Morphogenesis Dynamics in Leishmania Differentiation

Abstract

Leishmania, the causative agent of leishmaniasis, is an obligatory intracellular parasite that cycles between phagolysosome of mammalian macrophages, where it resides as round intracellular amastigotes, and the midgut of female sandflies, where it resides as extracellular elongated promastigotes. This protozoan parasite cytoskeleton is composed of stable and abundant subpellicular microtubules (SPMT). This study aims to determine the kinetics of developmental morphogenesis and assess whether microtubules remodelling is involved in this process. Using image-streaming technology, we observed that rounding of promastigotes during differentiation into amastigotes was initiated promptly after exposure to the differentiation signal. Stabilizing microtubules with taxol sped rounding, but later killed differentiating parasites if taxol was not removed. Microtubule destabilizers such as vinblastine had no effect on the rate of rounding, nor on the viability of differentiating parasites. In the reverse process, elongation is initiated after a delay of 7.5 and completed 72 h after exposure to the amastigote to the promastigote differentiation signal. During the delay, parasites became highly sensitive to treatment with microtubule destabilizers. The addition of vinblastine during the first 7.5 h halted differentiation and killed parasites. Between 8 and 24 h, parasites gradually became resistant to vinblastine and, in parallel, started to elongate. In contrast, taxol had no effect on parasite elongation, nor on the viability of these cells. In a parallel study, we showed that the Leishmania-specific protein kinase A (PKA) holoenzyme containing the LdPKAR3-C3 complex is essential for promastigote elongation. Mutant promastigotes lacking either of these proteins are round, but maintain their flagella. Here, we observed that during differentiation into amastigotes, these mutants round at the same rate as the wild type, but never exceed the WT density of round amastigotes. In the reverse process, these mutants undergo the same initial delay and then elongate at the same rate as the WT. They stop elongating when they reach 20% of elongated cells in mature promastigotes. Our analysis indicates that while promastigote rounding into amastigotes did not require microtubule remodelling, morphogenesis of round amastigotes into elongated promastigotes required microtubule rearrangement before elongation was initiated. This is the first study that investigates the dynamics of microtubules during parasite development.

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

“…Overall, our application of IFC to studying the cell cycle in Leishmania provides an extra dimension to the growing body of IFC applications for studying the cell biology of parasites. IFC has been used to assess morphological changes occurring during promastigote to amastigote differentiation in L. donovani (72), to assess colocalisation of mCLING and lysotracker fluorescent stains in T. brucei (36) and to assess PI live/dead staining and for morphological measurements in L. mexicana (38). IFC has also been employed extensively to study host-parasite interactions for Leishmania and other intracellular pathogens (21, 73–76) as well as to detect Leishmania infections in blood and parasite dissemination to organs around the body (77).…”

Modern Aspects of Leishmaniasis: Basis of Development New Approaches against Infection

“…Nutritional requirements, growth rate and ability to divide, the regulated expression of their surface molecules, and morphology are the factors that distinguish the developmental forms. The procyclic to metacyclic differentiation of promastigotes (also called metacyclogenesis) and the metacyclic promastigote are the two differentiation events studied with Leishmania to amastigote transformation [18] inside the host macrophage (Figure 2).…”

“…They are characterized in vitro by low pH, lack of oxygen, and nutritional depletion of tetrahydrobiopterin. The conditions such as low pH, 37°C temperature, and high CO 2 that mimic a phagolysosome-like environment can cause promastigote to amastigote differentiation [18]. Although environmental factors influencing Leishmania differentiation are known, little is known about the molecular processes that mediate cellular remodeling.…”

Disruption of Leishmania flagellum attachment zone architecture causes flagellum loss

“…have a complex digenetic life cycle with developmental forms in both the mammalian host and insect vector. To adapt to the different environmental conditions of these ecological niches, Leishmania parasites have the ability to differentiate into different cell morphologies and cell types (Dandugudumula et al., 2022 ; Dostálová & Volf, 2012 ; Gossage et al., 2003 ; Sunter & Gull, 2017 ; Wheeler et al., 2013 ). There are two key cell morphologies, the promastigote form, found in the sand fly, which has an elongated ovoid cell body and a long motile flagellum, and the amastigote form, found in the mammalian host, characterised by a smaller, spherical cell body and a short immotile flagellum (Hoare & Wallace, 1966 ; Sunter & Gull, 2017 ).…”