Formation and three-dimensional architecture ofLeishmaniaadhesion in the sand fly vector

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 parasiteLeishmaniaadheres to the stomodeal valve, as the specialised haptomonad form. Dissection of haptomonad adhesion is a critical step for understanding parasite transmission. 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, we identified five stages of haptomonad differentiation, and showed that calcium is necessary for haptomonad adhesion to the surface. This study provides the structural and regulatory foundations of the haptomonad form, which are critical for a holistic understanding ofLeishmaniatransmission.

Analysis of theLeishmania mexicanapromastigote cell cycle using imaging flow cytometry provides new insights into cell cycle flexibility and events of short duration

“…Propidium iodide (PI) quantitatively stains DNA (S2A,B Fig), but requires cell fixation, and is thus unsuitable for use in live cells. Hoechst 33342 is commonly used to stain the nucleus and kinetoplast in live Leishmania (40–42), but IFC indicated that it does not bind proportionally to DNA (S2C Fig). Images of Hoechst-stained cells revealed staining outside of the nucleus that may account for it being non-quantitative (S2C Fig).…”

Discovery of essential kinetoplastid-insect adhesion proteins and their function inLeishmania-sand fly interactions

“…Haptomonad adhesion occurs via the heavily modified parasite flagellum, in which an attachment plaque is positioned adjacent to the flagellum membrane from which filaments extend towards the cell body and specific connections to the stomodeal valve are formed 11 . Leishmania is closely related to other kinetoplastid parasites that cause disease in humans.…”

“…Leishmania adhesion proceeds through a series of defined steps, from initial adhesion, to flagellum disassembly and final maturation of the plaque 11 . To understand the point at which the different KIAPs appeared, we examined cells at different stages of adhesion (Fig.…”

“…Given the non-uniform distribution of KIAP1 and 3 across the attachment plaque, we revisited our electron tomograms of Leishmania attached in the sand fly and to plastic 11 (Fig. 2g–i and Extended Data Fig.…”

“…The capacity of haptomonads to differentiate is unknown but the stability of the attachment plaque suggests that the attached cell is terminally differentiated. Interestingly, we previously observed that ~1% of haptomonads were dividing 11 , which may generate a free-swimming daughter and such asymmetric differentiation divisions are found in other kinetoplastid parasites 20 .…”