Live-cell imaging and ultrastructure expansion microscopy reveal the dynamic intracellular life cycle of Encephalitozoon intestinalis

Microsporidia are divergent fungal pathogens that infect a wide range of hosts. Microsporidia have evolved highly reduced genomes, resulting in the loss of many metabolic pathways. Encephalitozoon intestinalis is a human-infecting microsporidian species with the smallest known eukaryotic genome (2.3 Mbp). As an obligate intracellular parasite, E. intestinalis is dependent on its host for replication. Our current understanding of E. intestinalis cell biology remains limited. This is largely due to genetic intractability, which has made live-cell imaging challenging, and its small size (~2 m), which is within the diffraction limit of conventional microscopy. Here we develop a live-cell imaging technique, "silhouette microscopy", that does not rely on genetic manipulation of microsporidia, and enables us to study the live cell dynamics of E. intestinalis replication in host cells, from entry to exit. We report the timing of cell division, kinetics of the parasite life cycle, and dynamics of the parasitophorous vacuole within which E. intestinalis resides. The parasitophorous vacuole undergoes frequent fission and fusion, and ultimately ruptures to facilitate parasite release from the host cell. To gain a higher resolution understanding into the cell biology of E. intestinalis development, we performed ultrastructure expansion microscopy (U-ExM) on infected cells. U-ExM reveals the organization of the E. intestinalis cytoskeleton and development of the specialized invasion organelle, the polar tube. The combination of live-cell imaging and U-ExM provide a platform to study the E. intestinalis life cycle, and sheds light on the dynamics and cell biology of E. intestinalis in host cells.