A Plasmodium falciparum lysophospholipase regulates fatty acid acquisition for membrane biogenesis to enable schizogonic asexual division

Phospholipid metabolism is crucial for membrane biogenesis and homeostasis during the intracellular life cycle of Plasmodium falciparum. To generate large amounts of phospholipids required during blood stages, the parasite massively scavenge, recycle and reassemble host lipids. P. falciparum possesses an unusual large number of lysophospholipases. However, their functional roles and importance remain to be elucidated. Here, we functionally characterized one of P. falciparum lysophospholipase (PfLPL3) (Gene ID PF3D7_1476800), to reveal its critical role in parasite propagation during asexual blood stages. We generated a transgenic parasite line using GFP-glmS C-terminal tagging approach, for localization as well as inducible knockdown of PfLPL3. PfLPL3 displayed a dynamic localization throughout asexual stages, mainly localizing in the host parasite interface: parasitophorous vacuole space and expanding into the tubulovesicular network within the host cell. Inducible knock-down of PfLPL3 hindered normal intraerythrocytic cycle, specifically causing disruption in parasite development from trophozoites to schizont, as well as reduction in number of merozoites progenies. Thus, down-regulation of PfLPL3 significantly inhibited parasite growth suggesting its critical role for proper parasite propagation during blood stages. Detailed lipidomic analyses showed that PfLPL3 generates fatty-acids for the synthesis of neutral lipids DAG and TAG, whilst controlling the timely synthesis of phospholipids that are crucial for membrane biogenesis required for merozoite development during asexual cycle. Setting up an in vitro activity based screening of Malaria Box allowed identification of specific inhibitors of PfLPL3 having potent parasitical efficacies. These compounds are pertinent both as anti-malarial drug candidates and chemical tools specifically targeting membrane biogenesis during asexual blood stages.

Expression patterns of Plasmodium falciparum clonally variant genes at the onset of a blood infection in non-immune humans

Clonally variant genes (CVGs) play fundamental roles in the adaptation of Plasmodium falciparum parasites to the fluctuating conditions of the human host. However, their expression patterns under the natural conditions of the blood circulation have been characterized in detail only for a few specific gene families. Here we provide a detailed characterization of the complete P. falciparum transcriptome across the full intraerythrocytic development cycle (IDC) at the onset of a blood infection in non-immune human volunteers. We found that the vast majority of transcriptional differences between parasites obtained from the volunteers and the parental parasite line maintained in culture occur in CVGs. Specifically, we observed a major increase in the transcript levels of most members of the pfmc-2tm and gbp families and of specific genes of other families, in addition to previously reported changes in var and clag3 genes expression. Large transcriptional differences correlate with changes in the distribution of heterochromatin, confirming their epigenetic nature. The analysis of parasites collected at different time points along the infection indicates that when parasites pass through transmission stages, the epigenetic memory at CVG loci is lost, resulting in a reset of their expression state and reestablishment of new epigenetic patterns.ImportanceThe ability of malaria parasites to adapt to changes in the human blood environment, where they produce long term infection associated with clinical symptoms, is fundamental for their survival. Clonally variant genes, regulated at the epigenetic level, play a major role in this adaptive process, as changes in the expression of these genes result in antigenic and functional alterations that enable immune evasion and provide phenotypic plasticity. However, the way these genes are expressed under the natural conditions of the human circulation or how their expression is affected by passage through transmission stages is not well understood. Here we provide a comprehensive characterization of the expression patterns of these genes at the onset of human blood infections, which reveals major differences with in vitro cultured parasites and also distinctive alterations between different families of clonally variant genes. We also show that epigenetic patterns are erased and reestablished during transmission stages.

A Novel Trichomonas vaginalis Surface Protein Modulates Parasite Attachment via Protein:Host Cell Proteoglycan Interaction

ABSTRACT Trichomonas vaginalis is a highly prevalent, sexually transmitted parasite which adheres to mucosal epithelial cells to colonize the human urogenital tract. Despite adherence being crucial for this extracellular parasite to thrive within the host, relatively little is known about the mechanisms or key molecules involved in this process. Here, we have identified and characterized a T. vaginalis hypothetical protein, TVAG_157210 (TvAD1), as a surface protein that plays an integral role in parasite adherence to the host. Quantitative proteomics revealed TvAD1 to be ∼4-fold more abundant in parasites selected for increased adherence (MA parasites) than the isogenic parental (P) parasite line. De novo modeling suggested that TvAD1 binds N-acetylglucosamine (GlcNAc), a sugar comprising host glycosaminoglycans (GAGs). Adherence assays utilizing GAG-deficient cell lines determined that host GAGs, primarily heparan sulfate (HS), mediate adherence of MA parasites to host cells. TvAD1 knockout (KO) parasites, generated using CRISPR-Cas9, were found to be significantly reduced in host cell adherence, a phenotype that is rescued by overexpression of TvAD1 in KO parasites. In contrast, there was no significant difference in parasite adherence to GAG-deficient lines by KO parasites compared with wild-type, which is contrary to that observed for KO parasites overexpressing TvAD1. Isothermal titration calorimetric (ITC) analysis showed that TvAD1 binds to HS, indicating that TvAD1 mediates host cell adherence via HS interaction. In addition to characterizing the role of TvAD1 in parasite adherence, these studies reveal a role for host GAG molecules in T. vaginalis adherence. IMPORTANCE The ability of the sexually transmitted parasite Trichomonas vaginalis to adhere to its human host is critical for establishing and maintaining an infection. Yet how parasites adhere to host cells is poorly understood. In this study, we employed a novel adherence selection method to identify proteins involved in parasite adherence to the host. This method led to the identification of a protein, with no previously known function, that is more abundant in parasites with increased capacity to bind host cells. Bioinformatic modeling and biochemical analyses revealed that this protein binds a common component on the host cell surface proteoglycans. Subsequent creation of parasites that lack this protein directly demonstrated that the protein mediates parasite adherence via an interaction with host cell proteoglycans. These findings both demonstrate a role for this protein in T. vaginalis adherence to the host and shed light on host cell molecules that participate in parasite colonization.

Disruption of Plasmodium falciparum histidine-rich protein 2 may affect haem metabolism in the blood stage

Background Haem is a key metabolic factor in the life cycle of the malaria parasite. In the blood stage, the parasite acquires host haemoglobin to generate amino acids for protein synthesis and the by-product haem for metabolic use. The malaria parasite can also synthesize haem de novo on its own. Plasmodium falciparum-specific histidine-rich protein 2 (PfHRP2) has a haem-binding site to mediate the formation of haemozoin, a biocrystallized form of haem aggregates. Notably, the gene regulates the mechanism of haemoglobin-derived haem metabolism and the de novo haem biosynthetic pathway in the Pfhrp2-disrupted parasite line during the intraerythrocytic stages. Methods The CRISPR/Cas9 system was used to disrupt the gene locus of Pfhrp2. DNA was extracted from the transgenic parasite, and PCR, Southern blotting and Western blotting were used to confirm the establishment of transgenic parasites. RNA-sequencing and comparative transcriptome analysis were performed to identify differences in gene expression between 3D7 and Pfhrp2--3D7 parasites. Results Pfhrp2- transgenic parasites were successfully established by the CRISPR/Cas9 system. A total of 964, 1261, 3138, 1064, 2512 and 1778 differentially expressed genes (DEGs) were identified in the six comparison groups, respectively, with 373, 520, 1499, 353, 1253 and 742 of these DEGs upregulated and 591, 741, 1639, 711, 1259 and 1036 of them downregulated, respectively. Five DEGs related to haem metabolism and synthesis were identified in the comparison groups at six time points (0, 8, 16, 24, 32, and 40 h after merozoite invasion). The genes encoding delta-aminolevulinic acid synthetase and ferrochelatase, both related to haem biosynthesis, were found to be significantly upregulated in the comparison groups, and those encoding haem oxygenase, stromal-processing peptidase and porphobilinogen deaminase were found to be significantly downregulated. No GO terms were significantly enriched in haem-related processes (Q value = 1). Conclusion Our data revealed changes in the transcriptome expression profile of the Pfhrp2--3D7 parasite during the intraerythrocytic stages. The findings provide insight at the gene transcript level that will facilitate further research on and development of anti-malaria drugs.

Disruption of Plasmodium falciparum histidine-rich protein II may affect haem metabolism in the blood stage

Background: Haem is a key metabolic factor in the life cycle of the malaria parasite. In the blood stage, the parasite acquires host haemoglobin to generate amino acids for protein synthesis and the by-product haem for metabolic use. The malaria parasite can also synthesize haem de novo by itself. Plasmodium falciparum-specific histidine-rich protein 2 (PfHRP2) has a haem-binding site to mediate the formation of haemozoin, a biocrystallized form of haem aggregates. Notably, the gene regulates the mechanism of haemoglobin-derived haem metabolism and the de novo haem biosynthetic pathway in the Pfhrp2-disrupted parasite line during the intraerythrocytic stages. Methods: The CRISPR/Cas9 system was used to disrupt the gene locus of Pfhrp2. DNA was extracted from the transgenic parasite, and polymerase chain reaction (PCR), Southern blotting and Western blotting were used to confirm the establishment of transgenic parasites. RNA-Seq and comparative transcriptome analysis were performed to identify differences in gene expression between 3D7 and Pfhrp2- 3D7 parasites.Results: Pfhrp2- transgenic parasites were successfully established by the CRISPR/Cas9 system. A total of 964, 1261, 3138, 1064, 2512, and 1778 differentially expressed genes (DEGs) were identified in the six comparison groups, and a total of 373, 520, 1499, 353, 1253, and 742 of the DEGs were upregulated, and 591, 741, 1639, 711, 1259, and 1036 of the DEGs were downregulated, respectively. Five DEGs related to haem metabolism and synthesis were identified in the comparison groups of six time points (0, 8, 16, 24, 32, and 40 h after merozoite invasion). The genes encoding ALAS and FC, related to haem biosynthesis, were found to be significantly upregulated in the comparison groups, and the HO, SPP, and PBGD genes were found to be significantly downregulated. No GO terms were significantly enriched in haem-related processes (Q value=1).Conclusion: Our data revealed changes in the transcriptome expression profile of the Pfhrp2-3D7 parasite during the intraerythrocytic stages. The above findings provide insight at the gene transcript level for further research and development of anti-malaria drugs.

An Effective Live Vaccine Strain Of Trypanosoma Cruzi Prevents Chagas Disease In The Mouse Model

Trypanosoma cruzi is the etiologic agent of Chagas disease for which there are no prophylactic vaccines. Cyclophilin 19 is a secreted cis-trans peptidyl isomerase expressed in all life stages of Trypanosoma cruzi, which in the insect stage leads to the inactivation of insect anti-parasitic peptides and parasite transformation and in intracellular amastigotes participates in generating ROS enhancing parasite growth. We have generated a parasite knock-out mutant of Cyp19 which fails to replicate in cell culture or in mice indicating that lack of Cyp19 is critical for infectivity. Knock-out parasites fail to replicate in or cause clinical disease in immune-deficient mice further validating their lack of virulence. Repeated inoculation of knock-out parasites into immuno-competent mice elicits parasite-specific antibodies and T-cell responses. Challenge of immunized mice with wild-type parasites is 100% effective at preventing disease. These results indicate that the knock-out parasite line is a live vaccine candidate for Chagas disease.

Disruption of Plasmodium falciparum histidine-rich protein II may affect haem metabolism in the blood stage

Background. Haem is a key metabolic factor in the life cycle of the malaria parasite. In the blood stage, the parasite acquires host haemoglobin to generate amino acids for protein synthesis and the by-product haem for metabolic use. The malaria parasite can also synthesize haem de novo by itself. Plasmodium falciparum-specific histidine-rich protein 2 (PfHRP2) has a haem-binding site to mediate the formation of haemozoin, a biocrystallized form of haem aggregates. Notably, the gene regulates the mechanism of haemoglobin-derived haem metabolism and the de novo haem biosynthetic pathway in the Pfhrp2-disrupted parasite line during the intraerythrocytic stages. Methods. The CRISPR/Cas9 system was used to disrupt the gene locus of Pfhrp2. DNA was extracted from the transgenic parasite, and polymerase chain reaction (PCR), Southern blotting and Western blotting were used to confirm the establishment of transgenic parasites. RNA-Seq and comparative transcriptome analysis were performed to identify differences in gene expression between 3D7 and Pfhrp2- 3D7 parasites.Results. Pfhrp2- transgenic parasites were successfully established by the CRISPR/Cas9 system. A total of 964, 1261, 3138, 1064, 2512, and 1778 differentially expressed genes (DEGs) were identified in the six comparison groups, and a total of 373, 520, 1499, 353, 1253, and 742 of the DEGs were upregulated, and 591, 741, 1639, 711, 1259, and 1036 of the DEGs were downregulated, respectively. Five DEGs related to haem metabolism and synthesis were identified in the comparison groups of six time points (0, 8, 16, 24, 32, and 40 h after merozoite invasion). The genes encoding ALAS and FC, related to haem biosynthesis, were found to be significantly upregulated in the comparison groups, and the HO, SPP, and PBGD genes were found to be significantly downregulated. No GO terms were significantly enriched in haem-related processes (Q value=1).Conclusion: In this study, our findings revealed changes in the transcriptome expression profile of the Pfhrp2-3D7 parasite during the intraerythrocytic stages. The results suggested that disruption of Pfhrp2 alters the parasite’s haem metabolic and biosynthesis pathways at the gene transcript level. A cooperative mechanism exists between the haem biosynthesis and metabolic pathways for parasite growth and survival in the blood stage. It is difficult to treat malaria patients by inhibiting only one pathway with traditional antimalarial drugs. The above findings provide insight at the gene transcript level for further research and development of anti-malaria drugs.

Atypical molecular basis for drug resistance to mitochondrial function inhibitors in Plasmodium falciparum

The continued emergence of drug-resistant Plasmodium falciparum parasites hinders global attempts to eradicate malaria, emphasizing the need to identify new antimalarial drugs. Attractive targets for chemotherapeutic intervention are the cytochrome (cyt) bc1 complex, which is an essential component of the mitochondrial electron transport chain (mtETC) necessary for ubiquinone recycling and mitochondrially localized dihydroorotate dehydrogenase (DHODH) critical for de novo pyrimidine synthesis. Despite the essentiality of this complex, resistance to a novel acridone class of compounds targeting cyt bc1 was readily attained, resulting in a parasite strain (SB1-A6) that was pan-resistant to both mtETC and DHODH inhibitors. Here we describe the molecular mechanism behind the resistance of the SB1-A6 parasite line which lacks the common cyt bc1 point mutations characteristic of resistance to mtETC inhibitors. Using Illumina whole-genome sequencing, we have identified both a copy number variation (∼2x) and a single-nucleotide polymorphism (C276F) associated with pfdhodh in SB1-A6. We have characterized the role of both genetic lesions by mimicking the copy number variation via episomal expression of pfdhodh and introducing the identified SNP using CRISPR/Cas9 and assessed their contributions to drug resistance. Although both of these genetic polymorphisms have been previously identified as contributing to both DSM-1 (1) and atovaquone resistance (2, 3), SB1-A6 represents a unique genotype in which both alterations are present in a single line, suggesting that the combination contributes to the pan-resistant phenotype. This novel mechanism of resistance to mtETC inhibition has critical implications for the development of future drugs targeting the bc1 complex or de novo pyrimidine synthesis that could help guide future anti-malarial combination therapies and reduce the rapid development of drug resistance in the field.

A Conditional Protein Degradation System To Study Essential Gene Function in Cryptosporidium parvum

ABSTRACT Cryptosporidium spp., protozoan parasites, are a leading cause of global diarrhea-associated morbidity and mortality in young children and immunocompromised individuals. The limited efficacy of the only available drug and lack of vaccines make it challenging to treat and prevent cryptosporidiosis. Therefore, the identification of essential genes and understanding their biological functions are critical for the development of new therapies. Currently, there is no genetic tool available to investigate the function of essential genes in Cryptosporidium spp. Here, we describe the development of the first conditional system in Cryptosporidium parvum. Our system utilizes the Escherichia coli dihydrofolate reductase degradation domain (DDD) and the stabilizing compound trimethoprim (TMP) for conditional regulation of protein levels in the parasite. We tested our system on the calcium-dependent protein kinase-1 (CDPK1), a leading drug target in C. parvum. By direct knockout strategy, we establish that cdpk1 is refractory to gene deletion, indicating its essentiality for parasite survival. Using CRISPR/Cas9, we generated transgenic parasites expressing CDPK1 with an epitope tag, and localization studies indicate its expression during asexual parasite proliferation. We then genetically engineered C. parvum to express CDPK1 tagged with DDD. We demonstrate that TMP can regulate CDPK1 levels in this stable transgenic parasite line, thus revealing the critical role of this kinase in parasite proliferation. Further, these transgenic parasites show TMP-mediated regulation of CDPK1 levels in vitro and an increased sensitivity to kinase inhibitor upon conditional knockdown. Overall, this study reports the development of a powerful conditional system that can be used to study essential genes in Cryptosporidium. IMPORTANCE Cryptosporidium parvum and Cryptosporidium hominis are leading pathogens responsible for diarrheal disease (cryptosporidiosis) and deaths in infants and children below 5 years of age. There are no effective treatment options and no vaccine for cryptosporidiosis. Therefore, there is an urgent need to identify essential gene targets and uncover their biological function to accelerate the development of new and effective anticryptosporidial drugs. Current genetic tool allows targeted disruption of gene function but leads to parasite lethality if the gene is essential for survival. In this study, we have developed a genetic tool for conditional degradation of proteins in Cryptosporidium spp., thus allowing us to study the function of essential genes. Our conditional system expands the molecular toolbox for Cryptosporidium, and it will help us to understand the biology of this important human diarrheal pathogen for the development of new drugs and vaccines.

Piperaquine resistant Cambodian Plasmodium falciparum clinical isolates: in vitro genotypic and phenotypic characterization

Background: High rates of dihydroartemisinin-piperaquine (DHA-PPQ) treatment failures have been documented for uncomplicated Plasmodium falciparum in Cambodia. It is essential to establish sensitive and reliable markers of PPQ resistance which can be adopted for monitoring the prevalence of drug resistance and guide drug policy change. Several genetic markers have been proposed such as copy numbers of P. falciparum multidrug resistance 1 ( pfmdr1 ) and plasmepsin 2 (PM2), and mutations in exonuclease (exo-E415G) and P. falciparum chloroquine resistance transporter (PfCRT) genes.Methods: To examine the relative contribution of each marker to PPQ resistance, in vitro culture and the PPQ survival assay were performed on seventeen P. falciparum isolates from northern Cambodia and the presence of exo-E415G and PfCRT mutations as well as PM2 copy number polymorphisms were determined. Parasites were then cloned by limiting dilution and the cloned parasites were tested for drug susceptibility. The efficacy of several drug combinations between standard clones and newly cloned P. falciparum Cambodian isolates was also measured.Results: The characterization of culture-adapted isolates revealed that exo-E415G and novel PfCRT mutations can confer PPQ-resistance, in the absence of PM2 amplification. In vitro testing of PPQ resistant parasites showed bimodal dose-response phenotype as previously reported in both Cambodian isolates and genome-edited Dd2 strains. Our finding is the first PPQ resistant clinical isolate with documented swollen digestive vacuole phenotype. From the field isolates, we were able to clone a new parasite line, 14-B5, which is sensitive to arteminsinin and piperaquine but resistant to chloroquine. Most of the drug combinations tested revealed antagonistic interaction against the 14-B5 line, indicating its different genetic background from other P. falciparum laboratory reference lines.Conclusions: Surveillance for PPQ resistance in regions that rely on DHA-PPQ as the first line treatment should depend on the monitoring of the reflective molecular markers. Bimodal dose response curves and swelling of the food vacuole can be used as PPQ resistant phenotype. The use of currently circulating parasite isolates is necessary for relevant drug combination development against current P. falciparum resistance profiles.