no toxic effects. PPIX, metabolized from exogenous ALA, can persist in human sera for several hours before complete elimination, typically occurring within 35\u0026ndash;48 hours. Detailed information regarding ALA's drug use, safety profile, and human pharmacodynamics studies is provided in Supplementary Table S5. Collectively, ALA's widespread use across diverse populations suggests its safety for use as a public health intervention against infectious diseases.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003e\u003cstrong\u003eSummary of ALA dosage for safety and efficacy in human clinical studies.\u003c/strong\u003e (An extensive set of results are provided in Supplemental Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e)\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eExperimental system\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eALA dosage\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eDisease model\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eALA effect\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eReferences\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMouse model\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e40\u0026ndash;600 mg/kg\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eImaging,\u003c/p\u003e\n\u003cp\u003eCancer treatment,\u003c/p\u003e\n\u003cp\u003eMalaria treatment\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNo toxicity.\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUnpublished data\u003c/p\u003e\n\u003cp\u003ePredina et al [\u003cspan class=\"CitationRef\"\u003e78\u003c/span\u003e]\u003c/p\u003e\n\u003cp\u003eStowers et al [\u003cspan class=\"CitationRef\"\u003e79\u003c/span\u003e]\u003c/p\u003e\n\u003cp\u003eSuzuki et al [\u003cspan class=\"CitationRef\"\u003e80\u003c/span\u003e]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHuman\u003c/p\u003e\n\u003cp\u003eImaging\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(FDA approved)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e20 mg/kg\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBrain cancer surgery imaging\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNo toxicity.\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFDA Gleolan report NDA 208630\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHuman\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHigh dose\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eup to 2250 mg/day for 3\u0026ndash;7 days\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCOVID treatment in Japan\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNo toxicity.\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eKaketani and Nakajima [\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHuman\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLong term\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e200 mg/day for 3 months\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eType 2 diabetes treatment\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNo toxicity.\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAl-Saber et al [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHuman\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePharmacodynamics\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e40\u0026ndash;60 mg/kg\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eALA pharmacodynamics study\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNo toxicity.\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eWebber et al [\u003cspan class=\"CitationRef\"\u003e57\u003c/span\u003e]\u003c/p\u003e\n\u003cp\u003eRick et al [\u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eAbbreviations:\u003c/strong\u003e ALA, 5-aminolevulinate; DHA, dihydroartemisinin; PPIX, protoporphyrin IX\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003eTo evaluate the Bait-and-Kill Strategy against artemisinin-resistant clinical isolates, we utilized the clinical isolate F09A44 (a Southeast Asian clinical isolate) carrying the K13 (C469Y) mutation associated with drug resistance. Characterized as artemisinin-resistant, the Ring Survival Assay (RSA) revealed that 700 nM DHA alone resulted in approximately 10% parasite survival (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA). Subsequently, we applied a combination drug therapy comprising ALA and DHA. Initially, 1 mM ALA was administered to facilitate PPIX accumulation, followed by the addition of 700 nM DHA for parasite eradication. ALA was replenished at only 6 hours (wash out time of DHA), or two days or all three days until 72 hours, the readout time for RSA. Our results demonstrated that the combination of ALA and DHA when ALA was replenished everyday resulted in complete parasite elimination by day 3, with no parasite recurrence observed even after 3 weeks of culture. The experiment was conducted in biological replicates (n\u0026thinsp;=\u0026thinsp;3), providing evidence that this strategy effectively eradicates resistant parasites without recrudescence from parasite dormancy.\u003c/p\u003e\n\u003cp\u003eIn summary, our proposed Bait-and-Kill Strategy targets artemisinin-resistant parasites (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB). We show that uninfected red blood cells (RBCs) do not uptake ALA and therefore do not produce PPIX, remaining unharmed by the drug combination. In contrast, infected RBCs uptake ALA, leading to PPIX production, sensitizing DHA to kill the parasite. This approach selectively targets malaria-infected red blood cells, as ALA is exclusively taken up by these cells and cannot enter normal RBCs.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eAmidst the persistent threat of malaria and the rise of artemisinin resistance, urgent and innovative solutions are imperative. Drawing from our recent discoveries concerning cancer heme metabolic vulnerability, we have introduced a novel therapeutic strategy termed the \"bait-and-kill\" approach. This strategy targets analogous metabolic susceptibilities found in both malaria-infected red blood cells (iRBCs) and cancer cells. By exploiting the truncated heme biosynthesis pathway in mature RBCs and the heightened permeability of infected cells to the heme precursor ALA, we have effectively demonstrated the potent synergy between ALA and artemisinin in eradicating artemisinin-resistant Southeast Asian clinical isolates. Significantly, our strategy provides a targeted intervention that selectively eliminates infected RBCs while preserving uninfected cells, thereby minimizing collateral damage. These findings offer promise for the development of large-scale public health interventions leveraging clinically safe drugs.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn our study, we show the importance of considering human host metabolism as a pivotal battleground in the fight against malaria infection, especially given the emerging resistance to artemisinin [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], the primary treatment for malaria. Malaria parasites, as obligate intracellular organisms, heavily rely on reshaping host cells to ensure their survival. Notably, the occurrence of metabolic polymorphisms in malaria-endemic regions, such as G6PD deficiency [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e], highlights how natural selection has historically provided solutions to combat malaria infection through adjustments in host metabolism [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRBCs are functionally robust cells, constituting the most abundant yet metabolically minimalist cell type in the human body [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Upon release from bone marrow development, RBCs lack nuclei and organelles for most cellular repair processes, yet circulate for approximately three months [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], equipped with a robust antioxidant system, including high levels of hemoproteins like catalase and cytochrome b5 reductase [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In the context of malaria infections, natural selection has favored mechanisms that increase redox stress to kill parasites while preserving normal RBC function, as evidenced by the worldwide high prevalence of hematological diseases like G6PD deficiency and sickle cell anemia [\u003cspan additionalcitationids=\"CR62\" citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Our aim is to exploit this redox stress using bait-and-kill approaches. Our unique approach selectively enhances host redox stress in infected cells. To further elucidate the mechanisms, we are investigating a range of human malaria-related hematological diseases to understand the role of inherent host metabolic polymorphisms in determining infection outcomes.\u003c/p\u003e \u003cp\u003eHere, we leverage the distinctive metabolic characteristics of human mature red blood cells (RBCs), which possess a truncated heme biosynthesis pathway. Drawing inspiration from recent discoveries targeting similar pathways in cancer cells [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], we harness the resulting accumulation of redox-active heme intermediates [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e] to effectively induce cell death. Moreover, there is potential for further exploration into strategies that exploit host redox stress during infection, offering novel ways in treating other types of intracellular pathogens. The precise mode of action underlying the synergy between ALA and DHA in parasite killing remains to be elucidated in future research.\u003c/p\u003e \u003cp\u003eArtemisinin's mechanism of action relies on its activation by heme present within infected cells, triggering redox damage and ultimately killing the parasite [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Recent advances have demonstrated that porphyrins, similar to heme, can activate artemisinin [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e](unpublished). By inducing infected cells to accumulate porphyrins, we sensitize artemisinin, providing a novel approach to combat artemisinin-resistant parasites. Furthermore, this combinatorial approach could potentially extend to testing artemisinin in combination therapy with other frontline antimalarials [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], thereby revitalizing existing drugs that have encountered resistance.\u003c/p\u003e \u003cp\u003eThe quantification of synergy conducted in HC-04 cells, a liver cancer cell line utilized as a host for liver stage malaria assays [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], demonstrates the potential of targeting aberrant heme biosynthesis in malaria-infected liver cells. Given that malaria can infect diverse host cells, including liver [\u003cspan additionalcitationids=\"CR67\" citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e] and bone marrow cells [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e], which undergo parasite-induced remodeling, the bait-and-kill strategy holds promise for application across various stages of malaria infection. Evidence indicates that the heme biosynthesis pathway is dysregulated in infected liver and marrow niches, presenting opportunities to develop interventions that target multiple life stages of malaria beyond red blood cell infection. Further investigation could explore the efficacy of the bait-and-kill strategy other phases of malaria infection, thereby taking initial steps for therapeutic interventions against malaria by targeting different host niches.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of Cancer Gene Essentiality in Heme Biosynthesis Using CRISPR KO data\u003c/h2\u003e \u003cp\u003eThe assessment of cancer gene essentiality via CRISPR/Cas9 was conducted by leveraging data available on the DepMap Portal, using established methodologies [\u003cspan additionalcitationids=\"CR72\" citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e], similar to methods we previously used [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Whole-genome CRISPR/Cas9 datasets were utilized to identify notable reductions in the growth of mutant cells following targeted gene knockouts in pooled experiments. Gene essentiality was inferred based on the dependency of a particular gene, determined through CRISPR/Cas9 gRNA-mediated gene knockout. Essential scores were employed to evaluate cell growth fitness, with lower scores indicating a more significant impact on cell viability upon gene loss. Specifically, scores of 0, \u0026lt; 0, and \u0026gt;\u0026thinsp;0 represented no change in fitness, loss of fitness, and gain in fitness (suggesting potential growth advantage for the cell line) under the assay conditions. Identification of commonly essential genes was based on their significance for the fitness of most cell lines across various cancer types [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of human mature red blood cell (RBC) heme biosynthesis and malaria parasite host remodeling data\u003c/b\u003e \u003c/p\u003e \u003cp\u003eHemopoiesis gene expression data were sourced from previously published datasets [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], focusing on various stages of erythropoiesis, such as common myeloid progenitor (MYE_0), megakaryocyte/erythroid progenitor (MYE_1 and MYE_2), and erythroid cells (ERY1-4). Quantitative proteomic data of human mature red blood cells (RBCs) were obtained from comprehensive datasets [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] comprising a total of 18,581 proteins present in RBCs. Within this dataset, approximately 1200 proteins constitute the canonical RBC proteome, including mid-steps of heme biosynthesis enzymes ALAD, HMBS, UROS and UROD. The full sets of RBC proteome exhibit a broad range of protein abundances, from high to low, with the lowest abundance represented by a single peptide, for example, the mitochondrial hemoprotein COX10. Proteins considered abundantly present were those ranking above the 0.75 percentile in peptide counts within RBC proteomes. Differential expression levels between early and late stages were assessed using the Wilcoxon test, with P values adjusted using the Benjamini-Hochberg method for multiple hypothesis testing.\u003c/p\u003e \u003cp\u003eFor malaria host modeling protein analysis, the set of host-targeted proteins were retrieved from PlasmoDB v68 [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e] by combining searches for the 'PEXEL' motif and 'Host-Targeted' motif in the reference P. falciparum 3D7 genome [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Gene essentiality data, represented by the Mutagenesis Index score, were obtained based on saturation transposon mutagenesis phenotypic data of parasite in vitro survival in blood stages.\u003c/p\u003e \u003cp\u003eHost permeability proteins were identified based on the Malaria Parasite Metabolic Pathway (MPMP) v2023 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://mpmp.huji.ac.il\u003c/span\u003e\u003cspan address=\"http://mpmp.huji.ac.il\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), specifically focusing on the permeability of the membrane of infected RBCs. Malaria gene expression data were retrieved from transcriptomes of seven sexual and asexual life stages [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], including two gametocyte stages (II and V), ookinete, and four time points of erythrocytic stages (ring, early trophozoite, late trophozoite, and schizont). Bimodal distribution estimation was performed using histograms displaying two prominent peaks, and Kernel Density Estimation (KDE) was employed to estimate the probability density function of the data, providing a smoothed estimate of the underlying distribution and identifying multiple modes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePrimary Liver Cell and Hepatoma Cell Line Preparation\u003c/h2\u003e \u003cp\u003eIn vitro culture of primary human hepatocytes began by sterilizing 384-well plates (Greiner, Cat No. 781091) in a class II biosafety cabinet and placing them in a secondary container to prevent evaporation. The wells were coated with 40 \u0026micro;L of 15 \u0026micro;g \u0026micro;L-1 rat tail collagen I (Corning, Cat No. 354236) in sterile filtered 0.02 M acetic acid (Thermo Fisher Scientific) and kept at 37\u0026deg;C overnight. Before seeding, the wells were washed thrice with sterile phosphate-buffered saline (PBS) and filled with 20 \u0026micro;L of in vitro GRO\u0026reg; CP plate medium (BioIVT, Cat No. Z99029), supplemented with 1x Pen-Strep-Neo solution (100x, Fisher, Cat No. 15640-055) and 20 \u0026micro;M gentamicin (1000x, Fisher, Cat No. 15-710-072). Cryopreserved primary human hepatocytes (BioIVT, Cat No. M00995-P) were thawed by immersion in a 37\u0026deg;C water bath for 2 minutes, sterilized with 70% ethanol in a sterile field, and added directly to 4 mL plate medium. Live and dead cells were quantified using trypan blue exclusion on a Neubauer improved hemocytometer. The hepatocyte density was adjusted to 1 \u0026times; 103 live cells \u0026micro;L-1, and 18 \u0026micro;L of the cell suspension was added to each well. Medium exchange with the GRO\u0026reg; CP plating medium occurred thrice weekly.\u003c/p\u003e \u003cp\u003eThree hepatoma cell lines, namely HC-04, HepG2, and SNU449, were used as liver cancer cell lines for comparative analysis. These cryopreserved cell lines were thawed, suspended in a hepatocyte culture medium previously prepared, and transferred to T75 flasks coated with collagen (Corning, Cat No. 354236) at a density of 5 \u0026micro;g/cm\u0026sup2;. The culture medium consisted of a 1:1 (v/v) mixture of F12 base medium (Invitrogen, Cat No. 11765-054) and MEM base medium (Invitrogen, Cat No. A10490-01), supplemented with 10% FBS (Hyclone, Cat No. SH30070), 1.0 M HEPES (Invitrogen, Cat No. 15630-080), and 200 mM glutamine (Invitrogen, Cat No. 25030-081). The cells were cultured until reaching 70% confluence, with the medium being changed every other day. Once the desired confluence was attained, the cells were trypsinized using TrypLE\u0026trade; Express Enzyme (1X) (Gibco, Cat No. 12605028), washed with hepatocyte culture medium, and then seeded at a density of 6000 cells/well in 384-well plates (Greiner, Cat No. 781091), with each well receiving 20 \u0026micro;L of the aforementioned medium. Cells were then incubated either in the absence or presence of 1.0 mM ALA at 37\u0026deg;C for 4 hours under very low light conditions. During the last 45 minutes of incubation, a staining solution diluted in phenol-free, serum-free RPMI (Gibco, Cat No. 11835055), containing Hoechst 33342 (Life Technologies, Cat No. H3570) at a final concentration of 10 \u0026micro;M, was added to the cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCellular PPIX Quantification\u003c/h2\u003e \u003cp\u003eQuantification of intracellular protoporphyrin IX (PPIX) accumulation was performed using fluorescence-activated cell sorting (FACS), following established protocols [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. Cells were cultured in medium as described in the Cell Culture section, supplemented with 1.0 mM 5-aminolevulinic acid (ALA), and maintained at 37\u0026deg;C for 4 hours under low light conditions. After incubation, cells underwent triple washing with Dulbecco's phosphate-buffered saline (DPBS, 1X, Ca2+- and Mg2+-free; Corning, Cat No. 21-031-CV) and were resuspended in 250 \u0026micro;L of 1x DPBS. Subsequently, cells were washed once with serum-free medium (Gibco, Cat No. 11835055) and seeded in 6-well plates with the designated medium (as described in the Cell Culture section), with or without the addition of ALA, followed by incubation at 37\u0026deg;C. Intracellular PPIX concentration was evaluated 18 hours later using FACS. FACS analyses were conducted using a BD LSR II Analyzer (Becton, Dickinson, and Company) equipped with FACSDiva Version 6.1.3 software. To minimize background red fluorescence, the 633 nm-red laser was deactivated during PPIX emission data collection. PPIX emission within the 619 nm and 641 nm range (630/22BP filter) was measured following excitation with the 405 nm laser. Forward-scatter (FSC) versus side-scatter (SSC) dot plots were utilized to gate the entire cell population while excluding cell debris. A minimum of 10,000 cells within the gated region were then represented in dot plots of SSC vs. PPIX fluorescence, with the gate defined using cells lacking perturbation as negative controls.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMalaria Parasite Culture\u003c/h2\u003e \u003cp\u003eFor the cultivation of asexual blood-stage parasites, O\u0026thinsp;+\u0026thinsp;human red blood cells (RBCs) were utilized and maintained in a controlled environment at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e humidity. The parasite culture medium consisted of RPMI 1640 supplemented with 25 mM NaHCO\u003csub\u003e3\u003c/sub\u003e, 11 mM glucose, 25 mM HEPES (pH 7.4), 0.367 mM hypoxanthine, and 5 \u0026micro;g/liter gentamicin. Additionally, 0.5% AlbuMAX II lipid-rich bovine serum albumin from Thermo Fisher Scientific, MA, was added to enhance the lipid content. To synchronize the parasites at the ring stage, a 5% D-sorbitol treatment method was employed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMalaria Ring-Stage Survival Assays\u003c/h2\u003e \u003cp\u003eThe Ring-Stage Survival Assay (RSA) was conducted following established protocols. Initially, schizonts were purified from tightly synchronized cultures using a 75% Percoll gradient (Sigma-Aldrich). After purification, the schizonts were washed once in RPMI 1640 incomplete medium and allowed to rupture, invading fresh red blood cells (RBCs) for a duration of 3 hours. Following invasion, the cultures underwent another synchronization process using sorbitol to select for early rings and eliminate any remaining schizonts. For the RSA\u003csub\u003e0 \u0026ndash;3\u003c/sub\u003e h assay, ring-stage parasites (0 to 3 hours post-invasion) at a parasitemia of 1% and hematocrit of 1% were exposed to 700 nM Dihydroartemisinin (DHA) for a period of 6 hours, followed by a single wash.\u003c/p\u003e \u003cp\u003eSubsequently, the cultures were allowed to incubate for 66 hours, after which approximately 10,000 RBCs were blindly counted on thin blood smears to determine the number of viable parasites. For ALA combination assays, 1 mM ALA was added along with 700 nM DHA to the 0\u0026ndash;3 hours rings and aliquoted in four distinct wells. 1mM ALA was either replenished after DHA washout on day 1 or day 1 \u0026amp; 2 or all 3 days until readout.\u003c/p\u003e \u003cp\u003eSmears were prepared at 72 hours, and further parasite growth was monitored through Geimsa stained smears for over 3 weeks. For all assays, parallel dimethyl sulfoxide (DMSO)-treated controls (0.1% concentration) were included, and survival rates were expressed as ratios of viable parasites in DHA-exposed or DHA\u0026thinsp;+\u0026thinsp;ALA exposed Vs DMSO-exposed samples. All assays were performed in 3 biological replicates.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplemental files containing the datasets necessary to interpret, replicate, and build upon the methods or findings are available. The raw data of plate readouts from the drug synergy studies, conducted in experimental triplicates, are provided in the supplemental materials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRJ contributed to project design, data analysis, writing, and fund acquisition and management. FS and SA conducted the experiments, performed analysis, and contributed to the writing. LC contributed to the discussion and project management.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe gratefully acknowledge the College of Public Health, University of South Florida, for providing research funding for this project. Additionally, RJ received funding from Women\u0026rsquo;s Philanthropy Leadership (WLP) FY23-25 and FLDOH 9BC14 to support research related to the manuscript.\u003cstrong\u003e\u003cbr /\u003e\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBryk AH, Wiśniewski JR: Quantitative Analysis of Human Red Blood Cell Proteome. 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Antimicrob Agents Chemother 2015, 59(11):6960\u0026ndash;6967.\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":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-antimicrobials-and-resistance","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjamar","sideBox":"Learn more about [npj Antimicrobials and Resistance](http://www.nature.com/npjamar/)","snPcode":"44259","submissionUrl":"https://submission.springernature.com/new-submission/44259/3","title":"npj Antimicrobials and Resistance","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4535885/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4535885/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMalaria remains a global health challenge, exacerbated by artemisinin resistance. Inspired by our recent study targeting aberrant cancer heme metabolism, we propose a novel \"bait-and-kill\" strategy, focusing on the unique metabolic vulnerability of infected Red Blood Cells (iRBCs) to destroy host niche. We exploit three key factors: 1) mature RBCs inherently possess a truncated heme biosynthesis pathway capable of accumulating heme intermediates, \u003cem\u003ei.e\u003c/em\u003e., porphyrins, 2) Uninfected RBCs exhibit impermeability to the heme precursor ALA (Aminolaevulinic acid), while infected RBCs demonstrate increased permeability, and 3) heme/porphyrin mediated activation of artemisinin has been established as the primary mechanism of action for their antimalarial activity. Utilizing the heightened membrane permeability of iRBCs, we employ the heme precursor ALA as \u0026ldquo;bait\u0026rdquo;, inducing heme intermediates accumulation. This synergizes with artemisinin, acting as the 'kill' agent, to effectively eradicate parasites. Uninfected RBCs do not uptake ALA, avoiding collateral damage. We present experimental characterization of drug-drug synergy in a malaria liver stage host cell line and successful elimination of artemisinin-resistant parasites during the blood stage, particularly parasites from the Great Mekong sub-region, a hotspot for antimalarial drug resistance. Leveraging safe drugs like ALA and artemisinin, tested globally, this synergistic strategy holds promise for large-scale deployment in malaria control.\u003c/p\u003e","manuscriptTitle":"Targeting Host Metabolic Niche to Kill Malaria Parasites","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-12 15:09:23","doi":"10.21203/rs.3.rs-4535885/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"withdrawn","date":"2025-03-23T08:23:49+00:00","index":"","fulltext":""},{"type":"decision","content":"revise","date":"2024-07-30T05:21:13+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-07-26T21:31:40+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-07-09T06:31:12+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-07-07T22:11:08+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-06-22T12:34:12+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-06-22T09:41:25+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-06-19T10:44:12+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-13T15:49:02+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Antimicrobials and Resistance","date":"2024-06-10T16:28:45+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2024-06-07T03:19:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-05T18:50:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-antimicrobials-and-resistance","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjamar","sideBox":"Learn more about [npj Antimicrobials and Resistance](http://www.nature.com/npjamar/)","snPcode":"44259","submissionUrl":"https://submission.springernature.com/new-submission/44259/3","title":"npj Antimicrobials and Resistance","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c517d9ec-6d32-4704-99a2-4ac3e6fae8a0","owner":[],"postedDate":"July 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":33453041,"name":"Health sciences/Diseases/Infectious diseases/Malaria"},{"id":33453042,"name":"Biological sciences/Drug discovery"}],"tags":[],"updatedAt":"2024-07-30T05:25:19+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-12 15:09:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4535885","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4535885","identity":"rs-4535885","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}