Correlative light-electron microscopy methods to characterize the ultrastructural features of the replicative and dormant liver stages of Plasmodium parasites.

BACKGROUND: The infection of the liver by Plasmodium parasites is an obligatory step leading to malaria disease. Following hepatocyte invasion, parasites differentiate into replicative liver stage schizonts and, in the case of Plasmodium species causing relapsing malaria, into hypnozoites that can lie dormant for extended periods of time before activating. The liver stages of Plasmodium remain elusive because of technical challenges, including low infection rate. This has been hindering experimentations with well-established technologies, such as electron microscopy. A deeper understanding of hypnozoite biology could prove essential in the development of radical cure therapeutics against malaria. RESULTS: The liver stages of the rodent parasite Plasmodium berghei, causing non-relapsing malaria, and the simian parasite Plasmodium cynomolgi, causing relapsing malaria, were characterized in human Huh7 cells or primary non-human primate hepatocytes using Correlative Light-Electron Microscopy (CLEM). Specifically, CLEM approaches that rely on GFP-expressing parasites (GFP-CLEM) or on an immunofluorescence assay (IFA-CLEM) were used for imaging liver stages. The results from P. berghei showed that host and parasite organelles can be identified and imaged at high resolution using both CLEM approaches. While IFA-CLEM was associated with more pronounced extraction of cellular content, samples' features were generally well preserved. Using IFA-CLEM, a collection of micrographs was acquired for P. cynomolgi liver stage schizonts and hypnozoites, demonstrating the potential of this approach for characterizing the liver stages of Plasmodium species causing relapsing malaria. CONCLUSIONS: A CLEM approach that does not rely on parasites expressing genetically encoded tags was developed, therefore suitable for imaging the liver stages of Plasmodium species that lack established protocols to perform genetic engineering. This study also provides a dataset that characterizes the ultrastructural features of liver stage schizonts and hypnozoites from the simian parasite species P. cynomolgi.

Autophagy restricts Mycobacterium tuberculosis during acute infection in mice.

Whether or not autophagy has a role in defence against Mycobacterium tuberculosis infection remains unresolved. Previously, conditional knockdown of the core autophagy component ATG5 in myeloid cells was reported to confer extreme susceptibility to M. tuberculosis in mice, whereas depletion of other autophagy factors had no effect on infection. We show that doubling cre gene dosage to more robustly deplete ATG16L1 or ATG7 resulted in increased M. tuberculosis growth and host susceptibility in mice, although ATG5-depleted mice are more sensitive than ATG16L1- or ATG7-depleted mice. We imaged individual macrophages infected with M. tuberculosis and identified a shift from apoptosis to rapid necrosis in autophagy-depleted cells. This effect was dependent on phagosome permeabilization by M. tuberculosis. We monitored infected cells by electron microscopy, showing that autophagy protects the host macrophage by partially reducing mycobacterial access to the cytosol. We conclude that autophagy has an important role in defence against M. tuberculosis in mammals.

The ultrastructure of a Chlamydomonas reinhardtii mutant strain lacking phytoene synthase resembles that of a colorless alga.

Chlamydomonas reinhardtii strains lacking phytoene synthase, the first enzyme of carotenoid biosynthesis, are white. They lack carotenoid pigments, have very low levels of chlorophyll, and can grow only heterotrophically in the dark. Our electron and fluorescence microscopic studies showed that such a mutant strain (lts1-204) had a proliferated plastid envelope membrane but no stacks of thylakoid membranes within the plastid. It accumulated cytoplasmic compartments that appeared to be autophagous vacuoles filled with membranous material. The lts1 mutants apparently lacked pyrenoid bodies, which normally house ribulose bisphosphate carboxylase-oxygenase (Rubisco), and accumulated many starch granules. Although these mutant strains cannot synthesize the carotenoid and carotenoid-derived pigments present in the phototactic organelle (eyespot), the mutant we examined made a vestigial eyespot that was disorganized and often mislocalized to the posterior end of the cell. The absence of a pyrenoid body, the accumulation of starch, and the disorganization of the eyespot may all result from the absence of thylakoids. The ultrastructure of lts1 mutant strains is similar to but distinct from that of previously described white and yellow mutant strains of C. reinhardtii and is similar to that of naturally colorless algae of the Polytoma group.