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1.
Mol Microbiol ; 115(5): 1005-1024, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33368818

RESUMEN

Differentiation from asexual blood stages to mature sexual gametocytes is required for the transmission of malaria parasites. Here, we report that the ApiAP2 transcription factor, PfAP2-G2 (PF3D7_1408200) plays a critical role in the maturation of Plasmodium falciparum gametocytes. PfAP2-G2 binds to the promoters of a wide array of genes that are expressed at many stages of the parasite life cycle. Interestingly, we also find binding of PfAP2-G2 within the gene body of almost 3,000 genes, which strongly correlates with the location of H3K36me3 and several other histone modifications as well as Heterochromatin Protein 1 (HP1), suggesting that occupancy of PfAP2-G2 in gene bodies may serve as an alternative regulatory mechanism. Disruption of pfap2-g2 does not impact asexual development, but the majority of sexual parasites are unable to mature beyond stage III gametocytes. The absence of pfap2-g2 leads to overexpression of 28% of the genes bound by PfAP2-G2 and none of the PfAP2-G2 bound genes are downregulated, suggesting that it is a repressor. We also find that PfAP2-G2 interacts with chromatin remodeling proteins, a microrchidia (MORC) protein, and another ApiAP2 protein (PF3D7_1139300). Overall our data demonstrate that PfAP2-G2 establishes an essential gametocyte maturation program in association with other chromatin-related proteins.


Asunto(s)
Células Germinativas/crecimiento & desarrollo , Malaria Falciparum/parasitología , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Factores de Transcripción/metabolismo , Gametogénesis , Regulación del Desarrollo de la Expresión Génica , Células Germinativas/metabolismo , Humanos , Estadios del Ciclo de Vida , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Factores de Transcripción/genética
2.
Malar J ; 20(1): 317, 2021 Jul 14.
Artículo en Inglés | MEDLINE | ID: mdl-34261498

RESUMEN

BACKGROUND: Gene Regulatory Networks (GRN) produce powerful insights into transcriptional regulation in cells. The power of GRNs has been underutilized in malaria research. The Arboreto library was incorporated into a user-friendly web-based application for malaria researchers ( http://malboost.bi.up.ac.za ). This application will assist researchers with gaining an in depth understanding of transcriptomic datasets. METHODS: The web application for MALBoost was built in Python-Flask with Redis and Celery workers for queue submission handling, which execute the Arboreto suite algorithms. A submission of 5-50 regulators and total expression set of 5200 genes is permitted. The program runs in a point-and-click web user interface built using Bootstrap4 templates. Post-analysis submission, users are redirected to a status page with run time estimates and ultimately a download button upon completion. Result updates or failure updates will be emailed to the users. RESULTS: A web-based application with an easy-to-use interface is presented with a use case validation of AP2-G and AP2-I. The validation set incorporates cross-referencing with ChIP-seq and transcriptome datasets. For AP2-G, 5 ChIP-seq targets were significantly enriched with seven more targets presenting with strong evidence of validated targets. CONCLUSION: The MALBoost application provides the first tool for easy interfacing and efficiently allows gene regulatory network construction for Plasmodium. Additionally, access is provided to a pre-compiled network for use as reference framework. Validation for sexually committed ring-stage parasite targets of AP2-G, suggests the algorithm was effective in resolving "traditionally" low-level signatures even in bulk RNA datasets.


Asunto(s)
Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes , Plasmodium falciparum/genética , Programas Informáticos , Biología Computacional , Internet , Malaria/parasitología
3.
BMC Genomics ; 20(1): 920, 2019 Dec 03.
Artículo en Inglés | MEDLINE | ID: mdl-31795940

RESUMEN

BACKGROUND: Malaria pathogenesis relies on sexual gametocyte forms of the malaria parasite to be transmitted between the infected human and the mosquito host but the molecular mechanisms controlling gametocytogenesis remains poorly understood. Here we provide a high-resolution transcriptome of Plasmodium falciparum as it commits to and develops through gametocytogenesis. RESULTS: The gametocyte-associated transcriptome is significantly different from that of the asexual parasites, with dynamic gene expression shifts characterizing early, intermediate and late-stage gametocyte development and results in differential timing for sex-specific transcripts. The transcriptional dynamics suggest strict transcriptional control during gametocytogenesis in P. falciparum, which we propose is mediated by putative regulators including epigenetic mechanisms (driving active repression of proliferation-associated processes) and a cascade-like expression of ApiAP2 transcription factors. CONCLUSIONS: The gametocyte transcriptome serves as the blueprint for sexual differentiation and will be a rich resource for future functional studies on this critical stage of Plasmodium development, as the intraerythrocytic transcriptome has been for our understanding of the asexual cycle.


Asunto(s)
Gametogénesis/genética , Regulación de la Expresión Génica , Plasmodium falciparum/genética , Transcripción Genética , Células Germinativas/metabolismo , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/metabolismo , Reproducción/genética , Reproducción Asexuada/genética , Diferenciación Sexual/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
4.
J Antimicrob Chemother ; 73(5): 1279-1290, 2018 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-29420756

RESUMEN

Objectives: Novel chemical tools to eliminate malaria should ideally target both the asexual parasites and transmissible gametocytes. Several imidazopyridazines (IMPs) and 2-aminopyridines (2-APs) have been described as potent antimalarial candidates targeting lipid kinases. However, these have not been extensively explored for stage-specific inhibition of gametocytes in Plasmodium falciparum parasites. Here we provide an in-depth evaluation of the gametocytocidal activity of compounds from these chemotypes and identify novel starting points for dual-acting antimalarials. Methods: We evaluated compounds against P. falciparum gametocytes using several assay platforms for cross-validation and stringently identified hits that were further profiled for stage specificity, speed of action and ex vivo efficacy. Physicochemical feature extraction and chemogenomic fingerprinting were applied to explore the kinase inhibition susceptibility profile. Results: We identified 34 compounds with submicromolar activity against late stage gametocytes, validated across several assay platforms. Of these, 12 were potent at <100 nM (8 were IMPs and 4 were 2-APs) and were also active against early stage gametocytes and asexual parasites, with >1000-fold selectivity towards the parasite over mammalian cells. Front-runner compounds targeted mature gametocytes within 48 h and blocked transmission to mosquitoes. The resultant chemogenomic fingerprint of parasites treated with the lead compounds revealed the importance of targeting kinases in asexual parasites and gametocytes. Conclusions: This study encompasses an in-depth evaluation of the kinase inhibitor space for gametocytocidal activity. Potent lead compounds have enticing dual activities and highlight the importance of targeting the kinase superfamily in malaria elimination strategies.


Asunto(s)
Aminopiridinas/farmacología , Antimaláricos/farmacología , Fosfotransferasas/antagonistas & inhibidores , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/enzimología , Inhibidores de Proteínas Quinasas/farmacología , Aminopiridinas/química , Aminopiridinas/aislamiento & purificación , Antimaláricos/química , Antimaláricos/aislamiento & purificación , Supervivencia Celular/efectos de los fármacos , Concentración 50 Inhibidora , Pruebas de Sensibilidad Parasitaria , Plasmodium falciparum/química , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/aislamiento & purificación
5.
Bioorg Med Chem ; 23(16): 5131-43, 2015 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-25684422

RESUMEN

A new series of potent potent aryl/alkylated (bis)urea- and (bis)thiourea polyamine analogues were synthesized and evaluated in vitro for their antiplasmodial activity. Altering the carbon backbone and terminal substituents increased the potency of analogues in the compound library 3-fold, with the most active compounds, 15 and 16, showing half-maximal inhibitory concentrations (IC50 values) of 28 and 30 nM, respectively, against various Plasmodium falciparum parasite strains without any cross-resistance. In vitro evaluation of the cytotoxicity of these analogues revealed marked selectivity towards targeting malaria parasites compared to mammalian HepG2 cells (>5000-fold lower IC50 against the parasite). Preliminary biological evaluation of the polyamine analogue antiplasmodial phenotype revealed that (bis)urea compounds target parasite asexual proliferation, whereas (bis)thiourea compounds of the same series have the unique ability to block transmissible gametocyte forms of the parasite, indicating pluripharmacology against proliferative and non-proliferative forms of the parasite. In this manuscript, we describe these results and postulate a refined structure-activity relationship (SAR) model for antiplasmodial polyamine analogues. The terminally aryl/alkylated (bis)urea- and (bis)thiourea-polyamine analogues featuring a 3-5-3 or 3-6-3 carbon backbone represent a structurally novel and distinct class of potential antiplasmodials with activities in the low nanomolar range, and high selectivity against various lifecycle forms of P. falciparum parasites.


Asunto(s)
Antimaláricos/química , Malaria Falciparum/tratamiento farmacológico , Plasmodium falciparum/efectos de los fármacos , Poliaminas/química , Tiourea/análogos & derivados , Urea/análogos & derivados , Alquilación , Antimaláricos/farmacología , Humanos , Estadios del Ciclo de Vida/efectos de los fármacos , Malaria Falciparum/parasitología , Plasmodium falciparum/crecimiento & desarrollo , Poliaminas/farmacología , Relación Estructura-Actividad , Tiourea/farmacología , Urea/farmacología
6.
ACS Infect Dis ; 7(10): 2904-2916, 2021 10 08.
Artículo en Inglés | MEDLINE | ID: mdl-34569223

RESUMEN

Kinase-focused inhibitors previously revealed compounds with differential activity against different stages of Plasmodium falciparum gametocytes. MMV666810, a 2-aminopyrazine, is more active on late-stage gametocytes, while a pyrazolopyridine, MMV674850, preferentially targets early-stage gametocytes. Here, we probe the biological mechanisms underpinning this differential stage-specific killing using in-depth transcriptome fingerprinting. Compound-specific chemogenomic profiles were observed with MMV674850 treatment associated with biological processes shared between asexual blood stage parasites and early-stage gametocytes but not late-stage gametocytes. MMV666810 has a distinct profile with clustered gene sets associated primarily with late-stage gametocyte development, including Ca2+-dependent protein kinases (CDPK1 and 5) and serine/threonine protein kinases (FIKK). Chemogenomic profiling therefore highlights essential processes in late-stage gametocytes, on a transcriptional level. This information is important to prioritize compounds that preferentially compromise late-stage gametocytes for further development as transmission-blocking antimalarials.


Asunto(s)
Antimaláricos , Malaria , Parásitos , Animales , Antimaláricos/farmacología , Humanos , Plasmodium falciparum/genética
7.
Nat Commun ; 11(1): 1503, 2020 03 20.
Artículo en Inglés | MEDLINE | ID: mdl-32198457

RESUMEN

In the malaria parasite Plasmodium falciparum, the switch from asexual multiplication to sexual differentiation into gametocytes is essential for transmission to mosquitos. The transcription factor PfAP2-G is a key determinant of sexual commitment that orchestrates this crucial cell fate decision. Here we identify the direct targets of PfAP2-G and demonstrate that it dynamically binds hundreds of sites across the genome. We find that PfAP2-G is a transcriptional activator of early gametocyte genes, and identify differences in PfAP2-G occupancy between gametocytes derived via next-cycle and same-cycle conversion. Our data implicate PfAP2-G not only as a transcriptional activator of gametocyte genes, but also as a potential regulator of genes important for red blood cell invasion. We also find that regulation by PfAP2-G requires interaction with a second transcription factor, PfAP2-I. These results clarify the functional role of PfAP2-G during sexual commitment and early gametocytogenesis.


Asunto(s)
Malaria/parasitología , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Sistemas CRISPR-Cas , Eritrocitos/parasitología , Regulación de la Expresión Génica , Genes Protozoarios/genética , Malaria/transmisión , Malaria Falciparum/parasitología , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/sangre , Factores de Transcripción/metabolismo
8.
Sci Rep ; 8(1): 16581, 2018 11 08.
Artículo en Inglés | MEDLINE | ID: mdl-30409996

RESUMEN

The life cycle of the malaria parasite Plasmodium falciparum is tightly regulated, oscillating between stages of intense proliferation and quiescence. Cyclic 48-hour asexual replication of Plasmodium is markedly different from cell division in higher eukaryotes, and mechanistically poorly understood. Here, we report tight synchronisation of malaria parasites during the early phases of the cell cycle by exposure to DL-α-difluoromethylornithine (DFMO), which results in the depletion of polyamines. This induces an inescapable cell cycle arrest in G1 (~15 hours post-invasion) by blocking G1/S transition. Cell cycle-arrested parasites enter a quiescent G0-like state but, upon addition of exogenous polyamines, re-initiate their cell cycle. This ability to halt malaria parasites at a specific point in their cell cycle, and to subsequently trigger re-entry into the cell cycle, provides a valuable framework to investigate cell cycle regulation in these parasites. We subsequently used gene expression analyses to show that re-entry into the cell cycle involves expression of Ca2+-sensitive (cdpk4 and pk2) and mitotic kinases (nima and ark2), with deregulation of the pre-replicative complex associated with expression of pk2. Changes in gene expression could be driven through transcription factors MYB1 and two ApiAP2 family members. This new approach to parasite synchronisation therefore expands our currently limited toolkit to investigate cell cycle regulation in malaria parasites.


Asunto(s)
Eflornitina/farmacología , Plasmodium falciparum/fisiología , Proteínas Protozoarias/genética , Ciclo Celular/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica/efectos de los fármacos , Quinasas Relacionadas con NIMA/genética , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Poliaminas/metabolismo , Factores de Transcripción/genética
9.
Sci Rep ; 7(1): 607, 2017 04 04.
Artículo en Inglés | MEDLINE | ID: mdl-28377601

RESUMEN

Gene expression in Plasmodia integrates post-transcriptional regulation with epigenetic marking of active genomic regions through histone post-translational modifications (PTMs). To generate insights into the importance of histone PTMs to the entire asexual and sexual developmental cycles of the parasite, we used complementary and comparative quantitative chromatin proteomics to identify and functionally characterise histone PTMs in 8 distinct life cycle stages of P. falciparum parasites. ~500 individual histone PTMs were identified of which 106 could be stringently validated. 46 individual histone PTMs and 30 co-existing PTMs were fully quantified with high confidence. Importantly, 15 of these histone PTMs are novel for Plasmodia (e.g. H3K122ac, H3K27me3, H3K56me3). The comparative nature of the data revealed a highly dynamic histone PTM landscape during life cycle development, with a set of histone PTMs (H3K4ac, H3K9me1 and H3K36me2) displaying a unique and conserved abundance profile exclusively during gametocytogenesis (P < 0.001). Euchromatic histone PTMs are abundant during schizogony and late gametocytes; heterochromatic PTMs mark early gametocytes. Collectively, this data provides the most accurate, complete and comparative chromatin proteomic analyses of the entire life cycle development of malaria parasites. A substantial association between histone PTMs and stage-specific transition provides insights into the intricacies characterising Plasmodial developmental biology.


Asunto(s)
Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Histonas/metabolismo , Plasmodium falciparum/fisiología , Procesamiento Proteico-Postraduccional , Proteómica , Secuencia de Aminoácidos , Biología Computacional/métodos , Proteínas de Unión al ADN/química , Perfilación de la Expresión Génica , Histonas/química , Estadios del Ciclo de Vida , Plasmodium falciparum/crecimiento & desarrollo , Proteómica/métodos , Transcriptoma
10.
Eur J Med Chem ; 90: 33-44, 2015 Jan 27.
Artículo en Inglés | MEDLINE | ID: mdl-25461309

RESUMEN

A series of dihydroartemisinyl-chalcone esters were synthesized through esterification of chalcones with dihydroartemisinin (DHA). The hybrids were screened against chloroquine (CQ) sensitive (3D7) and CQ resistant (W2) strains of intraerythrocytic Plasmodium falciparum parasites, and were all found to be active, with IC50 values ranging between 1.5 and 11 nM against both strains, with SI values over 5800. The esters featuring oxygenated aryl rings (7, 10 and 11), were found to be equipotent to DHA, but were 2-3 times more active than artesunate against the 3D7 and W2 strains of the malaria parasites. They were also screened in vitro against a panel of three cancer cell lines consisting of TK-10, UACC-62 and MCF-7. Compound 7, bearing a furan ring, displayed the most potent overall antitumor activity against all three cancer cell lines. TGA revealed that the targeted hybrids were all thermally more stable than DHA, which may be beneficial to the high temperature storage conditions that prevail in malaria endemic countries. During this study, ester 7 was identified as the best candidate for further investigation as a potential drug in search for new, safe and effective antimalarial drugs.


Asunto(s)
Antimaláricos/farmacología , Antineoplásicos/farmacología , Artemisininas/farmacología , Chalcona/farmacología , Ésteres/farmacología , Plasmodium falciparum/efectos de los fármacos , Antimaláricos/síntesis química , Antimaláricos/química , Antineoplásicos/síntesis química , Antineoplásicos/química , Artemisininas/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Chalcona/química , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Ésteres/síntesis química , Ésteres/química , Humanos , Células MCF-7 , Estructura Molecular , Pruebas de Sensibilidad Parasitaria , Relación Estructura-Actividad
11.
PLoS Negl Trop Dis ; 9(5): e0003711, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-25955414

RESUMEN

Human babesiosis, especially caused by the cattle derived Babesia divergens parasite, is on the increase, resulting in renewed attentiveness to this potentially life threatening emerging zoonotic disease. The molecular mechanisms underlying the pathophysiology and intra-erythrocytic development of these parasites are poorly understood. This impedes concerted efforts aimed at the discovery of novel anti-babesiacidal agents. By applying sensitive cell biological and molecular functional genomics tools, we describe the intra-erythrocytic development cycle of B. divergens parasites from immature, mono-nucleated ring forms to bi-nucleated paired piriforms and ultimately multi-nucleated tetrads that characterizes zoonotic Babesia spp. This is further correlated for the first time to nuclear content increases during intra-erythrocytic development progression, providing insight into the part of the life cycle that occurs during human infection. High-content temporal evaluation elucidated the contribution of the different stages to life cycle progression. Moreover, molecular descriptors indicate that B. divergens parasites employ physiological adaptation to in vitro cultivation. Additionally, differential expression is observed as the parasite equilibrates its developmental stages during its life cycle. Together, this information provides the first temporal evaluation of the functional transcriptome of B. divergens parasites, information that could be useful in identifying biological processes essential to parasite survival for future anti-babesiacidal discoveries.


Asunto(s)
Babesia/crecimiento & desarrollo , Babesiosis/parasitología , Enfermedades de los Bovinos/parasitología , Eritrocitos/parasitología , Estadios del Ciclo de Vida/genética , Animales , Bovinos/parasitología , Humanos , Garrapatas/parasitología , Transcriptoma/genética , Zoonosis/parasitología
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