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Ultrastructural insights into the microsporidian infection apparatus reveal the kinetics and morphological transitions of polar tube and cargo during host cell invasion [1]

['Himanshu Sharma', 'Department Of Molecular Biology', 'The Laboratory For Molecular Infection Medicine Sweden', 'Mims', 'Umeå Centre For Microbial Research', 'Ucmr', 'Science For Life Laboratory', 'Umeå University', 'Umeå', 'Department Of Medical Biochemistry']

Date: 2024-03

During host cell invasion, microsporidian spores translocate their entire cytoplasmic content through a thin, hollow superstructure known as the polar tube. To achieve this, the polar tube transitions from a compact spring-like state inside the environmental spore to a long needle-like tube capable of long-range sporoplasm delivery. The unique mechanical properties of the building blocks of the polar tube allow for an explosive transition from compact to extended state and support the rapid cargo translocation process. The molecular and structural factors enabling this ultrafast process and the structural changes during cargo delivery are unknown. Here, we employ light microscopy and in situ cryo-electron tomography to visualize multiple ultrastructural states of the Vairimorpha necatrix polar tube, allowing us to evaluate the kinetics of its germination and characterize the underlying morphological transitions. We describe a cargo-filled state with a unique ordered arrangement of microsporidian ribosomes, which cluster along the thin tube wall, and an empty post-translocation state with a reduced diameter but a thicker wall. Together with a proteomic analysis of endogenously affinity-purified polar tubes, our work provides comprehensive data on the infection apparatus of microsporidia and uncovers new aspects of ribosome regulation and transport.

Data Availability: The sub-tomogram averages can be accessed at the Electron Microscopy Data Bank with the following accession codes EMD-17391, EMD-17468 and EMD-17467. Representative tomograms and the raw tilt series have been uploaded to the Electron Microscopy Public Image Archive (EMPIAR) and can be accessed with the deposition EMPIAR-11557. Mass-spectrometry data has been uploaded to PRIDE under project accession PXD042571.

To overcome these challenges, we employed light microscopy and cryo-electron tomography (cryo-ET) to examine the dynamics and ultrastructure remodeling of the Vairimorpha necatrix polar tube during germination in a near-native environment. We capture snapshots of the infectious sporoplasm seen as arrays of organized ribosomes and densely packed cargo passing through the polar tube. Further, 2 distinct states of the extruded polar tube, discernible in the protein layer lining its outer wall, are also described. We further characterize the outer proteinaceous layer of the tube using affinity purification and mass spectrometry, thus unraveling potential new protein–protein interactions of the PTPs. Overall, our results shed new light on the events underpinning cargo delivery by the polar tube and uncover protein factors that may assist host invasion.

During germination, the entire infectious cellular content, known as the sporoplasm, is pushed through the narrow polar tube and delivered into or close to the host cell [ 26 ]. These events, including tube eversion and passage of sporoplasm cargo through this constricted tube, are extremely fast and occur within 2 s [ 25 ]. To achieve successful firing, sporoplasm delivery, and host infection, the polar tube and the cargo undergo drastic remodeling during this discharge [ 25 , 27 , 28 ]. These swift ultrastructure changes in the polar tube evince its extraordinary mechanical properties likely conferred by its components, the PTPs. However, the mechanisms driving polar tube firing, sporoplasm discharge, and the dynamics of polar tube eversion are not well understood [ 25 , 28 – 30 ]. Further, the swift nature of germination events creates challenges in capturing polar tube dynamics and contributes to the generally understudied nature of these parasites. Additionally, most in vitro studies on the microsporidian infection apparatus using electron microscopy (EM) employ denaturing purification of polar tube components or have relied on non-native staining and resin embedding methods, thus limiting their overall resolution.

While a general trend for reductive evolution is pervasive, microsporidia have also evolved specialized mechanisms for invading and hijacking host cell systems [ 16 , 17 ]. These mechanisms include, for example, an expanded repertoire of nucleotide transporters to steal energy and metabolic precursors from host cells [ 18 , 19 ]. However, the most drastic invention and specialization is the polar tube, a microsporidia-specific organelle used for host invasion. The polar tube comprises at least 6 polar tube proteins (PTPs) that may localize to its outermost layer or the terminal tip [ 20 – 22 ], but the structure and organization of PTPs and the exact composition of the tube is still a mystery. Inside the spore, this organelle adopts a spring-like arrangement and is referred to as the polar filament [ 23 – 25 ]. Polar filament coils can range from a couple to dozens, depending on the organism [ 16 ]. Upon exposure to the right environmental stimuli, the spores germinate. This includes an explosive firing at the apical pole of the spore that transforms the polar filament into the extended, tube-like state referred to as the polar tube.

Microsporidia are a group of obligate intracellular parasites that infect hosts across the animal kingdom [ 1 – 4 ]. To propagate from host to host, these fungi-like pathogens form stable environmental spores of prokaryotic size [ 5 ]. In addition to their small size, these parasites exemplify reductive evolution in eukaryotes [ 6 – 8 ]. The deletion of many genes considered essential for eukaryotic function has produced some of the smallest known genomes of the kingdom [ 9 ], streamlined biochemical pathways [ 10 ], and minimized cellular macromolecular complexes. Recent structural insights into microsporidian ribosomes and proteasomes bound to dormancy factors in the extracellular spore stage have highlighted the importance of mechanisms to enter and exit dormancy [ 11 – 14 ]. This trend of reductive evolution might have emerged from an obligate intracellular lifestyle, while the atypical regulatory factors support pathogen survival in nutrient-limiting environments [ 15 ].

Results

Cryo-ET captures assorted states of the polar tube and cellular content during cargo delivery The microsporidian infection apparatus rapidly transitions from a tightly packaged polar filament to an extended polar tube. The dynamics of germination and polar tube length have been studied in 3 human-infecting microsporidia; however, spore firing is known to vary between species, and firing efficiencies can also vary greatly even for uniform-looking spores [25]. We set out to identify and optimize conditions for V. necatrix germination and quantify the dynamics of tube firing in this agriculturally important parasite of Lepidoptera [31,32]. We used live microscopy to record 53 germination events, which enabled us to measure the polar tube length and firing velocity (S1 Fig). We observed that polar tubes could attain a maximum length of approximately 142 μm, a mean length of 113 μm, and extend with a mean maximum velocity of approximately 281 μm/s. Similar to the previous observations in distant microsporidia [25], V. necatrix everts its tube to the maximum length in less than 1 s and expels the sporoplasm from the tip of the nascent polar tube. Additionally, tube firing follows a typically observed triphasic mode where the tube undergoes observable states of elongation, followed by stasis during sporoplasm passage and a refractory period where the tube shortens after cargo emergence [25] (S1 Fig). Akin to tube remodeling events, the sporoplasm also likely transforms from a restricted spore state to extended conformation during extrusion [30], into a spherical shape upon emergence (S1a Fig), and these events may impose immediate reorganization of subcellular structures. The optimal alkaline priming conditions (see Methods) for polar tube firing were used to cryogenically preserve on-grid germinated spores (S2a Fig) for in situ characterization of polar tubes and cargo using cryo-ET. On-grid germination was optimized with spores consistently displaying high germination efficiencies (>80%). Spores were applied onto cryo-grids immediately after resuspension in the germination buffer, followed by blotting and plunge freezing. Various grid types were screened during freezing attempts, and polar tubes frequently interacted strongly with the regular grid support regions while avoiding thin carbon films or holes. To overcome this, we utilized lacey carbon grids with an ultra-thin carbon film where the variability in hole size and support mesh increased the chance of trapping sections of the tubes on larger thin-carbon areas. Subsequently, we collected 50 tilt series from different sections of polar tubes (S1 Table), of which 45 were suitable for tomogram reconstruction using the IMOD package [33]. The reconstructed tomograms were denoised using IsoNet [34] and auto-segmented using CNN in Eman2 [35] to better visualize the spatial organization of the cargo and polar tubes. This allowed us to capture a wide range of polar tube ultrastructural states, with differences in diameter, outer layer thickness, and interior composition, most likely resulting from the differential passage of reorganized sporoplasm. The diameter of the analyzed polar tubes ranges from 60 nm to 190 nm depending on their internal content, suggesting we captured a range of different phases during germination or after sporoplasm translocation (Fig 1). Polar tubes filled with dense cellular cargo (PTcargo) (Fig 1A–1C) had diameters of more than 120 nm and are enveloped by a thin layer; in contrast, the electron-lucent or empty tubes (PTempty) (Fig 1D and 1E) have a diameter of significantly less than 120 nm but were enveloped by a thick outer layer (S2b Fig). Here, it is worthwhile to note that the PTcargo or PTempty states represent static snapshots after firing and could represent states before or after sporoplasm translocation. Among the 2 states, of note was an apparent inverse correlation between the PT diameter and the thickness of the tube wall, which is composed of a lipid bilayer (pink arrows, Figs 1 and S2), flanked by an outer protein layer (light blue and magenta arrows, Figs 1 and S2). These observations are also in line with previous reports of an outer proteinaceous layer in germinated PTs [29,36,37]. Membrane-less PTs were not observed, and the PT membrane appeared continuous in all raw tomograms (small discontinuities in the membrane auto-segmentation likely being the result of mis-annotation due to limited signal-to-noise ratio). Most interestingly, across all our tomograms, we observed different internal cargo, including electron-dense material inside membranous compartments, randomly oriented or highly ordered large molecular complexes such as ribosomes and proteasome-like particles, and empty vesicles or empty tubes. The electron-dense cellular material seemed free-flowing or sometimes enveloped inside vesicle-like organelles (orange arrows, Figs 1A and S2d), while distinct densities corresponding to macromolecular complexes were randomly distributed or arranged in a very regular fashion (Fig 1B and 1C). Vesicle-containing tubes were reminiscent of the frequently observed tube-inside-tube architecture of the polar tube [29]. Notably, macromolecular complexes were completely missing from PTempty, which mostly housed electron-lucent vesicles (dark blue arrows, Figs 1D and S2c) or no vesicles (Figs 1E and S2c). Collectively, these tomograms capture the myriad heterogeneous states the polar tubes and the cellular cargo adopt immediately upon firing. PPT PowerPoint slide

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TIFF original image Download: Fig 1. Ultrastructural states of the polar tubes during or after delivering cellular cargo. (a–e) Slices through cryo-tomograms of germinated polar tubes above the respective neural network aided 3D segmentation. The central slice of a tomogram is represented at the top in each panel, as seen from the z-axis view. An additional section from the same tomogram is marked by a boxed region and shown below at axis z + 20 nm. The neural network-aided 3D segmentations corresponding to each tomogram are presented in the lower panel. Selected regions of interest in the tomogram slices, such as cellular complexes (a–c), the outer protein layer (a–e), or empty vesicles (d), are indicated with arrows colored as the labels and segmentations below. The black scale bars correspond to 100 nm. https://doi.org/10.1371/journal.pbio.3002533.g001

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