DAB-APT: a Fluorescence-Based Assay for Determining Aminopropyl Transferase Activity and Inhibition.

Polyamines are polycationic molecules that are crucial in a wide array of cellular functions. Their biosynthesis is mediated by aminopropyl transferases (APTs), promising targets in antimicrobial, antineoplastic and antineurodegenerative therapies. A major limitation, however, is the lack of high-throughput assays to measure their activity. We developed the first fluorescence-based assay, DAB-APT, for measurement of APT activity using 1,2-diacetyl benzene, which forms fluorescent conjugates with putrescine, spermidine and spermine with fluorescence intensity increasing with increasing carbon chain length. The assay has been validated using APT enzymes from S. cerevisiae and P. falciparum and is suitable for high-throughput screening of large chemical libraries. Given the importance of APTs in infectious diseases, cancer and neurobiology, our DAB-APT assay has broad applications, holding promise for advancing research and drug discovery efforts.

Tafenoquine-Atovaquone Combination Achieves Radical Cure and Confers Sterile Immunity in Experimental Models of Human Babesiosis.

Human babesiosis is a potentially fatal tick-borne disease caused by intraerythrocytic Babesia parasites. The emergence of resistance to recommended therapies highlights the need for new and more effective treatments. Here we demonstrate that the 8-aminoquinoline antimalarial drug tafenoquine inhibits the growth of different Babesia species in vitro, is highly effective against Babesia microti and Babesia duncani in mice and protects animals from lethal infection caused by atovaquone-sensitive and -resistant B. duncani strains. We further show that a combination of tafenoquine and atovaquone achieves cure with no recrudescence in both models of human babesiosis. Interestingly, elimination of B. duncani infection in animals following drug treatment also confers immunity to subsequent challenge. Altogether, the data demonstrate superior efficacy of tafenoquine plus atovaquone combination over current therapies for the treatment of human babesiosis and highlight its potential in providing protective immunity against Babesia following parasite clearance.

Babesia duncani multi-omics identifies virulence factors and drug targets.

Babesiosis is a malaria-like disease in humans and animals that is caused by Babesia species, which are tick-transmitted apicomplexan pathogens. Babesia duncani causes severe to lethal infection in humans, but despite the risk that this parasite poses as an emerging pathogen, little is known about its biology, metabolic requirements or pathogenesis. Unlike other apicomplexan parasites that infect red blood cells, B. duncani can be continuously cultured in vitro in human erythrocytes and can infect mice resulting in fulminant babesiosis and death. We report comprehensive, detailed molecular, genomic, transcriptomic and epigenetic analyses to gain insights into the biology of B. duncani. We completed the assembly, 3D structure and annotation of its nuclear genome, and analysed its transcriptomic and epigenetics profiles during its asexual life cycle stages in human erythrocytes. We used RNA-seq data to produce an atlas of parasite metabolism during its intraerythrocytic life cycle. Characterization of the B. duncani genome, epigenome and transcriptome identified classes of candidate virulence factors, antigens for diagnosis of active infection and several attractive drug targets. Furthermore, metabolic reconstitutions from genome annotation and in vitro efficacy studies identified antifolates, pyrimethamine and WR-99210 as potent inhibitors of B. duncani to establish a pipeline of small molecules that could be developed as effective therapies for the treatment of human babesiosis.

Epitope profiling of monoclonal antibodies to the immunodominant antigen BmGPI12 of the human pathogen Babesia microti.

The significant rise in the number of tick-borne diseases represents a major threat to public health worldwide. One such emerging disease is human babesiosis, which is caused by several protozoan parasites of the Babesia genus of which B. microti is responsible for most clinical cases reported to date. Recent studies have shown that during its intraerythrocytic life cycle, B. microti exports several antigens into the mammalian host using a novel vesicular-mediated secretion mechanism. One of these secreted proteins is the immunodominant antigen BmGPI12, which has been demonstrated to be a reliable biomarker of active B. microti infection. The major immunogenic determinants of this antigen remain unknown. Here we provide a comprehensive molecular and serological characterization of a set of eighteen monoclonal antibodies developed against BmGPI12 and a detailed profile of their binding specificity and suitability in the detection of active B. microti infection. Serological profiling and competition assays using synthetic peptides identified five unique epitopes on the surface of BmGPI12 which are recognized by a set of eight monoclonal antibodies. ELISA-based antigen detection assays identified five antibody combinations that specifically detect the secreted form of BmGPI12 in plasma samples from B. microti-infected mice and humans but not from other Babesia species or P. falciparum.

An Alternative Culture Medium for Continuous In Vitro Propagation of the Human Pathogen Babesia duncani in Human Erythrocytes.

Continuous propagation of Babesia duncani in vitro in human erythrocytes and the availability of a mouse model of B. duncani lethal infection make this parasite an ideal model to study Babesia biology and pathogenesis. Two culture media, HL-1 and Claycomb, with proprietary formulations are the only culture media known to support the parasite growth in human erythrocytes; however, the HL-1 medium has been discontinued and the Claycomb medium is often unavailable leading to major interruptions in the study of this pathogen. To identify alternative media conditions, we evaluated the growth of B. duncani in various culture media with well-defined compositions. We report that the DMEM-F12 culture medium supports the continuous growth of the parasite in human erythrocytes to levels equal to those achieved in the HL-1 and Claycomb media. We generated new clones of B. duncani from the parental WA-1 clinical isolate after three consecutive subcloning events in this medium. All clones showed a multiplication rate in vitro similar to that of the WA-1 parental isolate and cause fatal infection in C3H/HeJ mice. The culture medium, which can be readily reconstituted from its individual components, and the tools and resources developed here will facilitate the study of B. duncani.

Babesia duncani in Culture and in Mouse (ICIM) Model for the Advancement of Babesia Biology, Pathogenesis, and Therapy.

Babesiosis is a tick-borne disease caused by pathogens belonging to the genus Babesia. In humans, the disease presents as a malaria-like illness and can be fatal in immunocompromised and elderly people. In the past few years, human babesiosis has been a rising concern worldwide. The disease is transmitted through tick bite, blood transfusion, and transplacentally in rare cases, with several species of Babesia causing human infection. Babesia microti, Babesia duncani, and Babesia divergens are of particular interest because of their important health impact and amenability to research inquiries. B. microti, the most commonly reported Babesia pathogen infecting humans, can be propagated in immunocompetent and immunocompromised mice but so far has not been successfully continuously propagated in vitro in human red blood cells (hRBCs). Conversely, B. divergens can be propagated in vitro in human red blood cells but lacks a mouse model to study its virulence. Recent studies have highlighted the uniqueness of B. duncani as an ideal model organism to study intraerythrocytic parasitism in vitro and in vivo. An optimized B. duncani in culture and in mouse (ICIM) model has recently been described, combining long-term continuous in vitro culture of the parasite in hRBCs with an animal model of parasitemia (P) and lethal infection in C3H/HeJ mice. Here, we provide a detailed protocol for the use of the B. duncani ICIM model in research. This model provides a unique and sound foundation to gain further insights into the biology, pathogenesis, and virulence of Babesia and other intraerythrocytic parasites, and has been validated as an efficient system to evaluate novel strategies for the treatment of human babesiosis and possibly other parasitic diseases. This protocol was validated in: J Infect Dis (2022), DOI: 10.1093/infdis/jiac181 Graphical abstract ICIM model [Adapted and modified from Pal et al. (2022)].

Effective Therapy Targeting Cytochrome bc1 Prevents Babesia Erythrocytic Development and Protects from Lethal Infection.

An effective strategy to control blood-borne diseases and prevent outbreak recrudescence involves targeting conserved metabolic processes that are essential for pathogen viability. One such target for Plasmodium and Babesia, the infectious agents of malaria and babesiosis, respectively, is the mitochondrial cytochrome bc1 protein complex, which can be inhibited by endochin-like quinolones (ELQ) and atovaquone. We used the tick-transmitted and culturable blood-borne pathogen Babesia duncani to evaluate the structure-activity relationship, safety, efficacy, and mode of action of ELQs. We identified a potent and highly selective ELQ prodrug (ELQ-502), which, alone or in combination with atovaquone, eliminates B. microti and B. duncani infections in vitro and in mouse models of parasitemia and lethal infection. The strong efficacy at low dose, excellent safety, bioavailability, and long half-life of this experimental therapy make it an ideal clinical candidate for the treatment of human infections caused by Babesia and its closely related apicomplexan parasites.

Tick extracellular vesicles enable arthropod feeding and promote distinct outcomes of bacterial infection.

Extracellular vesicles are thought to facilitate pathogen transmission from arthropods to humans and other animals. Here, we reveal that pathogen spreading from arthropods to the mammalian host is multifaceted. Extracellular vesicles from Ixodes scapularis enable tick feeding and promote infection of the mildly virulent rickettsial agent Anaplasma phagocytophilum through the SNARE proteins Vamp33 and Synaptobrevin 2 and dendritic epidermal T cells. However, extracellular vesicles from the tick Dermacentor andersoni mitigate microbial spreading caused by the lethal pathogen Francisella tularensis. Collectively, we establish that tick extracellular vesicles foster distinct outcomes of bacterial infection and assist in vector feeding by acting on skin immunity. Thus, the biology of arthropods should be taken into consideration when developing strategies to control vector-borne diseases.

Evidence for SARS-CoV-2 Spike Protein in the Urine of COVID-19 Patients.

Background: SARS-CoV-2 infection has, as of April 2021, affected >133 million people worldwide, causing >2.5 million deaths. Because the large majority of individuals infected with SARS-CoV-2 are asymptomatic, major concerns have been raised about possible long-term consequences of the infection. Methods: Wedeveloped an antigen capture assay to detect SARS-CoV-2 spike protein in urine samples from patients with COVID-19whose diagnosis was confirmed by positive PCR results from nasopharyngeal swabs (NP-PCR+) forSARS-CoV-2. We used a collection of 233 urine samples from 132 participants from Yale New Haven Hospital and the Children's Hospital of Philadelphia that were obtained during the pandemic (106 NP-PCR+ and 26 NP-PCR-), and a collection of 20 urine samples from 20 individuals collected before the pandemic. Results: Our analysis identified 23 out of 91 (25%) NP-PCR+ adult participants with SARS-CoV-2 spike S1 protein in urine (Ur-S+). Interestingly, although all NP-PCR+ children were Ur-S-, one child who was NP-PCR- was found to be positive for spike protein in their urine. Of the 23 adults who were Ur-S+, only one individual showed detectable viral RNA in urine. Our analysis further showed that 24% and 21% of adults who were NP-PCR+ had high levels of albumin and cystatin C, respectively, in their urine. Among individuals with albuminuria (>0.3 mg/mg of creatinine), statistical correlation could be found between albumin and spike protein in urine. Conclusions: Together, our data showed that one of four individuals infected with SARS-CoV-2 develop renal abnormalities, such as albuminuria. Awareness about the long-term effect of these findings is warranted.

Palmitoylated Proteins in Plasmodium falciparum-Infected Erythrocytes: Investigation with Click Chemistry and Metabolic Labeling.

The examination of the complex cell biology of the human malaria parasite Plasmodium falciparum usually relies on the time-consuming generation of transgenic parasites. Here, metabolic labeling and click chemistry are employed as a fast transfection-independent method for the microscopic examination of protein S-palmitoylation, an important post-translational modification during the asexual intraerythrocytic replication of P. falciparum. Applying various microscopy approaches such as confocal, single-molecule switching, and electron microscopy, differences in the extent of labeling within the different asexual developmental stages of P. falciparum and the host erythrocytes over time are observed.

Crystal structures of Babesia microti lactate dehydrogenase BmLDH reveal a critical role for Arg99 in catalysis.

The tick- and transfusion-transmitted human pathogen Babesia microti infects host erythrocytes to cause the pathologic symptoms associated with human babesiosis, an emerging disease with worldwide distribution and potentially fatal clinical outcome. Drugs currently recommended for the treatment of babesiosis are associated with a high failure rate and significant adverse events, highlighting the urgent need for more-effective and safer babesiosis therapies. Unlike other apicomplexan parasites, B. microti lacks a canonical lactate dehydrogenase (LDH) but instead expresses a unique enzyme, B. microti LDH (BmLDH), acquired through evolution by horizontal transfer from a mammalian host. Here, we report the crystal structures of BmLDH in apo state and ternary complex (enzyme-NADH-oxamate) solved at 2.79 and 1.89 Å. Analysis of these structures reveals that upon binding to the coenzyme and substrate, the active pocket of BmLDH undergoes a major conformational change from an opened and disordered to a closed and stabilized state. Biochemical assays using wild-type and mutant B. microti and human LDHs identified Arg99 as a critical residue for the catalytic activity of BmLDH but not its human counterpart. Interestingly, mutation of Arg99 to Ala had no impact on the overall structure and affinity of BmLDH to NADH but dramatically altered the closure of the enzyme's active pocket. Together, these structural and biochemical data highlight significant differences between B. microti and human LDH enzymes and suggest that BmLDH could be a suitable target for the development of selective antibabesial inhibitors.-Yu, L., Shen, Z., Liu, Q., Zhan, X., Luo, X., An, X., Sun, Y., Li, M., Wang, S., Nie, Z., Ao, Y., Zhao, Y., Peng, G., Ben Mamoun, C., He, L., Zhao, J. Crystal structures of Babesia microti lactate dehydrogenase BmLDH reveal a critical role for Arg99 in catalysis.

Selection and identification of a precocious line of Eimeria intestinalis with enlarged oocysts and deletion of one generation of schizogony.

Rabbit coccidiosis is a common parasitic disease and responsible for enormous economic losses in the rabbit industry. Eimeria intestinalis, one of the highly pathogenic and common Eimeria species infecting rabbits, is considered as an indispensable species for the development of live oocyst vaccines against rabbit coccidiosis. In this study, we report the successful selection of a precocious line (EIP8) from a wild-type strain of E. intestinalis (WT) by successively collecting and propagating the early excreted progeny oocysts. The EIP8 line had a prepatent period of only 132 h compared to 204 h for the WT. Oocysts of EIP8 were notably different from those produced by the WT strain by their significantly larger size (mean length: 29.3 vs 27.6 µm and mean width 20.5 vs 19.8 µm). Examination of tissue sections prepared from EIP8-infected rabbits revealed that this precocious line undergoes only two generations of schizogony before differentiating into gametocytes by 120 h post-infection. In contrast, WT parasites undergo three generations of schizogony and gametocytes are present by 168 h post-infection. EIP8 multiplication capacity reduced by more than 35-fold and a concomitant decrease in pathogenicity was detected. Interestingly, immunization with 103 or 104 EIP8 oocysts provided sufficient protection against homologous challenge with wild-type parasites, as body weight gain of immunized and challenged rabbits was similar to that of untreated animals, as well as more than 90% reduction of oocyst output was detected in immunized and challenged animals when compared to unimmunized and challenged animals. Together, these results show that the EIP8 precocious line of E. intestinalis is an attenuated immunogenic strain and a suitable candidate for the development of a live vaccine against rabbit coccidiosis.

Stable transfection of Eimeria necatrix through nucleofection of second generation merozoites.

Eimeria spp., the causative agents of coccidiosis, are the most common protozoan pathogens of chickens. Infection with these parasites can result in poor development or death of animals leading to a devastating economic impact on poultry production. The establishment of transfection protocols for genetic manipulation of Eimeria species and stable expression of genes would help advance the biology of these parasites as well as establish these organisms as novel vaccine delivery vehicles. Here, we report the selection of the first stable transgenic E. necatrix population, EnHA1, consitutively expressing the EYFP reporter following transfection of the 2nd generation merozoites with a linear DNA fragment harboring the EYFP reporter gene, the HA1 gene from the avian influenza virus H9N2 and the TgDHFR-TS selectable marker, which confers resistance to pyrimethamine. Transfected merozoites were inoculated into chickens via the cloacal route, and feces from 18 h to 72 h post inoculation were collected and subjected to subsequent serial passages, FACS sorting and pyrimethamine selection. A gradual increase in the number of EYFP-expressing sporulated oocysts was noticed with more than 90% EYFP + oocysts obtained after five passages. Immunofluorescence assay confirmed successful expression of the HA1 antigen in the EnHA1 population. The ability to genetically manipulate E. necatrix merozoites and express heterologous genes in this parasite will pave the way for possible use of this organism as a vaccine-delivery vehicle.

Insights into the evolution and drug susceptibility of Babesia duncani from the sequence of its mitochondrial and apicoplast genomes.

Babesia microti and Babesia duncani are the main causative agents of human babesiosis in the United States. While significant knowledge about B. microti has been gained over the past few years, nothing is known about B. duncani biology, pathogenesis, mode of transmission or sensitivity to currently recommended therapies. Studies in immunocompetent wild type mice and hamsters have shown that unlike B. microti, infection with B. duncani results in severe pathology and ultimately death. The parasite factors involved in B. duncani virulence remain unknown. Here we report the first known completed sequence and annotation of the apicoplast and mitochondrial genomes of B. duncani. We found that the apicoplast genome of this parasite consists of a 34 kb monocistronic circular molecule encoding functions that are important for apicoplast gene transcription as well as translation and maturation of the organelle's proteins. The mitochondrial genome of B. duncani consists of a 5.9 kb monocistronic linear molecule with two inverted repeats of 48 bp at both ends. Using the conserved cytochrome b (Cytb) and cytochrome c oxidase subunit I (coxI) proteins encoded by the mitochondrial genome, phylogenetic analysis revealed that B. duncani defines a new lineage among apicomplexan parasites distinct from B. microti, Babesia bovis, Theileria spp. and Plasmodium spp. Annotation of the apicoplast and mitochondrial genomes of B. duncani identified targets for development of effective therapies. Our studies set the stage for evaluation of the efficacy of these drugs alone or in combination against B. duncani in culture as well as in animal models.

Functional analysis of Plasmodium falciparum subpopulations associated with artemisinin resistance in Cambodia.

BACKGROUND: Plasmodium falciparum malaria is one of the most widespread parasitic infections in humans and remains a leading global health concern. Malaria elimination efforts are threatened by the emergence and spread of resistance to artemisinin-based combination therapy, the first-line treatment of malaria. Promising molecular markers and pathways associated with artemisinin drug resistance have been identified, but the underlying molecular mechanisms of resistance remains unknown. The genomic data from early period of emergence of artemisinin resistance (2008-2011) was evaluated, with aim to define k13 associated genetic background in Cambodia, the country identified as epicentre of anti-malarial drug resistance, through characterization of 167 parasite isolates using a panel of 21,257 SNPs. RESULTS: Eight subpopulations were identified suggesting a process of acquisition of artemisinin resistance consistent with an emergence-selection-diffusion model, supported by the shifting balance theory. Identification of population specific mutations facilitated the characterization of a core set of 57 background genes associated with artemisinin resistance and associated pathways. The analysis indicates that the background of artemisinin resistance was not acquired after drug pressure, rather is the result of fixation followed by selection on the daughter subpopulations derived from the ancestral population. CONCLUSIONS: Functional analysis of artemisinin resistance subpopulations illustrates the strong interplay between ubiquitination and cell division or differentiation in artemisinin resistant parasites. The relationship of these pathways with the P. falciparum resistant subpopulation and presence of drug resistance markers in addition to k13, highlights the major role of admixed parasite population in the diffusion of artemisinin resistant background. The diffusion of resistant genes in the Cambodian admixed population after selection resulted from mating of gametocytes of sensitive and resistant parasite populations.

The antimalarial activity of the pantothenamide α-PanAm is via inhibition of pantothenate phosphorylation.

The biosynthesis of the major acyl carrier Coenzyme A from pantothenic acid (PA) is critical for survival of Plasmodium falciparum within human erythrocytes. Accordingly, a PA analog α-PanAm showed potent activity against blood stage parasites in vitro; however, its efficacy in vivo and its mode of action remain unknown. We developed a new synthesis route for α-PanAm and showed that the compound is highly effective against blood stages of drug-sensitive and -resistant P. falciparum strains, inhibits development of P. berghei in hepatocytes, and at doses up to 100 mg/kg also inhibits blood stage development of P. chabaudi in mice. We used yeast and its pantothenate kinase Cab1 as models to characterize mode of action of α-PanAm and found that α-PanAm inhibits yeast growth in a PA-dependent manner, and its potency increases dramatically in a yeast mutant with defective pantothenate kinase activity. Biochemical analyses using 14C-PA as a substrate demonstrated that α-PanAm is a competitive inhibitor of Cab1. Interestingly, biochemical and mass spectrometry analyses also showed that the compound is phosphorylated by Cab1. Together, these data suggest that α-PanAm exerts its antimicrobial activity by direct competition with the natural substrate PA for phosphorylation by the pantothenate kinase.

Babesia microti from humans and ticks hold a genomic signature of strong population structure in the United States.

BACKGROUND: Babesia microti is an emerging tick-borne apicomplexan parasite with increasing geographic range and incidence in the United States. The rapid expansion of B. microti into its current distribution in the northeastern USA has been due to the range expansion of the tick vector, Ixodes scapularis, upon which the causative agent is dependent for transmission to humans. RESULTS: To reconstruct the history of B. microti in the continental USA and clarify the evolutionary origin of human strains, we used multiplexed hybrid capture of 25 B. microti isolates obtained from I. scapularis and human blood. Despite low genomic variation compared with other Apicomplexa, B. microti was strongly structured into three highly differentiated genetic clusters in the northeastern USA. Bayesian analyses of the apicoplast genomes suggest that the origin of the current diversity of B. microti in northeastern USA dates back 46 thousand years with a signature of recent population expansion in the last 1000 years. Human-derived samples belonged to two rarely intermixing clusters, raising the possibility of highly divergent infectious phenotypes in humans. CONCLUSIONS: Our results validate the multiplexed hybrid capture strategy for characterizing genome-wide diversity and relatedness of B. microti from ticks and humans. We find strong population structure in B. microti samples from the Northeast indicating potential barriers to gene flow.

Whole genome mapping and re-organization of the nuclear and mitochondrial genomes of Babesia microti isolates.

Babesia microti is the primary causative agent of human babesiosis, an emerging pathogen that causes a malaria-like illness with possible fatal outcome in immunocompromised patients. The genome sequence of the B. microti R1 strain was reported in 2012 and revealed a distinct evolutionary path for this pathogen relative to that of other apicomplexa. Lacking from the first genome assembly and initial molecular analyses was information about the terminal ends of each chromosome, and both the exact number of chromosomes in the nuclear genome and the organization of the mitochondrial genome remained ambiguous. We have now performed various molecular analyses to characterize the nuclear and mitochondrial genomes of the B. microti R1 and Gray strains and generated high-resolution Whole Genome maps. These analyses show that the genome of B. microti consists of four nuclear chromosomes and a linear mitochondrial genome present in four different structural types. Furthermore, Whole Genome mapping allowed resolution of the chromosomal ends, identification of areas of misassembly in the R1 genome, and genomic differences between the R1 and Gray strains, which occur primarily in the telomeric regions. These studies set the stage for a better understanding of the evolution and diversity of this important human pathogen.

(1)H, (13)C, and (15)N chemical shift assignments for PfPMT, a phosphoethanolamine methyltransferase from Plasmodium falciparum.

Phosphoethanolamine methyltransferases (PMTs also known as PEAMTs) catalyze the three-step s-adenosyl-methionione-dependent methylation of phosphoethanolamine to form phosphocholine. These enzymes play an important function in the synthesis of phosphatidylcholine, the major phospholipid in the membranes of lower and higher eukaryotes, as well as in the production of the compatible solute and osmoprotectant glycine betaine in plants. Genetic studies in plants, Caenhorhabditis elegans and Plasmodium falciparum have demonstrated that disruption of PMT activity results in severe defects in important cellular processes such as development, replication, survival and sexual maturation and differentiation. Here we report chemical shift assignments for PfPMT, the PMT from Plasmodium falciparum. X-ray crystal structures have been recently reported for complexes of PfPMT, but the structure of the apoenzyme remains unknown. The solution structure of the apoenzyme will help to elucidate important details of the mechanism of substrate binding by PfPMT, as residues comprising the substrate binding site are inaccessible to solvent in the conformation evident in the available crystal structures. In addition to enabling determination of the solution structure of the apoenzyme, the assignments will facilitate additional investigations into the interaction of PfPMT with its substrates and inhibitors.

Identification of gene encoding Plasmodium knowlesi phosphatidylserine decarboxylase by genetic complementation in yeast and characterization of in vitro maturation of encoded enzyme.

The 23-megabase genome of Plasmodium falciparum, the causative agent of severe human malaria, contains ∼5300 genes, most of unknown function or lacking homologs in other organisms. Identification of these gene functions will help in the discovery of novel targets for the development of antimalarial drugs and vaccines. The P. falciparum genome is unusually A+T-rich, which hampers cloning and expressing these genes in heterologous systems for functional analysis. The large repertoire of genetic tools available for Saccharomyces cerevisiae makes this yeast an ideal system for large scale functional complementation analyses of parasite genes. Here, we report the construction of a cDNA library from P. knowlesi, which has a lower A+T content compared with P. falciparum. This library was applied in a yeast complementation assay to identify malaria genes involved in the decarboxylation of phosphatidylserine. Transformation of a psd1Δpsd2Δdpl1Δ yeast strain, defective in phosphatidylethanolamine synthesis, with the P. knowlesi library led to identification of a new parasite phosphatidylserine decarboxylase (PkPSD). Unlike phosphatidylserine decarboxylase enzymes from other eukaryotes that are tightly associated with membranes, the PkPSD enzyme expressed in yeast was equally distributed between membrane and soluble fractions. In vitro studies reveal that truncated forms of PkPSD are soluble and undergo auto-endoproteolytic maturation in a phosphatidylserine-dependent reaction that is inhibited by other anionic phospholipids. This study defines a new system for probing the function of Plasmodium genes by library-based genetic complementation and its usefulness in revealing new biochemical properties of encoded proteins.