Publications by authors named "Manoj T Duraisingh"

124 Publications

Deconstructing the parasite multiplication rate of Plasmodium falciparum.

Trends Parasitol 2021 Jun 9. Epub 2021 Jun 9.

Center for Communicable Diseases Dynamics, Department of Epidemiology, Harvard T.H. Chan School of Public Health, Boston, MA, USA. Electronic address:

Epidemiological indicators describing population-level malaria transmission dynamics are widely used to guide policy recommendations. However, the determinants of malaria outcomes within individuals are still poorly understood. This conceptual gap partly reflects the fact that there are few indicators that robustly predict the trajectory of individual infections or clinical outcomes. The parasite multiplication rate (PMR) is a widely used indicator for the Plasmodium intraerythrocytic development cycle (IDC), for example, but its relationship to clinical outcomes is complex. Here, we review its calculation and use in P. falciparum malaria research, as well as the parasite and host factors that impact it. We also provide examples of metrics that can help to link within-host dynamics to malaria clinical outcomes when used alongside the PMR.
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http://dx.doi.org/10.1016/j.pt.2021.05.001DOI Listing
June 2021

The molecular basis of antimalarial drug resistance in Plasmodium vivax.

Int J Parasitol Drugs Drug Resist 2021 Apr 26;16:23-37. Epub 2021 Apr 26.

Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, Boston, 02115, MA, USA. Electronic address:

Plasmodium vivax is the most geographically widespread cause of human malaria and is responsible for the majority of cases outside of the African continent. While great progress has been made towards eliminating human malaria, drug resistant parasite strains pose a threat towards continued progress. Resistance has arisen to multiple antimalarials in P. vivax, including to chloroquine, which is currently the first line therapy for P. vivax in most regions. Despite its importance, an understanding of the molecular mechanisms of drug resistance in this species remains elusive, in large part due to the complex biology of P. vivax and the lack of in vitro culture. In this review, we will cover the extent and challenges of measuring clinical and in vitro drug resistance in P. vivax. We will consider the roles of candidate drug resistance genes. We will highlight the development of molecular approaches for studying P. vivax biology that provide the opportunity to validate the role of putative drug resistance mutations as well as identify novel mechanisms of drug resistance in this understudied parasite. Validated molecular determinants and markers of drug resistance are essential for the rapid and cost-effective monitoring of drug resistance in P. vivax, and will be useful for optimizing drug regimens and for informing drug policy in control and elimination settings.
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http://dx.doi.org/10.1016/j.ijpddr.2021.04.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8113647PMC
April 2021

The Modular Circuitry of Apicomplexan Cell Division Plasticity.

Front Cell Infect Microbiol 2021 12;11:670049. Epub 2021 Apr 12.

Department of Biology, Boston College, Chestnut Hill, MA, United States.

The close-knit group of apicomplexan parasites displays a wide variety of cell division modes, which differ between parasites as well as between different life stages within a single parasite species. The beginning and endpoint of the asexual replication cycles is a 'zoite' harboring the defining apical organelles required for host cell invasion. However, the number of zoites produced per division round varies dramatically and can unfold in several different ways. This plasticity of the cell division cycle originates from a combination of hard-wired developmental programs modulated by environmental triggers. Although the environmental triggers and sensors differ between species and developmental stages, widely conserved secondary messengers mediate the signal transduction pathways. These environmental and genetic input integrate in division-mode specific chromosome organization and chromatin modifications that set the stage for each division mode. Cell cycle progression is conveyed by a smorgasbord of positively and negatively acting transcription factors, often acting in concert with epigenetic reader complexes, that can vary dramatically between species as well as division modes. A unique set of cell cycle regulators with spatially distinct localization patterns insert discrete check points which permit individual control and can uncouple general cell cycle progression from nuclear amplification. Clusters of expressed genes are grouped into four functional modules seen in all division modes: 1. mother cytoskeleton disassembly; 2. DNA replication and segregation (D&S); 3. karyokinesis; 4. zoite assembly. A plug-and-play strategy results in the variety of extant division modes. The timing of mother cytoskeleton disassembly is hard-wired at the species level for asexual division modes: it is either the first step, or it is the last step. In the former scenario zoite assembly occurs at the plasma membrane (external budding), and in the latter scenario zoites are assembled in the cytoplasm (internal budding). The number of times each other module is repeated can vary regardless of this first decision, and defines the modes of cell division: schizogony, binary fission, endodyogeny, endopolygeny.
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http://dx.doi.org/10.3389/fcimb.2021.670049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8072463PMC
April 2021

Plasmodium vivax infection compromises reticulocyte stability.

Nat Commun 2021 03 12;12(1):1629. Epub 2021 Mar 12.

Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, Boston, MA, USA.

The structural integrity of the host red blood cell (RBC) is crucial for propagation of Plasmodium spp. during the disease-causing blood stage of malaria infection. To assess the stability of Plasmodium vivax-infected reticulocytes, we developed a flow cytometry-based assay to measure osmotic stability within characteristically heterogeneous reticulocyte and P. vivax-infected samples. We find that erythroid osmotic stability decreases during erythropoiesis and reticulocyte maturation. Of enucleated RBCs, young reticulocytes which are preferentially infected by P. vivax, are the most osmotically stable. P. vivax infection however decreases reticulocyte stability to levels close to those of RBC disorders that cause hemolytic anemia, and to a significantly greater degree than P. falciparum destabilizes normocytes. Finally, we find that P. vivax new permeability pathways contribute to the decreased osmotic stability of infected-reticulocytes. These results reveal a vulnerability of P. vivax-infected reticulocytes that could be manipulated to allow in vitro culture and develop novel therapeutics.
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http://dx.doi.org/10.1038/s41467-021-21886-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7955053PMC
March 2021

A framework for signaling throughout the life cycle of Babesia species.

Mol Microbiol 2020 Dec 4. Epub 2020 Dec 4.

Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, MA, USA.

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.
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http://dx.doi.org/10.1111/mmi.14650DOI Listing
December 2020

Linking nutrient sensing and gene expression in Plasmodium falciparum blood-stage parasites.

Mol Microbiol 2020 Nov 25. Epub 2020 Nov 25.

Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, Boston, MA, USA.

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.
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http://dx.doi.org/10.1111/mmi.14652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8144236PMC
November 2020

Three Signatures of Adaptive Polymorphism Exemplified by Malaria-Associated Genes.

Mol Biol Evol 2021 04;38(4):1356-1371

Harvard T.H. Chan School of Public Health, Boston, MA.

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.
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http://dx.doi.org/10.1093/molbev/msaa294DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8042748PMC
April 2021

Transformative tools for parasitic flatworms.

Science 2020 09;369(6511):1562-1564

Harvard T.H. Chan School of Public Health, Boston, MA 02139, USA.

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http://dx.doi.org/10.1126/science.abe0710DOI Listing
September 2020

Plasmodium vivax Strains Use Alternative Pathways for Invasion.

J Infect Dis 2021 May;223(10):1817-1821

Harvard T. H. Chan School of Public Health, Boston, Massachusetts, USA.

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.
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http://dx.doi.org/10.1093/infdis/jiaa592DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8161644PMC
May 2021

Co-option of Plasmodium falciparum PP1 for egress from host erythrocytes.

Nat Commun 2020 07 15;11(1):3532. Epub 2020 Jul 15.

Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, Boston, 02115, MA, USA.

Asexual proliferation of the Plasmodium parasites that cause malaria follows a developmental program that alternates non-canonical intraerythrocytic replication with dissemination to new host cells. We carried out a functional analysis of the Plasmodium falciparum homolog of Protein Phosphatase 1 (PfPP1), a universally conserved cell cycle factor in eukaryotes, to investigate regulation of parasite proliferation. PfPP1 is indeed required for efficient replication, but is absolutely essential for egress of parasites from host red blood cells. By phosphoproteomic and chemical-genetic analysis, we isolate two functional targets of PfPP1 for egress: a HECT E3 protein-ubiquitin ligase; and GCα, a fusion protein composed of a guanylyl cyclase and a phospholipid transporter domain. We hypothesize that PfPP1 regulates lipid sensing by GCα and find that phosphatidylcholine stimulates PfPP1-dependent egress. PfPP1 acts as a key regulator that integrates multiple cell-intrinsic pathways with external signals to direct parasite egress from host cells.
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http://dx.doi.org/10.1038/s41467-020-17306-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7363832PMC
July 2020

Keras R-CNN: library for cell detection in biological images using deep neural networks.

BMC Bioinformatics 2020 Jul 11;21(1):300. Epub 2020 Jul 11.

The Broad Institute, Cambridge, MA, USA.

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.
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http://dx.doi.org/10.1186/s12859-020-03635-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7353739PMC
July 2020

Fussing About Fission: Defining Variety Among Mainstream and Exotic Apicomplexan Cell Division Modes.

Front Cell Infect Microbiol 2020 5;10:269. Epub 2020 Jun 5.

Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, Harvard University, Boston, MA, United States.

Cellular reproduction defines life, yet our textbook-level understanding of cell division is limited to a small number of model organisms centered around humans. The horizon on cell division variants is expanded here by advancing insights on the fascinating cell division modes found in the Apicomplexa, a key group of protozoan parasites. The Apicomplexa display remarkable variation in offspring number, whether karyokinesis follows each S/M-phase or not, and whether daughter cells bud in the cytoplasm or bud from the cortex. We find that the terminology used to describe the various manifestations of asexual apicomplexan cell division emphasizes either the number of offspring or site of budding, which are not directly comparable features and has led to confusion in the literature. Division modes have been primarily studied in two human pathogenic Apicomplexa, malaria-causing spp. and , a major cause of opportunistic infections. spp. divide asexually by schizogony, producing multiple daughters per division round through a cortical budding process, though at several life-cycle nuclear amplifications stages, are not followed by karyokinesis. divides by endodyogeny producing two internally budding daughters per division round. Here we add to this diversity in replication mechanisms by considering the cattle parasite and the pig parasite . produces two daughters per division round by a "binary fission" mechanism whereas produces daughters through both endodyogeny and multiple internal budding known as endopolygeny. In addition, we provide new data from the causative agent of equine protozoal myeloencephalitis (EPM), , which also undergoes endopolygeny but differs from by maintaining a single multiploid nucleus. Overall, we operationally define two principally different division modes: internal budding found in cyst-forming Coccidia (comprising endodyogeny and two forms of endopolygeny) and external budding found in the other parasites studied (comprising the two forms of schizogony, binary fission and multiple fission). Progressive insights into the principles defining the molecular and cellular requirements for internal vs. external budding, as well as variations encountered in sexual stages are discussed. The evolutionary pressures and mechanisms underlying apicomplexan cell division diversification carries relevance across Eukaryota.
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http://dx.doi.org/10.3389/fcimb.2020.00269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7289922PMC
June 2020

Accounting for red blood cell accessibility reveals distinct invasion strategies in Plasmodium falciparum strains.

PLoS Comput Biol 2020 04 21;16(4):e1007702. Epub 2020 Apr 21.

Center for Communicable Disease Dynamics, Department of Epidemiology, Harvard TH Chan School of Public Health, Boston, Massachusetts, United States of America.

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.
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http://dx.doi.org/10.1371/journal.pcbi.1007702DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7194430PMC
April 2020

Plasmodium vivax transcriptional profiling of low input cryopreserved isolates through the intraerythrocytic development cycle.

PLoS Negl Trop Dis 2020 03 2;14(3):e0008104. Epub 2020 Mar 2.

Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, Massachusetts, United States of America.

Approximately one-third of the global population is at risk of Plasmodium vivax infection, and an estimated 7.51 million cases were reported in 2017. Although, P. vivax research is currently limited by the lack of a robust continuous in vitro culture system for this parasite, recent work optimizing short-term ex vivo culture of P. vivax from cryopreserved isolates has facilitated quantitative assays on synchronous parasites. Pairing this improved culture system with low-input Smart-seq2 RNAseq library preparation, we sought to determine whether transcriptional profiling of P. vivax would provide insight into the differential survival of parasites in different culture media. To this end we probed the transcriptional signature of three different ex vivo P. vivax samples in four different culture media using only 1000 cells for each time point taken during the course of the intraerythrocytic development cycle (IDC). Using this strategy, we achieved similar quality transcriptional data to previously reported P. vivax transcriptomes. We found little effect with varying culture media on parasite transcriptional signatures, identified many novel gametocyte-specific genes from transcriptomes of FACS-isolated gametocytes, and determined invasion ligand expression in schizonts in biological isolates and across the IDC. In total, these data demonstrate the feasibility and utility of P. vivax RNAseq-based transcriptomic studies using minimal biomass input to maximize experimental capacity.
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http://dx.doi.org/10.1371/journal.pntd.0008104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7067476PMC
March 2020

Adaptation of Plasmodium falciparum to humans involved the loss of an ape-specific erythrocyte invasion ligand.

Nat Commun 2019 10 4;10(1):4512. Epub 2019 Oct 4.

Malaria Programme, Wellcome Sanger Institute, Wellcome Genome Campus, Cambridge, CB10 1SA, UK.

Plasmodium species are frequently host-specific, but little is currently known about the molecular factors restricting host switching. This is particularly relevant for P. falciparum, the only known human-infective species of the Laverania sub-genus, all other members of which infect African apes. Here we show that all tested P. falciparum isolates contain an inactivating mutation in an erythrocyte invasion associated gene, PfEBA165, the homologues of which are intact in all ape-infective Laverania species. Recombinant EBA165 proteins only bind ape, not human, erythrocytes, and this specificity is due to differences in erythrocyte surface sialic acids. Correction of PfEBA165 inactivating mutations by genome editing yields viable parasites, but is associated with down regulation of both PfEBA165 and an adjacent invasion ligand, which suggests that PfEBA165 expression is incompatible with parasite growth in human erythrocytes. Pseudogenization of PfEBA165 may represent a key step in the emergence and evolution of P. falciparum.
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http://dx.doi.org/10.1038/s41467-019-12294-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6778099PMC
October 2019

Quantitative comparative analysis of human erythrocyte surface proteins between individuals from two genetically distinct populations.

Commun Biol 2019 20;2:350. Epub 2019 Sep 20.

1Cambridge Institute for Medical Research, University of Cambridge, Hills Road, Cambridge, CB2 0XY UK.

Red blood cells (RBCs) play a critical role in oxygen transport, and are the focus of important diseases including malaria and the haemoglobinopathies. Proteins at the RBC surface can determine susceptibility to disease, however previous studies classifying the RBC proteome have not used specific strategies directed at enriching cell surface proteins. Furthermore, there has been no systematic analysis of variation in abundance of RBC surface proteins between genetically disparate human populations. These questions are important to inform not only basic RBC biology but additionally to identify novel candidate receptors for malarial parasites. Here, we use 'plasma membrane profiling' and tandem mass tag-based mass spectrometry to enrich and quantify primary RBC cell surface proteins from two sets of nine donors from the UK or Senegal. We define a RBC surface proteome and identify potential receptors based on either diminished protein abundance, or increased variation in RBCs from West African individuals.
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http://dx.doi.org/10.1038/s42003-019-0596-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6754445PMC
April 2020

Fetal hemoglobin does not inhibit growth.

Blood Adv 2019 07;3(14):2149-2152

Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, Harvard University, Boston, MA.

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http://dx.doi.org/10.1182/bloodadvances.2019000399DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6650726PMC
July 2019

Generation of an immortalized erythroid progenitor cell line from peripheral blood: A model system for the functional analysis of Plasmodium spp. invasion.

Am J Hematol 2019 09 18;94(9):963-974. Epub 2019 Jun 18.

Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, Massachusetts.

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.
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http://dx.doi.org/10.1002/ajh.25543DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6984401PMC
September 2019

Elucidating Host Cell Uptake by Malaria Parasites.

Trends Parasitol 2019 05 16;35(5):333-335. Epub 2019 Apr 16.

Harvard T.H. Chan School of Public Health, Boston, MA, USA. Electronic address:

The malaria parasite must digest host cytoplasm for normal growth, and many studies have revealed the essential role of proteases in hemoglobin digestion. Here, we discuss the results of Jonscher et al. (Cell Host Microbe 2019;25:166-173) who have, for the first time, identified a molecule, VPS45, involved in the uptake and trafficking of host cytoplasm to the digestive vacuole.
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http://dx.doi.org/10.1016/j.pt.2019.03.005DOI Listing
May 2019

Structure of Plasmodium falciparum Rh5-CyRPA-Ripr invasion complex.

Nature 2019 01 12;565(7737):118-121. Epub 2018 Dec 12.

Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia.

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 basigin, 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 β-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.
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http://dx.doi.org/10.1038/s41586-018-0779-6DOI Listing
January 2019

To kill a piroplasm: genetic technologies to advance drug discovery and target identification in Babesia.

Int J Parasitol 2019 02 1;49(2):153-163. Epub 2018 Nov 1.

Harvard T. H. Chan School of Public Health, 651 Huntington Ave, Boston, MA 02115, USA. Electronic address:

Babesia parasites infect a diverse range of vertebrate hosts, from penguins to pigs. Recently, the emergence of zoonotic Babesia infection has been increasing, and the list of species reported to infect humans continues to grow. Babesiosis represents a burgeoning veterinary and medical threat, and the need for novel therapeutic drugs to effectively target this diverse group of parasites is pressing. Here, we review the current culture systems that exist to study and manipulate Babesia parasites, and identify the scope and methods for target discovery and validation to identify novel, potent anti-babesial inhibitors. Challenges exist including difficulties in the culture systems of important zoonotic parasites, and there is a lack of integrated morphological and molecular data. While molecular approaches in several Babesia spp. has become a reality, the ability to rapidly identify and validate drug targets is hindered by a lack of sophisticated genetic tools to probe parasite biology. The minimal genome size and haploid nature of blood-stage Babesia parasites presents an opportunity to adapt techniques from related systems and characterise the druggable genomic space in a high-throughput way. The considerable diversity of parasites within the genus suggests the existence of highly divergent biology and polymorphism that could present a formidable barrier to the development of a pan-babesiacidal therapeutic strategy.
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http://dx.doi.org/10.1016/j.ijpara.2018.09.005DOI Listing
February 2019

Molecular and cellular interactions defining the tropism of Plasmodium vivax for reticulocytes.

Curr Opin Microbiol 2018 12 23;46:109-115. Epub 2018 Oct 23.

Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, MA, USA. Electronic address:

Plasmodium vivax is uniquely restricted to invading reticulocytes, the youngest of red blood cells. Parasite invasion relies on the sequential deployment of multiple parasite invasion ligands. Correct targeting of the host reticulocyte is mediated by two families of invasion ligands: the reticulocyte binding proteins (RBPs) and erythrocyte binding proteins (EBPs). The Duffy receptor has long been established as a key determinant for P. vivax invasion. However, recently, the RBP protein PvRBP2b has been shown to bind to transferrin receptor, which is expressed on reticulocytes but lost on normocytes, implicating the ligand-receptor in the reticulocyte tropism of P. vivax. Furthermore there is increasing evidence for P. vivax growth and sexual development in reticulocyte-enriched tissues such as the bone marrow.
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http://dx.doi.org/10.1016/j.mib.2018.10.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6688184PMC
December 2018

Erythrocytes lacking the Langereis blood group protein ABCB6 are resistant to the malaria parasite .

Commun Biol 2018 3;1:45. Epub 2018 May 3.

Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, 02115, MA, USA.

The ATP-binding cassette transporter was recently discovered to encode the Langereis (Lan) blood group antigen. Lan null individuals are asymptomatic, and the function of ABCB6 in mature erythrocytes is not understood. Here, we assessed ABCB6 as a host factor for malaria parasites during erythrocyte invasion. We show that Lan null erythrocytes are highly resistant to invasion by , in a strain-transcendent manner. Although both Lan null and Jr(a-) erythrocytes harbor excess porphyrin, only Lan null erythrocytes exhibit a invasion defect. Further, the zoonotic parasite invades Lan null and control cells with similar efficiency, suggesting that ABCB6 may mediate invasion through species-specific molecular interactions. Using tandem mass tag-based proteomics, we find that the only consistent difference in membrane proteins between Lan null and control cells is absence of ABCB6. Our results demonstrate that a newly identified naturally occurring blood group variant is associated with resistance to .
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http://dx.doi.org/10.1038/s42003-018-0046-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6123700PMC
May 2018

Resistance to in sickle cell trait erythrocytes is driven by oxygen-dependent growth inhibition.

Proc Natl Acad Sci U S A 2018 07 26;115(28):7350-7355. Epub 2018 Jun 26.

Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, Boston, MA 02115;

Sickle cell trait (AS) confers partial protection against lethal 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 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.
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http://dx.doi.org/10.1073/pnas.1804388115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6048551PMC
July 2018

Epigenetic Variation and Regulation in Malaria Parasites.

Annu Rev Microbiol 2018 Sep 21;72:355-375. Epub 2018 Jun 21.

Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, Massachusetts 02115, USA; email: ,

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.
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http://dx.doi.org/10.1146/annurev-micro-090817-062722DOI Listing
September 2018

Bone Marrow Is a Major Parasite Reservoir in Plasmodium vivax Infection.

mBio 2018 05 8;9(3). Epub 2018 May 8.

Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts, USA

causes heavy burdens of disease across malarious regions worldwide. Mature asexual and transmissive gametocyte stages occur in the blood circulation, and it is often assumed that accumulation/sequestration in tissues is not an important phase in their development. Here, we present a systematic study of stage distributions in infected tissues of nonhuman primate (NHP) malaria models as well as in blood from human infections. In a comparative analysis of the transcriptomes of and blood-stage parasites, we found a conserved cascade of stage-specific gene expression despite the greatly different gametocyte maturity times of these two species. Using this knowledge, we validated a set of conserved asexual- and gametocyte-stage markers both by quantitative real-time PCR and by antibody assays of peripheral blood samples from infected patients and NHP ( sp.). Histological analyses of parasites in organs of 13 infected NHP ( and species) demonstrated a major fraction of immature gametocytes in the parenchyma of the bone marrow, while asexual schizont forms were enriched to a somewhat lesser extent in this region of the bone marrow as well as in sinusoids of the liver. These findings suggest that the bone marrow is an important reservoir for gametocyte development and proliferation of malaria parasites. malaria continues to cause major public health burdens worldwide. Yet, significant knowledge gaps in the basic biology and epidemiology of malaria remain, largely due to limited available tools for research and diagnostics. Here, we present a systematic examination of tissue sequestration during infection. Studies of nonhuman primates and malaria patients revealed enrichment of developing sexual stages (gametocytes) and mature replicative stages (schizonts) in the bone marrow and liver, relative to those present in peripheral blood. Identification of the bone marrow as a major tissue reservoir has important implications for parasite diagnosis and treatment.
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http://dx.doi.org/10.1128/mBio.00625-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5941073PMC
May 2018

Enhanced Ex Vivo Plasmodium vivax Intraerythrocytic Enrichment and Maturation for Rapid and Sensitive Parasite Growth Assays.

Antimicrob Agents Chemother 2018 04 27;62(4). Epub 2018 Mar 27.

Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA

chloroquine resistance has been documented in nearly every region where this malaria-causing parasite is endemic. Unfortunately, resistance surveillance and drug discovery are challenging due to the low parasitemias of patient isolates and poor parasite survival through maturation that reduce the sensitivity and scalability of current antimalarial assays. Using cryopreserved patient isolates from Brazil and fresh patient isolates from India, we established a robust enrichment method for parasites. We next performed a medium screen for formulations that enhance survival. Finally, we optimized an isotopic metabolic labeling assay for measuring maturation and its sensitivity to antimalarials. A KCl Percoll density gradient enrichment method increased parasitemias from small-volume isolates by an average of >40-fold. The use of Iscove's modified Dulbecco's medium for culture approximately doubled the parasite survival through maturation. Coupling these with [H]hypoxanthine metabolic labeling permitted sensitive and robust measurements of parasite maturation, which was used to measure the sensitivities of Brazilian isolates to chloroquine and several novel antimalarials. These techniques can be applied to rapidly and robustly assess the isolate sensitivities to antimalarials for resistance surveillance and drug discovery.
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http://dx.doi.org/10.1128/AAC.02519-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5913983PMC
April 2018

Transferrin receptor 1 is a reticulocyte-specific receptor for .

Science 2018 01;359(6371):48-55

The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia.

shows a strict host tropism for reticulocytes. We identified transferrin receptor 1 (TfR1) as the receptor for reticulocyte-binding protein 2b (PvRBP2b). We determined the structure of the N-terminal domain of PvRBP2b involved in red blood cell binding, elucidating the molecular basis for TfR1 recognition. We validated TfR1 as the biological target of PvRBP2b engagement by means of TfR1 expression knockdown analysis. TfR1 mutant cells deficient in PvRBP2b binding were refractory to invasion of but not to invasion of Using Brazilian and Thai clinical isolates, we show that PvRBP2b monoclonal antibodies that inhibit reticulocyte binding also block entry into reticulocytes. These data show that TfR1-PvRBP2b invasion pathway is critical for the recognition of reticulocytes during invasion.
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http://dx.doi.org/10.1126/science.aan1078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5788258PMC
January 2018

Targeting Plasmodium Proteases to Block Malaria Parasite Escape and Entry.

Trends Parasitol 2018 02 18;34(2):95-97. Epub 2017 Dec 18.

Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, 651 Huntington Avenue, FXB, Room 202, Boston, Massachusetts 02115, USA. Electronic address:

Proliferation of malaria parasites in a host requires mechanisms to spread between red blood cells (RBCs). We discuss here the implications for biology and antimalarial drug development of companion studies that establish the requirement of two Plasmodium spp. proteases of the plasmepsin family in parasite egress from, and invasion into, RBCs.
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http://dx.doi.org/10.1016/j.pt.2017.11.012DOI Listing
February 2018