Genome-Scale Identification of Essential Metabolic Processes for Targeting the Plasmodium Liver Stage.

Plasmodium gene functions in mosquito and liver stages remain poorly characterized due to limitations in the throughput of phenotyping at these stages. To fill this gap, we followed more than 1,300 barcoded P. berghei mutants through the life cycle. We discover 461 genes required for efficient parasite transmission to mosquitoes through the liver stage and back into the bloodstream of mice. We analyze the screen in the context of genomic, transcriptomic, and metabolomic data by building a thermodynamic model of P. berghei liver-stage metabolism, which shows a major reprogramming of parasite metabolism to achieve rapid growth in the liver. We identify seven metabolic subsystems that become essential at the liver stages compared with asexual blood stages: type II fatty acid synthesis and elongation (FAE), tricarboxylic acid, amino sugar, heme, lipoate, and shikimate metabolism. Selected predictions from the model are individually validated in single mutants to provide future targets for drug development.

Interplay between liver and blood stages of Plasmodium infection dictates malaria severity via γδ T cells and IL-17-promoted stress erythropoiesis.

Plasmodium replicates within the liver prior to reaching the bloodstream and infecting red blood cells. Because clinical manifestations of malaria only arise during the blood stage of infection, a perception exists that liver infection does not impact disease pathology. By developing a murine model where the liver and blood stages of infection are uncoupled, we showed that the integration of signals from both stages dictated mortality outcomes. This dichotomy relied on liver stage-dependent activation of Vγ4+ γδ T cells. Subsequent blood stage parasite loads dictated their cytokine profiles, where low parasite loads preferentially expanded IL-17-producing γδ T cells. IL-17 drove extra-medullary erythropoiesis and concomitant reticulocytosis, which protected mice from lethal experimental cerebral malaria (ECM). Adoptive transfer of erythroid precursors could rescue mice from ECM. Modeling of γδ T cell dynamics suggests that this protective mechanism may be key for the establishment of naturally acquired malaria immunity among frequently exposed individuals.

Malaria: Biology and Disease.

Malaria has been a major global health problem of humans through history and is a leading cause of death and disease across many tropical and subtropical countries. Over the last fifteen years renewed efforts at control have reduced the prevalence of malaria by over half, raising the prospect that elimination and perhaps eradication may be a long-term possibility. Achievement of this goal requires the development of new tools including novel antimalarial drugs and more efficacious vaccines as well as an increased understanding of the disease and biology of the parasite. This has catalyzed a major effort resulting in development and regulatory approval of the first vaccine against malaria (RTS,S/AS01) as well as identification of novel drug targets and antimalarial compounds, some of which are in human clinical trials.

A Potent Anti-Malarial Human Monoclonal Antibody Targets Circumsporozoite Protein Minor Repeats and Neutralizes Sporozoites in the Liver.

Discovering potent human monoclonal antibodies (mAbs) targeting the Plasmodium falciparum circumsporozoite protein (PfCSP) on sporozoites (SPZ) and elucidating their mechanisms of neutralization will facilitate translation for passive prophylaxis and aid next-generation vaccine development. Here, we isolated a neutralizing human mAb, L9 that preferentially bound NVDP minor repeats of PfCSP with high affinity while cross-reacting with NANP major repeats. L9 was more potent than six published neutralizing human PfCSP mAbs at mediating protection against mosquito bite challenge in mice. Isothermal titration calorimetry and multiphoton microscopy showed that L9 and the other most protective mAbs bound PfCSP with two binding events and mediated protection by killing SPZ in the liver and by preventing their egress from sinusoids and traversal of hepatocytes. This study defines the subdominant PfCSP minor repeats as neutralizing epitopes, identifies an in vitro biophysical correlate of SPZ neutralization, and demonstrates that the liver is an important site for antibodies to prevent malaria.

In vitro production of infectious Plasmodium falciparum sporozoites.

An effective vaccine is needed for the prevention and elimination of malaria. The only immunogens that have been shown to have a protective efficacy of more than 90% against human malaria are Plasmodium falciparum (Pf) sporozoites (PfSPZ) manufactured in mosquitoes (mPfSPZ)1-7. The ability to produce PfSPZ in vitro (iPfSPZ) without mosquitoes would substantially enhance the production of PfSPZ vaccines and mosquito-stage malaria research, but this ability is lacking. Here we report the production of hundreds of millions of iPfSPZ. iPfSPZ invaded human hepatocytes in culture and developed to mature liver-stage schizonts expressing P. falciparum merozoite surface protein 1 (PfMSP1) in numbers comparable to mPfSPZ. When injected into FRGhuHep mice containing humanized livers, iPfSPZ invaded the human hepatocytes and developed to PfMSP1-expressing late liver stage parasites at 45% the quantity of cryopreserved mPfSPZ. Human blood from FRGhuHep mice infected with iPfSPZ produced asexual and sexual erythrocytic-stage parasites in culture, and gametocytes developed to PfSPZ when fed to mosquitoes, completing the P. falciparum life cycle from infectious gametocyte to infectious gametocyte without mosquitoes or primates.

A spatiotemporally resolved single-cell atlas of the Plasmodium liver stage.

Malaria infection involves an obligatory, yet clinically silent liver stage1,2. Hepatocytes operate in repeating units termed lobules, exhibiting heterogeneous gene expression patterns along the lobule axis3, but the effects of hepatocyte zonation on parasite development at the molecular level remain unknown. Here we combine single-cell RNA sequencing4 and single-molecule transcript imaging5 to characterize the host and parasite temporal expression programmes in a zonally controlled manner for the rodent malaria parasite Plasmodium berghei ANKA. We identify differences in parasite gene expression in distinct zones, including potentially co-adaptive programmes related to iron and fatty acid metabolism. We find that parasites develop more rapidly in the pericentral lobule zones and identify a subpopulation of periportally biased hepatocytes that harbour abortive infections, reduced levels of Plasmodium transcripts and parasitophorous vacuole breakdown. These 'abortive hepatocytes', which appear predominantly with high parasite inoculum, upregulate immune recruitment and key signalling programmes. Our study provides a resource for understanding the liver stage of Plasmodium infection at high spatial resolution and highlights the heterogeneous behaviour of both the parasite and the host hepatocyte.

Two chemoattenuated PfSPZ malaria vaccines induce sterile hepatic immunity.

The global decline in malaria has stalled1, emphasizing the need for vaccines that induce durable sterilizing immunity. Here we optimized regimens for chemoprophylaxis vaccination (CVac), for which aseptic, purified, cryopreserved, infectious Plasmodium falciparum sporozoites (PfSPZ) were inoculated under prophylactic cover with pyrimethamine (PYR) (Sanaria PfSPZ-CVac(PYR)) or chloroquine (CQ) (PfSPZ-CVac(CQ))-which kill liver-stage and blood-stage parasites, respectively-and we assessed vaccine efficacy against homologous (that is, the same strain as the vaccine) and heterologous (a different strain) controlled human malaria infection (CHMI) three months after immunization ( https://clinicaltrials.gov/ , NCT02511054 and NCT03083847). We report that a fourfold increase in the dose of PfSPZ-CVac(PYR) from 5.12 × 104 to 2 × 105 PfSPZs transformed a minimal vaccine efficacy (low dose, two out of nine (22.2%) participants protected against homologous CHMI), to a high-level vaccine efficacy with seven out of eight (87.5%) individuals protected against homologous and seven out of nine (77.8%) protected against heterologous CHMI. Increased protection was associated with Vδ2 γδ T cell and antibody responses. At the higher dose, PfSPZ-CVac(CQ) protected six out of six (100%) participants against heterologous CHMI three months after immunization. All homologous (four out of four) and heterologous (eight out of eight) infectivity control participants showed parasitaemia. PfSPZ-CVac(CQ) and PfSPZ-CVac(PYR) induced a durable, sterile vaccine efficacy against a heterologous South American strain of P. falciparum, which has a genome and predicted CD8 T cell immunome that differs more strongly from the African vaccine strain than other analysed African P. falciparum strains.

Flp/FRT-mediated disruption of ptex150 and exp2 in Plasmodium falciparum sporozoites inhibits liver-stage development.

Plasmodium falciparum causes severe malaria and assembles a protein translocon (PTEX) complex at the parasitophorous vacuole membrane (PVM) of infected erythrocytes, through which several hundred proteins are exported to facilitate growth. The preceding liver stage of infection involves growth in a hepatocyte-derived PVM; however, the importance of protein export during P. falciparum liver infection remains unexplored. Here, we use the FlpL/FRT system to conditionally excise genes in P. falciparum sporozoites for functional liver-stage studies. Disruption of PTEX members ptex150 and exp2 did not affect sporozoite development in mosquitoes or infectivity for hepatocytes but attenuated liver-stage growth in humanized mice. While PTEX150 deficiency reduced fitness on day 6 postinfection by 40%, EXP2 deficiency caused 100% loss of liver parasites, demonstrating that PTEX components are required for growth in hepatocytes to differing degrees. To characterize PTEX loss-of-function mutations, we localized four liver-stage Plasmodium export element (PEXEL) proteins. P. falciparum liver specific protein 2 (LISP2), liver-stage antigen 3 (LSA3), circumsporozoite protein (CSP), and a Plasmodium berghei LISP2 reporter all localized to the periphery of P. falciparum liver stages but were not exported beyond the PVM. Expression of LISP2 and CSP but not LSA3 was reduced in ptex150-FRT and exp2-FRT liver stages, suggesting that expression of some PEXEL proteins is affected directly or indirectly by PTEX disruption. These results show that PTEX150 and EXP2 are important for P. falciparum development in hepatocytes and emphasize the emerging complexity of PEXEL protein trafficking.

Winners vs. losers: Schistosoma mansoni intestinal and liver eggs exhibit striking differences in gene expression and immunogenicity.

The eggs of the blood fluke Schistosoma mansoni are the main cause of the clinical manifestations of chronic schistosomiasis. After laying, the egg "winners" attach to the endothelium of the mesenteric vein and, after a period of development, induce the growth of a small granuloma, which facilitates their passage to the intestinal lumen. Egg "losers" carried by the bloodstream to non-specific tissues also undergo full development and induce large granuloma formation, but their life ends there. Although these trapped eggs represent a dead end in the parasite life cycle, the vast majority of studies attempting to describe the biology of the S. mansoni eggs have studied these liver-trapped "losers" instead of migrating intestinal "winners". This raises the fundamental question of how these eggs differ. With robust comparative transcriptomic analysis performed on S. mansoni eggs isolated 7 weeks post infection, we show that gene expression is critically dependent on tissue localization, both in the early and late stages of development. While mitochondrial genes and venom allergen-like proteins are significantly upregulated in mature intestinal eggs, well-described egg immunomodulators IPSE/alpha-1 and omega-1, together with micro-exon genes, are predominantly expressed in liver eggs. In addition, several proteases and protease inhibitors previously implicated in egg-host interactions display clear tissue-specific gene expression patterns. These major differences in gene expression could be then reflected in the observed different ability of liver and intestinal soluble egg antigens to elicit host immune responses and in the shorter viability of miracidia hatched from liver eggs. Our comparative analysis provides a new perspective on the biology of parasite's eggs in the context of their development and tissue localization. These findings could contribute to a broader and more accurate understanding of parasite eggs interactions with the host, which have historically been often restricted to liver eggs and sometimes inaccurately generalized.

Plasmodium vivax Latent Liver Stage Infection and Relapse: Biological Insights and New Experimental Tools.

Plasmodium vivax is the most widespread human malaria parasite, in part because it can form latent liver stages known as hypnozoites after transmission by female anopheline mosquitoes to human hosts. These persistent stages can activate weeks, months, or even years after the primary clinical infection; replicate; and initiate relapses of blood stage infection, which causes disease and recurring transmission. Eliminating hypnozoites is a substantial obstacle for malaria treatment and eradication since the hypnozoite reservoir is undetectable and unaffected by most antimalarial drugs. Importantly, in some parts of the globe where P. vivax malaria is endemic, as many as 90% of P. vivax blood stage infections are thought to be relapses rather than primary infections, rendering the hypnozoite a major driver of P. vivax epidemiology. Here, we review the biology of the hypnozoite and recent discoveries concerning this enigmatic parasite stage. We discuss treatment and prevention challenges, novel animal models to study hypnozoites and relapse, and hypotheses related to hypnozoite formation and activation.

Moving on: How malaria parasites exit the liver.

An essential step in the life cycle of malaria parasites is their egress from hepatocytes, which enables the transition from the asymptomatic liver stage to the pathogenic blood stage of infection. To exit the liver, Plasmodium parasites first disrupt the parasitophorous vacuole membrane that surrounds them during their intracellular replication. Subsequently, parasite-filled structures called merosomes emerge from the infected cell. Shrouded by host plasma membrane, like in a Trojan horse, parasites enter the vasculature undetected by the host immune system and travel to the lung where merosomes rupture, parasites are released, and the blood infection stage begins. This complex, multi-step process must be carefully orchestrated by the parasite and requires extensive manipulation of the infected host cell. This review aims to outline the known signaling pathways that trigger exit, highlight Plasmodium proteins that contribute to the release of liver-stage merozoites, and summarize the accompanying changes to the hepatic host cell.

Stearoyl-CoA desaturase regulates organelle biogenesis and hepatic merozoite formation in Plasmodium berghei.

Plasmodium is an obligate intracellular parasite that requires intense lipid synthesis for membrane biogenesis and survival. One of the principal membrane components is oleic acid, which is needed to maintain the membrane's biophysical properties and fluidity. The malaria parasite can modify fatty acids, and stearoyl-CoA Δ9-desaturase (Scd) is an enzyme that catalyzes the synthesis of oleic acid by desaturation of stearic acid. Scd is dispensable in P. falciparum blood stages; however, its role in mosquito and liver stages remains unknown. We show that P. berghei Scd localizes to the ER in the blood and liver stages. Disruption of Scd in the rodent malaria parasite P. berghei did not affect parasite blood stage propagation, mosquito stage development, or early liver-stage development. However, when Scd KO sporozoites were inoculated intravenously or by mosquito bite into mice, they failed to initiate blood-stage infection. Immunofluorescence analysis revealed that organelle biogenesis was impaired and merozoite formation was abolished, which initiates blood-stage infections. Genetic complementation of the KO parasites restored merozoite formation to a level similar to that of WT parasites. Mice immunized with Scd KO sporozoites confer long-lasting sterile protection against infectious sporozoite challenge. Thus, the Scd KO parasite is an appealing candidate for inducing protective pre-erythrocytic immunity and hence its utility as a GAP.

LC3B labeling of the parasitophorous vacuole membrane of Plasmodium berghei liver stage parasites depends on the V-ATPase and ATG16L1.

The protozoan parasite Plasmodium, the causative agent of malaria, undergoes an obligatory stage of intra-hepatic development before initiating a blood-stage infection. Productive invasion of hepatocytes involves the formation of a parasitophorous vacuole (PV) generated by the invagination of the host cell plasma membrane. Surrounded by the PV membrane (PVM), the parasite undergoes extensive replication. During intracellular development in the hepatocyte, the parasites provoke the Plasmodium-associated autophagy-related (PAAR) response. This is characterized by a long-lasting association of the autophagy marker protein, and ATG8 family member, LC3B with the PVM. LC3B localization at the PVM does not follow the canonical autophagy pathway since upstream events specific to canonical autophagy are dispensable. Here, we describe that LC3B localization at the PVM of Plasmodium parasites requires the V-ATPase and its interaction with ATG16L1. The WD40 domain of ATG16L1 is crucial for its recruitment to the PVM. Thus, we provide new mechanistic insight into the previously described PAAR response targeting Plasmodium liver stage parasites.

Zonal human hepatocytes are differentially permissive to Plasmodium falciparum malaria parasites.

Plasmodium falciparum (Pf) is a major cause of human malaria and is transmitted by infected Anopheles mosquitoes. The initial asymptomatic infection is characterized by parasite invasion of hepatocytes, followed by massive replication generating schizonts with blood-infective merozoites. Hepatocytes can be categorized by their zonal location and metabolic functions within a liver lobule. To understand specific host conditions that affect infectivity, we studied Pf parasite liver stage development in relation to the metabolic heterogeneity of fresh human hepatocytes. We found selective preference of different Pf strains for a minority of hepatocytes, which are characterized by the particular presence of glutamine synthetase (hGS). Schizont growth is significantly enhanced by hGS uptake early in development, showcasing a novel import system. In conclusion, Pf development is strongly determined by the differential metabolic status in hepatocyte subtypes. These findings underscore the importance of detailed understanding of hepatocyte host-Pf interactions and may delineate novel pathways for intervention strategies.

Same, same but different: Exploring Plasmodium cell division during liver stage development.

Plasmodium parasites have a complex life cycle alternating between a mosquito and a vertebrate host. Following the bite of an Anopheles female mosquito, Plasmodium sporozoites are transmitted from the skin to the liver; their first place of replication within the host. Successfully invaded sporozoites undergo a massive replication and growth involving asynchronous DNA replication and division that results in the generation of tens of thousands or even hundreds of thousands of merozoites depending on the Plasmodium species. The generation of a high number of daughter parasites requires biogenesis and segregation of organelles to finally reach a relatively synchronous cytokinesis event. At the end of liver stage (LS) development, merozoites are packed into merosomes and released into the bloodstream. They are then liberated and infect red blood cells to again produce merozoites by schizogony for the erythrocytic stage of the life cycle. Although parasite LS and asexual blood stage (ABS) differ in many respects, important similarities exist between the two. This review focuses on the cell division of Plasmodium parasite LS in comparison with other life cycle stages especially the parasite blood stage.

Liver-Resident Memory CD8+ T Cells Form a Front-Line Defense against Malaria Liver-Stage Infection.

In recent years, various intervention strategies have reduced malaria morbidity and mortality, but further improvements probably depend upon development of a broadly protective vaccine. To better understand immune requirement for protection, we examined liver-stage immunity after vaccination with irradiated sporozoites, an effective though logistically difficult vaccine. We identified a population of memory CD8+ T cells that expressed the gene signature of tissue-resident memory T (Trm) cells and remained permanently within the liver, where they patrolled the sinusoids. Exploring the requirements for liver Trm cell induction, we showed that by combining dendritic cell-targeted priming with liver inflammation and antigen recognition on hepatocytes, high frequencies of Trm cells could be induced and these cells were essential for protection against malaria sporozoite challenge. Our study highlights the immune potential of liver Trm cells and provides approaches for their selective transfer, expansion, or depletion, which may be harnessed to control liver infections or autoimmunity.

ATF3 is involved in rSjP40-mediated inhibition of HSCs activation in Schistosoma japonicum-infected mice.

Schistosomiasis is a parasitic disease characterized by liver fibrosis, a process driven by the activation of hepatic stellate cells (HSCs) and subsequent collagen production. Previous studies from our laboratory have demonstrated the ability of Schistosoma japonicum protein P40 (SjP40) to inhibit HSCs activation and exert an antifibrotic effect. In this study, we aimed to elucidate the molecular mechanism underlying the inhibitory effect of recombinant SjP40 (rSjP40) on HSCs activation. Using a cell model in which rSjP40 inhibited LX-2 cell activation, we performed RNA-seq analyses and identified ATF3 as the most significantly altered gene. Further investigation revealed that rSjP40 inhibited HSCs activation partly by suppressing ATF3 activation. Knockdown of ATF3 in mouse liver significantly alleviated S. japonicum-induced liver fibrosis. Moreover, our results indicate that ATF3 is a direct target of microRNA-494-3p, a microRNA associated with anti-liver fibrosis effects. rSjP40 was found to downregulate ATF3 expression by upregulating microRNA-494-3p in LX-2 cells. This downregulation led to the inhibition of the expression of liver fibrosis proteins α-SMA and COL1A1, ultimately alleviating liver fibrosis caused by S. japonicum.

Effect of hepatocyte damage in hepatic fibrogenesis of patients infected with Schistosoma japonicum.

Schistosomiasis is a serious public health problem, and previous studies found that liver function and hepatic cells are damaged. To evaluate the serum parameters of liver function and fibrosis in schistosomiasis patients infected with Schistosoma japonicum (Schistosoma J.) and analyze the correlations between liver function and serum fibrosis markers in patients infected with Schistosoma J., this retrospective study enrolled 133 patients. The study population was divided into four groups: healthy people control group (n = 20), chronic schistosomiasis without liver cirrhosis (CS) group (n = 21), schistosomiasis cirrhosis without hypoalbuminemia (SC-HA) group (n = 68), and schistosomiasis cirrhosis with hypoalbuminemia (SC +HA) group (n = 24). Clinical and laboratory data were collected for analysis. In the multiple comparison of abnormal rates of aspartate aminotransferase (AST) and total bilirubin (TBIL), the abnormal rate of the SC +HA group was significantly higher than that of the other three groups (P < 0.05), and the abnormal rate of γ-GT in the SC +HA group was significantly higher than that in the control group (P < 0.05). Multiple comparison results of serum levels of fibrosis markers showed that the SC group had a significantly higher level of indexes than other groups (P < 0.05). The levels of TGF-ß1 in the CS group, SC-HA group and SC +HA group were significantly higher than those in the control group (P < 0.001). Our study demonstrated that the liver function and hepatic cells were damaged with the progression of liver disease in patients infected with Schistosoma J., and they played an important role in the occurrence and development of liver fibrosis.

Metabolic mechanism and pharmacological study of albendazole in secondary hepatic alveolar echinococcosis (HAE) model rats.

Albendazole (ABZ) is the primary treatment for alveolar echinococcosis (AE); however, its limited solubility impacts oral bioavailability, affecting therapeutic outcomes. In this study, various ABZ-solubilizing formulations, including albendazole crystal dispersion system (ABZ-CSD), albendazole hydrochloride-hydroxypropyl methylcellulose phthalate composite (TABZ-HCl-H), and albendazole hydroxyethyl sulfonate-hydroxypropyl methylcellulose phthalate composite (TABZ-HES-H), were developed and evaluated. Physicochemical properties as well as liver enzyme activity were analyzed and their pharmacodynamics in an anti-secondary hepatic alveolar echinococcosis (HAE) rat model were investigated. The formulations demonstrated improved solubility, exhibiting enhanced inhibitory effects on microcysts in HAE model rats compared to albendazole tablets. However, altered hepatic drug-metabolizing enzymes in HAE model rats led to increased ABZ levels and reduced ABZ-SO production, potentially elevating drug toxicity. These findings emphasize the importance of dose adjustments in patient administration, considering the impact of alveolar echinococcosis on rat hepatic drug metabolism.

Efficacy of ivermectin and its metabolites against Plasmodium falciparum liver stages in primary human hepatocytes.

Ivermectin, a broad-spectrum anti-parasitic drug, has been proposed as a novel vector control tool to reduce malaria transmission by mass drug administration. Ivermectin and some metabolites have mosquito-lethal effect, reducing Anopheles mosquito survival. Ivermectin inhibits liver stage development in a rodent malaria model, but no inhibition was observed in a primate malaria model or in a human malaria challenge trial. In the liver, cytochrome P450 3A4 and 3A5 enzymes metabolize ivermectin, which may impact drug efficacy. Thus, understanding ivermectin metabolism and assessing this impact on Plasmodium liver stage development is critical. Using primary human hepatocytes (PHHs), we characterized ivermectin metabolism and evaluated the efficacy of ivermectin and its primary metabolites M1 (3″-O-demethyl ivermectin) and M3 (4-hydroxymethyl ivermectin) against Plasmodium falciparum liver stages. Two different modes of ivermectin exposure were evaluated: prophylactic mode (days 0-3 post-infection) and curative mode (days 3-5 post-infection). We used two different PHH donors and modes to determine the inhibitory concentration (IC50) of ivermectin, M1, M3, and the known anti-malarial drug pyrimethamine, with IC50 values ranging from 1.391 to 14.44, 9.95-23.71, 4.767-8.384, and 0.9073-5.416 µM, respectively. In our PHH model, ivermectin and metabolites M1 and M3 demonstrated inhibitory activity against P. falciparum liver stages in curative treatment mode (days 3-5) and marginal activity in prophylactic treatment mode (days 0-3). Ivermectin had improved efficacy when co-administered with ketoconazole, a specific inhibitor of cytochrome P450 3A4 enzyme. Further studies should be performed to examine ivermectin liver stage efficacy when co-administered with CYP3A4 inhibitors and anti-malarial drugs to understand the pharmacokinetic and pharmacodynamic drug-drug interactions that enhance efficacy against human malaria parasites in vitro.