Importing statistical measures into Artemis enhances gene identification in the Leishmania genome project

BACKGROUND: Seattle Biomedical Research Institute (SBRI) as part of the Leishmania Genome Network (LGN) is sequencing chromosomes of the trypanosomatid protozoan species Leishmania major. At SBRI, chromosomal sequence is annotated using a combination of trained and untrained non-consensus gene-prediction algorithms with ARTEMIS, an annotation platform with rich and user-friendly interfaces. RESULTS: Here we describe a methodology used to import results from three different protein-coding gene-prediction algorithms (GLIMMER, TESTCODE and GENESCAN) into the ARTEMIS sequence viewer and annotation tool. Comparison of these methods, along with the CODONUSAGE algorithm built into ARTEMIS, shows the importance of combining methods to more accurately annotate the L. major genomic sequence. CONCLUSION: An improvised and powerful tool for gene prediction has been developed by importing data from widely-used algorithms into an existing annotation platform. This approach is especially fruitful in the Leishmania genome project where there is large proportion of novel genes requiring manual annotation

The genome of Leishmania panamensis: insights into genomics of the L. (Viannia) subgenus.

Kinetoplastid parasites of the Leishmania genus cause several forms of leishmaniasis. Leishmania species pathogenic to human are separated into two subgenera, Leishmania (Leishmania) and L. (Viannia). Species from the Viannia subgenus cause predominantly cutaneous leishmaniasis in Central and South America, occasionally leading to more severe clinical presentations. Although the genomes of several species of Leishmania have been sequenced to date, only one belongs to this rather different subgenus. Here we explore the unique features of the Viannia subgenus by sequencing and analyzing the genome of L. (Viannia) panamensis. Against a background of conservation in gene content and synteny, we found key differences at the genomic level that may explain the occurrence of molecular processes involving nucleic acid manipulation and differential modification of surface glycoconjugates. These differences may in part explain some phenotypic characteristics of the Viannia parasites, including their increased adaptive capacity and enhanced metastatic ability.Kinetoplastid parasites of the Leishmania genus cause several forms of leishmaniasis. Leishmania species pathogenic to human are separated into two subgenera, Leishmania (Leishmania) and L. (Viannia). Species from the Viannia subgenus cause predominantly cutaneous leishmaniasis in Central and South America, occasionally leading to more severe clinical presentations. Although the genomes of several species of Leishmania have been sequenced to date, only one belongs to this rather different subgenus. Here we explore the unique features of the Viannia subgenus by sequencing and analyzing the genome of L. (Viannia) panamensis. Against a background of conservation in gene content and synteny, we found key differences at the genomic level that may explain the occurrence of molecular processes involving nucleic acid manipulation and differential modification of surface glycoconjugates. These differences may in part explain some phenotypic characteristics of the Viannia parasites, including their increased adaptive capacity and enhanced metastatic ability

Translational Research for Zoonotic Parasites: New Findings toward Improved Diagnostics, Therapy and Prevention

In this book is reported novel information on diagnosis, treatment, and control of parasites that are naturally transmitted from animal reservoirs to humans. Subjects: Public Health and Healthcare: Prevention; Medicine and Pharmacology: Therapy

Life in the nucleus, the genomic basis of energy exploitation by intranuclear microsporidia

The Microsporidia are obligate intracellular parasites that have jettisoned oxidation phosphorylative capabilities during their early evolutionary history and so rely on ATP import from their host and glycolysis for their energy needs. Some species form tight associations with the host’s mitochondria and this is thought to facilitate ATP sequestration by the developing intracellular microsporidian. The human parasite, Enterocytozoon bieneusi has however lost glycolytic capabilities and may rely entirely on ATP import from its host for energy. E. bieneusi belongs to the Enterocytozoonidae microsporidian family and recent rDNA-based phylogenetic studies have suggested it has close evolutionary ties with Enterospora canceri, a crab-infecting intranuclear parasite. Such a close evolutionary relationship implied that glycolysis might also be absent in the intranuclear parasite raising questions as to how this parasite obtains energy from its unusual niche that is physically walled off from the host mitochondria, the main source of ATP in the host cell. In this study, draft genomes of four species of the Enterocytozoonidae namely, Ent. canceri, E. hepatopenaei, Hepatospora eriocheir and Hepatospora eriocheir canceri and one non-Enterocytozoonidae species, Thelohania sp. were assembled and annotated (The genome assembly of Hepatospora eriocheir was provided by Dr. Bryony Williams). Phylogenomics performed with this and publicly available genomic data confirmed the close evolutionary ties between Ent. canceri and E. bieneusi. Comparative genomic analyses also revealed that glycolysis is indeed lost in all members of the Enterocytozoonidae family sequenced in this study, hinting to the relaxation of evolutionary pressures to maintain this pathway at the base of this microsporidian family. Despite this absence, the hexokinase gene was retained in all aglycolytic genomes analysed, and that of Ent. canceri was fused to a PTPA gene. Functional assays and yeast complementation assays suggest that this chimera is able to recognise glucose as a substrate but the heterologously expressed homolog of H. eriocheir cannot. Finally, phylogenomics have been used here to demonstrate that despite the morphological differences between three Hepatospora-like organisms parasitizing different crab hosts, they are the same species. This finding adds more weight to current evidence suggesting that morphology is not an ideal marker for taxonomical classification in the Microsporidia.University of Exeter Centre for Environment Fisheries and Aquaculture Scienc

Examining epigenetic variation in the brain in mental illness

Mental health represents one of the most significant and increasing burdens to global public health. Depression and schizophrenia, among other mental illnesses, constitute strong risk factors for suicidality which results in over 800,000 deaths every year. The majority of suicides worldwide are indeed related to psychiatric diseases. A growing body of genetic, epigenetic and epidemiological evidence suggests that psychiatric disorders are highly complex phenotypes originating from the multilevel interplay between the strong genetic component and a range of environmental and psychosocial factors. Deeper understanding about the biology of the genome has led to increased interest for the role of non-sequence-based variation in the etiology of neuropsychiatric phenotypes, including suicidality. Epigenetic alterations and gene expression dysregulation have been repetitively reported in post-mortem brain of individuals who died by suicide. To date, however, studies characterizing disease-associated methylomic and transcriptomic variation in the brain have been limited by screening performed in bulk tissue and by the assessment of a single marker at a time. The main aim of this thesis was to investigate DNA methylation and miRNA expression differences in post-mortem brain associated with suicidality and unravel the complexity of epigenetic signals in a heterogeneous tissue like the human brain by developing a method to profile genomic variation at the resolution of individual neural cell types. The results here reported, provide further support for a suicide-specific epigenetic signature, independent from comorbidity with other psychiatric phenotypes, as well as confirming the strong bias perpetrated by bulk tissue studies hence the need to examine genomic variations in purified cell types. In summary, this thesis has identified a) a suicide-specific signal in two different epigenetic markers (DNA methylation and miRNA expression) and b) a protocol to simultaneously profile DNA methylation levels across three purified cell types in the healthy brain highlighting the utility of cell sorting for identifying cell type-driven epigenetic differences associated with etiological variation in complex psychiatric phenotypes.1) ARUK-PPG2018A-010 – “Developing approaches to address neural cell heterogeneity in genomic studies of Alzheimer's disease”. 2) SBF001\1011 - “Using functional epigenomics to dissect the molecular architecture of schizophrenia

Études chimiques et immunologiques des capsules polysaccharidiques de Streptococcus suis

Streptococcus suis est l’un des plus importants pathogènes bactériens du porc, causant des pertes économiques substantielles à l’industrie porcine. De plus, c’est un agent zoonotique représentant de sérieux risques pour la santé humaine. Cette bactérie cause diverses pathologies, dont la méningite, la mort subite et le choc septique sont les plus fréquentes. De plus, il demeure qu’à ce jour, aucun vaccin efficace n’est disponible pour prévenir les infections à S. suis. Étant encapsulé, S. suis est classifié en 35 sérotypes définis par l’antigénicité de sa capsule polysaccharidique (CPS). Malgré cela, seule la structure de la CPS du sérotype 2 est connue à présent. Immunologiquement parlant, les CPS purifiées sont généralement des antigènes T-indépendant de par leur nature, faisant d’elles de très pauvres immunogènes. Ceci a déjà été démontré pour la CPS du sérotype 2. Paradoxalement, les anticorps anti-CPS sont fortement opsonisants et protecteurs, ce qui les rend désirables. Ainsi, les objectifs de cette thèse sont, d’abord, de poursuivre la caractérisation chimique des structures des CPS de différents sérotypes de S. suis, puis d’étudier les propriétés immunostimulatrices et immunogènes de ces nouvelles CPS. De plus, les travaux de cette thèse ont tenté d’améliorer l’immunogénicité de la CPS du sérotype 2 pour pouvoir l’utiliser comme antigène vaccinal. Dans un premier temps, un recensement de la littérature a permis de dresser un portrait à jour et mondial des infections chez le porc et chez l’homme, et d’identifier les sérotypes les plus importants. À l’aide de ces données épidémiologiques, les CPS des sérotypes choisis ont été purifiées, ce qui a permis de déterminer la structure pour huit CPS additionnelles (1, 1/2, 3, 7, 8, 9, 14 et 18). D’autre part, ces nouvelles connaissances sur la structure nous ont permis d’évaluer l’antigénicité et l’immunogénicité de ces nouvelles CPS. Ainsi, nous avons pu observer pour la première fois que la CPS du sérotype 3 de S. suis est hautement immunogène en produisant une réponse opsonisante d’IgG et d’IgM par un mécanisme T-indépendant. De plus, nous avons développé pour la première fois un vaccin glycoconjugué constitué de CPS du sérotype 2 couplé au toxoïde tétanique afin d’obtenir une réponse anticorps T-dépendante protectrice. Une preuve de concept chez le porc a permis de démontrer la protection conférée par notre vaccin glycoconjugué contre S. suis, et plus largement le potentiel des vaccins glycoconjugués pour combattre les infections bactériennes invasives en médecine vétérinaire. Enfin, l’étude de l’immunogénicité du vaccin glycoconjugué chez la souris a permis d’évaluer le rôle du choix du modèle animal et des adjuvants sur la réponse anti-CPS, en plus de produire trois nouveaux anticorps monoclonaux qui ont permis notamment d’étudier les mécanismes humoraux impliqués dans l’élimination de S. suis par opsonophagocytose. L’ensemble de ces études va permettre de souligner l’importance des anticorps anti-CPS dans la protection face aux infections bactériennes invasives.Streptococcus suis is one of the most important porcine bacterial pathogens and is responsible for substantial economic losses to the swine industry. Moreover, it is also a zoonotic agent representing serious risks to human health. This bacterium causes a variety of clinical signs, of which meningitis, sudden death, and septic shock are the most frequent. Furthermore, no efficient vaccine is available to protect against infections caused by S. suis. Being encapsulated, S. suis is classified into 35 serotypes based on the antigenicity of their capsular polysaccharide (CPS). However, only the structure of the serotype 2 CPS is known to date. Immunologically, purified CPSss generally behave as T-independent antigens, making them very poor immunogens. Indeed, this has been previously demonstrated to be the case for the serotype 2 CPS. Paradoxically, anti-CPS antibodies are strongly opsonizing and protective, making them attractive targets for vaccine development. Therefore, the objectives of this thesis are, firstly, to further characterize the chemical composition and structure of the S. suis CPSs from different serotypes, and secondly, to study the immunostimulatory and immunogenic properties of these additional CPSs. Moreover, this thesis will attempt to increase the serotype 2 CPS immunogenicity for potential use it as a vaccine antigen. In a first step, a review of the literature provided an updated and global view of S. suis infections in swine and humans and identified the most important serotypes. With these epidemiological data in hand, we then purified the CPSs of selected serotypes, which allowed us to determine the structures for eight additional CPSs (1, 1/2, 3, 7, 8, 9, 14, and 18). The knowledge obtained regarding the structure of theses CPSs allowed us to then evaluate their antigenicity and immunogenicity. We demonstrated for the first time that serotype 3 is highly immunogenic S. suis CPS, as it induces an opsonizing response composed of IgG and IgM by a T-independent mechanism. In addition, we have developped for the first time a glycoconjugate vaccine composed of serotype 2 CPS conjugated to tetanus toxoid in order to obtain a protective T-dependent antibody response. A proof of concept performed with pigs demonstrated protection conferred by our glycoconjugate vaccine against S. suis, and more importantly the potential of glycoconjugate vaccines in the fight against invasive bacterial infections in veterinary medicine. Finally, immunogenicity studies performed in mice with this glycoconjugate vaccine allowed us to evaluate the effect of the animal model and adjuvant choice on the anti-CPS response. It also allowed us to produce three new monoclonal antibodies that enabled us to study, among other aspects, the humoral mechanisms involved in S. suis clearance by opsonophagocytosis. Consequently, the work conducted during this thesis will serve to highlight the importance of anti-CPS antibodies in the protection against invasive bacterial diseases