Cryo-ET reveals the in situ architecture of the polar tube invasion apparatus from microsporidian parasites.

Microsporidia are divergent fungal pathogens that employ a harpoon-like apparatus called the polar tube (PT) to invade host cells. The PT architecture and its association with neighboring organelles remain poorly understood. Here, we use cryo-electron tomography to investigate the structural cell biology of the PT in dormant spores from the human-infecting microsporidian species, Encephalitozoon intestinalis . Segmentation and subtomogram averaging of the PT reveal at least four layers: two protein-based layers surrounded by a membrane, and filled with a dense core. Regularly spaced protein filaments form the structural skeleton of the PT. Combining cryo-electron tomography with cellular modeling, we propose a model for the 3-dimensional organization of the polaroplast, an organelle that is continuous with the membrane layer that envelops the PT. Our results reveal the ultrastructure of the microsporidian invasion apparatus in situ , laying the foundation for understanding infection mechanisms.

Mechanics of Microsporidian Polar Tube Firing.

As obligate intracellular parasites with reduced genomes, microsporidia must infect host cells in order to replicate and cause disease. They can initiate infection by utilizing a harpoon-like invasion organelle called the polar tube (PT). The PT is both visually and functionally a striking organelle and is a characteristic feature of the microsporidian phylum. Outside the host, microsporidia exist as transmissible, single-celled spores. Inside each spore, the PT is arranged as a tight coil. Upon germination, the PT undergoes a large conformational change into a long, linear tube and acts as a tunnel for the delivery of infectious cargo from the spore to a host cell. The firing process is extremely rapid, occurring on a millisecond timescale, and the emergent tube may be as long as 20 times the size of the spore body. In this chapter, we discuss what is known about the structure of the PT, the mechanics of the PT firing process, and how it enables movement of material from the spore body.

3-Dimensional organization and dynamics of the microsporidian polar tube invasion machinery.

Microsporidia, a divergent group of single-celled eukaryotic parasites, harness a specialized harpoon-like invasion apparatus called the polar tube (PT) to gain entry into host cells. The PT is tightly coiled within the transmissible extracellular spore, and is about 20 times the length of the spore. Once triggered, the PT is rapidly ejected and is thought to penetrate the host cell, acting as a conduit for the transfer of infectious cargo into the host. The organization of this specialized infection apparatus in the spore, how it is deployed, and how the nucleus and other large cargo are transported through the narrow PT are not well understood. Here we use serial block-face scanning electron microscopy to reveal the 3-dimensional architecture of the PT and its relative spatial orientation to other organelles within the spore. Using high-speed optical microscopy, we also capture and quantify the entire PT germination process of three human-infecting microsporidian species in vitro: Anncaliia algerae, Encephalitozoon hellem and E. intestinalis. Our results show that the emerging PT experiences very high accelerating forces to reach velocities exceeding 300 µm⋅s-1, and that firing kinetics differ markedly between species. Live-cell imaging reveals that the nucleus, which is at least 7 times larger than the diameter of the PT, undergoes extreme deformation to fit through the narrow tube, and moves at speeds comparable to PT extension. Our study sheds new light on the 3-dimensional organization, dynamics, and mechanism of PT extrusion, and shows how infectious cargo moves through the tube to initiate infection.