Flow Cytometry Based Detection and Isolation of Plasmodium falciparum Liver Stages In Vitro

Dumoulin, Peter C.; Trop, Stefanie A.; Ma, Jinxia; Zhang, Hao; Sherman, Matthew A.; Levitskaya, Jelena

doi:10.1371/journal.pone.0129623

Cited by 16 publications

(20 citation statements)

References 57 publications

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“…The traversal activity of sporozoites was measured using the standard cell-wounding assay described in (Dumoulin et al, 2015). Briefly, 5 × 10 4 HC-04 cells were seeded into each well of a 48-well plate (Corning, Sigma-Aldrich) that had been coated with type IV rat-tail collagen.…”

Section: Cell Traversal Assaymentioning

confidence: 99%

“…Although P. falciparum sporozoites traverse human macrophages and hepatocytes in vitro (Behet et al, 2014;Dumoulin et al, 2015;van Schaijk et al, 2008;, there is a paucity of knowledge surrounding the molecular basis for these events (reviewed by Yang & Boddey, 2017). Recently, it was shown that GAPDH on the sporozoite surface interacts with CD68 on Kupffer cells to facilitate traversal through the macrophages (Cha, Kim, Pandey, & Jacobs-Lorena, 2016) and that SPECT and PLP1 are essential for P. falciparum traversal of hepatocytes .…”

Section: Introductionmentioning

confidence: 99%

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AMA1 and MAEBL are important forPlasmodium falciparumsporozoite infection of the liver

Yang

Lopaticki

O'Neill

et al. 2017

Cellular Microbiology

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The malaria sporozoite injected by a mosquito migrates to the liver by traversing host cells. The sporozoite also traverses hepatocytes before invading a terminal hepatocyte and developing into exoerythrocytic forms. Hepatocyte infection is critical for parasite development into merozoites that infect erythrocytes, and the sporozoite is thus an important target for antimalarial intervention. Here, we investigated two abundant sporozoite proteins of the most virulent malaria parasite Plasmodium falciparum and show that they play important roles during cell traversal and invasion of human hepatocytes. Incubation of P. falciparum sporozoites with R1 peptide, an inhibitor of apical merozoite antigen 1 (AMA1) that blocks merozoite invasion of erythrocytes, strongly reduced cell traversal activity. Consistent with its inhibitory effect on merozoites, R1 peptide also reduced sporozoite entry into human hepatocytes. The strong but incomplete inhibition prompted us to study the AMA-like protein, merozoite apical erythrocyte-binding ligand (MAEBL). MAEBL-deficient P. falciparum sporozoites were severely attenuated for cell traversal activity and hepatocyte entry in vitro and for liver infection in humanized chimeric liver mice. This study shows that AMA1 and MAEBL are important for P. falciparum sporozoites to perform typical functions necessary for infection of human hepatocytes. These two proteins therefore have important roles during infection at distinct points in the life cycle, including the blood, mosquito, and liver stages.

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Section: Cell Traversal Assaymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

AMA1 and MAEBL are important forPlasmodium falciparumsporozoite infection of the liver

Yang

Lopaticki

O'Neill

et al. 2017

Cellular Microbiology

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“…falciparum in cryopreserved primary human hepatocytes has recently been reported [51] [52] Based on or results, and other published reports, we infer that primary human hepatocytes may be a better system than the HCO-4 cell line to study the development and growth of liver form P . falciparum parasites [53]. …”

Section: Discussionmentioning

confidence: 99%

Molecular Markers of Radiation Induced Attenuation in Intrahepatic Plasmodium falciparum Parasites

et al. 2016

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Experimental immunization with radiation attenuated sporozoites (RAS) and genetically attenuated sporozoites has proved to be a promising approach for malaria vaccine development. However, parasite biomarkers of growth attenuation and enhanced immune protection in response to radiation remain poorly understood. Here, we report on the effect of an attenuating dose of γ-irradiation (15 krad) on the Plasmodium falciparum sporozoite (PfSPZ) ultrastructure by electron microscopy, growth rate of liver stage P. falciparum in liver cell cultures, and genome-wide transcriptional profile of liver stage parasites by microarray. We find that γ-irradiation treated PfSPZ retained a normal cellular structure except that they were vacuous with a partially disrupted plasma membrane and inner membrane complex. A similar infection rate was observed by γ-irradiation-treated and untreated PfSPZ in human HCO-4 liver cells (0.47% versus 0.49%, respectively) on day 3 post-infection. In the microarray studies, cumulatively, 180 liver stage parasite genes were significantly transcriptionally altered on day 3 and/or 6 post-infection. Among the transcriptionally altered biomarkers, we identified a signature of seven candidate parasite genes that associated with functionally diverse pathways that may regulate radiation induced cell cycle arrest of the parasite within the hepatocyte. A repertoire of 14 genes associated with protein translation is transcriptionally overexpressed within the parasite by radiation. Additionally, 37 genes encode proteins expressed on the cell surface or exported into the host cell, 4 encode membrane associated transporters, and 10 encode proteins related to misfolding and stress-related protein processing. These results have significantly increased the repertoire of novel targets for 1) biomarkers of safety to define proper attenuation, 2) generating genetically attenuated parasite vaccine candidates, and 3) subunit candidate vaccines against liver stage malaria.

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“…Primary mouse hepatocytes and mouse cell lines, have had infectivity's lower than 6 and 2%, respectively, using RMPs, based on immunofluorescence microscopy or flow cytometry ( Table 3). The lower infectivity's observed are a typical feature of malaria liver-stage infection and depend not only on the origin of the host cells and strain of parasite, but also on the expression of CD81 receptor, which has been described as essential for primary human hepatocyte invasion (107,109). The infectivity may vary due to the expression levels of EhpA2 (110) and the class B, type I scavenger receptor (SR-BI) (111).…”

Section: Infectivitymentioning

confidence: 99%

Current Challenges in the Identification of Pre-Erythrocytic Malaria Vaccine Candidate Antigens

Bettencourt

2020

Front. Immunol.

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Plasmodium spp.-infected mosquitos inject sporozoites into the skin of a mammalian host during a blood meal. These enter the host's circulatory system and establish an infection in the liver. After a silent metamorphosis, merozoites invade the blood leading to the symptomatic and transmissible stages of malaria. The silent pre-erythrocytic malaria stage represents a bottleneck in the disease which is ideal to block progression to clinical malaria, through chemotherapeutic and immunoprophylactic interventions. RTS,S/AS01, the only malaria vaccine close to licensure, although with poor efficacy, blocks the sporozoite invasion mainly through the action of antibodies against the CSP protein, a major component of the pellicle of the sporozoite. Strikingly, sterile protection against malaria can be obtained through immunization with radiation-attenuated sporozoites, genetically attenuated sporozoites or through chemoprophylaxis with infectious sporozoites in animals and humans, but the deployability of sporozoite-based live vaccines pose tremendous challenges. The protection induced by sporozoites occurs in the pre-erythrocytic stages and is mediated mainly by antibodies against the sporozoite and CD8 + T cells against peptides presented by MHC class I molecules in infected hepatocytes. Thus, the identification of malaria antigens expressed in the sporozoite and liver-stage may provide new vaccine candidates to be included, alone or in combination, as recombinant protein-based, virus-like particles or sub-unit virally-vectored vaccines. Here I review the efforts being made to identify Plasmodium falciparum antigens expressed during liver-stage with focus on the development of parasite, hepatocyte, mouse models, and resulting rate of infection in order to identify new vaccine candidates and to improve the efficacy of the current vaccines. Finally, I propose new approaches for the identification of liver-stage antigens based on immunopeptidomics.

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Flow Cytometry Based Detection and Isolation of Plasmodium falciparum Liver Stages In Vitro

Cited by 16 publications

References 57 publications

AMA1 and MAEBL are important forPlasmodium falciparumsporozoite infection of the liver

AMA1 and MAEBL are important forPlasmodium falciparumsporozoite infection of the liver

Molecular Markers of Radiation Induced Attenuation in Intrahepatic Plasmodium falciparum Parasites

Current Challenges in the Identification of Pre-Erythrocytic Malaria Vaccine Candidate Antigens

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