Coupling cryo-electron tomography with mixed-scale dense neural networks reveals re-organization of the invasion machinery of Toxoplasma gondii upon ionophore-stimulation

bioRxiv (Cold Spring Harbor Laboratory)

Abstract

Abstract Host cell invasion by intracellular, eukaryotic parasites within the phylum Apicomplexa, is a remarkable and active process involving the coordinated action of apical organelles and other structures. To date, capturing how these structures interact during invasion has been difficult to observe in detail. Here, we used cryogenic electron tomography to image the apical complex of Toxoplasma gondii tachyzoites under conditions that mimic resting parasites and those primed to invade through stimulation with calcium ionophore. Through the application of Mixed Scale Dense networks for image-processing, we developed a highly efficient pipeline for annotation of tomograms, enabling us to identify and extract densities of relevant subcellular organelles and accurately analyze features in 3D. The results reveal a dramatic change in the shape of the anteriorly located apical vesicle upon its apparent fusion with a rhoptry, that occurs only in the stimulated parasites. We also present information indicating that this vesicle originates from the vesicles that parallel the intraconoidal microtubules and that the latter two structures are linked by a novel tether. We show that a rosette structure previously proposed to be involved in rhoptry secretion is associated with apical vesicles beyond just the most anterior one. This result, suggesting multiple vesicles are primed to enable rhoptry secretion, may shed light on the mechanisms Toxoplasma employs to enable repeated invasion attempts. Using the same approach, we examine Plasmodium falciparum merozoites and show that they too possess an apical vesicle just beneath a rosette, demonstrating evolutionary conservation of this overall subcellular organization. Significance Statement Parasites in the phylum Apicomplexa are responsible for some of the most important parasitic diseases of humans, such as malaria and toxoplasmosis. Invasion by these obligatory, intracellular parasites depends on protein injection into the host cell. Using cryogenic electron tomography, we reveal evolutionarily conserved features shared by the invasive forms of Plasmodium falciparum and Toxoplasma gondii . By comparing resting Toxoplasma tachyzoites with those primed to invade we also gain new insight into the very first steps in invasion. For this work, we take an interdisciplinary approach, adopting a mixed-scale dense neural network that enables efficient and objective processing of the data. Combined, the results provide new information on how these important parasites accomplish the essential step of invasion.

Authors 9

1. Li-av T. Segev-Zarko

   Stanford Medicine · Stanford University

   Affiliation as printed

   Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA

2. Peter D. Dahlberg

   Stanford University

   Affiliation as printed

   Department of Chemistry, Stanford University, Stanford, CA, USA

3. Stella Sun

   University of Pittsburgh · Stanford University

   Affiliation as printed

   Department of Bioengineering, Stanford University, Stanford, CA, USA

   Department of Structural Biology, University of Pittsburgh, Pittsburgh, PA, USA

4. Daniël M. Pelt Aachen Leiden Institute of Advanced Computer Science

   Leiden University

   Affiliation as printed

   Leiden Institute of Advanced Computer Science, Leiden University, Leiden, The Netherlands

5. Chi Yong Kim

   Stanford Medicine · Stanford University

   Affiliation as printed

   Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA

   Department of Pediatrics - Infectious Diseases, Stanford University School of Medicine, Stanford, CA, USA

6. Elizabeth S. Egan

   Stanford Medicine · Stanford University

   Affiliation as printed

   Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA

   Department of Pediatrics - Infectious Diseases, Stanford University School of Medicine, Stanford, CA, USA

7. James A. Sethian

   Lawrence Berkeley National Laboratory · University of California, Berkeley

   Affiliation as printed

   Center for Advanced Mathematics for Energy Research Application (CAMERA), Lawrence Berkeley National Laboratory, Berkeley, CA, USA

   Department of Mathematics, University of California, Berkeley, CA, USA

8. Wah Keung Chiu corresponding

   SLAC National Accelerator Laboratory · Stanford Synchrotron Radiation Lightsource · Stanford Medicine · Stanford University

   Affiliation as printed

   Department of Bioengineering, Stanford University, Stanford, CA, USA

   Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA

   Division of Cryo-EM and Bioimaging, SSRL, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, CA, USA

9. John C. Boothroyd corresponding

   Stanford Medicine · Stanford University

   Affiliation as printed

   Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA