Actomyosin forces and the energetics of red blood cell invasion by the malaria parasite Plasmodium falciparum
Preprint posted on 25 June 2020 https://www.biorxiv.org/content/10.1101/2020.06.25.171900v1
Article now published in PLOS Pathogens at http://dx.doi.org/10.1371/journal.ppat.1009007
Malaria is caused by single-celled, obligate intracellular parasites from the genus Plasmodium, the most virulent of which is Plasmodium falciparum. All symptoms associated with malaria are the result of the asexual erythrocytic stages, involving cycles of red blood cell (RBC) invasion and egress by the Plasmodium spp. merozoite. Merozoites are small cells around 1 µm in size, that use substrate-dependent gliding motility. Merozoites are primed for invasion by phosphorylation of the motility apparatus before RBC egress, and have a short window of viability to invade a RBC. Plasmodium merozoites rely on a conserved molecular motor for gliding and RBC invasion, centred around a MyoA motor complex (MMC) or glideosome, situated between the parasite plasma membrane and the double membrane inner membrane complex (IMC). However, the precise function of PfMyoA in invasion, its regulation, the role of other myosins and overall energetics of invasion remain unclear. Questions still remain open, for example, regarding PfMyoA organisation and function, how motor force is integrated with retrograde flow of parasite plasma membrane and how force is applied across the parasite. Altogether, a greater understanding of the mechanical function of actomyosin during merozoite invasion is important. In their work, Blake et al (1) combine a conditional mutagenesis strategy with live video microscopy to gain insight into the energetics of invasion and probe PfMyoA function, and that of the auxiliary motor PfMyoB, during merozoite invasion.
Key findings and developments
Parasite line generation and characterization
The authors began by generating an ectopic expression platform for Plasmodium myosins. They modified a previously existing line, a conditional KO for PfMyoA (PfMyoA-cKO) that excises a terminal portion of the PfMyoA gene on rapamycin (RAP) treatment rendering it non-functional (2), to further investigate the role of PfMyoA.
Altogether, they used a) the PfMyoA-cKO parasite line and generated de novo b) a line for PfMyoA-complementation (PfMyoA-comp); c) a line carrying a K764E mutation, which is designed to probe phospho-regulation of PfMyoA –(hypothetically, this mutation should leave merozoites unaffected if they only need PfMyoA for maximal force production during invasion); d) a mutant for the PfMyoA light chain ELC (PfELC-cKO), as previous studies have suggested that the absence of PfELC leaves a functional but strongly weakened motor; and e) a conditional KO for PfMyoB, as myosin redundancy has been shown to contribute to residual invasion in T. gondii MyoA-cKO parasites (3). All cKO phenotypes, like PfMyoA-cKO, could be initiated with RAP treatment (activating DiCre-dependent gene excision).
Time-lapse microscopy-based phenotype characterization
WT and DMSO-treated parasites
Time-lapse imaging in live cells has been consistently used to describe merozoite invasion and to characterize Plasmodium mutants at various stages. The authors went on to use live imaging to analyse merozoite invasion by the various mutant lines generated in this work. Parasites were synchronized in the schizont stage, and merozoite invasion characterized. Invasion phenotypes were characterized based on separate stages generally observed in WT parasites, namely, attachment to RBC, deformation of the RBC surface, internalization into the RBC, and RBC echinocytosis. Mutants were classified into 4 types: Type A: successful invasion; Type B: incomplete internalization and merozoite ejection; Type C: deformation present, but no internalization; and Type D: neither deformation not internalization present, just attachment. Moreover, a score was used to assign each event based on the intensity of deformation.
PfMyoA-cKO and PfMyoA-comp
PfMyoA-cKO parasites after RAP treatment showed zero successful invasion events, showing no deformation or internalization (Type D failure), and therefore this mutant could not be used to probe the role of the motor in internalization directly. For PfMyoA-comp parasites, whilst a moderate drop in invasion success was observed, no significant differences in deformation strength or phase timings were observed.
Like PfMyoA-cKO parasites, rapamycin-treated PfELC-cKO parasites did not achieve any successful invasion. However, when observed in detail a significant proportion of events were nonetheless able to deform the RBC. This shows that a partially functional motor can achieve inefficient deformation. Furthermore, no PfELC-cKO merozoites were able to initiate internalization supporting a critical role for PfMyoA in driving merozoite internalisation, as well as deformation, and suggesting that the process of internalisation has a higher energetic barrier than deformation. Altogether, the results from PfMyoA-cKO and PfELC-cKO mutants shows that without PfMyoA or PfELC, merozoites cannot strongly deform or internalize.
Understanding the role of motor force during internalisation depends on finding intermediate-strength motor mutants able to initiate internalisation (namely, a gradient effect). PfMyoA-K764E merozoites could initiate invasion, but showed a significant increase in Type B failures (being ejected from the RBC). PfMyoA-K764E parasites are more likely to fail at initiation of internalisation (an increase in Type C failures) and, when they can initiate it, they take longer to do so (a longer pre-internalisation pause). These parasites are also more likely to fail to complete internalisation (causing the increase in Type B failures) and they take much longer to internalise when they fail, and slightly longer even when successful.
Video microscopy of RAP-treated PfMyoB-cKO parasites showed only a moderate reduction in successful invasion and increase in Type C failures, while the distribution of deformation scores was unchanged. The durations of some invasion phases were affected by PfMyoB-cKO. The significant delay in initiation of internalisation is consistent with a model of PfMyoB supporting the first stages of translocating the tight junction, but overall, PfMyoB is not required for internalization.
The study concludes overall, that while PfMyoB-cKO merozoites are delayed in initiation of internalisation, and PfMyoA-cKO and PfELC-cKO merozoites have insufficient force production to overcome the steps of deformation or internalisation, PfMyoA-K764E merozoites show a distinct defect at a third energetic barrier: completion of internalisation. These data therefore support a three-step model for the energetics of red blood cell entry by the merozoite: surface deformation of the RBC; initiation of internalisation/entry; and completion of internationalisation/closure of the tight junction around the entering merozoite.
What I like about this preprint
I find the question approached an interesting part of the puzzle to understand the invasion process. I like also that the authors took vast advantage of microscopy tools to explore the question addressed.
- Blake TCA et al, Actomyosin forces and the energetics of red blood cell invasion by the malaria parasite Plasmodium falciparum, bioRxiv, 2020.
- Birnbaum J et al, A genetic system to study Plasmodium falciparum protein function, Nature Methods, 2017.
- Frenal K et al, Plasticity between MyoC and MyoA- glideosomes: an example of functional compensation in Toxoplasma gondii PloS Pathogens, 2014.
Posted on: 13 July 2020Read preprint
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