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

A novel Leishmania infantum reference strain for gene editing and the study of visceral leishmaniasis

Parasites of the Leishmania donovani complex are responsible for visceral leishmaniasis, a vector-borne disease transmitted through the bite of female phlebotomine sand flies. As well as the human hosts, these parasites infect many mammals which can serve as reservoirs. Dogs are particularly important reservoirs. Transmission is widespread across Asia, Africa, the Americas, and the Mediterranean basin, including South of France. Visceral leishmaniasis poses a fatal threat if left untreated. Research into the pathophysiology of this neglected disease is of prime importance, as is the development of new drugs. In this study, we evaluated the growth, differentiation, and macrophage infectivity of four L. donovani complex strains and identified L. infantum S9F1 (MHOM/MA/67/ITMAP263, clone S9F1) as a well-adapted strain for genetic engineering studies. We present here the genome sequence and annotation of L infantum S9F1 T7 Cas9, providing the scientific community with easy access to its genomic information. The data has been integrated into the LeishGEdit online resource to support primer design for CRISPR-Cas9 experiments. We now aim to make this strain widely available to foster studies of visceral leishmaniasis

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

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.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

Involvement of the Adenovirus protease in the partial disassembly of the capsid and the release of the membrane lytic protein 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

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

Biopolitics

The cofactors nicotinamide adenine dinucleotide (NAD+) and its phosphate form, NADP+, are crucial molecules present in all living cells. The delicate balance between the oxidized and reduced forms of these molecules is tightly regulated by intracellular metabolism assuring the maintenance of homeostatic conditions, which are essential for cell survival and proliferation. A recent cluster of data has highlighted the importance of the intracellular NAD+/NADH and NADP+/NADPH ratios during host–pathogen interactions, as fluctuations in the levels of these cofactors and in precursors’ bioavailability may condition host response and, therefore, pathogen persistence or elimination. Furthermore, an increasing interest has been given towards how pathogens are capable of hijacking host cell proteins in their own advantage and, consequently, alter cellular redox states and immune function. Here, we review the basic principles behind biosynthesis and subcellular compartmentalization of NAD+ and NADP+, as well as the importance of these cofactors during infection, with a special emphasis on pathogen-driven modulation of host NAD+/NADP+ levels and contribution to the associated immune response

Alterations on cellular redox states upon infection and implications for host cell homeostasis

The cofactors nicotinamide adenine dinucleotide (NAD+) and its phosphateform, NADP+, are crucial molecules present in all living cells. The delicate balancebetween the oxidized and reduced forms of these molecules is tightly regulated byintracellular metabolism assuring the maintenance of homeostatic conditions, whichare essential for cell survival and proliferation. A recent cluster of data has highlightedthe importance of the intracellular NAD+/NADH and NADP+/NADPH ratios duringhost–pathogen interactions, as fluctuations in the levels of these cofactors and inprecursors’ bioavailability may condition host response and, therefore, pathogenpersistence or elimination. Furthermore, an increasing interest has been given towardshow pathogens are capable of hijacking host cell proteins in their own advantage and,consequently, alter cellular redox states and immune function. Here, we review thebasic principles behind biosynthesis and subcellular compartmentalization of NAD+and NADP+, as well as the importance of these cofactors during infection, with aspecial emphasis on pathogen-driven modulation of host NAD+/NADP+ levels andcontribution to the associated immune response

Protozoan Parasite Auxotrophies and Metabolic Dependencies

International audienc

Functional divergence of SIR2 orthologs between trypanosomatid parasites

SIR2 proteins are NAD+-dependent deacetylases involved in epigenetic control of gene expression and metabolic regulation through post-translational modification of diverse target proteins. In pathogens, these enzymes are considered as attractive drug targets involved in key aspects of the infectious cycle. Leishmania infantum LiSIR2rp1 was among the first non-nuclear and essential SIR2 deacetylases described in eukaryotes. Here, we show that the two other LiSIR2rp2 and LiSIRrp3 paralogs are both located in mitochondria. Gene deletion experiments show that LiSIR2rp3 is not required for parasite survival. Surprisingly, multiple extrachromosomal amplicons bearing the LiSIR2rp2 gene are constitutively produced in wild type strains. Consequently, a knockout of this gene could not be obtained, even after episomal rescue experiments. We further provide genetic and biochemical evidence showing that SIR2rp2 protein directly affects parasite proliferation in relation to NAD+ bioavailability. Together, these results highlight unexpected genus-specific divergence of the SIR2 machinery among trypanosomatid parasites