Reporter lines based on the gexp02 promoter enable early quantification of sexual conversion rates in the malaria parasite Plasmodium falciparum

Abstract Transmission of malaria parasites from humans to mosquito vectors requires that some asexual parasites differentiate into sexual forms termed gametocytes. The balance between proliferation in the same host and conversion into transmission forms can be altered by the conditions of the environment. The ability to accurately measure the rate of sexual conversion under different conditions is essential for research addressing the mechanisms underlying sexual conversion, and to assess the impact of environmental factors. Here we describe new Plasmodium falciparum transgenic lines with genome-integrated constructs in which a fluorescent reporter is expressed under the control of the promoter of the gexp02 gene. Using these parasite lines, we developed a sexual conversion assay that shortens considerably the time needed for an accurate determination of sexual conversion rates, and dispenses the need to add chemicals to inhibit parasite replication. Furthermore, we demonstrate that gexp02 is expressed specifically in sexual parasites, with expression starting as early as the sexual ring stage, which makes it a candidate marker for circulating sexual rings in epidemiological studies.

Download Full-text

Stable Expression of a New Chimeric Fluorescent Reporter in the Human Malaria Parasite Plasmodium falciparum

Infection and Immunity ◽

10.1128/iai.68.4.2328-2332.2000 ◽

2000 ◽

Vol 68 (4) ◽

pp. 2328-2332 ◽

Cited By ~ 28

Author(s):

Madhusudan Kadekoppala ◽

Kimberly Kline ◽

Thomas Akompong ◽

Kasturi Haldar

Keyword(s):

Plasmodium Falciparum ◽

Malaria Parasite ◽

Stable Transfection ◽

Green Fluorescence ◽

Fluorescent Reporter ◽

Human Malaria Parasite ◽

Human Malaria ◽

Methotrexate Resistance ◽

Parasite Plasmodium ◽

Parasite Plasmodium Falciparum

ABSTRACT Stable transfection of a new, chimeric reporter in the human malaria parasite Plasmodium falciparum confers green fluorescence and methotrexate resistance that can be quantitated by Western blotting and flow cytometry. This provides a sensitive, live reporter for exploitation of genomic and high-throughput assays for the identification of new pathogenic determinants.

Download Full-text

Actomyosin forces and the energetics of red blood cell invasion by the malaria parasite Plasmodium falciparum

10.1101/2020.06.25.171900 ◽

2020 ◽

Cited By ~ 1

Author(s):

Thomas C. A. Blake ◽

Silvia Haase ◽

Jake Baum

Keyword(s):

Plasmodium Falciparum ◽

Blood Cell ◽

Red Blood Cell ◽

Force Production ◽

Essential Light Chain ◽

Parasite Plasmodium ◽

Parasite Plasmodium Falciparum ◽

Parasite Replication ◽

Malaria Species

SummaryAll symptoms of malaria disease are associated with the asexual blood stages of development, involving cycles of red blood cell (RBC) invasion and egress by the Plasmodium spp. merozoite. Merozoite invasion is rapid and is actively powered by a parasite actomyosin motor. The current accepted model for actomyosin force generation envisages arrays of parasite myosins, pushing against short actin filaments connected to the external milieu that drive the merozoite forwards into the RBC. In Plasmodium falciparum, the most virulent human malaria species, Myosin A (PfMyoA) is critical for parasite replication. However, the precise function of PfMyoA in invasion, its regulation, the role of other myosins and overall energetics of invasion remain unclear. Here, we developed a conditional mutagenesis strategy combined with live video microscopy to probe PfMyoA function and that of the auxiliary motor PfMyoB in invasion. By imaging conditional mutants with increasing defects in force production, based on disruption to a key PfMyoA phospho-regulation site, the absence of the PfMyoA essential light chain, or complete motor absence, we define three distinct stages of incomplete RBC invasion. These three defects reveal three energetic barriers to successful entry: RBC deformation (pre-entry), mid-invasion initiation, and completion of internalisation, each requiring an active parasite motor. In defining distinct energetic barriers to invasion, these data illuminate the mechanical challenges faced in this remarkable process of protozoan parasitism, highlighting distinct myosin functions and identifying potential targets for preventing malaria pathogenesis.

Download Full-text

Actomyosin forces and the energetics of red blood cell invasion by the malaria parasite Plasmodium falciparum

PLoS Pathogens ◽

10.1371/journal.ppat.1009007 ◽

2020 ◽

Vol 16 (10) ◽

pp. e1009007 ◽

Cited By ~ 1

Author(s):

Thomas C. A. Blake ◽

Silvia Haase ◽

Jake Baum

Keyword(s):

Plasmodium Falciparum ◽

Blood Cell ◽

Red Blood Cell ◽

Force Production ◽

Essential Light Chain ◽

Parasite Plasmodium ◽

Parasite Plasmodium Falciparum ◽

Parasite Replication ◽

Malaria Species

All symptoms of malaria disease are associated with the asexual blood stages of development, involving cycles of red blood cell (RBC) invasion and egress by the Plasmodium spp. merozoite. Merozoite invasion is rapid and is actively powered by a parasite actomyosin motor. The current accepted model for actomyosin force generation envisages arrays of parasite myosins, pushing against short actin filaments connected to the external milieu that drive the merozoite forwards into the RBC. In Plasmodium falciparum, the most virulent human malaria species, Myosin A (PfMyoA) is critical for parasite replication. However, the precise function of PfMyoA in invasion, its regulation, the role of other myosins and overall energetics of invasion remain unclear. Here, we developed a conditional mutagenesis strategy combined with live video microscopy to probe PfMyoA function and that of the auxiliary motor PfMyoB in invasion. By imaging conditional mutants with increasing defects in force production, based on disruption to a key PfMyoA phospho-regulation site, the absence of the PfMyoA essential light chain, or complete motor absence, we define three distinct stages of incomplete RBC invasion. These three defects reveal three energetic barriers to successful entry: RBC deformation (pre-entry), mid-invasion initiation, and completion of internalisation, each requiring an active parasite motor. In defining distinct energetic barriers to invasion, these data illuminate the mechanical challenges faced in this remarkable process of protozoan parasitism, highlighting distinct myosin functions and identifying potential targets for preventing malaria pathogenesis.

Download Full-text

Analysis of Antibodies Directed against Merozoite Surface Protein 1 of the Human Malaria Parasite Plasmodium falciparum

Infection and Immunity ◽

10.1128/iai.74.2.1313-1322.2006 ◽

2006 ◽

Vol 74 (2) ◽

pp. 1313-1322 ◽

Cited By ~ 47

Author(s):

Ute Woehlbier ◽

Christian Epp ◽

Christian W. Kauth ◽

Rolf Lutz ◽

Carole A. Long ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Surface Protein ◽

Merozoite Surface Protein ◽

Merozoite Surface Protein 1 ◽

Erythrocyte Invasion ◽

Parasite Plasmodium ◽

Parasite Plasmodium Falciparum ◽

Parasite Multiplication ◽

Parasite Replication

ABSTRACT The 190-kDa merozoite surface protein 1 (MSP-1) of Plasmodium falciparum, an essential component in the parasite's life cycle, is a primary candidate for a malaria vaccine. Rabbit antibodies elicited by the heterologously produced MSP-1 processing products p83, p30, p38, and p42, derived from strain 3D7, were analyzed for the potential to inhibit in vitro erythrocyte invasion by the parasite and parasite growth. Our data show that (i) epitopes recognized by antibodies, which inhibit parasite replication, are distributed throughout the entire MSP-1 molecule; (ii) when combined, antibodies specific for different regions of MSP-1 inhibit in a strictly additive manner; (iii) anti-MSP-1 antibodies interfere with erythrocyte invasion as well as with the intraerythrocytic growth of the parasite; and (iv) antibodies raised against MSP-1 of strain 3D7 strongly cross-inhibit replication of the heterologous strain FCB-1. Accordingly, anti-MSP-1 antibodies appear to be capable of interfering with parasite multiplication at more than one level. Since the overall immunogenicity profile of MSP-1 in rabbits closely resembles that found in sera of Aotus monkeys immunized with parasite-derived MSP-1 and of humans semi-immune to malaria from whom highly inhibiting antigen-specific antibodies were recovered, we consider the findings reported here to be relevant for the development of MSP-1-based vaccines against malaria.

Download Full-text

Exposure to artemisinin at the trophozoite stage increases sexual conversion rates in the malaria parasite Plasmodium falciparum

10.1101/2020.06.15.151746 ◽

2020 ◽

Author(s):

Harvie P. Portugaliza ◽

Shinya Miyazaki ◽

Fiona J.A. Geurten ◽

Christopher Pell ◽

Anna Rosanas-Urgell ◽

...

Keyword(s):

Malaria Parasite ◽

Fold Increase ◽

Trophozoite Stage ◽

Conversion Rates ◽

Multiplication Cycle ◽

Parasite Plasmodium ◽

Parasite Plasmodium Falciparum ◽

The Impact ◽

Gametocytocidal Activity ◽

Potential Public Health

ABSTRACTMalaria transmission is dependent on formation of gametocytes in the human blood. The sexual conversion rate, the proportion of asexual parasites that convert into gametocytes at each multiplication cycle, is variable and reflects the relative parasite investment between transmission and maintaining the infection. The impact of environmental factors such as drugs on sexual conversion rates is not well understood. We developed a robust assay using gametocyte-reporter parasite lines to accurately measure the impact of drugs on conversion rates, independently from their gametocytocidal activity. We found that exposure to subcurative doses of the frontline antimalarial drug dihydroartemisinin (DHA) at the trophozoite stage resulted in a ~4-fold increase in sexual conversion. In contrast, no increase was observed when ring stages were exposed or in cultures in which sexual conversion was stimulated by choline depletion. Our results reveal a complex relationship between antimalarial drugs and sexual conversion, with potential public health implications.

Download Full-text