Characterization of apicomplexan amino acid transporters (ApiATs) in the malaria parasite Plasmodium falciparum

ABSTRACTDuring the symptomatic human blood phase, malaria parasites replicate within red blood cells. Parasite proliferation relies on the uptake of nutrients, such as amino acids, from the host cell and the blood plasma, requiring transport across multiple membranes. Amino acids are delivered to the parasite through the parasite surrounding vacuolar compartment by specialized nutrient-permeable channels of the erythrocyte membrane and the parasitophorous vacuole membrane (PVM). However, further transport of amino acid across the parasite plasma membrane (PPM) is currently not well characterized. In this study, we focused on a family of Apicomplexan amino acid transporters (ApiATs) that comprises five members in Plasmodium falciparum. First, we localized four of the PfApiATs at the PPM using endogenous GFP-tagging. Next, we applied reverse genetic approaches to probe into their essentiality during asexual replication and gametocytogenesis. Upon inducible knockdown and targeted gene disruption a reduced asexual parasite proliferation was detected for PfApiAT2 and PfApiAT4. Functional inactivation of individual PfApiATs targeted in this study had no effect on gametocyte development. Our data suggest that individual PfApiATs are partially redundant during asexual in vitro proliferation and fully redundant during gametocytogenesis of P. falciparum parasites.IMPORTANCEMalaria parasites live and multiply inside cells. To facilitate their extremely fast intracellular proliferation they hijack and transform their host cells. This also requires the active uptake of nutrients, such as amino acids, from the host cell and the surrounding environment through various membranes that are the consequence of the parasite’s intracellular lifestyle. In this manuscript we focus on a family of putative amino acid transporters termed ApiAT. We show expression and localization of four transporters in the parasite plasma membrane of Plasmodium falciparum-infected erythrocytes that represent one interface of the pathogen to its host cell. We probed into the impact of functional inactivation of individual transporters on parasite growth in asexual and sexual blood stages of P. falciparum and reveal that only two of them show a modest but significant reduction in parasite proliferation but no impact on gametocytogenesis pointing towards redundancy within this transporter family.

A 39-Amino-Acid C-Terminal Truncation of GDV1 Disrupts Sexual Commitment in Plasmodium falciparum

ABSTRACT Malaria is a mosquito-borne disease caused by apicomplexan parasites of the genus Plasmodium. Completion of the parasite’s life cycle depends on the transmission of sexual stages, the gametocytes, from an infected human host to the mosquito vector. Sexual commitment occurs in only a small fraction of asexual blood-stage parasites and is initiated by external cues. The gametocyte development protein 1 (GDV1) has been described as a key facilitator to trigger sexual commitment. GDV1 interacts with the silencing factor heterochromatin protein 1 (HP1), leading to its dissociation from heterochromatic DNA at the genomic locus encoding AP2-G, the master transcription factor of gametocytogenesis. How this process is regulated is not known. In this study, we have addressed the role of protein kinases implicated in gametocyte development. From a pool of available protein kinase knockout (KO) lines, we identified two kinase knockout lines which fail to produce gametocytes. However, independent genetic verification revealed that both kinases are not required for gametocytogenesis but that both lines harbor the same mutation that leads to a truncation in the extreme C terminus of GDV1. Introduction of the identified nonsense mutation into the genome of wild-type parasite lines replicates the observed phenotype. Using a GDV1 overexpression line, we show that the truncation in the GDV1 C terminus does not interfere with the nuclear import of GDV1 or its interaction with HP1 in vitro but appears to be important to sustain GDV1 protein levels and thereby sexual commitment. IMPORTANCE Transmission of malaria-causing Plasmodium species by mosquitos requires the parasite to change from a continuously growing asexual parasite form growing in the blood to a sexually differentiated form, the gametocyte. Only a small subset of asexual parasites differentiates into gametocytes that are taken up by the mosquito. Transmission represents a bottleneck in the life cycle of the parasite, so a molecular understanding of the events that lead to stage conversion may identify novel intervention points. Here, we screened a subset of kinases we hypothesized to play a role in this process. While we did not identify kinases required for sexual conversion, we identified a mutation in the C terminus of the gametocyte development 1 protein (GDV1), which abrogates sexual development. The mutation destabilizes the protein but not its interaction with its cognate binding partner HP1. This suggests an important role for the GDV1 C terminus beyond trafficking and protein stability.

Antibody Responses to Crude Gametocyte Extract Predict Plasmodium falciparum Gametocyte Carriage in Kenya

BackgroundMalaria caused by Plasmodium falciparum remains a serious global public health challenge especially in Africa. Interventions that aim to reduce malaria transmission by targeting the gametocyte reservoir are key to malaria elimination and/or eradication. However, factors that are associated with gametocyte carriage have not been fully explored. Consequently, identifying predictors of the infectious reservoir is fundamental in the elimination campaign.MethodsWe cultured P. falciparum NF54 gametocytes (to stage V) and prepared crude gametocyte extract. Samples from a total of 687 participants (aged 6 months to 67 years) representing two cross-sectional study cohorts in Kilifi, Kenya were used to assess IgG antibody responses by ELISA. We also analyzed IgG antibody responses to the blood-stage antigen AMA1 as a marker of asexual parasite exposure. Gametocytemia and asexual parasitemia data quantified by microscopy and molecular detection (QT-NASBA) were used to determine the relationship with antibody responses, season, age, and transmission setting. Multivariable logistic regression models were used to study the association between antibody responses and gametocyte carriage. The predictive power of the models was tested using the receiver operating characteristic (ROC) curve.ResultsMultivariable logistic regression analysis showed that IgG antibody response to crude gametocyte extract predicted both microscopic (OR=1.81 95% CI: 1.06–3.07, p=0.028) and molecular (OR=1.91, 95% CI: 1.11–3.29, p=0.019) P. falciparum gametocyte carriage. Antibody responses to AMA1 were also associated with both microscopic (OR=1.61 95% CI: 1.08–2.42, p=0.020) and molecular (OR=3.73 95% CI: 2.03–6.74, p<0.001) gametocytemia. ROC analysis showed that molecular (AUC=0.897, 95% CI: 0.868–0.926) and microscopic (AUC=0.812, 95% CI: 0.758–0.865) multivariable models adjusted for gametocyte extract showed very high predictive power. Molecular (AUC=0.917, 95% CI: 0.891–0.943) and microscopic (AUC=0.806, 95% CI: 0.755–0.858) multivariable models adjusted for AMA1 were equally highly predictive.ConclusionIn our study, it appears that IgG responses to crude gametocyte extract are not an independent predictor of gametocyte carriage after adjusting for AMA1 responses but may predict gametocyte carriage as a proxy marker of exposure to parasites. Serological responses to AMA1 or to gametocyte extract may facilitate identification of individuals within populations who contribute to malaria transmission and support implementation of transmission-blocking interventions.

A redox-active crosslinker reveals an essential and inhibitable oxidative folding network in the endoplasmic reticulum of malaria parasites

Malaria remains a major global health problem, creating a constant need for research to identify druggable weaknesses in P. falciparum biology. As important components of cellular redox biology, members of the Thioredoxin (Trx) superfamily of proteins have received interest as potential drug targets in Apicomplexans. However, the function and essentiality of endoplasmic reticulum (ER)-localized Trx-domain proteins within P. falciparum has not been investigated. We generated conditional mutants of the protein PfJ2—an ER chaperone and member of the Trx superfamily—and show that it is essential for asexual parasite survival. Using a crosslinker specific for redox-active cysteines, we identified PfJ2 substrates as PfPDI8 and PfPDI11, both members of the Trx superfamily as well, which suggests a redox-regulatory role for PfJ2. Knockdown of these PDIs in PfJ2 conditional mutants show that PfPDI11 may not be essential. However, PfPDI8 is required for asexual growth and our data suggest it may work in a complex with PfJ2 and other ER chaperones. Finally, we show that the redox interactions between these Trx-domain proteins in the parasite ER and their substrates are sensitive to small molecule inhibition. Together these data build a model for how Trx-domain proteins in the P. falciparum ER work together to assist protein folding and demonstrate the suitability of ER-localized Trx-domain proteins for antimalarial drug development.

4-Aminoquinoline-ferrocene Hybrids as Potential Antimalarials

Background: Malaria is a deadly disease. It is mostly treated using 4- aminoquinoline derivatives such as chloroquine etc. because it is well-tolerated, displays low toxicity, and after administration, it is rapidly absorbed. The combination of 4-aminoquinoline with other classes of antimalarial drugs has been reported to be an effective approach for the treatment of malaria. Furthermore, some patents reported hybrids 4-aminoquinolines containing ferrocene moiety with potent antimalarial activity. Objective: The objective of the current study is to prepare 4-aminoquinoline-ferrocene hybrids via esterification and amidation reactions. The compounds were characterized via FTIR, LC-MS and NMR spectroscopy. In vitro screening against chloroquine-sensitive P. falciparum parasite (NF54) at concentrations (1 μM and 5 μM) and an inhibitory concentration (full dose-response) was studied. Methods: The compounds were prepared via known reactions and monitored by Thin Layer Chromatography. The compounds were purified by column chromatography and characterized using FTIR, NMR and MS. In vitro antiplasmodial evaluation was performed against asexual parasite and chloroquine was used as a reference drug. Results: The percentage inhibition effects of the hybrid compounds were in a range of 97.9-102% at 5 μM and 36-96% at 1 μM. Furthermore, the IC50 values of the compounds were in the range of 0.7-1.6 μM when compared to the parent drug, 4-ferrocenylketobutanoic acid. Conclusion: The hybrid compounds displayed significant antimalarial activity when compared to the parent drug. However, they were not as effective as chloroquine on the drug-sensitive parasite. The findings revealed that 4-aminoquinolines and ferrocene are potential scaffolds for developing potent antimalarials.

Plasmodium falciparum sexual parasites regulate infected erythrocyte permeability

To ensure the transport of nutrients necessary for their survival, Plasmodium falciparum parasites increase erythrocyte permeability to diverse solutes. These new permeation pathways (NPPs) have been extensively characterized in the pathogenic asexual parasite stages, however the existence of NPPs has never been investigated in gametocytes, the sexual stages responsible for transmission to mosquitoes. Here, we show that NPPs are still active in erythrocytes infected with immature gametocytes and that this activity declines along gametocyte maturation. Our results indicate that NPPs are regulated by cyclic AMP (cAMP) signaling cascade, and that the decrease in cAMP levels in mature stages results in a slowdown of NPP activity. We also show that NPPs facilitate the uptake of artemisinin derivatives and that phosphodiesterase (PDE) inhibitors can reactivate NPPs and increase drug uptake in mature gametocytes. These processes are predicted to play a key role in P. falciparum gametocyte biology and susceptibility to antimalarials.

GDV1 C-terminal truncation of 39 amino acids disrupts sexual commitment in Plasmodium falciparum

Malaria is a mosquito-borne disease caused by apicomplexan parasites of the genus Plasmodium. Completion of the parasite’s life cycle depends on the transmission of sexual stages, the gametocytes, from an infected human host to the mosquito vector. Sexual commitment occurs in only a small fraction of asexual blood stage parasites and is initiated by external cues. The gametocyte development protein 1 (GDV1) has been described as a key facilitator to trigger sexual commitment. GDV1 interacts with the silencing factor heterochromatin protein 1 (HP1), leading to its dissociation from heterochromatic DNA at the genomic locus encoding AP2-G, the master transcription factor of gametocytogenesis. How this process is regulated is not known. In this study we have addressed the role of protein kinases implicated in gametocyte development. From a pool of available protein kinase KO lines, we identified two kinase knockout lines which fail to produce gametocytes. However, independent genetic verification revealed that both kinases are not required for gametocytogenesis but both lines harbour the same mutation that leads to a truncation in the extreme C-terminus of GDV1. Introduction of the identified nonsense mutation into the genome of wild type parasite lines replicates the observed phenotype. Using a GDV1 overexpression line we show that the truncation in the GDV1 C-terminus does neither interfere with the nuclear import of GDV1 nor its interaction with HP1 in vitro, but appears important to sustain GDV1 protein levels and thereby sexual commitment.ImportanceTransmission of malaria causing Plasmodium species by mosquitos requires the parasite to change from a continuously growing asexual parasite form growing in the blood, to a sexually differentiated form, the gametocyte. Only a small subset of asexual parasites differentiates into gametocytes that are taken up by the mosquito. Transmission represents a bottleneck in the lifecycle of the parasite, so a molecular understanding of the events that lead to stage conversion may identify novel intervention points. Here we screened a subset of kinases we hypothesized to play a role in this process. While we did not identify kinases required for sexual conversion, we identified a mutation in the C-terminus of the Gametocyte Development 1 protein (GDV1), which abrogates sexual development. The mutation destabilises the protein but not its interaction with its cognate binding partner HP1. This suggest an important role for the GDV1 C-terminus beyond trafficking and protein stability.

Genetic Diversity of Merozoite Surface Protein 2 (MSP2) Plasmodium falciparum Clinical Isolate in Wamena General Hospital

The genetic diversity of typical clinical isolated Plasmodium falciparum in the malaria population varies greatly, especially at the location where malaria disease were recorded at high incidence rate. MSP2 is known as glycoprotein expressed on the surface of merozoites, which is an antigenic protein and has a potential to act as vaccine candidate for malaria. The MSP2 gene has two main allelic groups called FC27 and 3D7/IC. Block 3 from MSP2 gene is the most polymorphic to describe the diversity of parasite populations. The P. falciparum parasite population is often characterized by wide genetic diversity in areas of high transmission intensity. Therefore, the study on P. falciparum diversity is useful to describe the level of malaria transmission. The study of genetic diversity focused on clinical isolated species at Wamena General Hospital was aimed to determine the presence of the MSP2 gene, variety of MSP2 gene allele and the dominant frequency of the MSP2 gene allele. This research has been carried out from March 2018 to February 2019 using a cross sectional approach. The research sample was taken and prepared from Wamena Regional Hospital and followed by the analyzing of DNA isolation, PCR, electrophoresis of the research samples was done at the genetic science laboratory in Jakarta, Indonesia. The samples studied were patients who met the inclusion criteria, namely a single P. falciparum infection with an asexual parasite density >1000 parasites/µl or >3+ (1-10 P/Lp), and were agreed to become respondents by signing an informed consent. A total of 26 clinical isolates of P. falciparum were isolated with the MSP2 gene distribution on the FC27 allele with the highest as many as 25 samples (96.2%), 22 samples (84.6%) of the 3D7 / IC allele while the mixture of the two alleles was 22 samples (84.6%). From a total of 26 samples, there were samples with the male gender category counted for 77.3% and female 41%. The results of the identification of clinical isolated P. falciparum at Wamena Hospital with a total of 26 samples were found in productive age, between 15-34 years with a single allele (95.8%), while 23 cases and mix (both alleles 87.5%) about 21 cases, meanwhile in cases of before-productive age, in which ages were 12 and 14 years of age with a single allele 100% (FC27) 2 cases and 50% (3D7/IC) found to be 1 case, The mixture of the two alleles is 50% was only 1 case and there was no sample at non-productive age observed. Key words: Malaria; MSP-2; P. falciparum; Wamena

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.