Abstract
Formation of gametes in the malaria parasite occurs in the midgut of the mosquito and is critical to onward parasite transmission. Transformation of the male gametocyte into microgametes, called microgametogenesis, is an explosive cellular event and one of the fastest eukaryotic DNA replication events known. The transformation of one microgametocyte into eight flagellated microgametes requires reorganisation of the parasite cytoskeleton, replication of the 22.9 Mb genome, axoneme formation and host erythrocyte egress, all of which occur simultaneously in <20 minutes. Whilst high-resolution imaging has been a powerful tool for defining stages of microgametogenesis, it has largely been limited to fixed parasite samples, given the speed of the process and parasite photosensitivity. Here, we have developed a live-cell fluorescence imaging workflow that captures the entirety of microgametogenesis. Using the most virulent human malaria parasite, Plasmodium falciparum, our live-cell approach captured early microgametogenesis with three-dimensional imaging through time (4D imaging) and microgamete release with two-dimensional (2D) fluorescence microscopy. To minimise the phototoxic impact to parasites, acquisition was alternated between 4D fluorescence, brightfield and 2D fluorescence microscopy. Combining live-cell dyes specific for DNA, tubulin and the host erythrocyte membrane, 4D and 2D imaging together enables definition of the positioning of newly replicated and segregated DNA. This combined approach also shows the microtubular cytoskeleton, location of newly formed basal bodies, elongation of axonemes and morphological changes to the erythrocyte membrane, the latter including potential echinocytosis of the erythrocyte membrane prior to microgamete egress. Extending the utility of this approach, the phenotypic effects of known transmission-blocking inhibitors on microgametogenesis were confirmed. Additionally, the effects of bortezomib, an untested proteasomal inhibitor, revealed a clear block of DNA replication, full axoneme nucleation and elongation. Thus, as well as defining a framework for broadly investigating microgametogenesis, these data demonstrate the utility of using live imaging to validate potential targets for transmission-blocking antimalarial drug development.
Highlights
Whilst microgametogenesis has been extensively studied using various microscopy techniques, efforts to date have been limited by the need for complex transgenic parasite generation and fixed-parasite protocols
Transmission is triggered by the uptake of sexual stage gametocytes during a mosquito feed that instantly activate, initiating a transformation in the mosquito midgut that has become a trademark in the cellular biology of these protozoan parasites [1]
We describe a protocol that captures the entire process of microgametogenesis in P. falciparum microgametocytes, utilising specific labels for microtubules, DNA and the host erythrocyte membrane and imaging using an approach that alternates between live-cell 3D fluorescence microscopy through time (4D imaging), brightfield imaging and 2D fluorescence microscopy
Summary
Malaria disease is caused by single-cell protozoan parasites from the genus Plasmodium. Over its complex two-host lifecycle, the Plasmodium cell demonstrates remarkable cellular plasticity as it transitions between multiple developmental stages. In the transition from mammalian to mosquito host, the parasite faces an extreme population bottleneck in numbers, which presents a natural target for novel antimalarial treatments aimed at blocking transmission. Transmission is triggered by the uptake of sexual stage gametocytes during a mosquito feed that instantly activate, initiating a transformation in the mosquito midgut that has become a trademark in the cellular biology of these protozoan parasites [1]. Dormant male (micro) and female (macro) gametocytes form a sub-population of between 0.2–1% of the circulating asexual blood stage parasite reservoir in the mammalian host. The signals that initiate commitment of asexual parasites to sexual differentiation are, poorly understood [1]. The transformation from gametocyte to gamete, a process termed gametogenesis, is activated by a decrease in temperature to 20–25 ̊C, rise in pH and the presence of the mosquito metabolite, xanthurenic acid in the mosquito midgut [4]
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