Conservation of the Toxoplasma conoid complex proteome reveals a cryptic conoid in Plasmodium that differentiates between blood- and vector-stage zoites

AbstractThe apical complex is the instrument of invasion used by apicomplexan parasites, and the conoid is a conspicuous feature of this apparatus found throughout this phylum. The conoid, however, is believed to be heavily reduced or missing from Plasmodium species and other members of the class Aconoidasida. Relatively few conoid proteins have previously been identified, making it difficult to address how conserved this feature is throughout the phylum, and whether it is genuinely missing from some major groups. Moreover, parasites such as Plasmodium species cycle through three invasive forms and there is the possibility of differential presence of the conoid between these stages. We have applied spatial proteomics and high-resolution microscopy to develop a more complete molecular inventory and understanding of the organisation of conoid-associated proteins in the model apicomplexan Toxoplasma gondii. These data revealed molecular conservation of all conoid substructures throughout Apicomplexa, including Plasmodium, and even in allied Myzozoa such as Chromera and dinoflagellates. We reporter-tagged and observed the expression and location of several conoid complex proteins in the malaria model P. berghei and revealed equivalent structures in all of its zoite forms, as well as evidence of molecular differentiation between blood-stage merozoites and the ookinetes and sporozoites of the mosquito vector. Collectively we show that the conoid is a conserved apicomplexan element at the heart of the invasion mechanisms of these highly successful and often devastating parasites.

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Molecular characterization of the conoid complex in Toxoplasma reveals its conservation in all apicomplexans, including Plasmodium species

PLoS Biology ◽

10.1371/journal.pbio.3001081 ◽

2021 ◽

Vol 19 (3) ◽

pp. e3001081 ◽

Cited By ~ 2

Author(s):

Ludek Koreny ◽

Mohammad Zeeshan ◽

Konstantin Barylyuk ◽

Eelco C. Tromer ◽

Jolien J. E. van Hooff ◽

...

Keyword(s):

Plasmodium Species ◽

Mosquito Vector ◽

Apicomplexan Parasites ◽

Invasion Mechanisms ◽

Molecular Differentiation ◽

Associated Proteins ◽

Molecular Conservation ◽

Malaria Model ◽

High Resolution Microscopy

The apical complex is the instrument of invasion used by apicomplexan parasites, and the conoid is a conspicuous feature of this apparatus found throughout this phylum. The conoid, however, is believed to be heavily reduced or missing fromPlasmodiumspecies and other members of the class Aconoidasida. Relatively few conoid proteins have previously been identified, making it difficult to address how conserved this feature is throughout the phylum, and whether it is genuinely missing from some major groups. Moreover, parasites such asPlasmodiumspecies cycle through 3 invasive forms, and there is the possibility of differential presence of the conoid between these stages. We have applied spatial proteomics and high-resolution microscopy to develop a more complete molecular inventory and understanding of the organisation of conoid-associated proteins in the model apicomplexanToxoplasma gondii. These data revealed molecular conservation of all conoid substructures throughout Apicomplexa, includingPlasmodium, and even in allied Myzozoa such asChromeraand dinoflagellates. We reporter-tagged and observed the expression and location of several conoid complex proteins in the malaria modelP.bergheiand revealed equivalent structures in all of its zoite forms, as well as evidence of molecular differentiation between blood-stage merozoites and the ookinetes and sporozoites of the mosquito vector. Collectively, we show that the conoid is a conserved apicomplexan element at the heart of the invasion mechanisms of these highly successful and often devastating parasites.

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High Resolution Microscopy of Multi-Layered Biological Crystals

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010005319x ◽

1982 ◽

Vol 40 ◽

pp. 80-83

Author(s):

H.A. Cohen ◽

T.W. Jeng ◽

W. Chiu

Keyword(s):

High Resolution ◽

Low Dose ◽

Three Dimensional ◽

Data Interpretation ◽

Two Dimensional ◽

Biological Objects ◽

Density Maps ◽

Density Map ◽

Complex Crystal ◽

High Resolution Microscopy

This tutorial will discuss the methodology of low dose electron diffraction and imaging of crystalline biological objects, the problems of data interpretation for two-dimensional projected density maps of glucose embedded protein crystals, the factors to be considered in combining tilt data from three-dimensional crystals, and finally, the prospects of achieving a high resolution three-dimensional density map of a biological crystal. This methodology will be illustrated using two proteins under investigation in our laboratory, the T4 DNA helix destabilizing protein gp32*I and the crotoxin complex crystal.

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High Resolution Observations of Ion-Milling Induced Transformation in Synthetic Hollandses

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100097259 ◽

1981 ◽

Vol 39 ◽

pp. 114-115 ◽

Cited By ~ 1

Author(s):

T. J. Headley

Keyword(s):

High Resolution ◽

Radioactive Waste Disposal ◽

Ion Milling ◽

Minor Elements ◽

Waste Forms ◽

Carbon Substrates ◽

Structural Differences ◽

Oxide Phases ◽

Argon Ions ◽

High Resolution Microscopy

Oxide phases having the hollandite structure have been identified in multiphase ceramic waste forms being developed for radioactive waste disposal. High resolution studies of phases in the waste forms described in Ref. [2] were initiated to examine them for fine scale structural differences compared to natural mineral analogs. Two hollandites were studied: a (Ba,Cs,K)-titan-ate with minor elements in solution that is produced in the waste forms, and a synthesized BaAl2Ti6O16 phase containing ∼ 4.7 wt% Cs2O. Both materials were consolidated by hot pressing at temperatures above 1100°C. Samples for high resolution microscopy were prepared both by ion-milling (7kV argon ions) and by crushing and dispersing the fragments on holey carbon substrates. The high resolution studies were performed in a JEM 200CX/SEG operating at 200kV.

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Design of a new objective lens for an HB-501A STEM

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100086386 ◽

1991 ◽

Vol 49 ◽

pp. 416-417

Author(s):

Earl J. Kirkland ◽

Robert J. Keyse

Keyword(s):

High Resolution ◽

Spherical Aberration ◽

Scanning Transmission Electron Microscope ◽

Dark Field ◽

Objective Lens ◽

Specimen Holder ◽

Transmission Electron ◽

Scanning Transmission ◽

Annular Dark Field ◽

High Resolution Microscopy

An ultra-high resolution pole piece with a coefficient of spherical aberration Cs=0.7mm. was previously designed for a Vacuum Generators HB-501A Scanning Transmission Electron Microscope (STEM). This lens was used to produce bright field (BF) and annular dark field (ADF) images of (111) silicon with a lattice spacing of 1.92 Å. In this microscope the specimen must be loaded into the lens through the top bore (or exit bore, electrons traveling from the bottom to the top). Thus the top bore must be rather large to accommodate the specimen holder. Unfortunately, a large bore is not ideal for producing low aberrations. The old lens was thus highly asymmetrical, with an upper bore of 8.0mm. Even with this large upper bore it has not been possible to produce a tilting stage, which hampers high resolution microscopy.

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Practical High Resolution Microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100101256 ◽

1968 ◽

Vol 26 ◽

pp. 302-303

Author(s):

P. A. Marsh ◽

T. Mullens ◽

D. Price

Keyword(s):

High Resolution ◽

Magnetic Fields ◽

Low Frequency ◽

Resolving Power ◽

Careful Attention ◽

Electron Microscopes ◽

The Relationship ◽

High Resolution Microscopy ◽

New Facilities

It is possible to exceed the guaranteed resolution on most electron microscopes by careful attention to microscope parameters essential for high resolution work. While our experience is related to a Philips EM-200, we hope that some of these comments will apply to all electron microscopes.The first considerations are vibration and magnetic fields. These are usually measured at the pre-installation survey and must be within specifications. It has been our experience, however, that these factors can be greatly influenced by the new facilities and therefore must be rechecked after the installation is completed. The relationship between the resolving power of an EM-200 and the maximum tolerable low frequency interference fields in milli-Oerstedt is 10 Å - 1.9, 8 Å - 1.4, 6 Å - 0.8.

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Automated high-resolution microscopy: the approaches so far

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100125312 ◽

1987 ◽

Vol 45 ◽

pp. 58-61

Author(s):

W. O. Saxton

Keyword(s):

High Resolution ◽

Early Stage ◽

Automatic Recording ◽

Microprocessor Control ◽

Alternative Systems ◽

Electron Image ◽

Complete Automation ◽

Analysis System ◽

High Resolution Microscopy

Recent commercial microscopes with internal microprocessor control of all major functions have already demonstrated some of the benefits anticipated from such systems, such as continuous magnification, rotation-free diffraction and magnification, automatic recording of mutually registered focal series, and fewer control knobs. Complete automation of the focusing, stigmating and alignment of a high resolution microscope, allowing focal series to be recorded at preselected focus values as well, is still imminent rather than accomplished, however; some kind of image pick-up and analysis system, fed with the electron image via a TV camera, is clearly essential for this, but several alternative systems and algorithms are still being explored. This paper reviews the options critically in turn, and stresses the need to consider alignment and focusing at an early stage, and not merely as an optional extension to a basic proposal.

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High-resolution microscopy of twin boundaries in gold

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100126202 ◽

1987 ◽

Vol 45 ◽

pp. 260-261

Author(s):

William Krakow ◽

David A. Smith

Keyword(s):

High Resolution ◽

Specimen Preparation ◽

Gold Films ◽

Boundary Area ◽

Transmission Electron ◽

Recent Developments ◽

Tilt Boundaries ◽

High Resolution Microscopy ◽

Twist Boundaries

Recent developments in specimen preparation, imaging and image analysis together permit the experimental determination of the atomic structure of certain, simple grain boundaries in metals such as gold. Single crystal, ∼125Å thick, (110) oriented gold films are vapor deposited onto ∼3000Å of epitaxial silver on (110) oriented cut and polished rock salt substrates. Bicrystal gold films are then made by first removing the silver coated substrate and placing in contact two suitably misoriented pieces of the gold film on a gold grid. Controlled heating in a hot stage first produces twist boundaries which then migrate, so reducing the grain boundary area, to give mixed boundaries and finally tilt boundaries perpendicular to the foil. These specimens are well suited to investigation by high resolution transmission electron microscopy.

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Untargeted Metabolomics Uncovers the Essential Lysine Transporter in Toxoplasma gondii

Metabolites ◽

10.3390/metabo11080476 ◽

2021 ◽

Vol 11 (8) ◽

pp. 476

Author(s):

Joachim Kloehn ◽

Matteo Lunghi ◽

Emmanuel Varesio ◽

David Dubois ◽

Dominique Soldati-Favre

Keyword(s):

Amino Acids ◽

Toxoplasma Gondii ◽

Rna Degradation ◽

Major Facilitator Superfamily ◽

Untargeted Metabolomics ◽

Apicomplexan Parasites ◽

Obligate Intracellular ◽

Intracellular Parasites ◽

Major Facilitator ◽

Plasma Membrane Transporter

Apicomplexan parasites are responsible for devastating diseases, including malaria, toxoplasmosis, and cryptosporidiosis. Current treatments are limited by emerging resistance to, as well as the high cost and toxicity of existing drugs. As obligate intracellular parasites, apicomplexans rely on the uptake of many essential metabolites from their host. Toxoplasma gondii, the causative agent of toxoplasmosis, is auxotrophic for several metabolites, including sugars (e.g., myo-inositol), amino acids (e.g., tyrosine), lipidic compounds and lipid precursors (cholesterol, choline), vitamins, cofactors (thiamine) and others. To date, only few apicomplexan metabolite transporters have been characterized and assigned a substrate. Here, we set out to investigate whether untargeted metabolomics can be used to identify the substrate of an uncharacterized transporter. Based on existing genome- and proteome-wide datasets, we have identified an essential plasma membrane transporter of the major facilitator superfamily in T. gondii—previously termed TgApiAT6-1. Using an inducible system based on RNA degradation, TgApiAT6-1 was depleted, and the mutant parasite’s metabolome was compared to that of non-depleted parasites. The most significantly reduced metabolite in parasites depleted in TgApiAT6-1 was identified as the amino acid lysine, for which T. gondii is predicted to be auxotrophic. Using stable isotope-labeled amino acids, we confirmed that TgApiAT6-1 is required for efficient lysine uptake. Our findings highlight untargeted metabolomics as a powerful tool to identify the substrate of orphan transporters.

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