Formation and three-dimensional architecture of Leishmania adhesion in the sand fly vector
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eLife assessment
This important study provides compelling observations of the organization and architecture of haptomonads, a distinct and poorly understood developmental form of Leishmania found in sand fly vectors at later stages of infection. The authors used 3D electron microscopy techniques, including serial block face scanning electron microscopy and electron tomography, to visualize the colonization sand fly by haptomonads in impressive detail.
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Abstract
Attachment to a substrate to maintain position in a specific ecological niche is a common strategy across biology, especially for eukaryotic parasites. During development in the sand fly vector, the eukaryotic parasite Leishmania adheres to the stomodeal valve, as the specialised haptomonad form. Dissection of haptomonad adhesion is a critical step for understanding the complete life cycle of Leishmania . Nevertheless, haptomonad studies are limited, as this is a technically challenging life cycle form to investigate. Here, we have combined three-dimensional electron microscopy approaches, including serial block face scanning electron microscopy (SBFSEM) and serial tomography to dissect the organisation and architecture of haptomonads in the sand fly. We showed that the attachment plaque contains distinct structural elements. Using time-lapse light microscopy of in vitro haptomonad-like cells, we identified five stages of haptomonad-like cell differentiation, and showed that calcium is necessary for Leishmania adhesion to the surface in vitro. This study provides the structural and regulatory foundations of Leishmania adhesion , which are critical for a holistic understanding of the Leishmania life cycle.
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Author Response
Reviewer #1 (Public Review):
The adhesion of Leishmania promastigotes to the stomodeal valve in the anterior region of the sandfly vector midgut is thought to be important to facilitate the transmission of the parasites by bite. The promastigote form found in attachment is termed a 'haptomonad', although its adhesion mechanism and role in facilitating transmission have not been well studied. Using 3D EM techniques, the paper provides detailed new information pertaining to the adhesion mechanism. Electron tomography was especially useful to reveal the ultrastructure of the attachment plaque and the extensive remodelling of the flagellum that occurs. A few of the attached haptomonads were found to be in division, which is a novel observation. The attachment of cultured promastigotes to plastic and glass surfaces in …
Author Response
Reviewer #1 (Public Review):
The adhesion of Leishmania promastigotes to the stomodeal valve in the anterior region of the sandfly vector midgut is thought to be important to facilitate the transmission of the parasites by bite. The promastigote form found in attachment is termed a 'haptomonad', although its adhesion mechanism and role in facilitating transmission have not been well studied. Using 3D EM techniques, the paper provides detailed new information pertaining to the adhesion mechanism. Electron tomography was especially useful to reveal the ultrastructure of the attachment plaque and the extensive remodelling of the flagellum that occurs. A few of the attached haptomonads were found to be in division, which is a novel observation. The attachment of cultured promastigotes to plastic and glass surfaces in vitro was found to involve a similar remodeling of the flagellum and was exploited to image the sequential steps in attachment, flagellar remodeling, and haptomonad differentiation. The in vitro attachment was found to be calcium2+ dependent. Based mainly on the in vitro observations, a sound model of the haptomonad attachment plaque and differentiation process is provided.
We thank the reviewer for highlighting the significant progress we have made in dissecting the adhesion mechanism and flagellum restructuring in the Leishmania haptomonad.
Reviewer #2 (Public Review):
The study by Yanase et al. investigated the details of the 3D architecture of Leishmania haptomonad promastigote's adhesion to the midgut of the insect vector. The authors generated a dataset of images that reveal intricate details of the formed adhesion plaque and expanded the study with in vitro alternatives for the exploration of how Leishmania promastigotes strong adhesion by hemidesmosomes to surfaces can happen and be maintained. They show with unprecedented detail the ultrastructure of the attachment plaque. The in vitro dataset of the paper adds to the specific literature important details on how to explore micro/nanostructures involved in an important attachment step for this eukaryotic parasite. However, the in vitro data should be reconsidered in its discussion and conclusions as it does not support direct comparison with in vivo Leishmania forms as pictured by the authors. In general, the dataset presented in this manuscript adds valuable data and resources for the study of Leishmania promastigotes to surfaces, especially to the thoracic midgut parts of its insect vector.
The dataset of this paper is well-collected and robust, but some aspects of image analysis need to be clarified and extended. Also, the in vitro data from the manuscript will benefit from an extensive adjustment in its discussion. Points to focus on:
We thank the reviewer for recognising the ultrastructural detail we have now provided of this cryptic parasite life cycle stage. Below we address each of your points in detail.
- The haptomonad promastigote is indeed a possible critical form for transmission, but it lacks formal demonstration still in all literature available. This should not be claimed without proper formal demonstration.
We agree with the reviewer that any relationship between transmission and the haptomonad form has yet to be formally demonstrated. Hence, we revised the descriptions referring to the relationship between transmission and the haptomonad form (Line 22-23, 31 and 113-114).
- Literature available and cited in this manuscript regarding in vitro adhesion of culture Leishmania promastigotes does not provide direct evidence for haptomonad differentiation. Haptomonads are still a largely unknown promastigote form with no defined ontogeny. With that, to propose an in vitro haptomonad differentiation protocol, more detailed direct evidence of in vivo haptomonads will be necessary. The in vitro experiments available show how cultured promastigotes attach to surfaces. Detailed studies in vivo will be needed still to attribute the findings in vitro to haptomonads.
We would like to highlight that promastigotes and haptomonads have morphological definitions within the literature and our cells are definitely more like haptomonads than promastigotes. As the reviewer highlights, the haptomonad-like cells we generate in vitro have an almost identical morphology and attachment plaque structure to those haptomonads we observed attached to the stomodeal valve. In addition, we have been able to watch individual cells that had a promastigote morphology acquire a haptomonad morphology and we believe this will provide future insights to the ontogeny of these forms. However, as there are currently no published molecular markers for haptomonads we have not been able to provide direct evidence other than the morphology and ultrastructure that in vitro attachment replicates in vivo haptomonad differentiation. Therefore, we have revised our nomenclature and now refer to the in vitro haptomonad-like cell. In the discussion, we have been careful to highlight that certain aspects of our model rely on in vitro data and therefore may not accurately reflect the situation in the sand fly.
- This manuscript will benefit by having a detailed description of how to analyze and get to the 3D models presented. This has a strong potential for usage beyond the Leishmania/sand fly field. Statistics should be made available with ease across the manuscript and with a dedicated section on methods.
We added a detailed description of how to analyse the 3D models (Line 756-763), and added videos showing a rotated view of each 3D model (Figure 1—video 3 and 4, Figure 2—video 2, and Figure 3—video 2 and 4). We have deposited the SBF-SEM and tomography data on the Electron Microscopy Public Image Archive (EMPIAR; https://www.ebi.ac.uk/empiar/), enabling access to the raw data (Line 763-766). We have added a statistics section into the Materials and Methods (Line 864-868).
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eLife assessment
This important study provides compelling observations of the organization and architecture of haptomonads, a distinct and poorly understood developmental form of Leishmania found in sand fly vectors at later stages of infection. The authors used 3D electron microscopy techniques, including serial block face scanning electron microscopy and electron tomography, to visualize the colonization sand fly by haptomonads in impressive detail.
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Reviewer #1 (Public Review):
The adhesion of Leishmania promastigotes to the stomodeal valve in the anterior region of the sandfly vector midgut is thought to be important to facilitate the transmission of the parasites by bite. The promastigote form found in attachment is termed a 'haptomonad', although its adhesion mechanism and role in facilitating transmission have not been well studied. Using 3D EM techniques, the paper provides detailed new information pertaining to the adhesion mechanism. Electron tomography was especially useful to reveal the ultrastructure of the attachment plaque and the extensive remodelling of the flagellum that occurs. A few of the attached haptomonads were found to be in division, which is a novel observation. The attachment of cultured promastigotes to plastic and glass surfaces in vitro was found to …
Reviewer #1 (Public Review):
The adhesion of Leishmania promastigotes to the stomodeal valve in the anterior region of the sandfly vector midgut is thought to be important to facilitate the transmission of the parasites by bite. The promastigote form found in attachment is termed a 'haptomonad', although its adhesion mechanism and role in facilitating transmission have not been well studied. Using 3D EM techniques, the paper provides detailed new information pertaining to the adhesion mechanism. Electron tomography was especially useful to reveal the ultrastructure of the attachment plaque and the extensive remodelling of the flagellum that occurs. A few of the attached haptomonads were found to be in division, which is a novel observation. The attachment of cultured promastigotes to plastic and glass surfaces in vitro was found to involve a similar remodeling of the flagellum and was exploited to image the sequential steps in attachment, flagellar remodeling, and haptomonad differentiation. The in vitro attachment was found to be calcium2+ dependent. Based mainly on the in vitro observations, a sound model of the haptomonad attachment plaque and differentiation process is provided.
-
Reviewer #2 (Public Review):
The study by Yanase et al. investigated the details of the 3D architecture of Leishmania haptomonad promastigote's adhesion to the midgut of the insect vector. The authors generated a dataset of images that reveal intricate details of the formed adhesion plaque and expanded the study with in vitro alternatives for the exploration of how Leishmania promastigotes strong adhesion by hemidesmosomes to surfaces can happen and be maintained. They show with unprecedented detail the ultrastructure of the attachment plaque. The in vitro dataset of the paper adds to the specific literature important details on how to explore micro/nanostructures involved in an important attachment step for this eukaryotic parasite. However, the in vitro data should be reconsidered in its discussion and conclusions as it does not …
Reviewer #2 (Public Review):
The study by Yanase et al. investigated the details of the 3D architecture of Leishmania haptomonad promastigote's adhesion to the midgut of the insect vector. The authors generated a dataset of images that reveal intricate details of the formed adhesion plaque and expanded the study with in vitro alternatives for the exploration of how Leishmania promastigotes strong adhesion by hemidesmosomes to surfaces can happen and be maintained. They show with unprecedented detail the ultrastructure of the attachment plaque. The in vitro dataset of the paper adds to the specific literature important details on how to explore micro/nanostructures involved in an important attachment step for this eukaryotic parasite. However, the in vitro data should be reconsidered in its discussion and conclusions as it does not support direct comparison with in vivo Leishmania forms as pictured by the authors. In general, the dataset presented in this manuscript adds valuable data and resources for the study of Leishmania promastigotes to surfaces, especially to the thoracic midgut parts of its insect vector.
The dataset of this paper is well-collected and robust, but some aspects of image analysis need to be clarified and extended. Also, the in vitro data from the manuscript will benefit from an extensive adjustment in its discussion. Points to focus on:
The haptomonad promastigote is indeed a possible critical form for transmission, but it lacks formal demonstration still in all literature available. This should not be claimed without proper formal demonstration.
Literature available and cited in this manuscript regarding in vitro adhesion of culture Leishmania promastigotes does not provide direct evidence for haptomonad differentiation. Haptomonads are still a largely unknown promastigote form with no defined ontogeny. With that, to propose an in vitro haptomonad differentiation protocol, more detailed direct evidence of in vivo haptomonads will be necessary. The in vitro experiments available show how cultured promastigotes attach to surfaces. Detailed studies in vivo will be needed still to attribute the findings in vitro to haptomonads.
This manuscript will benefit by having a detailed description of how to analyze and get to the 3D models presented. This has a strong potential for usage beyond the Leishmania/sand fly field. Statistics should be made available with ease across the manuscript and with a dedicated section on methods.
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