High-speed, three-dimensional imaging reveals chemotactic behaviour specific to human-infective Leishmania parasites
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Curated by eLife
Evaluation Summary:
This study utilizes holographic microscopy to study the swimming behaviour of flagellated forms of Leishmania mexicana in the presence or absence of host cell stimuli. Infective metacyclic promastigotes were found to swim faster than actively dividing procyclic promastigotes and to display different average trajectories. The swimming trajectories of these parasite stages were also altered in the presence of macrophages, promoting chemotaxis towards target host cells. The findings provide new insights into promastigote flagellar function and role of swimming behaviour in promoting pathogenesis.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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Abstract
Cellular motility is an ancient eukaryotic trait, ubiquitous across phyla with roles in predator avoidance, resource access, and competition. Flagellar motility is seen in various parasitic protozoans, and morphological changes in flagella during the parasite life cycle have been observed. We studied the impact of these changes on motility across life cycle stages, and how such changes might serve to facilitate human infection. We used holographic microscopy to image swimming cells of different Leishmania mexicana life cycle stages in three dimensions. We find that the human-infective (metacyclic promastigote) forms display ‘run and tumble’ behaviour in the absence of stimulus, reminiscent of bacterial motion, and that they specifically modify swimming direction and speed to target host immune cells in response to a macrophage-derived stimulus. Non-infective (procyclic promastigote) cells swim more slowly, along meandering helical paths. These findings demonstrate adaptation of swimming phenotype and chemotaxis towards human cells.
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Author Response:
Reviewer #2 (Public Review (required)):
Using high-speed holographic methodology, the swimming trajectories of two Leishmania life cycle stages are measured. Significant differences between the life stages become apparent. In addition, the authors show in a chemotaxis experiment that the infectious metacyclics respond chemotactically to the presence of macrophages.
The physics part of the study is flawless, and the holography is very impressive, especially in view of the comparatively simple setup. The analysis and presentation of the data is also flawless.
What is not so clear is the biological interpretation of the data. Chemotactic behavior has been repeatedly postulated for Leishmania, trypanosomes, and other parasites. However, there have been no experiments to date that allow conclusions to be drawn about …
Author Response:
Reviewer #2 (Public Review (required)):
Using high-speed holographic methodology, the swimming trajectories of two Leishmania life cycle stages are measured. Significant differences between the life stages become apparent. In addition, the authors show in a chemotaxis experiment that the infectious metacyclics respond chemotactically to the presence of macrophages.
The physics part of the study is flawless, and the holography is very impressive, especially in view of the comparatively simple setup. The analysis and presentation of the data is also flawless.
What is not so clear is the biological interpretation of the data. Chemotactic behavior has been repeatedly postulated for Leishmania, trypanosomes, and other parasites. However, there have been no experiments to date that allow conclusions to be drawn about in vivo relevance. Unfortunately, this does not really change with this study.
It has been shown in trypanosomes that the swimming behavior of different species and life stages are influenced by the mechanical conditions of their microenvironments. Viscosity, obstacles, and hydrodynamics can all play a critical role in determining motility. These factors are ignored in the study. Cell culture medium with the viscosity of water cannot image the situation in the vector or body fluids such as blood or lymph. A chemotactic gradient such as the one generated here by rather simple means cannot arise at all in vivo, simply because everything is in flux and parasites and macrophages move continuously. Moreover, one may wonder why Leishmania should actively move chemotactically toward macrophages when they come into contact with target cells much more rapidly by chance due to self-stirring properties of body fluids. I am not questioning the finding at all. I am merely questioning its biological relevance. Perhaps it would be better to describe this aspect of the paper more cautiously and to discuss it quite openly critically. Otherwise, the result might enter our knowledge as evidence for biologically relevant chemotaxis, and that would be problematic.
We thank the reviewer for their perspective and agree that providing formal evidence for chemotaxis in vivo is complicated. The reviewer is right that mechanical stimulus, viscosity, elasticity etc. are present in body tissues, and that they will affect the motion of the flagellum, and that there is evidence that physical obstructions interrupt the flagellar beat (though ‘stirring’ does not play a role in Leishmania’s motion through tissue). At any rate, we contend that an in vitro study such as ours decouples the mechanical heterogeneity of the in vivo environment from the parasite’s cellular response. If a chemotactic response is present in the parasite, then it will be most sensitively and uniquely tested in an isotropic environment such as a bulk Newtonian fluid - indeed, this is what we find. Chemical gradients are known to occur and persist in cutaneous infections, as damage to tissue, sand fly saliva and Leishmania-derived molecules have been shown to recruit immune cells by this mechanism - we have added references and words to this effect on lines 211-214.
Reviewer #3 (Public Review (required)):
The authors describe a clever and powerful assay to show chemotactic behavior in metacyclic Leishmania, which is an important result. The data seem mostly solid, but some results are confusing (perhaps partly an issue with presentation?) and overall conclusions seem like they need to be toned down a little. It is expected that this work will have long-lasting impact on the research community, and the new methods developed will be widely utilized.
Major concerns:
• "Pre-Adaptation", e.g. lines 149-150: A major message of the work is to suggest that motility behavior and chemotaxis is a "pre-adaptation". However, I don't agree that the current studies show that "…flagellar motility is a …preadaptation to infection of human hosts." What are the data to support this? The authors do a very good job of defining motility features of PCF and META forms, including quantitative analysis of motility features in 3D. They find that motility differs in PCF vs META forms. They also demonstrate chemotaxis in META forms. But, I don't see how these combined results demonstrate a "pre-adaptation" to infection of human hosts. As such, the "pre-adaptation" statement should be moved to speculation. Notably, I did not see tests for chemotaxis in PCF. Thus, it is even not formally demonstrated whether or not chemotaxis itself is an "adaptation" specific to META forma, or rather (and quite likely) is a fundamental property of all life cycle stages.
o To test if chemotaxis is an 'adaptation', the authors would need to provide an analysis of PCFs. To be an adaptation, one would expect to find either that PCFs do not exhibit chemotaxis, or that they do not chemotax toward macrophages in the assay used. Without this, the authors cannot say whether chemotaxis is a stage-specific behavior, much less a "pre"-adaptation.
We have moderated the language around claims of ‘pre-adaptation’ (please see next point for locations), and provided additional results from chemotaxis assays in PCF. Consistent with previous studies (e.g. Oliveira et al, Exp. Parasitol. (2000), Leslie et al., Exp. Parasitol. (2002), Barros et al., Exp. Parasitol. (2006)), we find a different chemo/osmotactic response in which PCF cells are drawn towards the agar in the pipette tip even in the absence of an embedded stimulant such as macrophages. We speculate that this result is due to the presence of small carbohydrate molecules from the unrefined agar - and note that the response is distinct to META, which show no such attraction. However, as suggested, this has been made more speculative in the revised discussion.
o Note, I think the work would not be negatively affected if the whole concept of "adaptation" were omitted and the work was framed around the very important results of developing a new and powerful approach to investigate Leishmania motility in 3D; quantitative definition of motility parameters; demonstration of chemotaxis in META forms.
We thank the reviewer for their suggestion (and their positive words), and have modified the language around claims of pre-adaptation. We have rephrased the claims in the abstract, and around lines 188-90 in the summary/conclusions.
• Chemotaxis: The work would benefit from some commentary on chemotaxis in kinetoplastids. A 'suggestion' for a potential advantage provided by chemotaxis (lines153-155) is not unwarranted, but that should be kept to speculation at this point, and implication that this is an 'adaptation' is not supported by the current data. With report of chemotaxis being a major message, the paper would benefit from a brief discussion on what's been demonstrated regarding chemotaxis in trypanosomatids, as this is an important, yet under-represented area of research on these organisms. Without this, the novelty and significance of the author's rigorous, novel and very interesting work are not brought out.
We thank the reviewer for this suggestion, and have added another paragraph to the introduction (lines 53-81), giving additional context to our results by providing an overview of more experiments in the field. We have also changed the word ‘suggest’ to ‘speculate’ in the summary and conclusions (line 243).
• Lines 125 - 129: How is it that tumble frequency decreases, but run duration is unaffacted? I would think that less frequent tumbles would lead to longer runs? This warrants more comment.
We thank the reviewer for pointing out the apparent confusion here. This stems from the fact that (as stated in the subsequent sentence) in the majority of the population, the tumble rate is significantly suppressed, to either one or zero tumbles per track. We require at least two tumbles per track to measure run duration, so the small fraction of the population unaffected by the stimulus contributes the bulk of the measurable runs. We have clarified this section of the text to clarify how we measure run duration.
• Fig 3 and Lines 135-139: How does one reconcile the finding that murine macrophages and human macrophages both induce taxis toward the pipet tip (3A), but there is opposite impact on speed profiles, with murine macrophages causing slower speeds, and human macrophages causing faster speeds (3H,K vs 3I,L)? Perhaps analysis done for human macrophages must also be done for murine macrophages. Some more commentary, and analysis needs to be provided on this point.
We thank the reviewer for this suggestion, and in the light of their comments, we have revised our description of the murine data, highlighting that the results are not statistically significant. To further emphasise this point to the reader, we have recast the error bars in figure 3a in terms of 95% confidence intervals rather than using the standard error on the mean, as in the previous version. Although one may be calculated directly from the other without any further assumptions, the 95% CI representation might be more familiar to the readership. In this light, the fairly modest decrease in average swimming speed (also seen in absolute terms in the DMEM case) reinforces the revised conclusion that the null hypothesis (META are not stimulated by mm\phi) cannot be rejected.
• Regarding replicates: While the number of cells tracked are clearly indicated, I did not see a description of how many different chambers were imaged for each condition, or how many different fields per chamber.
This has been amended in the Methods section, subheading “Chemotaxis Assay”
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Evaluation Summary:
This study utilizes holographic microscopy to study the swimming behaviour of flagellated forms of Leishmania mexicana in the presence or absence of host cell stimuli. Infective metacyclic promastigotes were found to swim faster than actively dividing procyclic promastigotes and to display different average trajectories. The swimming trajectories of these parasite stages were also altered in the presence of macrophages, promoting chemotaxis towards target host cells. The findings provide new insights into promastigote flagellar function and role of swimming behaviour in promoting pathogenesis.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share …
Evaluation Summary:
This study utilizes holographic microscopy to study the swimming behaviour of flagellated forms of Leishmania mexicana in the presence or absence of host cell stimuli. Infective metacyclic promastigotes were found to swim faster than actively dividing procyclic promastigotes and to display different average trajectories. The swimming trajectories of these parasite stages were also altered in the presence of macrophages, promoting chemotaxis towards target host cells. The findings provide new insights into promastigote flagellar function and role of swimming behaviour in promoting pathogenesis.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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Reviewer #1 (Public Review (required)):
The authors analyzed the swimming speeds and trajectories of ~500 procyclic (PCF) and purified metacyclic (META) promastigotes using 3D holographic microscopy. This approach allows measurement of speed, trajectories and chirality. Although the META fraction comprised a mixed population of promastigotes, they exhibit a distinctive run and tumble phenotype. The authors then developed an in vitro assay to assess the impact of a potential chemotactic signal, growing host cells, had on META swimming behaviour. They show that META exhibited significant affinity for human primary macrophages (and to a lesser extent J774 murine Mø) compared to medium alone. This was associated with a decrease in tumbling and increase in run duration, allowing directed swimming to the attractant. The use of holographic …
Reviewer #1 (Public Review (required)):
The authors analyzed the swimming speeds and trajectories of ~500 procyclic (PCF) and purified metacyclic (META) promastigotes using 3D holographic microscopy. This approach allows measurement of speed, trajectories and chirality. Although the META fraction comprised a mixed population of promastigotes, they exhibit a distinctive run and tumble phenotype. The authors then developed an in vitro assay to assess the impact of a potential chemotactic signal, growing host cells, had on META swimming behaviour. They show that META exhibited significant affinity for human primary macrophages (and to a lesser extent J774 murine Mø) compared to medium alone. This was associated with a decrease in tumbling and increase in run duration, allowing directed swimming to the attractant. The use of holographic microscopy to map promastigote swimming trajectories under different conditions is innovative and the finding highlight a novel virulence trait for these protists. There are only a number of minor points that need to be addressed.
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Reviewer #2 (Public Review (required)):
Using high-speed holographic methodology, the swimming trajectories of two Leishmania life cycle stages are measured. Significant differences between the life stages become apparent. In addition, the authors show in a chemotaxis experiment that the infectious metacyclics respond chemotactically to the presence of macrophages.
The physics part of the study is flawless, and the holography is very impressive, especially in view of the comparatively simple setup. The analysis and presentation of the data is also flawless.
What is not so clear is the biological interpretation of the data. Chemotactic behavior has been repeatedly postulated for Leishmania, trypanosomes, and other parasites. However, there have been no experiments to date that allow conclusions to be drawn about in vivo relevance. …
Reviewer #2 (Public Review (required)):
Using high-speed holographic methodology, the swimming trajectories of two Leishmania life cycle stages are measured. Significant differences between the life stages become apparent. In addition, the authors show in a chemotaxis experiment that the infectious metacyclics respond chemotactically to the presence of macrophages.
The physics part of the study is flawless, and the holography is very impressive, especially in view of the comparatively simple setup. The analysis and presentation of the data is also flawless.
What is not so clear is the biological interpretation of the data. Chemotactic behavior has been repeatedly postulated for Leishmania, trypanosomes, and other parasites. However, there have been no experiments to date that allow conclusions to be drawn about in vivo relevance. Unfortunately, this does not really change with this study.
It has been shown in trypanosomes that the swimming behavior of different species and life stages are influenced by the mechanical conditions of their microenvironments. Viscosity, obstacles, and hydrodynamics can all play a critical role in determining motility. These factors are ignored in the study. Cell culture medium with the viscosity of water cannot image the situation in the vector or body fluids such as blood or lymph. A chemotactic gradient such as the one generated here by rather simple means cannot arise at all in vivo, simply because everything is in flux and parasites and macrophages move continuously. Moreover, one may wonder why Leishmania should actively move chemotactically toward macrophages when they come into contact with target cells much more rapidly by chance due to self-stirring properties of body fluids. I am not questioning the finding at all. I am merely questioning its biological relevance. Perhaps it would be better to describe this aspect of the paper more cautiously and to discuss it quite openly critically. Otherwise, the result might enter our knowledge as evidence for biologically relevant chemotaxis, and that would be problematic.
-
Reviewer #3 (Public Review (required)):
The authors describe a clever and powerful assay to show chemotactic behavior in metacyclic Leishmania, which is an important result. The data seem mostly solid, but some results are confusing (perhaps partly an issue with presentation?) and overall conclusions seem like they need to be toned down a little. It is expected that this work will have long-lasting impact on the research community, and the new methods developed will be widely utilized.
Major concerns:
• "Pre-Adaptation", e.g. lines 149-150: A major message of the work is to suggest that motility behavior and chemotaxis is a "pre-adaptation". However, I don't agree that the current studies show that "...flagellar motility is a ...preadaptation to infection of human hosts." What are the data to support this? The authors do a very good job …
Reviewer #3 (Public Review (required)):
The authors describe a clever and powerful assay to show chemotactic behavior in metacyclic Leishmania, which is an important result. The data seem mostly solid, but some results are confusing (perhaps partly an issue with presentation?) and overall conclusions seem like they need to be toned down a little. It is expected that this work will have long-lasting impact on the research community, and the new methods developed will be widely utilized.
Major concerns:
• "Pre-Adaptation", e.g. lines 149-150: A major message of the work is to suggest that motility behavior and chemotaxis is a "pre-adaptation". However, I don't agree that the current studies show that "...flagellar motility is a ...preadaptation to infection of human hosts." What are the data to support this? The authors do a very good job of defining motility features of PCF and META forms, including quantitative analysis of motility features in 3D. They find that motility differs in PCF vs META forms. They also demonstrate chemotaxis in META forms. But, I don't see how these combined results demonstrate a "pre-adaptation" to infection of human hosts. As such, the "pre-adaptation" statement should be moved to speculation. Notably, I did not see tests for chemotaxis in PCF. Thus, it is even not formally demonstrated whether or not chemotaxis itself is an "adaptation" specific to META forma, or rather (and quite likely) is a fundamental property of all life cycle stages.
o To test if chemotaxis is an 'adaptation', the authors would need to provide an analysis of PCFs. To be an adaptation, one would expect to find either that PCFs do not exhibit chemotaxis, or that they do not chemotax toward macrophages in the assay used. Without this, the authors cannot say whether chemotaxis is a stage-specific behavior, much less a "pre"-adaptation.
o Note, I think the work would not be negatively affected if the whole concept of "adaptation" were omitted and the work was framed around the very important results of developing a new and powerful approach to investigate Leishmania motility in 3D; quantitative definition of motility parameters; demonstration of chemotaxis in META forms.
• Chemotaxis: The work would benefit from some commentary on chemotaxis in kinetoplastids. A 'suggestion' for a potential advantage provided by chemotaxis (lines153-155) is not unwarranted, but that should be kept to speculation at this point, and implication that this is an 'adaptation' is not supported by the current data. With report of chemotaxis being a major message, the paper would benefit from a brief discussion on what's been demonstrated regarding chemotaxis in trypanosomatids, as this is an important, yet under-represented area of research on these organisms. Without this, the novelty and significance of the author's rigorous, novel and very interesting work are not brought out.
• Lines 125 - 129: How is it that tumble frequency decreases, but run duration is unaffacted? I would think that less frequent tumbles would lead to longer runs? This warrants more comment.
• Fig 3 and Lines 135-139: How does one reconcile the finding that murine macrophages and human macrophages both induce taxis toward the pipet tip (3A), but there is opposite impact on speed profiles, with murine macrophages causing slower speeds, and human macrophages causing faster speeds (3H,K vs 3I,L)? Perhaps analysis done for human macrophages must also be done for murine macrophages. Some more commentary, and analysis needs to be provided on this point.
• Regarding replicates: While the number of cells tracked are clearly indicated, I did not see a description of how many different chambers were imaged for each condition, or how many different fields per chamber.
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