Experimental study of the function of the excreted/secreted Leishmania LmSIR2 protein by heterologous expression in eukaryotic cell line

BACKGROUND: In yeast and Caenorhabditis elegans, Silent Information Regulator (SIR2) proteins have been shown to be involved in ageing regulation. In Leishmania, the LmSIR2rp was originally isolated from the excreted/secreted material of the Leishmania parasites. Among the function(s) of this protein in Leishmania biology, we have documented its implication in parasite survival, and in particular in Leishmania amastigotes. In this paper we question the role of the excreted/secreted form of the protein. In particular we wonder if the Leishmania Sir2 homologue is involved in some aspect of its biological function(s), in various components and pathways, which could promote the host cell survival. To test this hypothesis we have mimicked an intracellular release of the protein through constitutive expression in mouse L929 fibrosarcoma cells. RESULTS: Our results demonstrate that the LmSIR2 protein was properly expressed by fibroblasts and that LmSIR2 is localized both in the cytoplasm and the nucleus of all the transformed cell clones. Unexpectedly, we found that cells expressing LmSIR2 presents reduced saturation cell density ranging from 40% to 60% and expressed an acidic (pH6.0) β-galactosidase activity, which is known to be a senescence biomarker. As a consequence, we observed that LmSIR2 positive fibroblasts were more permissive towards Leihmania infection. CONCLUSIONS: LmSIR2 is able to substantially interfere with the host cell physiology. Thus, it is tempting to speculate that these modifications could help Leishmania to survive for a long period in a cell with reduced capacity to multiply or respond to immunologic stimuli. The potential implications of our finding during the in vivo infection process are discussed

INsPECT, an Open-Source and Versatile Software for Automated Quantification of (Leishmania) Intracellular Parasites

Intracellular protozoan parasites are causative agents of infectious diseases that constitute major health problems for developing countries. Leishmania sp., Trypanosoma cruzi or Toxoplasma gondii are all obligate intracellular protozoan parasites that reside and multiply within the host cells of mammals, including humans. Following up intracellular parasite proliferation is therefore an essential and a quotidian task for many laboratories working on primary screening of new natural and synthetic drugs, analyzing drug susceptibility or comparing virulence properties of natural and genetically modified strains. Nevertheless, laborious manual microscopic counting of intracellular parasites is still the most commonly used approach. Here, we present INsPECT (Intracellular ParasitE CounTer), an open-source and platform independent software dedicated to automate infection level measurement based on fluorescent DNA staining. It offers the possibility to choose between different types of analyses (fluorescent DNA acquisitions only or in combination with phase contrast image set to further separate intra-from extracellular parasites), and software running modes (automatic or custom). A proof-of-concept study with intracellular Leishmania infantum parasites stained with DAPI (49,6-diamidino-2-phenylindole) confirms a good correspondence between digital results and the "gold standard" microscopic counting method with Giemsa. Interestingly, this software is versatile enough to accurately detect intracellular T. gondii parasites on images acquired with High Content Screening (HCS) systems. In conclusion, INsPECT software is proposed as a new fast and simple alternative to the classical intracellular Leishmania quantification methods and can be adapted for mid to large-scale drug screening against different intracellular parasites

Implication de la protéase de l'Adénovirus dans le désassemblage partiel de la capside et le relargage de la protéine VI

L’Adénovirus est un virus non-enveloppé qui entre dans les cellules par endocytose après reconnaissance aux récepteurs cellulaires. Pendant cette étape d’entrée, la capside subit un désassemblage partiel à l’intérieur de l’endosome qui permet la libération de la protéine VI. Cette protéine est responsable de la lyse de la membrane de l’endosome, permettant ainsi à la capside de s’échapper de l’endosome pour accéder au cytosol et être transportée vers l’enveloppe nucléaire via les microtubules. Les mécanismes cellulaires et / ou viraux permettant ce désassemblage partiel ne sont pas entièrement compris. Le projet vise à investiguer le rôle potentiel de la protéase de l’Adénovirus (AVP) dans cette étape d’entrée. L’AVP est une protéase à cystéine produite dans une forme inactive et incorporée dans les capsides comme une protéine de liaison à l’ADN, indépendamment de sa séquence. Une fois activée par deux cofacteurs, elle permet le clivage de différentes protéines via une réaction biochimique à une dimension. Cette étape, appelée maturation, se produit suite à / pendant l’assemblage de la capside dans la cellule infectée et rend la capside moins stable, ce qui est essentiel pour l’infectivité du virus. Un mutant de l’Adénovirus qui n’incorpore pas l‘AVP est donc composé de protéines non clivées et possède par conséquent une capside hyperstable. Ce mutant est ainsi incapable de libérer la protéine VI dans l’endosome et est dégradé par la voie lysosomale. Nous avons produit et purifié l’AVP afin de développer un test de maturation in vitro des protéines virales et apporter une meilleure compréhension de son rôle potentiel dans le désassemblage partiel de la capside. Nous avons identifié un nouveau site de clivage fonctionnel d’une protéine de la capside qui pourrait être un des déclencheurs de la libération de la protéine VI. Nous combinons des approches de biochimie et de biologie cellulaire afin de confirmer que l’activité de clivage de l’AVP facilite l’entrée du virus.The Adenovirus is a non-enveloped virus entering the cells by endocytosis after attachment to cell receptors. During entry, the capsid undergoes a partial disassembly inside the endosome, which is allowing the release of protein VI. This protein is responsible for the endosomal membrane damages that allow the capsid to escape the endosome to access the cytoplasm, to follow nuclear transport mediated by microtubules. The cellular and / or viral cues allowing this intra-endosomal disassembly are not fully understood. The project goal is to investigate the potential role of the Adenovirus protease (AVP) in this entry step. The AVP is produced as an inactive cysteine protease and is incorporated in the capsids as an unspecific DNA sequence binding protein. Once activated by two co-factors, it allows the cleavage of multiple proteins through a one dimensional biochemical reaction. This step, called maturation, is happening after / during the assembly of the capsids in the infected cell. It destabilizes the capsid, which is essential for the virus infectivity. An Adenovirus mutant, which is not incorporating the AVP, is composed of unprocessed proteins and thus, possesses a hyperstable capsid. Therefore, this mutant cannot release the protein VI in the endosome and thus, is degraded via the lysosomal pathway. We produced and purified the AVP to develop an in vitro maturation assay of the viral proteins, and bring a better understanding of its potential role in the partial disassembly of the capsid. We identified a new functional cleavage site of a capsid protein which could be one of the triggers of the protein VI release. We combined biochemical and cell biology approaches to confirm that the cleavage activity of the AVP is facilitating the virus entry

Ambivalent activity of nicotinamide against Leishmania parasites (therapeutic adjuvant and main NAD+ precursor)

Le nicotinamide est une vitamine fournie par l'alimentation et utilisée en thérapie dans le traitement de certaines pathologies humaines. Chez Leishmania, un protozoaire parasite responsable des leishmanioses, cette vitamine présente une action toxique contre le parasite et une action synergique avec l'antimoine, la principale molécule utilisée dans le traitement des leishmanioses. En recherchant le mode d'action de cette vitamine, nous avons observé qu'elle était en réalité un précurseur essentiel à la synthèse du NAD+ chez le parasite, un cofacteur responsable de la plupart des réactions d'oxydoréduction chez tous les êtres vivants. Leishmania étant en effet dépourvu des voies de synthèse de novo du NAD+, il doit le générer à partir de précurseurs qu'il importe depuis son environnement (nicotinamide, nicotinamide riboside, acide nicotinique). Cette auxotrophie du parasite pour le NAD+ révèle donc un rôle ambivalent du nicotinamide, à la fois toxique à fortes concentrations et pourtant essentiel à sa survie en tant que précurseur majeur du NAD+. À partir des bases de données, nous avons reconstitué l'ensemble de la voie de synthèse du NAD+ chez Leishmania. Parmi les enzymes impliquées, nous avons identifié une nicotinamidase qui n'a pas d'homologue chez les mammifères, et qui assure la conversion du nicotinamide en acide nicotinique, première étape à la synthèse du NAD+. Cette enzyme étant un candidat intéressant pour le développement de molécules ciblant spécifiquement le parasite, nous avons réalisé la caractérisation fonctionnelle de ce gène. Son inactivation induit une diminution importante de la concentration en NAD+ chez le parasite et provoque un arrêt de la prolifération en culture, ainsi qu'une incapacité des mutants à établir une infection durable chez la souris. L'obtention de la structure de la nicotinamidase de L. infantum nous offre désormais la possibilité de développer des inhibiteurs spécifiques contre cette nouvelle cible thérapeutique.Nicotinamide is a vitamin provided by food that is already used in human therapy. In Leishmania protozoan parasites, this molecule shows toxic activity against parasites and has synergistic activity with antimonials, the main drugs used to treat leishmaniasis. By investigating the mode of action of this cheap vitamin, we discovered that nicotinamide is in fact the main precursor of NAD+ synthesis in Leishmania, a redox cofactor essential for all living cells. Leishmania are indeed devoid of a de novo NAD+ pathway and must synthesize it by scavenging precursors from their environment (nicotinamide, nicotinic acid and nicotinamide riboside). This NAD+ auxotrophy reveals a mixed pattern of activity of nicotinamide in Leishmania, i.e. toxic at high concentrations but also essential for parasite survival through its role in NAD+ synthesis. All enzymes of the Leishmania NAD+ salvage pathway were then identified from genome databases. We focused on a putative nicotinamidase, which has no homolog in mammals and governs the conversion of nicotinamide to nicotinic acid, the first step in the NAD+ salvage pathway. Since this enzyme could be considered as an attractive therapeutic target to develop specific parasite inhibitors, we performed a functional analysis of the corresponding gene. Targeted deletion of the nicotinamidase encoding gene induced a marked drop in parasite NAD+ content and a phenotype with strongly delayed growth. Additionally, these mutants are unable to establish durable infections in mice. The crystal structure of the nicotinamidase from L. infantum will allow us to develop specific inhibitors against this new therapeutic target.MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Bicarbonate Concentration Induces Production of Exopolysaccharides by Arthrospira platensis That Mediate Bioflocculation and Enhance Flotation Harvesting Efficiency

International audienceArthrospira platensis, or spirulina, is a cyanobacterial species mainly exploited for its nutritional qualities and able to produce exopolysaccharides (EPS) under specific conditions. In this work we identify that the bicarbonate concentration is a key parameter that induces EPS production by the cells. Using flocculation/flotation harvesting experiments, in addition to atomic force microscopy to characterize the biophysical properties of EPS, we show that EPS produced by A. platensis cells form a soft, adhesive gel in the medium that forms aggregates in which cells are entrapped and thus bioflocculated. This results in the creation of microalgae flocs in which ascending microbubbles produced in the flotation process get trapped, thus enhancing the efficiency of the microalgae separation from water. This interdisciplinary study provides new insights into culture conditions that positively influence the EPS production, while allowing both a high final biomass concentration and a harvesting efficiency superior to 90% to be reached

Can pests in urban farms be naturally controlled by soil epigeic predators?

In cities of industrialized countries, urban agriculture is expanding in the townscape, both in surface and diversity of forms. In those highly populated areas a major challenge is to control plant pests without using pesticides. In rural context, soil epigeic generalist predators (EGP; mainly arthropods such as ground beetles or spiders) have an important role in biocontrol but little is known about their real impact and the subsequent ecosystem service in urban farms.The aim of this study is to better understand the biocontrol provided by EGP on various forms of urban farms. We surveyed 9 urban farms located in Paris and close suburb among (i) productive at ground-level, (ii) productive on rooftops and (iii) non-productive gardens. We measured the predation activity studying the consumption of sentinel eggs of the Mediterranean flour moth (Ephestia kuehniella) and pupae of the Housefly (Musca domestica) set on the ground. We also analyzed the density of potential EGP using pitfall traps.This study gave new insights about the biocontrol service in urban context and argue for the acknowledgement of EGP as ecosystem providers for urban farm

Can pests in urban farms be naturally controlled by soil epigeic predators?

In cities of industrialized countries, urban agriculture is expanding in the townscape, both in surface and diversity of forms. In those highly populated areas a major challenge is to control plant pests without using pesticides. In rural context, soil epigeic generalist predators (EGP; mainly arthropods such as ground beetles or spiders) have an important role in biocontrol but little is known about their real impact and the subsequent ecosystem service in urban farms.The aim of this study is to better understand the biocontrol provided by EGP on various forms of urban farms. We surveyed 9 urban farms located in Paris and close suburb among (i) productive at ground-level, (ii) productive on rooftops and (iii) non-productive gardens. We measured the predation activity studying the consumption of sentinel eggs of the Mediterranean flour moth (Ephestia kuehniella) and pupae of the Housefly (Musca domestica) set on the ground. We also analyzed the density of potential EGP using pitfall traps.This study gave new insights about the biocontrol service in urban context and argue for the acknowledgement of EGP as ecosystem providers for urban farm