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. 1A). 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. 1B) (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. 1C) (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. 2A) (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. 2B). 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. 2C). 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. 2D). 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. 3A). 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. 3B). 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. 3C). 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. 4D & E). Similarly, F09A44-infected RBCs with higher parasitemia showed no PPIX accumulation without ALA, but levels rose after ALA addition (Fig. 4D & 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. 5A). 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. 5B). 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. 5C). 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. 6A). 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. 6B). 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.