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

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?