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

Abstract

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.

Generation of a microsporidia species attribute database and analysis of the extensive ecological and phenotypic diversity of microsporidia

“…4C). In contrast to previous studies which have reported that the length of fired tubes is longer than the estimated length of unfired tubes, we see many examples of unfired polar tubes being both shorter and longer than those that are fired 38, 50 .…”

“…The following formula was used: Volume= 4/3πabc, where a, b, and c are the 3 semi-axes of the ovoid. Width/2 was used for both a and b as width and depth of spores have been shown to be similar (41). Length/2 was used for c. When possible, priority was given to the "fresh" measurements of documented spore for calculating volume.…”

“…Although they were once thought to be protists, it is now clear they belong to a lineage of early diverging fungi (1). Microsporidia was the first parasite to the two species for which such data is available (41). For Anncaliia algerae, we estimated a volume of 22.7 μm 3 and the volume was calculated to be 8.8 μm .…”

“…For Encephalitozoon hellem, we estimated a volume of 2.1 μm 3 , and the volume was calculated to be 3.6 μm 3 . We also used the same volume formula, but the length and width measurements from (41), and estimated A. algerae to be 12.8 μm 3 and E. hellem to be 4.1 μm 3 . These comparisons demonstrate, at least for this limited set, that our estimated volumes are similar to those calculated using three-dimensional methods and that the majority of the differences in volume arise from differences in spore length and width measurements.…”

Von Willebrand Factor Facilitates Intravascular Dissemination of Microsporidia Encephalitozoon hellem

“…Untreated or pre-incubated E. hellem spores were subjected to germination, triggered by germination buffer (140 mM NaCl, 5 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , pH 9.5) at 37°C for 10 min, and then 5% (v/v, final ratio) H 2 O 2 (Sangon Biotech) was added for 5 min (Leitch et al, 1993;He et al, 1996;Pattana Jaroenlak et al, 2020).…”