Dual Plasmepsin-Targeting Antimalarial Agents Disrupt Multiple Stages of the Malaria Parasite Life Cycle

ABSTRACT A role for the Plasmodium falciparum cyclic GMP (cGMP)-dependent protein kinase (PfPKG) in gametogenesis in the malaria parasite was elucidated previously. In the present study we examined the role of PfPKG in the asexual blood-stage of the parasite life cycle, the stage that causes malaria pathology. A specific PKG inhibitor (compound 1, a trisubstituted pyrrole) prevented the progression of P. falciparum schizonts through to ring stages in erythrocyte invasion assays. Addition of compound 1 to ring-stage parasites allowed normal development up to 30 h postinvasion, and segmented schizonts were able to form. However, synchronized schizonts treated with compound 1 for ≥6 h became large and dysmorphic and were unable to rupture or liberate merozoites. To conclusively demonstrate that the effect of compound 1 on schizogony was due to its selective action on PfPKG, we utilized genetically manipulated P. falciparum parasites expressing a compound 1-insensitive PfPKG. The mutant parasites were able to complete schizogony in the presence of compound 1 but not in the presence of the broad-spectrum protein kinase inhibitor staurosporine. This shows that PfPKG is the primary target of compound 1 during schizogony and provides direct evidence of a role for PfPKG in this process. Discovery of essential roles for the P. falciparum PKG in both asexual and sexual development demonstrates that cGMP signaling is a key regulator of both of these crucial life cycle phases and defines this molecule as an exciting potential drug target for both therapeutic and transmission blocking action against malaria.

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Enlightening the malaria parasite life cycle: bioluminescent Plasmodium in fundamental and applied research

Frontiers in Microbiology ◽

10.3389/fmicb.2015.00391 ◽

2015 ◽

Vol 6 ◽

Cited By ~ 20

Author(s):

Giulia Siciliano ◽

Pietro Alano

Keyword(s):

Life Cycle ◽

Malaria Parasite ◽

Applied Research ◽

Parasite Life Cycle

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Calcium in the Backstage of Malaria Parasite Biology

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.708834 ◽

2021 ◽

Vol 11 ◽

Author(s):

Lucas Silva de Oliveira ◽

Marcos Rodrigo Alborghetti ◽

Renata Garcia Carneiro ◽

Izabela Marques Dourado Bastos ◽

Rogerio Amino ◽

...

Keyword(s):

Life Cycle ◽

Protein Kinases ◽

Malaria Parasite ◽

Developmental Stages ◽

Receptor Gene ◽

Food Vacuole ◽

Parasite Development ◽

Cell Infection ◽

Parasite Life Cycle ◽

Downstream Signaling

The calcium ion (Ca2+) is a ubiquitous second messenger involved in key biological processes in prokaryotes and eukaryotes. In Plasmodium species, Ca2+ signaling plays a central role in the parasite life cycle. It has been associated with parasite development, fertilization, locomotion, and host cell infection. Despite the lack of a canonical inositol-1,4,5-triphosphate receptor gene in the Plasmodium genome, pharmacological evidence indicates that inositol-1,4,5-triphosphate triggers Ca2+ mobilization from the endoplasmic reticulum. Other structures such as acidocalcisomes, food vacuole and mitochondria are proposed to act as supplementary intracellular Ca2+ reservoirs. Several Ca2+-binding proteins (CaBPs) trigger downstream signaling. Other proteins with no EF-hand motifs, but apparently involved with CaBPs, are depicted as playing an important role in the erythrocyte invasion and egress. It is also proposed that a cross-talk among kinases, which are not members of the family of Ca2+-dependent protein kinases, such as protein kinases G, A and B, play additional roles mediated indirectly by Ca2+ regulation. This statement may be extended for proteins directly related to invasion or egress, such as SUB1, ERC, IMC1I, IMC1g, GAP45 and EBA175. In this review, we update our understanding of aspects of Ca2+-mediated signaling correlated to the developmental stages of the malaria parasite life cycle.

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The dimerisable Cre recombinase allows conditional genome editing in the mosquito stages of Plasmodium berghei

10.1101/2020.07.13.200402 ◽

2020 ◽

Author(s):

Priyanka Fernandes ◽

Sylvie Briquet ◽

Delphine Patarot ◽

Manon Loubens ◽

Bénédicte Hoareau-Coudert ◽

...

Keyword(s):

Life Cycle ◽

Dna Sequences ◽

Malaria Parasite ◽

Reverse Genetics ◽

High Efficiency ◽

Parasite Life Cycle ◽

Asexual Blood Stage ◽

Functional Studies ◽

Powerful Approach ◽

Regulatable Promoters

ABSTRACTAsexual blood stages of the malaria parasite are readily amenable to genetic modification via homologous recombination, allowing functional studies of parasite genes that are not essential in this part of the life cycle. However, conventional reverse genetics cannot be applied for the functional analysis of genes that are essential during asexual blood-stage replication. Various strategies have been developed for conditional mutagenesis of Plasmodium, including recombinase-based gene deletion, regulatable promoters, and mRNA or protein destabilization systems. Among these, the dimerisable Cre (DiCre) recombinase system has emerged as a powerful approach for conditional gene targeting in P. falciparum. In this system, the bacteriophage Cre is expressed in the form of two separate, enzymatically inactive polypeptides, each fused to a different rapamycin-binding protein. Rapamycin-induced heterodimerization of the two components restores recombinase activity. We have implemented the DiCre system in the rodent malaria parasite P. berghei, and show that rapamycin-induced excision of floxed DNA sequences can be achieved with very high efficiency in both mammalian and mosquito parasite stages. This tool can be used to investigate the function of essential genes not only in asexual blood stages, but also in other parts of the malaria parasite life cycle.

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Identification of aTheileria annulataAntigen Expressed in Multiple Stages of the Parasite Life Cycle

Experimental Parasitology ◽

10.1006/expr.1998.4303 ◽

1998 ◽

Vol 90 (1) ◽

pp. 110-121 ◽

Cited By ~ 5

Author(s):

P.A. Knight ◽

S.W. Williamson ◽

C.G.D. Brown ◽

L. Bell-Sakyi ◽

E. Kirvar ◽

...

Keyword(s):

Life Cycle ◽

Parasite Life Cycle ◽

Multiple Stages

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Supression of Perforin-like Protein pores inhibitPlasmodiummultistage-growth, transmission and erythrocyte senescence

10.1101/756197 ◽

2019 ◽

Cited By ~ 2

Author(s):

Swati Garg ◽

Abhishek Shivappagowdar ◽

Rahul S. Hada ◽

Rajagopal Ayana ◽

Chandramohan Bathula ◽

...

Keyword(s):

Life Cycle ◽

Pore Formation ◽

Host Cells ◽

Parasite Development ◽

Parasite Life Cycle ◽

Efficient Treatment ◽

Transmission Blocking ◽

Multi Stage ◽

Macpf Domain ◽

Multiple Stages

AbstractThe pore formingPlasmodiumperforin like proteins (PPLP), expressed in all stages of the parasite life cycle are central drivers for host interactions critical for completion of parasite life cycle and high transmission rates. The high sequence similarity in the central membrane attack complex/ perforin (MACPF) domain and consequent functional overlaps defines them as an attractive target for the development of multi-stage antimalarials. Herein we evaluated the mechanism of pan active function of central, highly conserved region of PPLPs, MACPF domain (PMD) and inhibitory potential of specifically designed anti-PMD chemo. TheE. coliexpressed rPMD interacts with erythrocyte membrane and form pores of ~10.5 nm height and ~24.3 nm diameter leading to haemoglobin release and dextran uptake. The treatment with PMD induced erythrocytes senescence at 48 hours which can account for the physiological effect of disseminated PLPs in loss of circulating erythrocytes inducing anemia during malaria infection. The anti-PMD inhibitors effectively blocked intraerythrocytic growth by suppressing invasion and egress of merozoites and protecting against erythrocyte senescence. Moreover, these inhibitors also blocked the hepatic stage and transmission stage parasite development suggesting multi-stage and transmission-blocking potential of these inhibitors. Additionally, the erythrocyte senescence protective potential of PMD inhibitors can be used to occlude PPLPs mediated severe malarial anemia. Further these inhibitors can be developed with a potential to protect against severity of the disease.Author SummaryMalaria continues to be a major global health threat despite of several exciting improvements in the treatment and prevention of the disease. One of the major concerns in the development of therapy is the emergence of the drug resistance. But for the efficient treatment regime, targeting multiple stages including host and vector would serve as an ideal therapy. Perforin like proteins (PLPs) are eukaryotic pore forming proteins that are highly conserved in the apicomplexan parasites. These play crucial roles in entry and exit of parasites from the host cells and establish infection at multiple stages ofPlasmodium spp.life cycle. Understanding the mechanism of pore formation by smaller, functional, pan-active scaffold of PLPs can serve as a target for development of cross stage protection. Here, using various biochemical, biophysical and pharmacological evidences, we validate the activity and characterize the pore formation of PLPs on erythrocytes. Further, our specifically designed inhibitors could restrict this pore formation and impede the exit/entry of the parasites. Moreover, these inhibitors could also exert multiple stage inhibition and rescue the uninfected erythrocytes from death. Together, this study highlights the mechanism of pore formation by PPLPs and evaluates their potential for the development of pan-active inhibitors to provide both symptomatic and transmission blocking cure for malaria.

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Kinesin-8B controls basal body function and flagellum formation and is key to malaria parasite transmission

10.1101/686568 ◽

2019 ◽

Cited By ~ 4

Author(s):

Mohammad Zeeshan ◽

David J. P. Ferguson ◽

Steven Abel ◽

Alana Burrrell ◽

Edward Rea ◽

...

Keyword(s):

Electron Microscopy ◽

Life Cycle ◽

Live Cell Imaging ◽

Malaria Parasite ◽

Cell Imaging ◽

Male Gamete ◽

Live Cell ◽

Basal Bodies ◽

Parasite Transmission ◽

Parasite Life Cycle

AbstractEukaryotic flagella are conserved microtubule-based organelles that drive cell motility. Plasmodium, the causative agent of malaria, has a single flagellate stage: the male gamete in the mosquito. Three rounds of endomitotic division together with an unusual mode of flagellum assembly rapidly produce eight motile gametes. These processes are tightly coordinated but their regulation is poorly understood. To understand this important developmental stage, we studied the function and location of the microtubule-based motor kinesin-8B, using gene-targeting, electron microscopy and live cell imaging. Deletion of the kinesin-8B gene showed no effect on mitosis but disrupted 9+2 axoneme assembly and flagellum formation during male gamete development and also completely ablated parasite transmission. Live cell imaging showed that kinesin-8B-GFP did not colocalise with kinetochores in the nucleus but instead revealed dynamic, cytoplasmic localisation with the basal bodies and the assembling axoneme during flagellum formation. We thus uncovered an unexpected role for kinesin-8B in parasite flagellum formation that is vital for the parasite life cycle.

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Targeting the Malaria Parasite cGMP-Dependent Protein Kinase to Develop New Drugs

Frontiers in Microbiology ◽

10.3389/fmicb.2020.602803 ◽

2020 ◽

Vol 11 ◽

Author(s):

David A. Baker ◽

Alexios N. Matralis ◽

Simon A. Osborne ◽

Jonathan M. Large ◽

Maria Penzo

Keyword(s):

Life Cycle ◽

Protein Kinase ◽

Malaria Parasite ◽

Public Health Problem ◽

Major Public Health Problem ◽

Parasite Life Cycle ◽

Dependent Protein Kinase ◽

Cgmp Dependent Protein Kinase ◽

Cgmp Signaling ◽

Dependent Protein

The single-celled apicomplexan parasite Plasmodium falciparum is responsible for the majority of deaths due to malaria each year. The selection of drug resistance has been a recurring theme over the decades with each new drug that is developed. It is therefore crucial that future generations of drugs are explored to tackle this major public health problem. Cyclic GMP (cGMP) signaling is one of the biochemical pathways that is being explored as a potential target for new antimalarial drugs. It has been shown that this pathway is essential for all of the key developmental stages of the complex malaria parasite life cycle. This gives hope that targeting cGMP signaling might give rise to drugs that treat disease, block its transmission and even prevent the establishment of infection. Here we review previous work that has been carried out to develop and optimize inhibitors of the cGMP-dependent protein kinase (PKG) which is a critical regulator of the malaria parasite life cycle.

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Structure and Function of an Atypical Homodimeric Actin Capping Protein from the Malaria Parasite

10.21203/rs.3.rs-905037/v1 ◽

2021 ◽

Author(s):

Ábris Ádám Bendes ◽

Petri Kursula ◽

Inari Kursula

Keyword(s):

Life Cycle ◽

Actin Filament ◽

Malaria Parasite ◽

Critical Concentration ◽

Binding Mode ◽

Actin Dynamics ◽

Β Subunit ◽

Parasite Life Cycle ◽

Capping Protein ◽

Actin Capping Protein

Abstract Apicomplexan parasites, such as Plasmodium spp., rely on an unusual actomyosin motor, termed glideosome, for motility and host cell invasion. The actin filaments are maintained by a small set of essential regulators, which provide control over actin dynamics in the different stages of the parasite life cycle. Actin filament capping proteins (CPs) are indispensable heterodimeric regulators of actin dynamics. CPs have been extensively characterized in higher eukaryotes, but their role and functional mechanism in Apicomplexa remain enigmatic. Here, we present the first crystal structure of a homodimeric CP from the malaria parasite and compare the homo- and heterodimeric CP structures in detail. Despite retaining several characteristics of a canonical CP, the homodimeric Plasmodium berghei (Pb)CP exhibits crucial differences to the canonical heterodimers. Both homo- and heterodimeric PbCPs regulate actin dynamics in an atypical manner, facilitating rapid turnover of parasite actin, without affecting its critical concentration. Homo- and heterodimeric PbCPs show partially redundant activities, possibly to rescue actin filament capping in life cycle stages where the β-subunit is downregulated,. Our data suggest that the homodimeric PbCP also influences actin kinetics by recruiting lateral actin dimers. This unusual function could arise from the absence of a β-subunit, as the asymmetric PbCP homodimer lacks structural elements essential for canonical barbed end interactions suggesting a novel CP binding mode. These findings will facilitate further studies aimed at elucidating the precise actin filament capping mechanism in Plasmodium.

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Identification and characterisation of a phospholipid scramblase in the malaria parasite Plasmodium falciparum

10.1101/2020.06.22.165258 ◽

2020 ◽

Author(s):

Silvia Haase ◽

Melanie Condron ◽

David Miller ◽

Dounia Cherkaoui ◽

Sarah Jordan ◽

...

Keyword(s):

Life Cycle ◽

Metal Ion ◽

Malaria Parasite ◽

Gene Knockout ◽

Parasite Development ◽

Asexual Parasite ◽

Parasite Life Cycle ◽

Phospholipid Scramblase ◽

Erythrocyte Invasion ◽

Parasite Plasmodium Falciparum

AbstractRecent studies highlight the emerging role of lipids as important messengers in malaria parasite biology. In an attempt to identify interacting proteins and regulators of these dynamic and versatile molecules, we hypothesised the involvement of phospholipid translocases and their substrates in the infection of the host erythrocyte by the malaria parasite Plasmodium spp. Here, using a data base mining approach, we have identified a putative phospholipid (PL) scramblase in P. falciparum (PfPLSCR) that is conserved across the genus and in closely related unicellular algae. By reconstituting recombinant PfPLSCR into liposomes, we demonstrate metal ion dependent PL translocase activity and substrate preference, confirming PfPLSCR as a bona fide scramblase. We confirm that PfPLSCR is expressed during asexual and sexual parasite development, localising to different membranous compartments of the parasite throughout the intra-erythrocytic life cycle. Two different gene knockout approaches, however, suggest that PfPLSCR is not essential for erythrocyte invasion and asexual parasite development, pointing towards a possible role in other stages of the parasite life cycle.

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