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bioRxiv · 10.1101/2025.08.29.673095

In situ architecture of the endosymbiont Wolbachia pipientis

Pujhari, S.·Heebner, J.·Raumann, E.·Zhong, T.·Rasgon, J. L.·Swulius, M. T.·Shaffer, C. L.·Kaplan, M.
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Abstract

Hidden within host cells, the endosymbiont Wolbachia pipientis is the most prevalent bacterial infection in the animal kingdom. Scientific breakthroughs over the past century yielded fundamental mechanisms by which Wolbachia controls arthropod reproduction to shape dynamic ecological and evolutionary trajectories. However, the structure and spatial organization of symbiont machineries that underpin intracellular colonization and orchestrate maternal inheritance remain unknown. Here, we used cryo-electron tomography to directly image the nanoscale architecture of bacterial tools deployed for host manipulation and germline transmission. We discovered that Wolbachia assembles multiple structures at the host-endosymbiont interface including a filamentous ladder-like framework hypothesized to serve as a specialized motility mechanism that enables bacterial translocation to specific host cell compartments during embryogenesis and somatic tissue dissemination. In addition, we present the first in situ structure of the Rickettsiales vir homolog type IV secretion system (rvh T4SS). We provide evidence that the rvh T4SS nanomachine exhibits architectural similarities to the pED208-encoded T4SS apparatus including the biogenesis of rigid conjugative pili extending hundreds of nanometers beyond the bacterial cell surface. Coupled with integrative structural modeling, we demonstrate that in contrast to canonical T4SS architectures, the -proteobacterial T4SS outer membrane complex assembles a periplasmic baseplate structure predicted to comprise VirB9 oligomers complexed with cognate VirB10 subunits that form extended antennae projections surrounding the translocation channel pore. Collectively, these studies provide an unprecedented view into Wolbachia structural cell biology and unveil the molecular blueprints for architectural paradigms that reinforce ancient host-microbe symbioses.

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Pujhari, S., Heebner, J., Raumann, E., Zhong, T., Rasgon, J. L., Swulius, M. T., Shaffer, C. L., Kaplan, M.. 2025-08-29. In situ architecture of the endosymbiont Wolbachia pipientis. https://doi.org/10.1101/2025.08.29.673095

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