Biology subjects

Jaroenlak, P.

Publications and source records attributed to Jaroenlak, P..

5 recordsLinked to original sources

bioRxiv2025

Polar tube firing dynamics and ultrastructure from the shrimp microsporidian Ecytonucleospora hepatopenaei (EHP)

Ecytonucleospora (Enterocytozoon) hepatopenaei or EHP is an obligate intracellular parasite that belongs to phylum Microsporidia. EHP infection in shrimp results in growth retardation and size variation, leading to severe economic loss to shrimp aquaculture worldwide. Similar to other microsporidian species, EHP utilizes a harpoon-like invasion apparatus called the polar tube in order to infect host cells. The polar tube typically coils inside the spore and rapidly transits to a long, extended tube after being activated with proper stimuli. However, the mechanism and physical conditions affecting the polar tube firing in EHP are poorly understood. Here, we screened several germination buffers and found that a buffer containing potassium hydrogen phthalate (KHP) at pH 3.0 yielded the highest germination rate. The optimal temperatures for EHP germination range between 25-28{degrees}C, similar to the shrimps body temperature. The spores require at least 30 seconds to be activated, suggesting that the stimuli could rapidly move across the spore wall. We utilized high-speed live-cell imaging to study the dynamics of the EHP polar tube firing and compared the dynamics when the spores were treated with KHP and a previously reported stimulus, phloxine B. The polar tube firing dynamics between these two conditions are different. The total firing time was [~]100 milliseconds with the maximum velocity of [~]300 m*s-1 in KHP condition. Further investigation on the EHP polar tube ultrastructure using cryo-electron microscopy revealed that the EHP polar tube was composed of a membrane layer with an additional repetitive protein-array on the outermost surface. The distance between each repetitive unit ranges between 52-66 [A]. These repetitive units could possibly be polar tube proteins (PTPs). Altogether, this study provides insights into EHP polar tube firing dynamics and its architecture which are important foundations for understanding the biophysical factors governing EHP pathogenesis and in developing EHP control strategies. Author summaryThe microsporidian Ecytonucleospora hepatopenaei (EHP) poses a significant threat to global shrimp aquaculture, causing substantial economic losses. Many studies on EHP have been focused on development of detection methods, and farm management. Little is known about EHP pathogenesis and how it infects shrimp cells. Here, we identified laboratory conditions to activate the parasites infection process, which occurs on a millisecond timescale. We utilized a high-speed live-cell imaging to further characterize the rapid EHP infection process. The architecture and ultrastructure of EHP invasion organelle were also investigated using cryo-electron microscopy. Our findings provide a better understanding of the EHP infection mechanisms that are crucial for developing effective control strategies.

microbiology↗

bioRxiv2024

scRNA-seq reveals transcriptional dynamics of Encephalitozoon intestinalis parasites in human macrophages

Microsporidia are single-celled intracellular parasites that cause opportunistic diseases in humans. Encephalitozoon intestinalis is a prevalent human-infecting species that invades the small intestine. Dissemination to other organ systems is also observed, and is potentially facilitated by macrophages. The macrophage response to infection and the developmental trajectory of the parasite are not well studied. Here we use single cell RNA sequencing to investigate transcriptional changes in both the host and parasite during infection. While a small population of infected macrophages mount a response, most remain transcriptionally unchanged, suggesting that the majority of parasites may avoid host detection. The parasite transcriptome reveals large transcriptional changes throughout the life cycle, providing a blueprint for parasite development. The stealthy microsporidian lifestyle likely allows these parasites to harness macrophages for replication and dissemination. Together, our data provide insights into the host response in primary human macrophages and the E. intestinalis developmental program.

microbiology↗

bioRxiv2023

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 {micro}m in size) shoot out a 100-nm-wide PT at a speed of 300 {micro}m/sec, creating a shear rate of 3000 sec-1. The infectious cargo, which contains two nuclei, is shot through this narrow tube for a distance of [~]60-140 {micro}m (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 ring 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. Statement of SignicanceMicrosporidia are a group of spore-forming, intracellular parasites that infect a wide range of hosts (including humans). Once triggered, microsporidian spores (3-4 {micro}m in size) shoot out a specialized organelle called the polar tube (PT) (60-140 {micro}m long, 100 nm wide) at ultrafast speed (300 {micro}m/sec), penetrating host cells and acting as a conduit for the transport of infectious cargo. Although this process has fascinated biologists for a century, the biophysical mechanisms underlying PT extrusion are not understood. We thus take a data-driven approach to generate models for the physical basis of PT ring and cargo transport through the PT. Our approach here demonstrates the extreme limits of cellular hydraulics and the potential applications of biophysical approaches to other cellular architectures.

biophysics↗

bioRxiv2020

Waffle method: A general and flexible approach for FIB-milling small and anisotropically oriented samples

Cryo-FIB/SEM combined with cryo-ET has emerged from within the field of cryo-EM as the method for obtaining the highest resolution structural information of complex biological samples in-situ in native and non-native environments. However, challenges remain in conventional cryo-FIB/SEM workflows, including milling thick specimens that do not vitrify well, specimens with preferred orientation, low-throughput when milling small and/or low concentration specimens, and cellular specimens that distribute poorly across grid squares. Here we present a general approach we call the Waffle Method which leverages high-pressure freezing to address these challenges. We illustrate the mitigation of these challenges by applying the Waffle Method and cryo-ET to reveal the macrostructure of the polar tube in microsporidian spores in multiple complementary orientations, which was previously not possible due to preferred orientation of the spores on the grid. We demonstrate the broadness of the Waffle Method by applying it to three additional cellular samples and a single particle sample using a variety of cryo-FIB-milling hardware, with both manual and automated approaches. We also present a unique and critical stress-relief gap designed specifically for waffled lamellae. Additionally, we describe applications of the Waffle Method which are currently being explored. We propose the Waffle Method as a way to achieve many of the advantages of cryo-liftout on the specimen grid while avoiding the long, challenging, and technically-demanding process required for cryo-liftout.

microbiology↗

bioRxiv2020

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 in vitro. Our results show that the emerging PT experiences very high accelerating forces to reach velocities exceeding 300 m.s-1, and that firing kinetics differ markedly between species. Live-cell imaging reveals that the nucleus, which is approximately 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.

microbiology↗