Sébastien Besteiro

University of Michigan, Ann Arbor, MI, United States

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Publications (33)229.18 Total impact

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    ABSTRACT: Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.
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    ABSTRACT: In the process of autophagy, the Atg8 protein is conjugated, through a ubiquitin-like system, to the lipid phosphatidylethanolamine (PE) to associate with the membrane of forming autophagosomes. There, it plays a crucial role in the genesis of these organelles and in autophagy in general. In most eukaryotes, the cysteine peptidase Atg4 processes the C terminus of cytosolic Atg8 to regulate its association with autophagosomal membranes and also delipidates Atg8 to release this protein from membranes. The parasitic protist Toxoplasma gondii contains a functional, yet apparently reduced, autophagic machinery. T. gondii Atg8 homolog, in addition to a cytosolic and occasionally autophagosomal localization, also localizes to the apicoplast, a nonphotosynthetic plastid bounded by four membranes. Our attempts to interfere with TgATG8 function showed that it appears to be essential for parasite multiplication inside its host cell. This protein also displays a peculiar C terminus that does not seem to necessitate processing prior to membrane association and yet an unusually large Toxoplasma homolog of ATG4 is predicted in the parasite genome. A TgATG4 conditional expression mutant that we have generated is severely affected in growth, and displays significant alterations at the organellar level, noticeably with a fragmentation of the mitochondrial network and a loss of the apicoplast. TgATG4-depleted parasites appear to be defective in the recycling of membrane-bound TgATG8. Overall, our data highlight a role for the TgATG8 conjugation pathway in maintaining the homeostasis of the parasite's organelles and suggest that Toxoplasma has evolved a specialized autophagic machinery with original regulation.
    Autophagy 06/2013; 9(9). · 12.04 Impact Factor
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    ABSTRACT: Plasmodium falciparum is the causative agent of malaria, a deadly infectious disease for which treatments are scarce and drug-resistant parasites are now increasingly found. A comprehensive method of identifying and quantifying metabolites of this intracellular parasite could expand the arsenal of tools to understand its biology, and be used to develop new treatments against the disease. Here, we present two methods based on liquid chromatography tandem mass spectrometry for reliable measurement of water-soluble metabolites involved in phospholipid biosynthesis, as well as several other metabolites that reflect the metabolic status of the parasite including amino acids, carboxylic acids, energy-related carbohydrates, and nucleotides. A total of 35 compounds was quantified. In the first method, polar compounds were retained by hydrophilic interaction chromatography (amino column) and detected in negative mode using succinic acid-(13)C(4) and fluorovaline as internal standards. In the second method, separations were carried out using reverse phase (C18) ion-pair liquid chromatography, with heptafluorobutyric acid as a volatile ion pairing reagent in positive detection mode, using d(9)-choline and 4-aminobutanol as internal standards. Standard curves were performed in P. falciparum-infected and uninfected red blood cells using standard addition method (r(2)>0.99). The intra- and inter-day accuracy and precision as well as the extraction recovery of each compound were determined. The lower limit of quantitation varied from 50pmol to 100fmol/3×10(7)cells. These methods were validated and successfully applied to determine intracellular concentrations of metabolites from uninfected host RBCs and isolated Plasmodium parasites.
    Analytica chimica acta 08/2012; 739:47-55. · 4.31 Impact Factor
  • Sébastien Besteiro
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    ABSTRACT: Autophagy is a life-sustaining process by which cytoplasmic components are sequestered in double-membrane vesicles called autophagosomes, and degraded after fusion with a lytic compartment. This process can be triggered under cellular stress conditions in order to recycle damaged organelles or provide nutrients to the cell, but may also be involved in cell remodelling during normal development. This catabolic process is conserved among most eukaryotes and characterisation of its molecular machinery has benefited greatly from functional genetic studies in yeast and mammalian models. Until recently, not much was known about the functions of autophagy in Apicomplexa, but recent data obtained in Toxoplasma have shed light on a very important role for this machinery, potentially at the crossroads between life and death decisions for the parasite. The possible roles for autophagy during the life cycles of other medically important apicomplexan parasites and the perspectives for discovering new drug targets in this pathway for combating these parasites are discussed in this review.
    Molecular and Biochemical Parasitology 04/2012; 184(1):1-8. · 2.73 Impact Factor
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    ABSTRACT: In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
    Autophagy 04/2012; 8(4):445. · 12.04 Impact Factor
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    Autophagy 04/2012; 8(4):1-100. · 12.04 Impact Factor
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    [Show abstract] [Hide abstract]
    ABSTRACT: In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process);5,6 thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
    Autophagy 04/2012; 8(4). · 12.04 Impact Factor
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    [Show abstract] [Hide abstract]
    ABSTRACT: In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
    Autophagy 04/2012; 8(4):445-544. · 12.04 Impact Factor
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    Autophagy 04/2012; 4454(8):445-544. · 12.04 Impact Factor
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    Sébastien Besteiro
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    ABSTRACT: Toxoplasma gondii belongs to the phylum Apicomplexa, a diverse group of early branching unicellular eukaryotes related to dinoflagellates and ciliates. Like several other Apicomplexa such as Plasmodium (the causative agent of malaria), T. gondii is a human pathogen responsible for a potentially lethal disease called toxoplasmosis. Most Apicomplexa have complex life cycles, involving intermediate hosts and vectors, which include obligatory intracellular developmental stages. In the case of malaria and toxoplasmosis, it is that replicative process, leading to the ultimate lysis of the host cell, which is causing the symptoms of the disease. For Toxoplasma, the invasive and fast-replicating form of the parasite is called the tachyzoite. While autophagy has been a fast-growing field of research in recent years, not much was known about the relevance of this catabolic process in medically important apicomplexan parasites. Vesicles resembling autophagosomes had been described in drug-treated Plasmodium parasites in the early 1970s and a potential role for autophagy in organelle recycling during differentiation between Plasmodium life stages has also been recently described. Interestingly, recent database searches have identified putative orthologs of the core machinery responsible for the formation of autophagosomes in several protists, including Toxoplasma. In spite of an apparently reduced machinery (only about one-third of the yeast ATG genes appear to be conserved), T. gondii seemed thus able to perform macroautophagy, but the cellular functions of the pathway for this parasite remained to be demonstrated.
    Autophagy 03/2012; 8(3):435-7. · 12.04 Impact Factor
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    Autophagy 01/2012; 4454(8):445-544. · 12.04 Impact Factor
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    ABSTRACT: Autophagy is a cellular process that is highly conserved among eukaryotes and permits the degradation of cellular material. Autophagy is involved in multiple survival-promoting processes. It not only facilitates the maintenance of cell homeostasis by degrading long-lived proteins and damaged organelles, but it also plays a role in cell differentiation and cell development. Equally important is its function for survival in stress-related conditions such as recycling of proteins and organelles during nutrient starvation. Protozoan parasites have complex life cycles and face dramatically changing environmental conditions; whether autophagy represents a critical coping mechanism throughout these changes remains poorly documented. To investigate this in Toxoplasma gondii, we have used TgAtg8 as an autophagosome marker and showed that autophagy and the associated cellular machinery are present and functional in the parasite. In extracellular T. gondii tachyzoites, autophagosomes were induced in response to amino acid starvation, but they could also be observed in culture during the normal intracellular development of the parasites. Moreover, we generated a conditional T. gondii mutant lacking the orthologue of Atg3, a key autophagy protein. TgAtg3-depleted parasites were unable to regulate the conjugation of TgAtg8 to the autophagosomal membrane. The mutant parasites also exhibited a pronounced fragmentation of their mitochondrion and a drastic growth phenotype. Overall, our results show that TgAtg3-dependent autophagy might be regulating mitochondrial homeostasis during cell division and is essential for the normal development of T. gondii tachyzoites.
    PLoS Pathogens 12/2011; 7(12):e1002416. · 8.14 Impact Factor
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    Sébastien Besteiro, Jean-François Dubremetz, Maryse Lebrun
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    ABSTRACT: Most Apicomplexa are obligate intracellular parasites and many are important pathogens of human and domestic animals. For a successful cell invasion, they rely on their own motility and on a firm anchorage to their host cell, depending on the secretion of proteins and the establishment of a structure called the moving junction (MJ). The MJ moves from the apical to the posterior end of the parasite, leading to the internalization of the parasite into a parasitophorous vacuole. Based on recent data obtained in Plasmodium and Toxoplasma, an emerging model emphasizes a cooperative role of secreted parasitic proteins in building the MJ and driving this crucial invasive process. More precisely, the parasite exports the microneme protein AMA1 to its own surface and the rhoptry neck RON2 protein as a receptor inserted into the host cell together with other RON partners. Ongoing and future research will certainly help refining the model by characterizing the molecular organization within the MJ and its interactions with both host and parasite cytoskeleton for anchoring of the complex.
    Cellular Microbiology 06/2011; 13(6):797-805. · 4.81 Impact Factor
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    ABSTRACT: Obligate intracellular Apicomplexa parasites share a unique invasion mechanism involving a tight interaction between the host cell and the parasite surfaces called the moving junction (MJ). The MJ, which is the anchoring structure for the invasion process, is formed by secretion of a macromolecular complex (RON2/4/5/8), derived from secretory organelles called rhoptries, into the host cell membrane. AMA1, a protein secreted from micronemes and associated with the parasite surface during invasion, has been shown in vitro to bind the MJ complex through a direct association with RON2. Here we show that RON2 is inserted as an integral membrane protein in the host cell and, using several interaction assays with native or recombinant proteins, we define the region that binds AMA1. Our studies were performed both in Toxoplasma gondii and Plasmodium falciparum and although AMA1 and RON2 proteins have diverged between Apicomplexa species, we show an intra-species conservation of their interaction. More importantly, invasion inhibition assays using recombinant proteins demonstrate that the RON2-AMA1 interaction is crucial for both T. gondii and P. falciparum entry into their host cells. This work provides the first evidence that AMA1 uses the rhoptry neck protein RON2 as a receptor to promote invasion by Apicomplexa parasites.
    PLoS Pathogens 01/2011; 7(2):e1001276. · 8.14 Impact Factor
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    ABSTRACT: SUMMARYPlasmodium falciparum, the agent responsible for malaria, is an obligate intracellular protozoan parasite. For proliferation, differentiation and survival, it relies on its own protein-encoding genes, as well as its host cells for nutrient sources. Nutrients and subsequent metabolites are required by the parasites to support their high rate of growth and replication, particularly in the intra-erythrocytic stages of the parasite that are responsible for the clinical symptoms of the disease. Advances in mass spectrometry have improved the analysis of endogenous metabolites and enabled a global approach to identify the parasite's metabolites by the so-called metabolomic analyses. This level of analysis complements the genomic, transcriptomic and proteomic data already available and should allow the identification of novel metabolites, original pathways and networks of regulatory interactions within the parasite, and between the parasite and its hosts. The field of metabolomics is just in its infancy in P. falciparum, hence in this review, we concentrate on the available methodologies and their potential applications for deciphering important biochemical processes of the parasite, such as the astonishingly diverse phospholipid biosynthesis pathways. Elucidating the regulation of the biosynthesis of these crucial metabolites could help design of future anti-malarial drugs.
    Parasitology 08/2010; 137(9):1343-56. · 2.36 Impact Factor
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    ABSTRACT: It has been proposed that the natural cysteine peptidase inhibitor ICP of Leishmania mexicana protects the protozoan parasite from insect host proteolytic enzymes, thereby promoting survival. To test this hypothesis, L. mexicana mutants deficient in ICP were evaluated for their ability to develop in the sand fly Lutzomyia longipalpis. No significant differences were found between the wild-type parasites, two independently derived ICP-deficient mutants, or mutants overexpressing ICP; all lines developed similarly in the sand fly midgut and produced heavy late-stage infections. In addition, recombinant L. mexicana ICP did not inhibit peptidase activity of the midgut extracts in vitro. We conclude that ICP has no major role in promoting survival of L. mexicana in the vectorial part of its life cycle in L. longipalpis.
    Journal of Medical Entomology 06/2009; 46(3):605-9. · 1.86 Impact Factor
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    ABSTRACT: Leishmania mexicana cysteine peptidases (CPs) have been identified as important parasite virulence factors. More recently, a natural inhibitor of CPs (ICP) from L. mexicana has been characterized, and ICP mutants have been created. Infection of BALB/c mice with ICP null mutants or ICP reexpressing mutants resulted in nonhealing, progressively growing lesions albeit slightly attenuated compared with the growth of lesions produced by wild-type parasites. In contrast, BALB/c mice infected with mutants overexpressing ICP were able to significantly control lesion growth or heal. While BALB/c mice infected with wild-type parasites, ICP null mutants, or ICP reexpressing mutants produced significant antibody responses, including immunoglobulin E (IgE), no Th1 response, as indicated by antigen-induced splenocyte gamma interferon (IFN-gamma) production, could be demonstrated. In contrast, BALB/c mice infected with mutants overexpressing ICP produced significantly less antibody, particularly IgE, as well as significantly reduced splenocyte interleukin-4 and enhanced IFN-gamma production. BALB/c mice were able to resolve infection following infection with one ICP overexpressing clone, which was subsequently used for vaccination studies with BALB/c mice. However, no protection was afforded these mice when they were challenged with wild-type parasites. Nevertheless, two other mouse strains susceptible to L. mexicana, C3H and C57BL/6, vaccinated with overexpressing ICP mutants were able to control challenge infection associated with an enhanced Th1 response. This study confirms that L. mexicana CPs are virulence factors and that ICPs have therapeutic potential.
    Infection and immunity 06/2009; 77(7):2971-8. · 4.21 Impact Factor
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    ABSTRACT: One of the most conserved features of the invasion process in Apicomplexa parasites is the formation of a moving junction (MJ) between the apex of the parasite and the host cell membrane that moves along the parasite and serves as support to propel it inside the host cell. The MJ was, up to a recent period, completely unknown at the molecular level. Recently, proteins originated from two distinct post-Golgi specialised secretory organelles, the micronemes (for AMA1) and the neck of the rhoptries (for RON2/RON4/RON5 proteins), have been shown to form a complex. AMA1 and RON4 in particular, have been localised to the MJ during invasion. Using biochemical approaches, we have identified RON8 as an additional member of the complex. We also demonstrated that all RON proteins are present at the MJ during invasion. Using metabolic labelling and immunoprecipitation, we showed that RON2 and AMA1 were able to interact in the absence of the other members. We also discovered that all MJ proteins are subjected to proteolytic maturation during trafficking to their respective organelles and that they could associate as non-mature forms in vitro. Finally, whereas AMA1 has previously been shown to be inserted into the parasite membrane upon secretion, we demonstrated, using differential permeabilization and loading of RON-specific antibodies into the host cell, that the RON complex is targeted to the host cell membrane, where RON4/5/8 remain associated with the cytoplasmic face. Globally, these results point toward a model of MJ organization where the parasite would be secreting and inserting interacting components on either side of the MJ, both at the host and at its own plasma membranes.
    PLoS Pathogens 03/2009; 5(2):e1000309. · 8.14 Impact Factor

Publication Stats

2k Citations
229.18 Total Impact Points

Institutions

  • 2012
    • University of Michigan
      • Life Sciences Institute
      Ann Arbor, MI, United States
  • 2011–2012
    • Université de Montpellier 1
      Montpelhièr, Languedoc-Roussillon, France
  • 2008–2012
    • French National Centre for Scientific Research
      • Unité de recherche Dynamique des Interactions Membranaires Normales et Pathologiques (DIMNP)
      Montpelhièr, Languedoc-Roussillon, France
  • 2005–2008
    • University of Glasgow
      • Wellcome Trust Centre for Molecular Parasitology
      Glasgow, SCT, United Kingdom
    • Anderson College
      Anderson, Indiana, United States
  • 2002–2006
    • Université Victor Segalen Bordeaux 2
      • Centre de Résonance Magnétique des Systèmes Biologiques
      Bordeaux, Aquitaine, France