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

Thomas C. A. Blake, Silvia Haase, Jake Baum

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

Modulating forces at play in malaria infection.

Selected by Mariana De Niz

Categories: cell biology

Background

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

Figure 1. A stepwise model for Plasmodium myosin force generation during merozoite invasion (Reproduced from Ref.1 (Figure 7) under CC-BY license)).

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.

PfELC-cKO

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.

PfMyoA-K764E

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.

PfMyoB-cKO

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.

References

  1. Blake TCA et al, Actomyosin forces and the energetics of red blood cell invasion by the malaria parasite Plasmodium falciparum, bioRxiv, 2020.
  2. Birnbaum J et al, A genetic system to study Plasmodium falciparum protein function, Nature Methods, 2017.
  3. 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 2020

doi: https://doi.org/10.1242/prelights.23049

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Author's response

Jake Baum and Thomas Blake shared

Open questions

1.You discuss in your work that the culture conditions that best imitate physiological conditions are not clear. Various strains capable of infecting rodents exist, and rodent models have been very useful for addressing various questions ranging from molecular biology to the in vivo pathology of malaria. Would rodent models be a suitable option to reproduce the mutants you have generated here, and explore in vivo the different invasion phenotypes?

This is certainly an interesting idea and could work in principle, however, if any mutant has a lethal phenotype (E.g. ELC or MyoA) then the parasite will simply not propagate in the mouse. A conditional system is possible, but timing this so that on addition of some signal all parasites would lose the target gene or gain a particular mutant is challenging, and, even then, the phenotype would be plus/minus, growth or no growth. Unfortunately, the other major limitation with mouse malaria models is they don’t propagate well in vitro so the ability to do a similar detailed video microscopy study is actually harder in these parasites. Staying with the human parasite also has its advantages in terms of relevance to human disease and setting the stage if we ever really wanted to try and target the motor as a way of targeting disease, which is certainly a long-term goal of this research.

2.You and others have described that suspension cultures significantly affects invasion phenotypes and adhesin expression. A lot of the work prior to this finding, were performed in static conditions. In your opinion, what observations and characterizations regarding invasion, might be worth re-visiting in suspension cultures?

The debate regarding static vs. suspension cultures is still very much alive (e.g. a recent study from the University of Ghana showed parasites invade RBCs using different receptor-ligand interactions under these conditions). This means we likely have only a semblance of understanding what really goes on in the blood stream under flow and shear forces. Yet the challenges of doing cell biology in a suspension environment are clear – you can’t exactly video a cell under these conditions. Some groups are exploring lab on a chip approaches so that moving cells can actually be videoed. This would be a potential game changer for understanding the fine-grained details of receptor usage, motor force and the stepwise events of invasion in a much more physiologically relevant setting. But for now, it’s a bit like where the world was at with 2D vs. 3D tissue culture a few years back – we learn from the 2D and eventually when 3D is possible, we re-test. And we may have to accept that some observations won’t hold up under these conditions! If we could do flow imaging I think receptor usage is the big one – since this is where a LOT of vaccines are targeting. If under flow it turns out receptor usage is different then that could change translational strategies entirely.

3.What other biophysical factors in physiological circulation, could further influence invasion success? For instance it is known that in vivo, while there is exponential parasitemia growth, a plateau is eventually reached, with less successful invasion events. Is there a threshold whereby maximum force generation by the merozoite still is not enough for invasion – for instance in a highly viscous environment?

Not sure how to answer this question – there’s so much we don’t know. One question we’ve had for a long time is where does invasion occur? We don’t really know this. It’s unlikely to be in the fast flowing arteries and veins of the body, much more likely in deep vascular capillary beds where flow is almost zero, so in fact it might be more like 2D static cultures after all. We also don’t really know which RBCs falciparum is actually choosing to invade. If some invasion is happening in the bone marrow milieu for example, this means reticulocytes may be being chosen. If this is the case the biophysical forces required to enter a reticulocyte are completely different from a discoid normocyte. There are some clinical studies that have tried to classify which RBCs are targeted in vivo but we probably need more work on that to really know. Also, you might imagine that physiological factors like blood lipids or cholesterol that affect the RBC membrane composition would affect parasite invasion success. To get back to your question regarding the plateau of growth, I’ve seen 50% parasitaemia in a clinical sample (the patient didn’t recover), so plateaus may be multi-factorial (e.g. including immune responses) so I’m not sure whether biophysical factors are the only, or even the major, force at play.

4.You discussed in your work the role of RBC biophysical properties. Could you expand further how this could alter invasion, and specifically whether and how MyoA-mediated force generation might be modulated in such case?

The purpose of RBC deformation may be to select a RBC with suitable biophysical properties for subsequent invasion. After this exclusion of RBCs that would be too difficult to invade, we’ve previously shown (Koch et al. PMID: 28373555) that parasite receptor binding induces biophysical changes in the RBC likely at (or just prior to) invasion. This means the parasite may be laying the table so to speak – preparing the RBC surface for invasion just before it drives in. There’s other data that supports this (e.g. from atomic force microscopy) as well as emerging data that RBC polymorphisms may have intrinsic differences in RBC biophysical properties that prevent invasion (or make it harder) – e.g. the Dantu blood type caused by a hybrid glycophorin receptor. So, in the case where the RBC is more rigid or the parasite has changed the dynamics of the RBC surface on binding, the biophysical barriers to invasion may be quite different. With the former, we could well imagine that subtle motor differences (e.g. effectiveness of Myosin B) might actually be MUCH more important. With the motor, we know its phospho-regulated. Is it possible the motor is tuned depending on how much force/load bearing is required? Possibly – and it would certainly fit with structural models of a responsive motor as and when its required to be either fast/slow, forceful or light. They are terrific molecules to study!

5.Following from this question, what are host-based factors affecting invasion? Namely, blood types, blood cell stage of maturation, RBC age? Is it known how these factors affect invasion preference?

Following some of our previous comments above, they ALL likely come into play. RBCs change radically in their biophysical properties as they mature from soft, malleable reticulocytes to hard discoid normocytes (which get harder with age as they lose RBC membrane). So, age is a massive factor – and it may be one of falciparum’s success stories. Unlike P. vivax which can only invade retics, P. falciparum can invade any cell – and likely does so by modulating host cell biophysical properties, a process we are only just understanding. But many other factors will likely also come into play, and (as mentioned above) a better understanding of where invasion occurs and under what conditions is a key piece of the puzzle which will better help us discern what are the key factors for in vivo invasion. Certainly in vitro, RBC receptors, age and physiology (oxygenation/ATP etc.) all play a role and can reduce invasion success.

6.Out of curiosity, what role do other myosins (aside of MyoA and MyoB) play in RBC invasion?

That was meant to be the rest of Tom’s PhD! We did make a start and, following some terrific work from Nottingham University, there’s a lot more information out there on the role of other myosins. We think there’s likely still one more involved in invasion, perhaps PfMyoE, and this is to facilitate getting the bulky nucleus through the junction. This would fit with the accumulation of actin behind the nucleus as seen in high resolution imaging of invasion (see Del Rosario et al 2019 PMID: 31584242).

7.From a technical point of view, and mostly in terms of phototoxicity to RBCs, could any artifacts related to invasion be introduced from imaging into your observations, and how do you prevent them?

That’s a very astute question. It’s the Schrodinger’s cat challenge of microscopy – we can’t help affecting the experiment when we look at it (with a warm bulb!). Light sources are much less phototoxic than they used to be, we use as little light as we can and we image a very large field of view to try and capture many cells (rather than focussing on one at a time). Critically, and full credit to Tom for this, we imaged hundreds of invasion events, so statistics is probably our best support with this work – if it was based on one or two movies (as we used to do when I was a PhD student) we’d be less sure, but having based our analysis on nearly 700 movies, we’re quite confident in the robustness of the observations made. Again, only when we can image under flow and have something more physiologically “real” will we truly understand what challenges the parasite is overcoming for invasion – our hunch is that it will be even more remarkable, and we’ll be even more impressed by this wily foe!

8.Is it known if any events related to invasion influence the parasite’s fate in terms of commitment to either sexual or asexual replication?

That’s a terrific question. we’re not sure if we know when commitment to sexual development is actually made, there is some data showing that merozoites already “know” if they are going to be asexual or sexual… The new frontier of Plasmodium cell biology is environmental sensing – how do parasites receive the cues from the external world and make the decisions required in terms of how to respond. This will likely increase a lot in the coming years and as it does, we will get a better sense of what the parasite is responding to, in order to make the decision to go sexual. Part of this may indeed be what the merozoite finds when it enters the RBC (or how hard it was to get in). “That was a tough RBC to enter – let’s get out of this host before its too late!”. Perhaps?

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