Malaria, a parasitic disease, was commonly associated with third world countries, with the highest mortality in nations in Sub-Saharan Africa and Asia. But, travel increases the risk of spread to more temperate regions, such as Western Europe and the United States where Malaria has been successfully eradicated. In the past 40 years, with a better understanding of the mosquito vector and the parasite itself, advancements in treatment and containment have been made. Understanding the parasite as well as its pathogenesis is vital in formulating effective treatments. Following the incidences of Plasmodium falciparum, knowlesi, vivax, malaria, ovale, and less commonly cynomolgi and simium over time as well as region helps to better illuminate the methods of Malarial transmission, interplay with environmental factors, and methods of treatment. While each species of parasite is similar in terms of mode of infection, they differ slightly when considering incubation periods and diagnostic and treatment techniques. Many drugs have been developed to treat Malaria and include Chloroquine, Primaquine, and derivatives of Artemisinin. While the discovery of these drugs was a significant breakthrough that dramatically reduced incidence and deaths caused by Malaria, improper administration of treatment has led to a recent increase in strains of the parasite which have developed drug resistance to Artemisinin Combination Therapies (ACT’s). Of these species, P. falciparum and P. vivax, the most common causes of malaria, are also so far the only species to have developed drug resistance. The goal of this thesis is to explore popular interventions, both drug and public health based, and how research focus has now shifted to better understanding the mechanism of parasitic drug resistance, specifically linked to mutations found in the Kelch protein in P. Falciparum. The recent findings of Kelch mutations pave the way towards addressing the growing problem of anti-Malarial resistance.

Malaria is a major health problem, mainly in developing countries, and causes an estimated 1 million deaths per year. Plasmodium falciparum is the major type of human malaria parasite, and causes the most infections and deaths. Malaria drugs, like any other drugs, suffer from possible side effects and the potential for emergence of resistance. Chloroquine, which was a very effective drug, has been used since about 1945, but its use is severely limited by resistance, even though it has mild side effects, and is otherwise very efficacious. Research has shown that there are chloroquine reversal agents, molecules that can reinstate antimalarial activity of chloroquine and chloroquine-like drugs; many such reversal agents are composed of two aromatic groups linked to a hydrogen bond acceptor several bonds away. By linking a chloroquine-like molecule to a reversal agent-like molecule, it was hoped that a hybrid molecule could be made with both antimalarial and reversal agent properties. In the Peyton Lab, such hybrid "Reversed Chloroquine" molecules have been synthesized and shown to have better antimalarial activity than chloroquine against the P. falciparum chloroquine-sensitive strain D6, as well as the P. falciparum chloroquine-resistant strains Dd2 and 7G8. The work reported in this manuscript involves simplifying the reversal agent head group of the Reversed Chloroquine molecules, to a single aromatic ring instead of the two rings groups described by others; this modification retained, or even enhanced, the antimalarial activity of the parent Reversed Chloroquine molecules. Of note was compound PL154, which had IC50 values of 0.3 nM and 0.5 nM against chloroquine-sensitive D6 and chloroquine-resistant Dd2. Compound PL106 was made to increase water solubility (a requirement for bioavailability) of the simplified Reversed Chloroquine molecules. Molecular modifications inherent to PL106 were not very detrimental to the antimalarial activity, and PL106 was found to be orally available in mice infected with P. yoelli, with an ED50 value of about 5.5 mg/kg/d. Varying the linker length between the quinoline ring and the protonatable nitrogen, or between the head group and the protonatable nitrogen, did not have adverse effects on the antimalarial activities of the simplified Reversed Chloroquine molecules, in accord with the trends observed for the original design of Reversed Chloroquine molecules as found from previous studies in the Peyton Lab. The simplified Reversed Chloroquine molecules even tolerated aliphatic head groups (rather than the original design which specified aromatic rings), showing that major modifications could be made on the Reversed Chloroquine molecules without major loss in activity. A bisquinoline compound, PL192, was made that contained secondary nitrogens at position 4 of the quinoline ring (PL192 is a modification of piperaquine, a known antimalarial drug that contains tertiary nitrogens at position 4 of the quinoline ring); this compound was more potent than piperaquine which had an IC50 value of 0.7 nM against CQS D6 and an IC50 of 1.5 nM against CQR Dd2, PL192 had IC50 values of 0.63 nM against chloroquine sensitive D6 and 0.02 nM against chloroquine resistant Dd2. Finally, the mechanism of action of these simplified "Reversed Chloroquines" was evaluated; it was found that the simplified "Reversed Chloroquines" behaved like chloroquine in inhibiting β-hematin formation and in heme binding. However, the simplified "Reversed Chloroquines" were found to inhibit chloroquine transport for chloroquine resistant P. falciparum chloroquine resistance transporter expressed in Xenopus oocytes to a lesser extant than the classical reversal agent verapamil. From these studies it was noted that the simplified "Reversed Chloroquines" may not behave as well as classical reversal agents would in restoring chloroquine efficacy, but they are very potent, and so could be a major step in developing drug candidates against malaria.

Background Promotion of job opportunities and tourism in African countries has led to an increase in expatriates in malaria endemic areas. A paucity of data exist on characteristics and numbers of expatriates and long-term travellers being evacuated from sub-Saharan Africa for suspected malaria infections diagnosed while still in Africa. Methods A retrospective flight record review of a South African fixed-wing air-ambulance provider from June 2006 through July 2009 was performed. Adult expatriates and long-term travellers with suspected malaria being evacuated from sub-Saharan African countries to Johannesburg, South Africa were included. Results Suspected malaria was the single most common diagnosis for dispatching airambulances with 81 (11.9%) of the 679 flights. Accuracy of the initial diagnosis, based on confirmation of malaria at the receiving facility was 78.4% for blood smears, 92.3% for rapid detection tests and 42.8% for clinical signs alone. P. falciparum (alone, or in combination with other Plasmodium species) was the most frequently isolated species at both the referring (100%) and receiving (88.2%) facilities in cases where the species was documented. The suspected malaria patients were predominantly male 69 (84.1%), with a mean age of 42.1 ±12.8 years, and were in sub-Saharan Africa for occupational reasons 65 (79.3%). Angola, the Democratic Republic of Congo and Mozambique were the countries of origin in 48 (58.5%) of the suspected malaria flights. Compliance on appropriate malaria chemoprophylaxis was documented in two (2.4%) suspected malaria patients. Intubation as a marker of severity was required for 15 (18.3%) patients, and one (1.2%) patient died inflight. No statistically significant difference (p=0.50) was shown for intubation requirements when comparing patients who had utilised malaria chemoprophylaxis with the patients who had not utilised chemoprophylaxis. Conclusions Patients presented in advanced stages of severe/complicated malaria with concurrent poor chemoprophylaxis utilisation and compliance. Appropriate chemoprophylaxis did not decrease the severity of presentation (based on intubation requirements) and did not guarantee complete malaria protection.

The strides made in malaria eradication efforts have been aided by a combination of vector control and chemoprevention. However, Plasmodium resistance to first-line artemisinin-based combination therapies (ACTs), and mosquito resistance to insecticides threatens the progress made. Innovative vector control measures, vaccines and antimalarial drugs with novel modes of action are key to disease eradication. High-throughput phenotypic screening of chemical libraries tested directly against all the stages of the Plasmodium lifecycle have been the mainstay of antimalarial drug discovery efforts and have identified compounds that are effective in parasite clearance. Unfortunately, these screens are handicapped in that they are unable to specify the actual compound targets in the Plasmodium parasites. As a result, many candidate hits have had to be re-screened in specific assays to determine putative mechanisms of antiplasmodial action. Predictably, this has elevated target-specific screens as the next frontier in drug discovery. This shift has been aided by a number of factors, including the cost effectiveness of these screens and the fact that target-specific screens do not always require specialized access to parasites. When combined with knowledge of the target’s structure, where known, target-specific screens have the potential to give lead compounds with impeccable potency and selectivity. This approach has already been successfully put to use, for example, in the identification of P. falciparum p-type ATPase 4 (PfATP4) and P. falciparum phosphatidylinositol 4-kinase (PfPI(4)K) inhibitors. The new challenge now is the identification of quality targets. Here, computational biology ‘omics’ tools have proved to be an invaluable resource. Two of the more commonly used of these tools are genomics and metabolomics. In-vitro evolution assays followed by whole genome sequencing analysis is a popular genomics approach and helps unveil novel target genes. Plasmodium parasites are exposed to sublethal doses of a compound until an upward shift in the half-maximal inhibitory concentration (IC50), indicative of resistant parasites, is observed in the culture. Sequenced genomes of the resistant parasite clones are compared to those of the drug-naive parent to reveal genetic changes, which include both single nucleotide polymorphisms (SNPs) and copy number variations (CNVs). While these genomic changes may point to genes encoding actual drug targets, they often reveal mediators of drug resistance or tolerance. Follow-up assays like SNP validation through gene editing are necessary to distinguish between actual targets, resistance mechanisms and random background mutations. Expectedly, genetic changes in uncharacterized Plasmodium genes are the bottle-necks in the identification of novel druggable targets. Even so, this genomics method has uncovered or reconfirmed novel antimalarial drug targets, including the proteasome, aminophospholipid-transporting P-type ATPase (PfAT-Pase2) and cGMP-dependent protein kinase (PfPKG). Metabolomic profiling and transcriptomics narrows down a compound’s mode of action. Here, parasites are treated with a compound of interest and the metabolites extracted and analyzed using liquid chromatography-mass spectrometry (LC-MS). The metabolomics fingerprint or metaprint is then compared to that of untreated parasites. While this method rarely provides the exact drug target, it narrows down the compound’s mode of action, which is valuable for target validation and characterization. The issue of non-specific or non-viable phenotype metabolite signals is easily filtered out by treating parasites with various drug concentrations and/or over a period of time. Other areas that limit the effectiveness of this tool and need to be addressed include the analysis of compounds that do not act through metabolic pathway disruption and potential host contamination. Nonetheless, metabolomics are a key player in drug discovery and have successfully been used in the study of pantothenamides (MMV689258) where the observed CoA analog buildup helped identify their mechanism of action in sequestering coenzyme A to block acetyl-CoA anabolism. Presented herein is a culmination of my graduate research in antimalarial drug discovery. Three independent projects are presented, and they all have either been published or are currently under reviewership. Chapter 1 is an introduction to malaria, a disease that has and continues to claim hundreds of thousands of lives, especially in my home continent of Africa. In chapter 2, I detail the experimental procedures used to generate the data presented in chapters 3-5. Chapter 3 is a detailed susceptibility profiling and metabolomic fingerprinting of Plasmodium falciparum asexual blood stages (ABS) to clinical and experimental antimalarials. This work, published in Cell Chemical Biology (2020), presents to the malaria research community a medium-throughput assay that can be utilized to identify new antimalarial lead compounds and novel assayable targets. Chapter 4 presents a detailed analysis of a novel ATP-binding cassette (ABC) transporter that confers pleiotropic antimalarial drug resistance in P. falciparum and that was first identified through in vitro evolution assays. This work is currently under review in Cell Chemical Biology. Chapter 5 presents work on an promising new preclinical compound, MMV688533, that provides single-dose cure and that was discovered using an innovative orthology-based screen by the Sanofi drug discovery team. In this chapter, I also present in detail the assays performed to better understand this compound’s mode of antiplasmodial action and the potential drivers of parasite resistance. This work has been accepted, pending minor textual revisions, in Science Translational Medicine. Finally in chapter 6, I summarize chapters 3-5 and share future follow-up work needed to strengthen and contextualize some of the experimental findings presented here.

En 2020, la malaria a provoqué 241 millions de cas d'infections et 627 000 morts. La faible efficacité du vaccin disponible et la résistance aux traitements rendent indispensable l'identification de cibles thérapeutiques. Plasmodium falciparum (Pf) envahit les érythrocytes pour s'y répliquer. Pour cela, de protéines contenues dans des organites d'invasion, tels que les micronèmes et les rhoptries rassemblés à un complexe apical, sont sécrétées. Le trafic des protéines entre l'appareil de Golgi et les organites d'invasion est médié par la PfSortiline. Dans les organismes modèles, la sortiline est recyclée entre les organites cibles et l'appareil de Golgi via le complexe protéique rétromère composé des protéines de tri vacuolaire (Vps) Vps26-29-35. Ce complexe est recruté via les complexes VpsC, composé de Vps11-16-18-33, CORVET composé de Vps3-8 et HOPS, composé de Vps39-41. Chez Pf, l'ensemble des composants des complexes VpsC et rétromère sont conservés. Seulement PfVps3 du complexe CORVET est retrouvée et le complexe HOPS est absent. L'hypothèse du projet est que ces protéines conservées sont impliquées dans la biogenèse des organites du complexe apical via le recyclage de la PfSortiline vers l'appareil de Golgi. Pour vérifier cela, des souches de parasites exprimant les protéines de fusion PfVps3-11-16-18-29 étiquetées à un domaine GFP ont été construites. Des techniques de Western Blot et de microscopie à fluorescence ont montré que ces protéines de fusion sont exprimées lors du cycle érythrocytaire. Il semble que PfVps29-GFP localise à des structures semblables aux endosomes et partiellement aux micronèmes. Les protéines PfVps16-18-GFP semblent localiser aux micronèmes et partiellement aux rhoptries et à l'appareil de Golgi. Finalement, des souches dans lesquelles les protéines PfVps3-16-29-GFP peuvent être délocalisées de façon conditionnelle ont été construites. Il a été montré que PfVps16-GFP semble essentielle à la survie de Pf. Ce projet participe à la caractérisation de nouvelles pistes thérapeutiques antipaludiques. / Malaria was responsible for 627,000 deaths and 241 million infections in 2020 alone. Drug resistance, and the poor efficacy of the only available vaccine, are strong arguments supporting the need to identify therapeutic targets. The malaria parasite Plasmodium falciparum (Pf) invades erythrocytes and multiplies inside them. To do so, it secretes invasion proteins located inside organelles, like micronemes and rhoptries, which are localised at an apical complex. Protein trafficking from the Golgi apparatus to these organelles is dependent on PfSortilin. In model organisms, this protein is recycled between the target organelles and the Golgi Apparatus by a protein complex called retromer. This complex is composed of Vacuolar Sorting Proteins (Vps)-26-29-35. The retromer complex is recruited by complexes, composed of Vps11-16-18-33, CORVET, composed of Vps3-8, and HOPS, composed of Vps39-41. In Pf, all components of the retromer and the VpsC complexes are conserved. However, only PfVps3 of the CORVET complex is conserved and the HOPS complex is absent. We hypothesized that conserved proteins play a key role in apical complex biogenesis by recycling PfSortilin to the Golgi apparatus. To verify the hypothesis, parasite strains coding the fusion proteins PfVps3-11-16-18-29 tagged with a GFP were generated. Western Blot and fluorescence microscopy showed that those proteins are expressed during the erythrocyte life cycle. PfVps29-GFP seemed to localize at endosome-like structures and partially at micronemes. PfVps16-18 seemed to localise at micronemes too and partially at rhoptries and at the Golgi apparatus. Finally, strains where PfVps3-16-29 could be functionally mislocalized have been generated. This technique showed that PfVps16-GFP were essential for Pf survival. Our work could lead to the characterization of new antimalarial drug targets.

The emergence and spread of drug resistance in Plasmodium falciparum has consistently been a major barrier to the control and eradication of malaria. Resistance to the affordable and fast-acting former first-line drug chloroquine (CQ) was first observed in the 1950s near the Thai-Cambodian border and in South America. Resistance later spread from Asia to highly endemic regions in Africa, with reports of up to 6-fold increases in regional malaria mortality rates. The replacement drug, sulfadoxine-pyrimethamine (SP), encountered resistance within one year of clinical use. Artemisinin-(ART) based combination therapies (ACTs), which consist of a fast-acting ART derivative and a slower-acting partner drug, became the global first-line standard in 2000 and, along with mosquito vector control measures, helped decrease mortality rates by 60%. Unfortunately, parasites resistant to ART derivatives arose in Southeast Asia. This compromised the effectiveness of the ACT partner drug piperaquine (PPQ) and resistance to this drug was first reported in 2015, around a decade after the introduction of dihydroartemisinin-PPQ. By 2019, PPQ resistance, driven primarily by a series of mutations in the P. falciparum chloroquine resistance transporter (PfCRT), was widespread in Southeast Asia, resulting in >50% failure upon treatment with dihydroartemisinin-PPQ. Malaria mortality rates have surged recently, causing an estimated 619,000 deaths in 2021. Sub-Saharan Africa is most heavily affected by this disease where 78.9% of deaths are of young children. To this date, PPQ remains effective in Africa. It is a major concern that PPQ resistance will arise on this continent, however, given the importance of PPQ in current efforts to expand the range of antimalarial interventions and reverse the current rise of malaria cases in Africa. Understanding PPQ resistance mechanisms and their effect on parasite biology is critical to creating effective treatments and minimizing the impact of drug-resistant P. falciparum malaria. This thesis aims to investigate the earliest reports of PPQ resistance, to define the PPQ susceptibility and parasite fitness of contemporary SE Asian parasite strains, and to predict future dominant strains in the field to further our understanding of parasite resistance mechanisms and combat the spread of drug-resistant malaria. In Chapter 3, we show that earlier reports of PPQ resistance in Yunnan Province, China could be explained by the unique China C PfCRT variant. Using gene editing, we reveal that this variant confers a loss of fitness and parasite re-sensitization to the chemically related former first-line antimalarial CQ, while acquiring PPQ resistance via drug efflux. We employ biochemical assays to measure mutant PfCRT-mediated drug transport and molecular dynamics simulations with the recently solved PfCRT structure to assess changes in the central drug-binding cavity. This study provides impetus for adding CQ into an antimalarial treatment regimen where PPQ has lost efficacy. In response to widespread treatment failures, PPQ was removed as a first-line partner drug. Recently, additional mutations have been observed on the highly-resistant Dd2+F145I PfCRT isoform. These mutations developed in parasites in long-term in vitro culture or in Southeast Asian field isolates. In Chapter 4, I characterized the impact of these mutations on parasite fitness and antimalarial susceptibility by editing asexual blood stage parasites to express these mutant PfCRT haplotypes. Competitive growth assays with a GFP-expressing reporter line revealed that these additional mutations reduce the fitness defect imposed by F145I, likely the primary driver of their emergence. I found that these mutations differentially impact parasite susceptibility to PPQ and CQ in in vitro dose-response assays. I used proteoliposome-based drug uptake studies, molecular dynamic simulations, and peptidomics to detail the molecular features of drug resistance and parasite physiology of these lines. These experiments provide insight into parasite responses to the changing drug selective pressures in SE Asia to inform treatment strategies in this region moving forward. In Chapter 5, I sought to determine whether Asian PPQ-resistant PfCRT mutations could also mediate PPQ resistance on African PfCRT haplotypes. Using zinc-finger nuclease-based gene editing, I introduce the most common African mutant pfcrt alleles with a SE Asian PfCRT mutation into Dd2 parasites. In PPQ survival assays, these mutations only confer high-grade PPQ resistance (defined as ≥10% survival at 200 nM) on the FCB PfCRT background. I assessed the susceptibility of these gene-edited isogenic lines to other clinical antimalarials and the relative fitness of these engineered lines with in vitro assays. These experiments clearly show that there is a genetic path to PPQ resistance in African parasites; however, they also suggest that fitness costs associated with these mutations may hinder the spread of resistance. Our data provide important insights into PPQ resistance. In chapter 6, these findings are summarized along with future studies to strengthen and expand on the findings presented herein.

La malaria est une maladie infectieuse causant plus de 500 000 morts chaque année. La maladie est causée par un protozoaire de la famille Plasmodium. L’apparition de souches résistantes aux traitements actuels et l’absence de vaccin efficace rendent la découverte de nouvelles cibles thérapeutiques urgente. Le parasite possède un complexe apical, un groupement de vacuoles sécrétoires spécialisées contenant les protéines responsables de l’invasion du globule rouge. Nous nous intéressons aux mécanismes gouvernant le transport intracellulaire de ces protéines et à la biogenèse du complexe apical lors de la formation des nouveaux parasites. Plus particulièrement, nous nous intéressons au rôle des phosphoinositides dans le recrutement des protéines à la membrane de l’appareil de Golgi. Par analyse bio-informatique du génome de P. falciparum, nous avons identifié plusieurs protéines effectrices liant potentiellement les phosphoinositides. Les travaux présentés dans ce mémoire concernent Mal13P1.188, une protéine possédant un domaine Pleckstrin homology. Nous proposons que Mal13P1.188 ait un rôle dans la génération du complexe apical en recrutant les protéines le constituant à la membrane du Golgi par la liaison avec les phosphoinositides. Afin de vérifier nos hypothèses, nous avons généré une lignée de parasite dont le gène de Mal13P1.188 est fusionné avec une GFP et une hémagglutinine. À l’aide de cette lignée de parasite, nous avons pu identifier Mal13P1.188 à proximité de l’appareil de Golgi lorsque les parasites étaient sous la forme schizont du cycle érythrocytaire. D’autres expériences ont permis de confirmer que le domaine Pleckstrin homology de Mal13P1.188 était capable de reconnaître les différentes formes de phosphoinositides. Finalement, d’autres travaux devront être faits sur Mal13P1.188 afin de déterminer si elle est essentielle à la survie du parasite. / Malaria is a deadly infectious disease taking more than 500,000 lives each year. The disease is caused by a protozoan of the Plasmodium family. Resistant strains beginning to spread and the inexistence of an efficient vaccine make the discovery of new targets urgent. The parasite secretes proteins to invade the red blood cell. Those proteins are regrouped in the apical complex, a group of organelles used for the invasion. Our research team focus on the transport mechanisms that drive the formation of the apical complex during the cellular division of new parasite. In other terms, we are interested on the role of phosphoinositide in the recruitment of protein inside the Golgi apparatus. After a bioinformatics analyse the P. falciparum genome, we identified many effectors protein that can bind phosphoinositides. Among them, we focused our work on Mal13P1.188, a protein with a Pleckstrin homology domain. We propose that Mal13P1.188 has a role in the recruitment of the apical proteins to the Golgi membrane using phosphoinositide as a marker on the membrane. To verify that hypothesis, we generated a strain of parasite with endogenous Mal13P1.188 tagged to a GFP and a hemagglutinin. With those parasites, we identified Mal13P1.188 near the Golgi apparatus during the Schizont stage of the blood cycle. Other experiment confirmed that the Pleckstrin homology domain of Mal13P1.188 is able to bind different form of phosphoinositides. Finally, more work has to be done to confirm if Mal13P1.188 is essential to the parasite survival.

No description available.

No description available.

La malaria est un des fléaux les plus dévastateurs dans les pays en voie de développement. L’absence d’un vaccin et la résistance aux agents antimalariaux disponibles démontrent le besoin urgent d’identifier de nouvelles cibles thérapeutiques. Les phosphoinositides (PIP) sont des composants essentiels des membranes cellulaires chez les eucaryotes jouant un rôle important dans la signalisation intracellulaire, la synthèse d’ADN et le trafic protéique, par exemple. Malgré leur importance chez les eucaryotes, on en connaît peu sur leurs fonctions chez le parasite de la malaria Plasmodium falciparum. Dans notre laboratoire, nous avons réalisé un criblage par inactivation génique de 36 effecteurs potentiels de la voie métabolique PIP pour identifier les gènes qui sont essentiels à la prolifération chez P. falciparum. Notre étude a montré que 72% des gènes potentiellement impliqués dans la voie métabolique des PIP ne pouvaient être inactivés et sont donc potentiellement essentiels pour la survie du parasite. L’analyse d’une souche knock-out pour la protéine PfPX, ayant un domaine de liaison aux PIP de type Phox, a démontré un ralentissement sévère de la croissance du parasite. La caractérisation de la protéine PfPX a révélé qu’elle se localisait à la membrane de la vacuole digestive, le site où le parasite digère l'hémoglobine (Hb) de l'hôte afin de subvenir à ses besoins en acides aminés. Nous avons montré que les parasites dépourvus de la protéine Phox accumulaient plus d'Hb et que celle-ci était piégée dans des vésicules à proximité de la vacuole digestive, suggérant un rôle pour cette protéine dans la fusion des vésicules d’Hb avec la membrane de la vacuole digestive. Globalement, nos résultats ont révélé que les PIP ont un rôle important dans le transport de l'Hb chez P. falciparum / Malaria is one of the most devastating curses in developing countries. The absence of a vaccine and resistance to available antimalarial agents demonstrate the urgent need to identify new therapeutic targets. Phosphoinositides (PIPs) are essential components of cell membranes in eukaryotes, playing an important role in intracellular signaling, DNA synthesis and protein trafficking, for example. Despite their importance in eukaryotes, little is known about their functions in the malaria parasite Plasmodium falciparum. In our laboratory, we screened 36 putative effectors of the PIP pathway by gene inactivation to identify the genes that are essential for proliferation in P. falciparum. Our studies showed that 72% of genes possibly involved in the PIP pathway could not be inactivated and are therefore potentially essential for parasite survival. Analysis of a knockout strain for PfPX protein, having a Phox-like PIP binding domain, demonstrated a severe slowdown in parasite growth. Characterization of the PfPX protein revealed that it was localized to the food vacuole membrane, the site where the parasite digests the hemoglobin (Hb) of the host in order to meet his needs in amino acids, and in vesicular type structures. We have shown that parasites lacking the Phox protein accumulate more Hb and that it is trapped in vesicles near the digestive vacuole, suggesting a role for this protein in the fusion of Hb vesicles with the membrane of the digestive vacuole. Overall, our results revealed that PIPs play an important role in the transport of P. falciparum Hb