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.