Biochemistry and Pharmacology - Research Publications
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ItemNo Preview AvailableNonsense-mediated decay machinery in Plasmodium falciparum is inefficient and non-essential(AMER SOC MICROBIOLOGY, 2023-08-24)Nonsense-mediated decay (NMD) is a conserved mRNA quality control process that eliminates transcripts bearing a premature termination codon. In addition to its role in removing erroneous transcripts, NMD is involved in post-transcriptional regulation of gene expression via programmed intron retention in metazoans. The apicomplexan parasite Plasmodium falciparum shows relatively high levels of intron retention, but it is unclear whether these variant transcripts are functional targets of NMD. In this study, we use CRISPR-Cas9 to disrupt and epitope-tag the P. falciparum orthologs of two core NMD components: PfUPF1 (PF3D7_1005500) and PfUPF2 (PF3D7_0925800). We localize both PfUPF1 and PfUPF2 to puncta within the parasite cytoplasm and show that these proteins interact with each other and other mRNA-binding proteins. Using RNA-seq, we find that although these core NMD orthologs are expressed and interact in P. falciparum, they are not required for degradation of nonsense transcripts. Furthermore, our work suggests that the majority of intron retention in P. falciparum has no functional role and that NMD is not required for parasite growth ex vivo. IMPORTANCE In many organisms, the process of destroying nonsense transcripts is dependent on a small set of highly conserved proteins. We show that in the malaria parasite, these proteins do not impact the abundance of nonsense transcripts. Furthermore, we demonstrate efficient CRISPR-Cas9 editing of the malaria parasite using commercial Cas9 nuclease and synthetic guide RNA, streamlining genomic modifications in this genetically intractable organism.
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ItemIn silico prediction of antimalarial drug target candidates(ELSEVIER SCI LTD, 2012-12)The need for new antimalarials is persistent due to the emergence of drug resistant parasites. Here we aim to identify new drug targets in Plasmodium falciparum by phylogenomics among the Plasmodium spp. and comparative genomics to Homo sapiens. The proposed target discovery pipeline is largely independent of experimental data and based on the assumption that P. falciparum proteins are likely to be essential if (i) there are no similar proteins in the same proteome and (ii) they are highly conserved across the malaria parasites of mammals. This hypothesis was tested using sequenced Saccharomycetaceae species as a touchstone. Consecutive filters narrowed down the potential target space of P. falciparum to proteins that are likely to be essential, matchless in the human proteome, expressed in the blood stages of the parasite, and amenable to small molecule inhibition. The final set of 40 candidate drug targets was significantly enriched in essential proteins and comprised proven targets (e.g. dihydropteroate synthetase or enzymes of the non-mevalonate pathway), targets currently under investigation (e.g. calcium-dependent protein kinases), and new candidates of potential interest such as phosphomannose isomerase, phosphoenolpyruvate carboxylase, signaling components, and transporters. The targets were prioritized based on druggability indices and on the availability of in vitro assays. Potential inhibitors were inferred from similarity to known targets of other disease systems. The identified candidates from P. falciparum provide insight into biochemical peculiarities and vulnerable points of the malaria parasite and might serve as starting points for rational drug discovery.
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ItemBiosynthesis, Localization, and Macromolecular Arrangement of the Plasmodium falciparum Translocon of Exported Proteins (PTEX)(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2012-03-09)To survive within its host erythrocyte, Plasmodium falciparum must export hundreds of proteins across both its parasite plasma membrane and surrounding parasitophorous vacuole membrane, most of which are likely to use a protein complex known as PTEX (Plasmodium translocon of exported proteins). PTEX is a putative protein trafficking machinery responsible for the export of hundreds of proteins across the parasitophorous vacuole membrane and into the human host cell. Five proteins are known to comprise the PTEX complex, and in this study, three of the major stoichiometric components are investigated including HSP101 (a AAA(+) ATPase), a protein of no known function termed PTEX150, and the apparent membrane component EXP2. We show that these proteins are synthesized in the preceding schizont stage (PTEX150 and HSP101) or even earlier in the life cycle (EXP2), and before invasion these components reside within the dense granules of invasive merozoites. From these apical organelles, the protein complex is released into the host cell where it resides with little turnover in the parasitophorous vacuole membrane for most of the remainder of the following cell cycle. At this membrane, PTEX is arranged in a stable macromolecular complex of >1230 kDa that includes an ∼600-kDa apparently homo-oligomeric complex of EXP2 that can be separated from the remainder of the PTEX complex using non-ionic detergents. Two different biochemical methods undertaken here suggest that PTEX components associate as EXP2-PTEX150-HSP101, with EXP2 associating with the vacuolar membrane. Collectively, these data support the hypothesis that EXP2 oligomerizes and potentially forms the putative membrane-spanning pore to which the remainder of the PTEX complex is attached.
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ItemNo Preview AvailableConservation, abundance, glycosylation profile, and localization of the TSP protein family in Cryptosporidium parvum(ELSEVIER, 2023-03)Cryptosporidium parvum is a zoonotic apicomplexan parasite and a common cause of diarrheal disease worldwide. The development of vaccines to prevent or limit infection remains an important goal for tackling cryptosporidiosis. At present, the only approved vaccine against any apicomplexan parasite targets a conserved adhesin possessing a thrombospondin repeat domain. C. parvum possesses 12 orthologous thrombospondin repeat domain-containing proteins known as CpTSP1-12, though little is known about these potentially important antigens. Here, we explore the architecture and conservation of the CpTSP protein family, as well as their abundance at the protein level within the sporozoite stage of the life cycle. We examine the glycosylation states of these proteins using a combination of glycopeptide enrichment techniques to demonstrate that these proteins are modified with C-, O-, and N-linked glycans. Using expansion microscopy, and an antibody against the C-linked mannose that is unique to the CpTSP protein family within C. parvum, we show that these proteins are found both on the cell surface and in structures that resemble the secretory pathway of C. parvum sporozoites. Finally, we generated a polyclonal antibody against CpTSP1 to show that it is found at the cell surface and within micronemes, in a pattern reminiscent of other apicomplexan motility-associated adhesins, and is present both in sporozoites and meronts. This work sheds new light on an understudied family of C. parvum proteins that are likely to be important to both parasite biology and the development of vaccines against cryptosporidiosis.
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Item3, 2, 1, go! Cryptosporidium counts down to sex(PUBLIC LIBRARY SCIENCE, 2022-05)Cryptosporidium is a leading cause of death from childhood diarrhea, but its biology is poorly understood. A recent study in PLOS Biology reveals hitherto unknown aspects of the parasite's life cycle that may lead to improvements in ex vivo culture.
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ItemNo Preview AvailableChromosome-level genome of Schistosoma haematobium underpins genome-wide explorations of molecular variation(PUBLIC LIBRARY SCIENCE, 2022-02)Urogenital schistosomiasis is caused by the blood fluke Schistosoma haematobium and is one of the most neglected tropical diseases worldwide, afflicting > 100 million people. It is characterised by granulomata, fibrosis and calcification in urogenital tissues, and can lead to increased susceptibility to HIV/AIDS and squamous cell carcinoma of the bladder. To complement available treatment programs and break the transmission of disease, sound knowledge and understanding of the biology and ecology of S. haematobium is required. Hybridisation/introgression events and molecular variation among members of the S. haematobium-group might effect important biological and/or disease traits as well as the morbidity of disease and the effectiveness of control programs including mass drug administration. Here we report the first chromosome-contiguous genome for a well-defined laboratory line of this blood fluke. An exploration of this genome using transcriptomic data for all key developmental stages allowed us to refine gene models (including non-coding elements) and annotations, discover 'new' genes and transcription profiles for these stages, likely linked to development and/or pathogenesis. Molecular variation within S. haematobium among some geographical locations in Africa revealed unique genomic 'signatures' that matched species other than S. haematobium, indicating the occurrence of introgression events. The present reference genome (designated Shae.V3) and the findings from this study solidly underpin future functional genomic and molecular investigations of S. haematobium and accelerate systematic, large-scale population genomics investigations, with a focus on improved and sustained control of urogenital schistosomiasis.
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ItemThe Plasmodium rhoptry associated protein complex is important for parasitophorous vacuole membrane structure and intraerythrocytic parasite growth(WILEY, 2017-08)Plasmodium parasites must invade erythrocytes in order to cause the disease malaria. The invasion process involves the coordinated secretion of parasite proteins from apical organelles that include the rhoptries. The rhoptry is comprised of two compartments: the neck and the bulb. Rhoptry neck proteins are involved in host cell adhesion and formation of the tight junction that forms between the invading parasite and erythrocyte, whereas the role of rhoptry bulb proteins remains ill-defined due to the lack of functional studies. In this study, we show that the rhoptry-associated protein (RAP) complex is not required for rhoptry morphology or erythrocyte invasion. Instead, post-invasion when the parasite is bounded by a parasitophorous vacuolar membrane (PVM), the RAP complex facilitates the survival of the parasite in its new intracellular environment. Consequently, conditional knockdown of members of the RAP complex leads to altered PVM structure, delayed intra-erythrocytic growth, and reduced parasitaemias in infected mice. This study provides evidence that rhoptry bulb proteins localising to the parasite-host cell interface are not simply by-products of the invasion process but contribute to the growth of Plasmodium in vivo.
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ItemNo Preview AvailableThe Novel bis-1,2,4-Triazine MIPS-0004373 Demonstrates Rapid and Potent Activity against All Blood Stages of the Malaria Parasite(AMER SOC MICROBIOLOGY, 2021-10)Novel bis-1,2,4-triazine compounds with potent in vitro activity against Plasmodium falciparum parasites were recently identified. The bis-1,2,4-triazines represent a unique antimalarial pharmacophore and are proposed to act by a novel but as-yet-unknown mechanism of action. This study investigated the activity of the bis-1,2,4-triazine MIPS-0004373 across the mammalian life cycle stages of the parasite and profiled the kinetics of activity against blood and transmission stage parasites in vitro and in vivo. MIPS-0004373 demonstrated rapid and potent activity against P. falciparum, with excellent in vitro activity against all asexual blood stages. Prolonged in vitro drug exposure failed to generate stable resistance de novo, suggesting a low propensity for the emergence of resistance. Excellent activity was observed against sexually committed ring stage parasites, but activity against mature gametocytes was limited to inhibiting male gametogenesis. Assessment of liver stage activity demonstrated good activity in an in vitro P. berghei model but no activity against Plasmodium cynomolgi hypnozoites or liver schizonts. The bis-1,2,4-triazine MIPS-0004373 efficiently cleared an established P. berghei infection in vivo, with efficacy similar to that of artesunate and chloroquine and a recrudescence profile comparable to that of chloroquine. This study demonstrates the suitability of bis-1,2,4-triazines for further development toward a novel treatment for acute malaria.
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ItemPlasmodium sexual differentiation: how to make a female(WILEY, 2019-12)Sexual development is integral to the transmission of Plasmodium parasites between vertebrates and mosquitos. Recent years have seen great advances in understanding the gene expression that underlies commitment of asexual parasites to differentiate into sexual gametocyte stages, then how they mature and form gametes once inside a mosquito. Less well understood is how parasites differentially control development to become males or females. Plasmodium parasites are haploid at the time of sexual differentiation, but a clonal haploid line can produce both male and female gametocytes, so they presumably lack the sex-determining alleles present in some other eukaryotes. Though the molecular switch to initiate male or female development remains hidden, recent studies reveal regulatory proteins needed for the sex-specific maturation of male and female gametocytes. Yuda and collaborators report the characterization of a transcription factor necessary for female gametocyte maturation. With renewed attention on malaria elimination, sex has been an increasing focus because transmission-blocking strategies are likely to be an important component of elimination efforts.
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ItemFunctional Characterization of the m6A-Dependent Translational Modulator PfYTH.2 in the Human Malaria Parasite(AMER SOC MICROBIOLOGY, 2021-04-27)Posttranscriptional regulation of gene expression is central to the development and replication of the malaria parasite, Plasmodium falciparum, within its human host. The timely coordination of RNA maturation, homeostasis, and protein synthesis relies on the recruitment of specific RNA-binding proteins to their cognate target mRNAs. One possible mediator of such mRNA-protein interactions is the N6-methylation of adenosines (m6A), a prevalent mRNA modification of parasite mRNA transcripts. Here, we used RNA protein pulldowns, RNA modification mass spectrometry, and quantitative proteomics to identify two P. falciparum YTH domain proteins (PfYTH.1 and PfYTH.2) as m6A-binding proteins during parasite blood-stage development. Interaction proteomics revealed that PfYTH.2 associates with the translation machinery, including multiple subunits of the eukaryotic initiation factor 3 (eIF3) and poly(A)-binding proteins. Furthermore, knock sideways of PfYTH.2 coupled with ribosome profiling showed that this m6A reader is essential for parasite survival and is a repressor of mRNA translation. Together, these data reveal an important missing link in the m6A-mediated mechanism controlling mRNA translation in a unicellular eukaryotic pathogen.IMPORTANCE Infection with the unicellular eukaryotic pathogen Plasmodium falciparum causes malaria, a mosquito-borne disease affecting more than 200 million and killing 400,000 people each year. Underlying the asexual replication within human red blood cells is a tight regulatory network of gene expression and protein synthesis. A widespread mechanism of posttranscriptional gene regulation is the chemical modification of adenosines (m6A), through which the fate of individual mRNA transcripts can be changed. Here, we report on the protein machinery that "reads" this modification and "translates" it into a functional outcome. We provide mechanistic insight into one m6A reader protein and show that it interacts with the translational machinery and acts as a repressor of mRNA translation. This m6A-mediated phenotype has not been described in other eukaryotes as yet, and the functional characterization of the m6A interactome will ultimately open new avenues to combat the disease.
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