Energetics of the microsporidian polar tube invasion machinery

Microsporidia are eukaryotic, obligate intracellular parasites that infect a wide range of hosts, leading to health and economic burdens worldwide. Microsporidia use an unusual invasion organelle called the polar tube (PT), which is ejected from a dormant spore at ultra-fast speeds, to infect host cells. The mechanics of PT ejection are impressive. Anncaliia algerae microsporidia spores (3–4 μm in size) shoot out a 100-nm-wide PT at a speed of 300 μm/s, creating a shear rate of 3000 s-1. The infectious cargo, which contains two nuclei, is shot through this narrow tube for a distance of ∼60–140 μm (Jaroenlak et al, 2020) and into the host cell. Considering the large hydraulic resistance in an extremely thin tube and the low-Reynolds-number nature of the process, it is not known how microsporidia can achieve this ultrafast event. In this study, we use Serial Block-Face Scanning Electron Microscopy to capture 3-dimensional snapshots of A. algerae spores in different states of the PT ejection process. Grounded in these data, we propose a theoretical framework starting with a systematic exploration of possible topological connectivity amongst organelles, and assess the energy requirements of the resulting models. We perform PT firing experiments in media of varying viscosity, and use the results to rank our proposed hypotheses based on their predicted energy requirement. We also present a possible mechanism for cargo translocation, and quantitatively compare our predictions to experimental observations. Our study provides a comprehensive biophysical analysis of the energy dissipation of microsporidian infection process and demonstrates the extreme limits of cellular hydraulics

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

Additional file 1: of Identification, characterization and heparin binding capacity of a spore-wall, virulence protein from the shrimp microsporidian, Enterocytozoon hepatopenaei (EHP)

Figure S1. Transcriptional pattern of EhSWP1 using one-step RT-PCR and nested RT-PCR analysis of RNA template from naĂŻve shrimp cohabitated with EHP-infected shrimp. (TIFF 497 kb

PCR primers used in this study.

<p>PCR primers used in this study.</p

Alignments of the SSU-PCR primer sequences and confirmation of cross reactions with closely related microsporidia.

<p>(A) Alignments of the SSU primer sequences (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166320#pone.0166320.t003" target="_blank">Table 3</a>) with homologous SSU regions of other microsporidia (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166320#pone.0166320.t001" target="_blank">Table 1</a>). Black highlights indicate matches with the primer sequences, while asterisks under the sequences indicate regions of 100% identity for all of the aligned sequences. (B) Agarose gel of SSU-PCR amplicons from EHP and other microsporidia. In addition to the pGEM-SSU plasmid (+ve) and water (-ve), total DNA obtained from EHP-infected shrimp (I) and naïve shrimp (U) were used as controls. PCR amplicons and false positive test results are marked with arrowheads and asterisks, respectively. The band at 226 bp show amplicons of residual primers ENR779 from the first PCR step and primers ENF176 from the second nested PCR step.</p

Higher sensitivity of first step SWP-PCR compared to first step SSU-PCR.

<p>(A) and (C) show agarose gels of amplicons from the first step PCR reactions, while (B) and (D) show agarose gels of amplicons from the nested step PCR reactions carried out using serial dilutions of the plasmid templates pGEM-SWP and pGEM-SSU, respectively.</p

Alignments of the SWP-PCR primer sequences and lack of cross reactions with closely related microsporidia.

<p>(A) Alignments of the SWP primer sequences (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166320#pone.0166320.t003" target="_blank">Table 3</a>) with homologous regions of spore wall protein genes of other microsporidia available in databases (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166320#pone.0166320.t002" target="_blank">Table 2</a>). Black highlights indicate matches with the primer sequences, and asterisks indicate regions of 100% identity for all of the aligned sequences. (B) Agarose gel of SWP-PCR amplicons from EHP and other microsporidia. In addition to the pGEM-SWP plasmid (+ve) and water (-ve), total DNA obtained from EHP-infected shrimp (I) and naïve shrimp (U) were used as controls. PCR amplicons are marked with arrowheads. The 180 bp band is PCR products from residual primers SWP1_R from the first PCR step and primers SWP_2F from the second nested PCR step.</p

Spore wall protein sequences used for multiple sequence alignment analysis.

<p>Spore wall protein sequences used for multiple sequence alignment analysis.</p

Small subunit rRNA sequences used for multiple sequence alignment analysis.

<p>Small subunit rRNA sequences used for multiple sequence alignment analysis.</p