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1.
Cell ; 171(7): 1532-1544.e15, 2017 Dec 14.
Artículo en Inglés | MEDLINE | ID: mdl-29129376

RESUMEN

Transmission represents a population bottleneck in the Plasmodium life cycle and a key intervention target of ongoing efforts to eradicate malaria. Sexual differentiation is essential for this process, as only sexual parasites, called gametocytes, are infective to the mosquito vector. Gametocyte production rates vary depending on environmental conditions, but external stimuli remain obscure. Here, we show that the host-derived lipid lysophosphatidylcholine (LysoPC) controls P. falciparum cell fate by repressing parasite sexual differentiation. We demonstrate that exogenous LysoPC drives biosynthesis of the essential membrane component phosphatidylcholine. LysoPC restriction induces a compensatory response, linking parasite metabolism to the activation of sexual-stage-specific transcription and gametocyte formation. Our results reveal that malaria parasites can sense and process host-derived physiological signals to regulate differentiation. These data close a critical knowledge gap in parasite biology and introduce a major component of the sexual differentiation pathway in Plasmodium that may provide new approaches for blocking malaria transmission.


Asunto(s)
Lisofosfatidilcolinas/metabolismo , Malaria/parasitología , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/metabolismo , Animales , Femenino , Humanos , Malaria/inmunología , Redes y Vías Metabólicas , Ratones , Ratones Endogámicos C57BL , Plasmodium berghei/fisiología , Reproducción
2.
Proc Natl Acad Sci U S A ; 121(9): e2312987121, 2024 Feb 27.
Artículo en Inglés | MEDLINE | ID: mdl-38377214

RESUMEN

Babesiosis is an emerging zoonosis and widely distributed veterinary infection caused by 100+ species of Babesia parasites. The diversity of Babesia parasites and the lack of specific drugs necessitate the discovery of broadly effective antibabesials. Here, we describe a comparative chemogenomics (CCG) pipeline for the identification of conserved targets. CCG relies on parallel in vitro evolution of resistance in independent populations of Babesia spp. (B. bovis and B. divergens). We identified a potent antibabesial, MMV019266, from the Malaria Box, and selected for resistance in two species of Babesia. After sequencing of multiple independently derived lines in the two species, we identified mutations in a membrane-bound metallodependent phosphatase (phoD). In both species, the mutations were found in the phoD-like phosphatase domain. Using reverse genetics, we validated that mutations in bdphoD confer resistance to MMV019266 in B. divergens. We have also demonstrated that BdPhoD localizes to the endomembrane system and partially with the apicoplast. Finally, conditional knockdown and constitutive overexpression of BdPhoD alter the sensitivity to MMV019266 in the parasite. Overexpression of BdPhoD results in increased sensitivity to the compound, while knockdown increases resistance, suggesting BdPhoD is a pro-susceptibility factor. Together, we have generated a robust pipeline for identification of resistance loci and identified BdPhoD as a resistance mechanism in Babesia species.


Asunto(s)
Antiinfecciosos , Babesia , Babesiosis , Humanos , Babesia/genética , Fosfatasa Alcalina , Antiparasitarios/farmacología , Antiparasitarios/uso terapéutico , Babesiosis/tratamiento farmacológico , Babesiosis/parasitología , Genómica , Antiinfecciosos/farmacología
3.
PLoS Biol ; 21(1): e3001997, 2023 01.
Artículo en Inglés | MEDLINE | ID: mdl-36696650

RESUMEN

Twenty years ago, the first transcriptome of the intraerythrocytic developmental cycle of the malaria parasite Plasmodium falciparum was published in PLOS Biology. Since then, transcriptomics studies have transformed the study of parasite biology.


Asunto(s)
Parásitos , Plasmodium falciparum , Animales , Plasmodium falciparum/genética , Transcriptoma/genética , Parásitos/genética , Perfilación de la Expresión Génica , Biología
4.
PLoS Pathog ; 19(11): e1011585, 2023 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-37939134

RESUMEN

Natural killer (NK) cells lyse virus-infected cells and transformed cells through polarized delivery of lytic effector molecules into target cells. We have shown that NK cells lyse Plasmodium falciparum-infected red blood cells (iRBC) via antibody-dependent cellular cytotoxicity (ADCC). A high frequency of adaptive NK cells, with elevated intrinsic ADCC activity, in people chronically exposed to malaria transmission is associated with reduced parasitemia and resistance to disease. How NK cells bind to iRBC and the outcome of iRBC lysis by NK cells has not been investigated. We applied gene ablation in inducible erythrocyte precursors and antibody-blocking experiments with iRBC to demonstrate a central role of CD58 and ICAM-4 as ligands for adhesion by NK cells via CD2 and integrin αMß2, respectively. Adhesion was dependent on opsonization of iRBC by IgG. Live imaging and quantitative flow cytometry of NK-mediated ADCC toward iRBC revealed that damage to the iRBC plasma membrane preceded damage to P. falciparum within parasitophorous vacuoles (PV). PV were identified and tracked with a P.falciparum strain that expresses the PV membrane-associated protein EXP2 tagged with GFP. After NK-mediated ADCC, PV were either found inside iRBC ghosts or released intact and devoid of RBC plasma membrane. Electron microscopy images of ADCC cultures revealed tight NK-iRBC synapses and free vesicles similar in size to GFP+ PV isolated from iRBC lysates by cell sorting. The titer of IgG in plasma of malaria-exposed individuals that bound PV was two orders of magnitude higher than IgG that bound iRBC. This immune IgG stimulated efficient phagocytosis of PV by primary monocytes. The selective NK-mediated damage to iRBC, resulting in release of PV, and subsequent phagocytosis of PV by monocytes may combine for efficient killing and removal of intra-erythrocytic P.falciparum parasite. This mechanism may mitigate the inflammation and malaria symptoms during blood-stage P. falciparum infection.


Asunto(s)
Malaria Falciparum , Malaria , Humanos , Monocitos , Ligandos , Vacuolas , Malaria Falciparum/parasitología , Eritrocitos/parasitología , Células Asesinas Naturales , Plasmodium falciparum , Malaria/metabolismo , Fagocitosis , Inmunoglobulina G/metabolismo
5.
PLoS Biol ; 20(9): e3001816, 2022 09.
Artículo en Inglés | MEDLINE | ID: mdl-36137068

RESUMEN

Babesia is a genus of apicomplexan parasites that infect red blood cells in vertebrate hosts. Pathology occurs during rapid replication cycles in the asexual blood stage of infection. Current knowledge of Babesia replication cycle progression and regulation is limited and relies mostly on comparative studies with related parasites. Due to limitations in synchronizing Babesia parasites, fine-scale time-course transcriptomic resources are not readily available. Single-cell transcriptomics provides a powerful unbiased alternative for profiling asynchronous cell populations. Here, we applied single-cell RNA sequencing to 3 Babesia species (B. divergens, B. bovis, and B. bigemina). We used analytical approaches and algorithms to map the replication cycle and construct pseudo-synchronized time-course gene expression profiles. We identify clusters of co-expressed genes showing "just-in-time" expression profiles, with gradually cascading peaks throughout asexual development. Moreover, clustering analysis of reconstructed gene curves reveals coordinated timing of peak expression in epigenetic markers and transcription factors. Using a regularized Gaussian graphical model, we reconstructed co-expression networks and identified conserved and species-specific nodes. Motif analysis of a co-expression interactome of AP2 transcription factors identified specific motifs previously reported to play a role in DNA replication in Plasmodium species. Finally, we present an interactive web application to visualize and interactively explore the datasets.


Asunto(s)
Babesia , Babesia/genética , Eritrocitos/parasitología , Factores de Transcripción/genética , Transcriptoma/genética
6.
Nature ; 565(7737): 118-121, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30542156

RESUMEN

Plasmodium falciparum causes the severe form of malaria that has high levels of mortality in humans. Blood-stage merozoites of P. falciparum invade erythrocytes, and this requires interactions between multiple ligands from the parasite and receptors in hosts. These interactions include the binding of the Rh5-CyRPA-Ripr complex with the erythrocyte receptor basigin1,2, which is an essential step for entry into human erythrocytes. Here we show that the Rh5-CyRPA-Ripr complex binds the erythrocyte cell line JK-1 significantly better than does Rh5 alone, and that this binding occurs through the insertion of Rh5 and Ripr into host membranes as a complex with high molecular weight. We report a cryo-electron microscopy structure of the Rh5-CyRPA-Ripr complex at subnanometre resolution, which reveals the organization of this essential invasion complex and the mode of interactions between members of the complex, and shows that CyRPA is a critical mediator of complex assembly. Our structure identifies blades 4-6 of the ß-propeller of CyRPA as contact sites for Rh5 and Ripr. The limited contacts between Rh5-CyRPA and CyRPA-Ripr are consistent with the dissociation of Rh5 and Ripr from CyRPA for membrane insertion. A comparision of the crystal structure of Rh5-basigin with the cryo-electron microscopy structure of Rh5-CyRPA-Ripr suggests that Rh5 and Ripr are positioned parallel to the erythrocyte membrane before membrane insertion. This provides information on the function of this complex, and thereby provides insights into invasion by P. falciparum.


Asunto(s)
Antígenos de Protozoos/ultraestructura , Proteínas Portadoras/ultraestructura , Microscopía por Crioelectrón , Complejos Multiproteicos/química , Complejos Multiproteicos/ultraestructura , Plasmodium falciparum , Proteínas Protozoarias/ultraestructura , Animales , Antígenos de Protozoos/química , Antígenos de Protozoos/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Drosophila , Membrana Eritrocítica/metabolismo , Membrana Eritrocítica/parasitología , Humanos , Modelos Moleculares , Complejos Multiproteicos/metabolismo , Plasmodium falciparum/química , Plasmodium falciparum/patogenicidad , Plasmodium falciparum/ultraestructura , Unión Proteica , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo
7.
Annu Rev Microbiol ; 72: 355-375, 2018 Sep 08.
Artículo en Inglés | MEDLINE | ID: mdl-29927705

RESUMEN

Eukaryotic pathogens must survive in different hosts, respond to changing environments, and exploit specialized niches to propagate. Plasmodium parasites cause human malaria during bloodstream infections, where they must persist long enough to be transmitted. Parasites have evolved diverse strategies of variant gene expression that control critical biological processes of blood-stage infections, including antigenic variation, erythrocyte invasion, innate immune evasion, and nutrient acquisition, as well as life-cycle transitions. Epigenetic mechanisms within the parasite are being elucidated, with discovery of epigenomic marks associated with gene silencing and activation, and the identification of epigenetic regulators and chromatin proteins that are required for the switching and maintenance of gene expression. Here, we review the key epigenetic processes that facilitate transition through the parasite life cycle and epigenetic regulatory mechanisms utilized by Plasmodium parasites to survive changing environments and consider epigenetic switching in the context of the outcome of human infections.


Asunto(s)
Adaptación Fisiológica , Epigénesis Genética , Plasmodium/crecimiento & desarrollo , Plasmodium/genética , Animales , Humanos
8.
Malar J ; 22(1): 250, 2023 Aug 31.
Artículo en Inglés | MEDLINE | ID: mdl-37653486

RESUMEN

BACKGROUND: Plasmodium vivax is the second most prevalent cause of malaria yet remains challenging to study due to the lack of a continuous in vitro culture system, highlighting the need to establish a biobank of clinical isolates with multiple freezes per sample for use in functional assays. Different methods for cryopreserving parasite isolates were compared and subsequently the most promising one was validated. Enrichment of early- and late-stage parasites and parasite maturation were quantified to facilitate assay planning. METHODS: In order to compare cryopreservation protocols, nine clinical P. vivax isolates were frozen with four glycerolyte-based mixtures. Parasite recovery post thaw, post KCl-Percoll enrichment and in short-term in vitro culture was measured via slide microscopy. Enrichment of late-stage parasites by magnetic activated cell sorting (MACS) was measured. Short and long-term storage of parasites at either - 80 °C or liquid nitrogen were also compared. RESULTS: Of the four cryopreservation mixtures, one mixture (glycerolyte:serum:RBC at a 2.5:1.5:1 ratio) resulted in improved parasite recovery and statistically significant (P < 0.05) enhancement in parasite survival in short-term in vitro culture. A parasite biobank was subsequently generated using this protocol resulting in a collection of 106 clinical isolates, each with 8 vials. The quality of the biobank was validated by measuring several factors from 47 thaws: the average reduction in parasitaemia post-thaw (25.3%); the average fold enrichment post KCl-Percoll (6.65-fold); and the average percent recovery of parasites (22.0%, measured from 30 isolates). During short-term in vitro culture, robust maturation of ring stage parasites to later stages (> 20% trophozoites, schizonts and gametocytes) was observed in 60.0% of isolates by 48 h. Enrichment of mature parasite stages via MACS showed good reproducibility, with an average of 30.0% post-MACS parasitaemia and an average of 5.30 × 105 parasites/vial. Finally, the effect of storage temperature was tested, and no large impacts from short-term (7 days) or long-term (7-10 years) storage at - 80 °C on parasite recovery, enrichment or viability was observed. CONCLUSIONS: Here, an optimized freezing method for P. vivax clinical isolates is demonstrated as a template for the generation and validation of a parasite biobank for use in functional assays.


Asunto(s)
Malaria Vivax , Plasmodium vivax , Humanos , Bancos de Muestras Biológicas , Reproducibilidad de los Resultados , Parasitemia
9.
Am J Hematol ; 98(10): 1598-1605, 2023 10.
Artículo en Inglés | MEDLINE | ID: mdl-37584425

RESUMEN

BACKGROUND: Sickle cell disease (SCD) remains prevalent because heterozygous carriers (HbAS) are partially resistant to Plasmodium falciparum malaria. Sickle hemoglobin (HbS) polymerization in low and intermediate oxygen (O2 ) conditions is the main driver of HbAS-driven resistance to P. falciparum malaria. However, epidemiological studies have reported mixed malaria morbidity and mortality outcomes in individuals with sickle cell disease (SCD). While maximum-tolerated dose hydroxyurea has been shown to lower malaria incidence, fetal hemoglobin (HbF), an inhibitor of HbS polymerization that is variably packaged in F-erythrocytes, might provide hemoglobin that is accessible to the parasite for feeding. METHODS: To explore that risk, we examined the effect of variable mean corpuscular fetal hemoglobin (MCHF) on P. falciparum proliferation, invasion, and development in HbSS RBCs. RESULTS: We found that greater MCHF in HbSS red blood cells (RBCs) is associated with increased P. falciparum proliferation in O2 environments comparable with the microcirculation. Moreover, both parasite invasion and intracellular growth, the major components of proliferation, occur predominantly in F-erythrocytes and are augmented with increasing MCHF. CONCLUSIONS: HbF modifies P. falciparum infection in HbSS RBCs, further highlighting the complexity of the molecular interactions between these two diseases. Other inhibitors of HbS polymerization that do not increase HbF or F-erythrocytes should be independently assessed for their effects on P. falciparum malaria proliferation in HbSS RBCs.


Asunto(s)
Anemia de Células Falciformes , Malaria Falciparum , Plasmodium falciparum , Humanos , Hemoglobina Fetal , Proliferación Celular , Eritrocitos
10.
Mol Biol Evol ; 38(4): 1356-1371, 2021 04 13.
Artículo en Inglés | MEDLINE | ID: mdl-33185667

RESUMEN

Malaria has been one of the strongest selective pressures on our species. Many of the best-characterized cases of adaptive evolution in humans are in genes tied to malaria resistance. However, the complex evolutionary patterns at these genes are poorly captured by standard scans for nonneutral evolution. Here, we present three new statistical tests for selection based on population genetic patterns that are observed more than once among key malaria resistance loci. We assess these tests using forward-time evolutionary simulations and apply them to global whole-genome sequencing data from humans, and thus we show that they are effective at distinguishing selection from neutrality. Each test captures a distinct evolutionary pattern, here called Divergent Haplotypes, Repeated Shifts, and Arrested Sweeps, associated with a particular period of human prehistory. We clarify the selective signatures at known malaria-relevant genes and identify additional genes showing similar adaptive evolutionary patterns. Among our top outliers, we see a particular enrichment for genes involved in erythropoiesis and for genes previously associated with malaria resistance, consistent with a major role for malaria in shaping these patterns of genetic diversity. Polymorphisms at these genes are likely to impact resistance to malaria infection and contribute to ongoing host-parasite coevolutionary dynamics.


Asunto(s)
Adaptación Biológica/genética , Técnicas Genéticas , Malaria/genética , Selección Genética , Estadística como Asunto , Humanos
11.
Mol Microbiol ; 115(5): 882-890, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33274587

RESUMEN

Babesia species are tick-borne intracellular parasites that infect the red blood cells of their mammalian host, leading to severe or fatal disease. Babesia spp. infect a wide range of mammalian species and cause a significant economic burden globally, predominantly through disease in cattle. Several Babesia spp. are increasingly being recognized as zoonotic pathogens of humans. Babesia spp. have complex life cycles involving multiple stages in the tick and the mammalian host. The parasite utilizes complex signaling pathways during replication, egress, and invasion in each of these stages. They must also rapidly respond to their environment when switching between the mammalian and tick stages. This review will focus on the signaling pathways and environmental stimuli that Babesia spp. utilize in the bloodstream and for transmission to the tick, with an emphasis on the role of phosphorylation- and calcium-based signaling during egress and invasion. The expanding availability of in vitro and in vivo culture systems, genomes, transcriptomes, and transgenic systems available for a range of Babesia spp. should encourage further biological and translational studies of these ubiquitous parasites.


Asunto(s)
Babesia/crecimiento & desarrollo , Babesia/metabolismo , Babesiosis/parasitología , Animales , Babesia/clasificación , Babesia/genética , Babesiosis/transmisión , Humanos , Estadios del Ciclo de Vida , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Transducción de Señal , Garrapatas/parasitología
12.
Mol Microbiol ; 115(5): 891-900, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33236377

RESUMEN

Malaria is one of the most life-threatening infectious diseases worldwide, caused by infection of humans with parasites of the genus Plasmodium. The complex life cycle of Plasmodium parasites is shared between two hosts, with infection of multiple cell types, and the parasite needs to adapt for survival and transmission through significantly different metabolic environments. Within the blood-stage alone, parasites encounter changing levels of key nutrients, including sugars, amino acids, and lipids, due to differences in host dietary nutrition, cellular tropism, and pathogenesis. In this review, we consider the mechanisms that the most lethal of malaria parasites, Plasmodium falciparum, uses to sense nutrient levels and elicit changes in gene expression during blood-stage infections. These changes are brought about by several metabolic intermediates and their corresponding sensor proteins. Sensing of distinct nutritional signals can drive P. falciparum to alter the key blood-stage processes of proliferation, antigenic variation, and transmission.


Asunto(s)
Malaria Falciparum/parasitología , Nutrientes/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Animales , Expresión Génica , Humanos , Estadios del Ciclo de Vida , Malaria Falciparum/sangre , Plasmodium falciparum/crecimiento & desarrollo
13.
J Infect Dis ; 223(10): 1817-1821, 2021 05 28.
Artículo en Inglés | MEDLINE | ID: mdl-32941614

RESUMEN

Plasmodium vivax has 2 invasion ligand/host receptor pathways (P. vivax Duffy-binding protein/Duffy antigen receptor for chemokines [DARC] and P. vivax reticulocyte binding protein 2b/transferrin receptor [TfR1]) that are promising targets for therapeutic intervention. We optimized invasion assays with isogenic cultured reticulocytes. Using a receptor blockade approach with multiple P. vivax isolates, we found that all strains utilized both DARC and TfR1, but with significant variation in receptor usage. This suggests that P. vivax, like Plasmodium falciparum, uses alternative invasion pathways, with implications for pathogenesis and vaccine development.


Asunto(s)
Antígenos CD , Sistema del Grupo Sanguíneo Duffy , Malaria Vivax , Plasmodium vivax , Receptores de Superficie Celular , Receptores de Transferrina , Células Cultivadas , Humanos , Plasmodium vivax/patogenicidad , Reticulocitos/parasitología
14.
PLoS Comput Biol ; 16(4): e1007702, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32315315

RESUMEN

The growth of the malaria parasite Plasmodium falciparum in human blood causes all the symptoms of malaria. To proliferate, non-motile parasites must have access to susceptible red blood cells, which they invade using pairs of parasite ligands and host receptors that define invasion pathways. Parasites can switch invasion pathways, and while this flexibility is thought to facilitate immune evasion, it may also reflect the heterogeneity of red blood cell surfaces within and between hosts. Host genetic background affects red blood cell structure, for example, and red blood cells also undergo dramatic changes in morphology and receptor density as they age. The in vivo consequences of both the accessibility of susceptible cells, and their heterogeneous susceptibility, remain unclear. Here, we measured invasion of laboratory strains of P. falciparum relying on distinct invasion pathways into red blood cells of different ages. We estimated invasion efficiency while accounting for red blood cell accessibility to parasites. This approach revealed different tradeoffs made by parasite strains between the fraction of cells they can invade and their invasion rate into them, and we distinguish "specialist" strains from "generalist" strains in this context. We developed a mathematical model to show that generalist strains would lead to higher peak parasitemias in vivo compared to specialist strains with similar overall proliferation rates. Thus, the ecology of red blood cells may play a key role in determining the rate of P. falciparum parasite proliferation and malaria virulence.


Asunto(s)
Eritrocitos/fisiología , Eritrocitos/parasitología , Malaria Falciparum/parasitología , Animales , Recuento de Eritrocitos , Humanos , Evasión Inmune/genética , Evasión Inmune/inmunología , Malaria/parasitología , Modelos Teóricos , Parásitos , Plasmodium falciparum/genética , Plasmodium falciparum/patogenicidad
15.
Proc Natl Acad Sci U S A ; 115(28): 7350-7355, 2018 07 10.
Artículo en Inglés | MEDLINE | ID: mdl-29946035

RESUMEN

Sickle cell trait (AS) confers partial protection against lethal Plasmodium falciparum malaria. Multiple mechanisms for this have been proposed, with a recent focus on aberrant cytoadherence of parasite-infected red blood cells (RBCs). Here we investigate the mechanistic basis of AS protection through detailed temporal mapping. We find that parasites in AS RBCs maintained at low oxygen concentrations stall at a specific stage in the middle of intracellular growth before DNA replication. We demonstrate that polymerization of sickle hemoglobin (HbS) is responsible for this growth arrest of intraerythrocytic P. falciparum parasites, with normal hemoglobin digestion and growth restored in the presence of carbon monoxide, a gaseous antisickling agent. Modeling of growth inhibition and sequestration revealed that HbS polymerization-induced growth inhibition following cytoadherence is the critical driver of the reduced parasite densities observed in malaria infections of individuals with AS. We conclude that the protective effect of AS derives largely from effective sequestration of infected RBCs into the hypoxic microcirculation.


Asunto(s)
Replicación del ADN , ADN Protozoario/biosíntesis , Eritrocitos Anormales/metabolismo , Oxígeno/metabolismo , Plasmodium falciparum/metabolismo , Rasgo Drepanocítico/metabolismo , Antidrepanocíticos/farmacología , Monóxido de Carbono/farmacología , Eritrocitos Anormales/parasitología , Humanos , Malaria Falciparum/metabolismo , Rasgo Drepanocítico/parasitología
16.
BMC Bioinformatics ; 21(1): 300, 2020 Jul 11.
Artículo en Inglés | MEDLINE | ID: mdl-32652926

RESUMEN

BACKGROUND: A common yet still manual task in basic biology research, high-throughput drug screening and digital pathology is identifying the number, location, and type of individual cells in images. Object detection methods can be useful for identifying individual cells as well as their phenotype in one step. State-of-the-art deep learning for object detection is poised to improve the accuracy and efficiency of biological image analysis. RESULTS: We created Keras R-CNN to bring leading computational research to the everyday practice of bioimage analysts. Keras R-CNN implements deep learning object detection techniques using Keras and Tensorflow ( https://github.com/broadinstitute/keras-rcnn ). We demonstrate the command line tool's simplified Application Programming Interface on two important biological problems, nucleus detection and malaria stage classification, and show its potential for identifying and classifying a large number of cells. For malaria stage classification, we compare results with expert human annotators and find comparable performance. CONCLUSIONS: Keras R-CNN is a Python package that performs automated cell identification for both brightfield and fluorescence images and can process large image sets. Both the package and image datasets are freely available on GitHub and the Broad Bioimage Benchmark Collection.


Asunto(s)
Aprendizaje Profundo , Procesamiento de Imagen Asistido por Computador/métodos , Programas Informáticos , Núcleo Celular , Humanos , Plasmodium vivax/crecimiento & desarrollo
17.
Proc Natl Acad Sci U S A ; 114(44): E9356-E9365, 2017 10 31.
Artículo en Inglés | MEDLINE | ID: mdl-29078358

RESUMEN

During malaria blood-stage infections, Plasmodium parasites interact with the RBC surface to enable invasion followed by intracellular proliferation. Critical factors involved in invasion have been identified using biochemical and genetic approaches including specific knockdowns of genes of interest from primary CD34+ hematopoietic stem cells (cRBCs). Here we report the development of a robust in vitro culture system to produce RBCs that allow the generation of gene knockouts via CRISPR/Cas9 using the immortal JK-1 erythroleukemia line. JK-1 cells spontaneously differentiate, generating cells at different stages of erythropoiesis, including terminally differentiated nucleated RBCs that we term "jkRBCs." A screen of small-molecule epigenetic regulators identified several bromodomain-specific inhibitors that promote differentiation and enable production of synchronous populations of jkRBCs. Global surface proteomic profiling revealed that jkRBCs express all known Pfalciparum host receptors in a similar fashion to cRBCs and that multiple Pfalciparum strains invade jkRBCs at comparable levels to cRBCs and RBCs. Using CRISPR/Cas9, we deleted two host factors, basigin (BSG) and CD44, for which no natural nulls exist. BSG interacts with the parasite ligand Rh5, a prominent vaccine candidate. A BSG knockout was completely refractory to parasite invasion in a strain-transcendent manner, confirming the essential role for BSG during invasion. CD44 was recently identified in an RNAi screen of blood group genes as a host factor for invasion, and we show that CD44 knockout results in strain-transcendent reduction in invasion. Furthermore, we demonstrate a functional interaction between these two determinants in mediating Pfalciparum erythrocyte invasion.


Asunto(s)
Sistemas CRISPR-Cas/genética , Eritrocitos/metabolismo , Eritrocitos/parasitología , Plasmodium falciparum/genética , Antígenos de Protozoos/metabolismo , Basigina/metabolismo , Proteínas Portadoras/metabolismo , Diferenciación Celular/fisiología , Línea Celular Tumoral , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/fisiología , Epigénesis Genética/fisiología , Técnicas de Inactivación de Genes/métodos , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/parasitología , Interacciones Huésped-Parásitos/fisiología , Humanos , Receptores de Hialuranos/metabolismo , Células K562 , Leucemia Eritroblástica Aguda/metabolismo , Leucemia Eritroblástica Aguda/parasitología , Ligandos , Malaria/parasitología , Malaria Falciparum/metabolismo , Malaria Falciparum/parasitología , Proteómica/métodos , Proteínas Protozoarias/metabolismo
18.
Am J Hematol ; 94(9): 963-974, 2019 09.
Artículo en Inglés | MEDLINE | ID: mdl-31148215

RESUMEN

Malaria pathogenesis is caused by the replication of Plasmodium parasites within the red blood cells (RBCs) of the vertebrate host. This selective pressure has favored the evolution of protective polymorphisms in erythrocyte proteins, a subset of which serve as cognate receptors for parasite invasion ligands. Recently, the generation of RBCs from immortalized hematopoietic stem cells (HSCs) has offered a more tractable system for genetic manipulation and long-term in vitro culture, enabling elucidation of the functional determinants of host susceptibility in vitro. Here we report the generation of an immortalized erythroid progenitor cell line (EJ cells) from as few as 100 000 peripheral blood mononuclear cells. It offers a robust method for the creation of customized model systems from small volumes of peripheral blood. The EJ cell differentiation mirrored erythropoiesis of primary HSCs, yielding orthochromatic erythroblasts and enucleated RBCs after eight days (ejRBCs). The ejRBCs supported invasion by both P. vivax and P. falciparum. To demonstrate the genetic tractability of this system, we used CRISPR/Cas9 to disrupt the Duffy Antigen/Receptor for Chemokines (DARC) gene, which encodes the canonical receptor of P. vivax in humans. Invasion of P. vivax into this DARC-knockout cell line was strongly inhibited providing direct genetic evidence that P. vivax requires DARC for RBC invasion. Further, genetic complementation of DARC restored P. vivax invasion. Taken together, the peripheral blood immortalization method presented here offers the capacity to generate biologically representative model systems for studies of blood-stage malaria invasion from the peripheral blood of donors harboring unique genetic backgrounds, or rare polymorphisms.


Asunto(s)
Células Precursoras Eritroides , Malaria Falciparum , Malaria Vivax , Modelos Biológicos , Células Madre de Sangre Periférica , Plasmodium falciparum/metabolismo , Plasmodium vivax/metabolismo , Línea Celular Transformada , Células Precursoras Eritroides/metabolismo , Células Precursoras Eritroides/parasitología , Células Precursoras Eritroides/fisiología , Humanos , Malaria Falciparum/metabolismo , Malaria Falciparum/patología , Malaria Vivax/metabolismo , Malaria Vivax/patología , Células Madre de Sangre Periférica/metabolismo , Células Madre de Sangre Periférica/parasitología , Células Madre de Sangre Periférica/patología
19.
Artículo en Inglés | MEDLINE | ID: mdl-29378713

RESUMEN

Plasmodium vivax chloroquine resistance has been documented in nearly every region where this malaria-causing parasite is endemic. Unfortunately, P. vivax resistance surveillance and drug discovery are challenging due to the low parasitemias of patient isolates and poor parasite survival through ex vivo maturation that reduce the sensitivity and scalability of current P. vivax antimalarial assays. Using cryopreserved patient isolates from Brazil and fresh patient isolates from India, we established a robust enrichment method for P. vivax parasites. We next performed a medium screen for formulations that enhance ex vivo survival. Finally, we optimized an isotopic metabolic labeling assay for measuring P. vivax maturation and its sensitivity to antimalarials. A KCl Percoll density gradient enrichment method increased parasitemias from small-volume ex vivo isolates by an average of >40-fold. The use of Iscove's modified Dulbecco's medium for P. vivax ex vivo culture approximately doubled the parasite survival through maturation. Coupling these with [3H]hypoxanthine metabolic labeling permitted sensitive and robust measurements of parasite maturation, which was used to measure the sensitivities of Brazilian P. vivax isolates to chloroquine and several novel antimalarials. These techniques can be applied to rapidly and robustly assess the P. vivax isolate sensitivities to antimalarials for resistance surveillance and drug discovery.


Asunto(s)
Antimaláricos/farmacología , Cloroquina/farmacología , Pruebas de Sensibilidad Parasitaria/métodos , Plasmodium vivax/efectos de los fármacos , Brasil , Humanos , India
20.
Proc Natl Acad Sci U S A ; 112(42): 13027-32, 2015 Oct 20.
Artículo en Inglés | MEDLINE | ID: mdl-26438871

RESUMEN

Malaria cases caused by the zoonotic parasite Plasmodium knowlesi are being increasingly reported throughout Southeast Asia and in travelers returning from the region. To test for evidence of signatures of selection or unusual population structure in this parasite, we surveyed genome sequence diversity in 48 clinical isolates recently sampled from Malaysian Borneo and in five lines maintained in laboratory rhesus macaques after isolation in the 1960s from Peninsular Malaysia and the Philippines. Overall genomewide nucleotide diversity (π = 6.03 × 10(-3)) was much higher than has been seen in worldwide samples of either of the major endemic malaria parasite species Plasmodium falciparum and Plasmodium vivax. A remarkable substructure is revealed within P. knowlesi, consisting of two major sympatric clusters of the clinical isolates and a third cluster comprising the laboratory isolates. There was deep differentiation between the two clusters of clinical isolates [mean genomewide fixation index (FST) = 0.21, with 9,293 SNPs having fixed differences of FST = 1.0]. This differentiation showed marked heterogeneity across the genome, with mean FST values of different chromosomes ranging from 0.08 to 0.34 and with further significant variation across regions within several chromosomes. Analysis of the largest cluster (cluster 1, 38 isolates) indicated long-term population growth, with negatively skewed allele frequency distributions (genomewide average Tajima's D = -1.35). Against this background there was evidence of balancing selection on particular genes, including the circumsporozoite protein (csp) gene, which had the top Tajima's D value (1.57), and scans of haplotype homozygosity implicate several genomic regions as being under recent positive selection.


Asunto(s)
Genoma de Protozoos , Plasmodium knowlesi/genética , Adaptación Fisiológica , Animales , Genética de Población , Plasmodium knowlesi/fisiología , Polimorfismo de Nucleótido Simple
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