Authors

Discussion

Within the sand fly vector, Leishmania parasites have two major morphological forms, a motile promastigote and a haptomonad, which is attached to the stomodeal valve through a shortened and modified flagellum. Dissecting haptomonad development and attachment is critical to understanding parasite transmission; however, studies of haptomonads are limited, as this is a technically challenging life cycle form to investigate. To gain an in-depth understanding of the in vivo haptomonad cellular architecture and organisation, we combined two volume electron microscopy techniques – serial block face-scanning electron microscopy (SBF-SEM) and serial electron microscopy tomography – generating high resolution 3D models of haptomonads attached to the stomodeal valve. Haptomonads were densely packed around the valve and were attached through the tip of a shortened flagellum. The attachment interface was filled, on the flagellum side, with an electron-dense plaque that connected to abundant filaments and filament bundles. Next, we generated attached L. mexicana haptomonads in vitro and confirmed that the fine ultrastructure of these forms was comparable to that of haptomonads found in vivo. Using comparative proteomic approaches, we identified proteins locating to either the attachment interface or the filaments within the attached flagellum, which we call Kinetoplastid-Insect Attachment Proteins (KIAPs). Importantly, a number of these proteins are present in other kinetoplastid parasites. Deletion analysis using CRISPR/Cas9 compromised Leishmania attachment both in vitro and in the sand fly, confirming that we have identified critical components of the parasite attachment mechanism. This provides the first molecular insights into a kinetoplastid parasite vector attachment interface, which will underpin our understanding of this crucial interaction and onward transmission.