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. Macrophages are potential reservoirs of infection, and dissemination to other organ systems is also observed. 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 parasite and the host during E. intestinalis infection of human macrophages in vitro. The parasite undergoes large transcriptional changes throughout the life cycle, providing a blueprint for parasite development. 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 stealthy microsporidian lifestyle likely allows these parasites to harness macrophages for replication. Together, our data provide insights into the host response in primary human macrophages and the E. intestinalis developmental program.

The outer membrane (OM) of Gram-negative bacteria is an asymmetric bilayer that protects the cell from various external stressors, such as antibiotics. The Mla transport system is implicated in the M aintenance of outer membrane L ipid A symmetry and is thought to mediate retrograde phospholipid transport across the cell envelope. This system uses a shuttle-like mechanism to move lipids between the MlaFEDB inner membrane complex and the MlaA-OmpF/C OM complex, via a periplasmic lipid-binding protein, MlaC. MlaC binds to MlaD and MlaA, but the underlying protein-protein interactions that facilitate lipid transfer are not well understood. Here, we take an unbiased deep mutational scanning approach to map the fitness landscape of MlaC, which provides insights into important functional sites. Combining this analysis with AlphaFold2 structure predictions and binding experiments, we map the MlaC-MlaA and MlaC-MlaD protein-protein interfaces. Our results suggest that the MlaD and MlaA binding surfaces on MlaC overlap to a large extent, leading to a model in which MlaC can only bind one of these proteins at a time. Low-resolution cryo-electron microscopy (cryo-EM) maps of MlaC bound to MlaFEDB suggest that at least two MlaC molecules can bind to MlaD at once, in a conformation consistent with AlphaFold2 predictions. These data lead us to a model for MlaC interaction with its binding partners and insights into lipid transfer steps that underlie phospholipid transport between the inner and outer membranes.

Microsporidia are an early-diverging group of fungal pathogens with a wide host range. Several microsporidian species cause opportunistic infections in humans that can be fatal. As obligate intracellular parasites with highly reduced genomes, microsporidia are dependent on host metabolites for successful replication and development. Our knowledge of microsporidian intracellular development remains rudimentary, and our understanding of the intracellular niche occupied by microsporidia has relied on 2D TEM images and light microscopy. Here, we use serial block-face scanning electron microscopy (SBF-SEM) to capture 3D snapshots of the human-infecting species, Encephalitozoon intestinalis , within host cells. We track E. intestinalis development through its life cycle, which allows us to propose a model for how its infection organelle, the polar tube, is assembled de novo in developing spores. 3D reconstructions of parasite-infected cells provide insights into the physical interactions between host cell organelles and parasitophorous vacuoles, which contain the developing parasites. The host cell mitochondrial network is substantially remodeled during E. intestinalis infection, leading to mitochondrial fragmentation. SBF-SEM analysis shows changes in mitochondrial morphology in infected cells, and live-cell imaging provides insights into mitochondrial dynamics during infection. Our data provide insights into parasite development, polar tube assembly, and microsporidia-induced host mitochondria remodeling.

Journal Article Visualizing the Intracellular Niche of Human-Infecting Microsporidia Using Serial Block Face Scanning Electron Microscopy Get access Noelle V Antao, Noelle V Antao Department of Cell Biology, NYU Grossman School of Medicine, New York, NY, USA Search for other works by this author on: Oxford Academic Google Scholar Cherry Lam, Cherry Lam Department of Cell Biology, NYU Grossman School of Medicine, New York, NY, USA Search for other works by this author on: Oxford Academic Google Scholar Alina Davydov, Alina Davydov Department of Cell Biology, NYU Grossman School of Medicine, New York, NY, USA Search for other works by this author on: Oxford Academic Google Scholar Joseph Sall, Joseph Sall Department of Cell Biology, NYU Grossman School of Medicine, New York, NY, USA Search for other works by this author on: Oxford Academic Google Scholar Feng-Xia Liang, Feng-Xia Liang Department of Cell Biology, NYU Grossman School of Medicine, New York, NY, USA Search for other works by this author on: Oxford Academic Google Scholar Damian Ekiert, Damian Ekiert Department of Cell Biology, NYU Grossman School of Medicine, New York, NY, USADepartment of Microbiology, NYU Grossman School of Medicine, New York, NY, USA Corresponding authors: gira.bhabha@gmail.com, damian.ekiert@ekiertlab.org Search for other works by this author on: Oxford Academic Google Scholar Gira Bhabha Gira Bhabha Department of Cell Biology, NYU Grossman School of Medicine, New York, NY, USA Corresponding authors: gira.bhabha@gmail.com, damian.ekiert@ekiertlab.org Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 28, Issue S1, 1 August 2022, Pages 1492–1493, https://doi.org/10.1017/S1431927622006031 Published: 01 August 2022