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Introduction
Lipidomics and Metabolism of infectious diseases and protists
Additional affiliations
January 2017 - present
University of Grenoble, CNRS
Position
- PostDoc Position
February 2013 - December 2016
University of Cambridge/University of Melbourne
Position
- PhD Student
December 2011 - January 2013
Publications
Publications (66)
Apicomplexa parasites cause major diseases such as toxoplasmosis and malaria that have major health and economic burdens. These unicellular pathogens are obligate intracellular parasites that heavily depend on lipid metabolism for the survival within their hosts. Their lipid synthesis relies on an essential combination of fatty acids (FAs) obtained...
Fundamental processes that govern the lytic cycle of the intracellular parasite Toxoplasma gondii are regulated by several signalling pathways. However, how these pathways are connected remains largely unknown. Here, we compare the phospho-signalling networks during Toxoplasma egress from its host cell by artificially raising cGMP or calcium levels...
Apicomplexan parasites have complex metabolic networks that coordinate acquisition of metabolites by de novo synthesis and by scavenging from the host. Toxoplasma gondii has a wide host range and may rely on the flexibility of this metabolic network. Currently, the literature categorizes genes as essential or dispensable according to their dispensa...
Apicomplexa are obligate intracellular parasites responsible for major human infectious diseases such as toxoplasmosis and malaria, which pose social and economic burdens around the world. To survive and propagate, these parasites need to acquire a significant number of essential biomolecules from their hosts. Among these biomolecules, lipids are a...
Apicomplexa are obligate intracellular parasites responsible for major human diseases. Their intracellular survival relies on intense lipid synthesis, which fuels membrane biogenesis. Parasite lipids are generated as an essential combination of fatty acids scavenged from the host and de novo synthesized within the parasite apicoplast. The molecular...
Apicomplexan parasites are causative agents of major human diseases. Calcium Dependent Protein Kinases (CDPKs) are crucial components for the intracellular development of apicomplexan parasites and are thus considered attractive drug targets. CDPK7 is an atypical member of this family, which initial characterization suggested to be critical for int...
Host cell invasion and subsequent egress by Toxoplasma parasites is regulated by a network of cGMP, cAMP, and calcium signalling proteins. Such eukaryotic signalling networks typically involve lipid second messengers including phosphatidylinositol phosphates (PIPs), diacylglycerol (DAG) and phosphatidic acid (PA). However, the lipid signalling netw...
Apicomplexan parasites are unicellular eukaryotic pathogens that must obtain and combine lipids from both host cell scavenging and de novo synthesis to maintain parasite propagation and survival within their human host. Major questions on the role and regulation of each lipid source upon fluctuating host nutritional conditions remain unanswered. Ch...
The myosin superfamily comprises of actin‐dependent eukaryotic molecular motors important in a variety of cellular functions. Although well studied in many systems, knowledge of their functions in Plasmodium, the causative agent of malaria, is restricted. Previously, six myosins were identified in this genus, including three Class XIV myosins found...
The myosin superfamily comprises of actin-dependent eukaryotic molecular motors important in a variety of cellular functions. Although well studied in many systems, knowledge of their functions in Plasmodium, the causative agent of malaria, is restricted. Previously, six myosins were identified in this genus, including three Class XIV myosins found...
Protozoan parasites of the phylum Apicomplexa actively move through tissue to initiate and perpetuate infection. The regulation of parasite motility relies on cyclic nucleotide-dependent kinases, but how these kinases are activated remains unknown. Here, using an array of biochemical and cell biology approaches, we show that the apicomplexan parasi...
Apicomplexan parasites are unicellular eukaryotes responsible for major human diseases including malaria and toxoplasmosis. Apicomplexan parasites must obtain and combine lipids both from host cell scavenging and de novo synthesis to maintain parasite propagation and survival within their human host. Major questions on the actual role for each lipi...
Apicomplexan parasites including Toxoplasma gondii and Plasmodium spp. manufacture a complex arsenal of secreted proteins used to interact with and manipulate their host environment. These proteins are organised into three principle exocytotic compartment types according to their functions: micronemes for extracellular attachment and motility, rhop...
The phylum Apicomplexa comprises a group of obligate intracellular parasites that alternate between intracellular replicating stages and actively motile extracellular forms that move through tissue. Parasite cytosolic Ca²⁺ signalling activates motility, but how this is switched off after invasion is complete to allow for replication to begin is not...
Primers used in this study.
(XLSX)
Individual GCaMP6/mCherry intensity traces of PKAc1 cKD/Δplp1 tachyzoites ±ATc treatment.
Individual invading tachyzoites were tracked using ImageJ and an intensity ratio between GCaMP6 and mCherry was derived, followed by normalising against the maximum value. Thirty-five percent of maximum is marked with a dotted line to arbitrarily signify ‘base...
Individual GCaMP6/mCherry intensity traces of PKAc1 cKD tachyzoites + 50 μM DCCD.
Individual invading tachyzoites were tracked using ImageJ and an intensity ratio between GCaMP6 and mCherry was derived, followed by normalising against the maximum value. Thirty-five percent of maximum is marked with a dotted line to arbitrarily signify ‘baseline’ le...
Quantitation of GCaMP6/mCherry-expressing parasites during motility.
Parental and PKAc1 cKD parasites ±ATc tachyzoites expressing GCaMP6/mCherry were resuspended in either EC or IC buffer and allowed to glide on a glass coverslip. Motile parasites were tracked and maximum and minimum values extracted and compared across conditions. Data represent m...
Live cell imaging of PKAc1 cKD treated with ATc.
Tachyzoite invasion precedes host cell collapse, followed by reactivation of motility. Movie corresponds to still images represented in top panels of Fig 3C. ATc, anhydrotetracycline; cKD, conditional knockdown; PKAc1, protein kinase A catalytic subunit 1.
(AVI)
Live cell imaging of PKAc1 cKD strain expressing GCaMP6/mCherry not treated with ATc.
An invading tachyzoite co-expressing GCaMP6 (green) and mCherry (red) is overlaid with transmitted light image. Movie corresponds to still images represented in panels of Fig 5Bi and GCaMP6/mCherry ratio graphed of Fig 5Bii. ATc, anhydrotetracycline; cKD, conditio...
Live cell imaging of PKAc1 cKD Δplp-1 strain expressing GCaMP6/mCherry not treated with ATc.
An invading tachyzoite co-expressing GCaMP6 (green) and mCherry (red) is overlaid with transmitted light image. Movie corresponds to still images represented in panels of Fig 6Ai and GCaMP6/mCherry ratio graphed of Fig 6Aii. ATc, anhydrotetracycline; cKD, c...
Live cell imaging of PKAc1 cKD Δplp-1 strain expressing GCaMP6/mCherry treated with ATc.
An invading tachyzoite co-expressing GCaMP6 (green) and mCherry (red) is overlaid with transmitted light image. Movie corresponds to still images represented in panels of Fig 6Bi and GCaMP6/mCherry ratio graphed of Fig 6Bii. ATc, anhydrotetracycline; cKD, condi...
Generation of a tet-off conditional knockout of the PKAc1 gene (TGME49_226030).
Two gene flanks for HDR were designed to replace the endogenous pkac1 promoter with the T7S4 ‘tet-off’ promoter. HDR fragments PCRed from genomic DNA and ligated into pPR2-HA3. The plasmid was linearised and transfected into Δku80:TATi and selected on Pyr to establish a...
Loss of PKAc1 leads to aberrant culture morphology.
Images at 100× and 200× of different fields of view over three biological replicates, highlighting the morphology of host cells and the presence or absence of intracellular tachyzoites. White arrows highlight examples of intracellular parasites, both late stage and recently invaded. Black arrows h...
Individual GCaMP6/mCherry intensity traces of Parental tachyzoites +ATc.
Individual invading tachyzoites were tracked using ImageJ and an intensity ratio between GCaMP and mCherry was derived, followed by normalising against the maximum value. Thirty-five percent of maximum is marked with a dotted line to arbitrarily signify ‘baseline’ level. Blue...
Individual GCaMP6/mCherry intensity traces of PKAc1 cKD tachyzoites with ATc treatment.
Individual invading tachyzoites were tracked using ImageJ and an intensity ratio between GCaMP6 and mCherry was derived, followed by normalising against the maximum value. Thirty-five percent of maximum is marked with a dotted line to arbitrarily signify ‘baseli...
Live cell imaging of parental strain (Δku80/TATi) not treated with ATc.
Slender arrow points to newly invaded tachyzoite (before filming commenced) and white arrow points to captured invasion event. Movie corresponds to still images represented in top panels of Fig 3B. ATc, anhydrotetracycline.
(AVI)
Live cell imaging of parental strain (Δku80/TATi) treated with ATc.
White arrowhead points to tachyzoites undergoing invasion. Movie corresponds to still images represented in middle panels of Fig 3B. ATc, anhydrotetracycline.
(AVI)
Live cell imaging of PKAc1 cKD strain not treated with ATc.
Tachyzoite captured invading in the centre of field of view. Movie corresponds to still images represented in bottom panels of Fig 3B. ATc, anhydrotetracycline; cKD, conditional knockdown; PKAc1, protein kinase A catalytic subunit 1.
(AVI)
Live cell imaging of parental strain (Δku80/TATi) expressing GCaMP6/mCherry treated with ATc.
An invading tachyzoite co-expressing GCaMP6 (green) and mCherry (red) is overlaid with transmitted light image. Movie corresponds to still images represented in panels of Fig 5Ai and GCaMP6/mCherry ratio graphed of Fig 5Aii. ATc, anhydrotetracycline; GCaMP...
Live cell imaging of PKAc1 cKD strain expressing GCaMP6/mCherry treated with ATc.
An invading tachyzoite co-expressing GCaMP6 (green) and mCherry (red) is overlaid with transmitted light image. Movie corresponds to still images represented in panels of Fig 5Ci and GCaMP6/mCherry ratio graphed of Fig 5Cii. ATc, anhydrotetracycline; cKD, conditional...
Loss of PKAc1 has no detectable effect on host cell invasion.
(A) Speed of host cell invasion of parental and PKAc1-deficient tachyzoites, measured as time taken from point of attachment to complete invasion. Data represent mean ± SEM over 9–11 separate invasion events. P values are calculated using an unpaired two-tailed t test. (B) Representative...
Loss of PKAc1 has no detectable defect in motility, host cell attachment, or microneme secretion.
(A) Two-dimensional quantitative motility assay of both parental and PKAc1 cKD ± ATc in either IC or EC buffer, in which the proportion of motile and nonmotile parasites were quantitated (i), as well as the type of motility (ii). (B) Quantitative host...
Generation of a PLP-1 knockout in PKAc1 cKD.
(A) Genetic strategy of gene disruption. Two regions of homology, upstream and downstream of the PLP-1, were PCR amplified from genomic DNA and ligated either side of an expression cassette encoding CAT gene. Parasites were selected upon transfection of linearised plasmid and chloramphenicol treatment. P...
Live cell imaging of PKAc1 cKD strain treated with ATc.
Tachyzoite invasion precedes host cell collapse, followed by reactivation of motility. Movie corresponds to still images represented in bottom panels of Fig 3C. ATc, anhydrotetracycline; cKD, conditional knockdown; PKAc1, protein kinase A catalytic subunit 1.
(AVI)
Live cell imaging of PKAc1 cKD Δplp-1 strain expressing GCaMP6/mCherry treated with ATc and DCCD.
An invading tachyzoite co-expressing GCaMP6 (green) and mCherry (red) is overlaid with transmitted light image. Movie corresponds to still images represented in panels of Fig 6Ci and GCaMP6/mCherry ratio graphed of Fig 6Cii. ATc, anhydrotetracycline; c...
Quantitative analysis of proteins secreted from extracellular tachyzoites.
Parental and PKAc1 tachyzoites were grown in heavy and light SILAC media, respectively. Samples were mixed and subjected to tandem mass spectrometry (MS/MS) and peptides identified from proteins using Mascot and quantitated using a custom pipeline. Proteins highlighted in bl...
Individual GCaMP6/mCherry intensity traces of PKAc1 cKD tachyzoites without ATc treatment.
Individual invading tachyzoites were tracked using ImageJ and an intensity ratio between GCaMP6 and mCherry was derived, followed by normalising against the maximum value. Thirty-five percent of maximum is marked with a dotted line to arbitrarily signify ‘bas...
Apicomplexan parasites including Toxoplasma gondii and Plasmodium spp. manufacture a complex arsenal of secreted proteins used to interact with and manipulate their host environment. These proteins are organised into three principle exocytotic compartment types according to their functions: micronemes for extracellular attachment and motility, rhop...
Apicomplexan parasites are pathogens responsible for major human diseases such as toxoplasmosis caused by Toxoplasma gondii and malaria caused by Plasmodium spp.. Throughout their intracellular division cycle, the parasites require vast and specific amounts of lipids to divide and survive. This demand for lipids relies on a fine balance between de...
The phylum Apicomplexa comprises a group of obligate intracellular parasites that alternate between intracellular replicating forms and actively motile extracellular forms that move through tissue. Parasite cytosolic Ca ²⁺ signalling activates motility, but how this is switched off after invasion is not understood. Here we show that the cAMP-depend...
Plasmodium parasites, the causative agents of malaria, possess a distinctive membranous structure of flattened alveolar vesicles supported by a proteinaceous network, and referred to as the inner membrane complex (IMC). The IMC has a role in actomyosin-mediated motility and host cell invasion. Here, we examine the location, protein interactome and...
Plasmodium parasites, the causative agents of malaria, possess a distinctive membranous structure of flattened alveolar vesicles supported by a proteinaceous network, and referred to as the inner membrane complex (IMC). The IMC has a role in actomyosin-mediated motility and host cell invasion. Here, we examine the location, protein interactome and...
The SAS6-like (SAS6L) protein, a truncated paralogue of the ubiquitous basal body/centriole protein SAS6, has been characterised recently as a flagellum protein in trypanosomatids, but associated with the conoid in apicomplexan Toxoplasma. The conoid has been suggested to derive from flagella parts, but is thought to have been lost from some apicom...
ELife digest
Single-celled parasites cause many severe diseases in humans and animals. The apicomplexans form probably the most successful group of these parasites and include the parasites that cause malaria. Apicomplexans infect a broad range of hosts, including humans, reptiles, birds, and insects, and often have complicated life cycles. For exa...
Genes encoding cytoskeletal components in the 26 species.Most of components in the actomyosin complex were predicted in silico with some manual curation for P. falciparum and T. gondii. Definitions of the categories and details of how these genes are compiled are provided in the materials and method section.DOI:
http://dx.doi.org/10.7554/eLife.0697...
RNA-seq libraries of Chromera velia under various growth conditions.The list of growth conditions and the file names are given.DOI:
http://dx.doi.org/10.7554/eLife.06974.023
Summary of the genome assembly and the annotated genes of Chromera velia, Vitrella brassicaformis. Details of transposable elements on the genome are shown in Supplementary file 2.
DOI:
http://dx.doi.org/10.7554/eLife.06974.026
Summary of transposable elements on the Chromera velia and Vitrella brassicaformis genomes.
DOI:
http://dx.doi.org/10.7554/eLife.06974.027
Domains of extracellular proteins and example genes in chromerids. (a) Species abbreviations: Perkinsus marinus, P. mar; Chromera velia, C. vel; Vitrella brassicaformis, V. bra; and Cryptosporidium parvum, C. par. (b) Domain accession identifiers. Domain information can be retrieved at the NCBI Conserved Domain website: (http://www.ncbi.nlm.nih.gov...
List of 24 species excluding Chomera and Vitrella used in this study and their data sources.DOI:
http://dx.doi.org/10.7554/eLife.06974.004
Genes encoding subunits of the endomembrane trafficking system.DOI:
http://dx.doi.org/10.7554/eLife.06974.008
List of genes implicated in invasion processes in apicomplexans.They were either annotated as implicated in invasion processes in P. falciparum or orthologs of those in other apicomplexans. Pubmed ID number or the database from which the annotation was obtained is listed in the last two columns.DOI:
http://dx.doi.org/10.7554/eLife.06974.024
Mitochondrial localization predictions of selected RAP genes. Various algorithmic methods were used to identify candidates for experimental validations in Toxoplasma. Classifications are given in the column ‘Loc’ as M-mitochondria; S- secreted; O-others.
DOI:
http://dx.doi.org/10.7554/eLife.06974.031
A list of 101 shared orthogroups with a single gene in all of the 26 species, used for the species phylogenetic tree.DOI:
http://dx.doi.org/10.7554/eLife.06974.005
Distribution of enzymes based on KEGG.This Excel spread sheet provides mapping of the curated set of extracellular numbers (and/or gene names when an extracellular number is not available) to each metabolic pathway listed for each of the 26 species that are compared here. All the extracellular numbers shown in black font color were assigned automat...
Genes encoding components of the flagellar apparatus in the 26 species.DOI:
http://dx.doi.org/10.7554/eLife.06974.014
Genes encoding extracellular proteins in P.falciparum and T. gondii.Details regarding how these genes are defined and compiled are provided in the ‘Materials and methods’ section.DOI:
http://dx.doi.org/10.7554/eLife.06974.016
Genes encoding proteins involved in forming photosystems in Chromera velia and Vitrella brassicaformis.
DOI:
http://dx.doi.org/10.7554/eLife.06974.028
Genes encoding enzymes involved in heme biosynthesis in chromerids.
DOI:
http://dx.doi.org/10.7554/eLife.06974.029
Evolutionary conservation of 12 orthogroups with RAP domains (for 'RAP' category in Figure 4A).IDs of the genes in Chromera and Plasmodium falciparum are list with odds-ratios and p-values based on Fisher's exact test.DOI:
http://dx.doi.org/10.7554/eLife.06974.034
The apical complex is the definitive cell structure of phylum Apicomplexa, and is the focus of the events of host cell penetration and the establishment of intracellular parasitism. Despite the importance of this structure, its molecular composition is relatively poorly known and few studies have experimentally tested its functions. We have charact...