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Rawat M, Srivastava A, Johri S, Gupta I, Karmodiya K. Single-Cell RNA Sequencing Reveals Cellular Heterogeneity and Stage Transition under Temperature Stress in Synchronized Plasmodium falciparum Cells. Microbiol Spectr 2021; 9:e0000821. [PMID: 34232098 PMCID: PMC8552519 DOI: 10.1128/spectrum.00008-21] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2021] [Accepted: 05/26/2021] [Indexed: 12/12/2022] Open
Abstract
The malaria parasite has a complex life cycle exhibiting phenotypic and morphogenic variations in two different hosts by existing in heterogeneous developmental states. To investigate this cellular heterogeneity of the parasite within the human host, we performed single-cell RNA sequencing of synchronized Plasmodium cells under control and temperature treatment conditions. Using the Malaria Cell Atlas (https://www.sanger.ac.uk/science/tools/mca) as a guide, we identified 9 subtypes of the parasite distributed across known intraerythrocytic stages. Interestingly, temperature treatment results in the upregulation of the AP2-G gene, the master regulator of sexual development in a small subpopulation of the parasites. Moreover, we identified a heterogeneous stress-responsive subpopulation (clusters 5, 6, and 7 [∼10% of the total population]) that exhibits upregulation of stress response pathways under normal growth conditions. We also developed an online exploratory tool that will provide new insights into gene function under normal and temperature stress conditions. Thus, our study reveals important insights into cell-to-cell heterogeneity in the parasite population under temperature treatment that will be instrumental toward a mechanistic understanding of cellular adaptation and population dynamics in Plasmodium falciparum. IMPORTANCE The malaria parasite has a complex life cycle exhibiting phenotypic variations in two different hosts accompanied by cell-to-cell variability that is important for stress tolerance, immune evasion, and drug resistance. To investigate cellular heterogeneity determined by gene expression, we performed single-cell RNA sequencing (scRNA-seq) of about 12,000 synchronized Plasmodium cells under physiologically relevant normal (37°C) and temperature stress (40°C) conditions phenocopying the cyclic bouts of fever experienced during malarial infection. In this study, we found that parasites exhibit transcriptional heterogeneity in an otherwise morphologically synchronized culture. Also, a subset of parasites is continually committed to gametocytogenesis and stress-responsive pathways. These observations have important implications for understanding the mechanisms of drug resistance generation and vaccine development against the malaria parasite.
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Affiliation(s)
- Mukul Rawat
- Department of Biology, Indian Institute of Science Education and Research, Pashan, Pune, Maharashtra, India
| | - Ashish Srivastava
- Department of Biology, Indian Institute of Science Education and Research, Pashan, Pune, Maharashtra, India
| | - Shreya Johri
- Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, New Delhi, India
| | - Ishaan Gupta
- Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, New Delhi, India
| | - Krishanpal Karmodiya
- Department of Biology, Indian Institute of Science Education and Research, Pashan, Pune, Maharashtra, India
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Correa R, Caballero Z, De León LF, Spadafora C. Extracellular Vesicles Could Carry an Evolutionary Footprint in Interkingdom Communication. Front Cell Infect Microbiol 2020; 10:76. [PMID: 32195195 PMCID: PMC7063102 DOI: 10.3389/fcimb.2020.00076] [Citation(s) in RCA: 21] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2019] [Accepted: 02/14/2020] [Indexed: 12/13/2022] Open
Abstract
Extracellular vesicles (EVs) are minute particles secreted by the cells of living organisms. Although the functional role of EVs is not yet clear, recent work has highlighted their role in intercellular communication. Here, we expand on this view by suggesting that EVs can also mediate communication among interacting organisms such as hosts, pathogens and vectors. This inter-kingdom communication via EVs is likely to have important evolutionary consequences ranging from adaptation of parasites to specialized niches in the host, to host resistance and evolution and maintenance of parasite virulence and transmissibility. A potential system to explore these consequences is the interaction among the human host, the mosquito vector and Plasmodium parasite involved in the malaria disease. Indeed, recent studies have found that EVs derived from Plasmodium infected red blood cells in humans are likely mediating the parasite's transition from the asexual to sexual stage, which might facilitate transmission to the mosquito vector. However, more work is needed to establish the adaptive consequences of this EV signaling among different taxa. We suggest that an integrative molecular approach, including a comparative phylogenetic analysis of the molecules (e.g., proteins and nucleic acids) derived from the EVs of interacting organisms (and their closely-related species) in the malaria system will prove useful for understanding interkingdom communication. Such analyses will also shed light on the evolution and persistence of host, parasite and vector interactions, with implications for the control of vector borne infectious diseases.
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Affiliation(s)
- Ricardo Correa
- Center of Cellular and Molecular Biology of Diseases, Instituto de Investigaciones Cientificas y Servicios de Alta Tecnologia (INDICASAT AIP), Panama, Panama.,Department of Biotechnology, Acharya Nagarjuna University, Guntur, India
| | - Zuleima Caballero
- Center of Cellular and Molecular Biology of Diseases, Instituto de Investigaciones Cientificas y Servicios de Alta Tecnologia (INDICASAT AIP), Panama, Panama
| | - Luis F De León
- Department of Biology, University of Massachusetts, Boston, MA, United States
| | - Carmenza Spadafora
- Center of Cellular and Molecular Biology of Diseases, Instituto de Investigaciones Cientificas y Servicios de Alta Tecnologia (INDICASAT AIP), Panama, Panama
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Correa R, Coronado L, Caballero Z, Faral-Tello P, Robello C, Spadafora C. Extracellular vesicles carrying lactate dehydrogenase induce suicide in increased population density of Plasmodium falciparum in vitro. Sci Rep 2019; 9:5042. [PMID: 30911042 PMCID: PMC6434017 DOI: 10.1038/s41598-019-41697-x] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2018] [Accepted: 03/14/2019] [Indexed: 11/29/2022] Open
Abstract
Even with access to sufficient nutrients and atmosphere, Plasmodium falciparum can barely be cultured at maximum growth capacity in vitro conditions. Because of this behavior, it has been suggested that P. falciparum has self-regulatory mechanisms in response to density stress. Only recently has this process begun to be acknowledged and characteristics of a programmed cell death been assigned to the parasite at high parasitaemia in vitro cultures. In searching for death signals within the parasite community, we have found that extracellular vesicles (EVs) of P. falciparum from high parasitaemia cultures are able to induce programmed cell death processes in the population. A comparative proteomic analysis of EVs from low (EVL) and high (EVH) parasitaemia cultures was conducted, pointing to lactate dehydrogenase from P. falciparum (PfLDH) as the only parasite protein overexpressed in the later. Although the major function of P. falciparum lactate dehydrogenase (PfLDH) is the conversion of pyruvate to lactate, a key process in the production of energy in most living organisms, we investigated its possible role in the mechanism of parasite density control by intercellular signaling, given that PfLDH had already been listed as a component of extracellular vesicles of P. falciparum. In this study we present evidence of the EV-associated PfLDH regulation of parasite population by inducing apoptosis in highly parasitized cultures.
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Affiliation(s)
- Ricardo Correa
- Center of Cellular and Molecular Biology of Diseases, Instituto de Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT AIP). City of Knowledge, Panama City, 0843-01103, Panama
- Department of Biotechnology, Acharya Nagarjuna University, Guntur, 522 510, A.P., India
- Sistema Nacional de Investigación, Secretaría Nacional de Ciencia, Tecnología e Innovación, Panama City, 0843-01103, Panama
| | - Lorena Coronado
- Center of Cellular and Molecular Biology of Diseases, Instituto de Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT AIP). City of Knowledge, Panama City, 0843-01103, Panama
- Sistema Nacional de Investigación, Secretaría Nacional de Ciencia, Tecnología e Innovación, Panama City, 0843-01103, Panama
| | - Zuleima Caballero
- Center of Cellular and Molecular Biology of Diseases, Instituto de Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT AIP). City of Knowledge, Panama City, 0843-01103, Panama
- Sistema Nacional de Investigación, Secretaría Nacional de Ciencia, Tecnología e Innovación, Panama City, 0843-01103, Panama
| | | | | | - Carmenza Spadafora
- Center of Cellular and Molecular Biology of Diseases, Instituto de Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT AIP). City of Knowledge, Panama City, 0843-01103, Panama.
- Sistema Nacional de Investigación, Secretaría Nacional de Ciencia, Tecnología e Innovación, Panama City, 0843-01103, Panama.
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Boulet C, Doerig CD, Carvalho TG. Manipulating Eryptosis of Human Red Blood Cells: A Novel Antimalarial Strategy? Front Cell Infect Microbiol 2018; 8:419. [PMID: 30560094 PMCID: PMC6284368 DOI: 10.3389/fcimb.2018.00419] [Citation(s) in RCA: 44] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2018] [Accepted: 11/14/2018] [Indexed: 12/13/2022] Open
Abstract
Malaria is a major global health burden, affecting over 200 million people worldwide. Resistance against all currently available antimalarial drugs is a growing threat, and represents a major and long-standing obstacle to malaria eradication. Like many intracellular pathogens, Plasmodium parasites manipulate host cell signaling pathways, in particular programmed cell death pathways. Interference with apoptotic pathways by malaria parasites is documented in the mosquito and human liver stages of infection, but little is known about this phenomenon in the erythrocytic stages. Although mature erythrocytes have lost all organelles, they display a form of programmed cell death termed eryptosis. Numerous features of eryptosis resemble those of nucleated cell apoptosis, including surface exposure of phosphatidylserine, cell shrinkage and membrane ruffling. Upon invasion, Plasmodium parasites induce significant stress to the host erythrocyte, while delaying the onset of eryptosis. Many eryptotic inducers appear to have a beneficial effect on the course of malaria infection in murine models, but major gaps remain in our understanding of the underlying molecular mechanisms. All currently available antimalarial drugs have parasite-encoded targets, which facilitates the emergence of resistance through selection of mutations that prevent drug-target binding. Identifying host cell factors that play a key role in parasite survival will provide new perspectives for host-directed anti-malarial chemotherapy. This review focuses on the interrelationship between Plasmodium falciparum and the eryptosis of its host erythrocyte. We summarize the current knowledge in this area, highlight the different schools of thoughts and existing gaps in knowledge, and discuss future perspectives for host-directed therapies in the context of antimalarial drug discovery.
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Affiliation(s)
- Coralie Boulet
- Molecular Parasitology Laboratory, Department of Physiology, Anatomy and Microbiology, La Trobe University, Bundoora, VIC, Australia
| | - Christian D Doerig
- Infection and Immunity Program, Biomedicine Discovery Institute, Department of Microbiology, Monash University, Clayton, VIC, Australia
| | - Teresa G Carvalho
- Molecular Parasitology Laboratory, Department of Physiology, Anatomy and Microbiology, La Trobe University, Bundoora, VIC, Australia
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Totino PRR, Daniel-Ribeiro CT, Ferreira-da-Cruz MDF. Evidencing the Role of Erythrocytic Apoptosis in Malarial Anemia. Front Cell Infect Microbiol 2016; 6:176. [PMID: 28018860 PMCID: PMC5145864 DOI: 10.3389/fcimb.2016.00176] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2016] [Accepted: 11/21/2016] [Indexed: 01/06/2023] Open
Abstract
In the last decade it has become clear that, similarly to nucleated cells, enucleated red blood cells (RBCs) are susceptible to programmed apoptotic cell death. Erythrocytic apoptosis seems to play a role in physiological clearance of aged RBCs, but it may also be implicated in anemia of different etiological sources including drug therapy and infectious diseases. In malaria, severe anemia is a common complication leading to death of children and pregnant women living in malaria-endemic regions of Africa. The pathogenesis of malarial anemia is multifactorial and involves both ineffective production of RBCs by the bone marrow and premature elimination of non-parasitized RBCs, phenomena potentially associated with apoptosis. In the present overview, we discuss evidences associating erythrocytic apoptosis with the pathogenesis of severe malarial anemia, as well as with regulation of parasite clearance in malaria. Efforts to understand the role of erythrocytic apoptosis in malarial anemia can help to identify potential targets for therapeutic intervention based on apoptotic pathways and consequently, mitigate the harmful impact of malaria in global public health.
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Affiliation(s)
- Paulo R R Totino
- Laboratory of Malaria Research, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz Rio de Janeiro, Brazil
| | - Cláudio T Daniel-Ribeiro
- Laboratory of Malaria Research, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz Rio de Janeiro, Brazil
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