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L’évolution des pangénomes de procaryotes sur des échelles de temps humainesN'Guessan, Arnaud 12 1900 (has links)
Le pangénome est l’ensemble des gènes uniques retrouvé chez une espèce. Dans le cas des espèces procaryotes, notamment celles qui sont présentes dans le microbiote intestinal humain, la variation du contenu en gène est caractérisée par des événements de gain de gènes principalement par transfert horizontal de gènes (THG) et de perte de gène. Cette variation du contenu en gène peut être plus rapide que le taux de mutation et permettre aux microbes de s’adapter rapidement à des pressions sélectives. Cela justifie donc l’étude de l’évolution pangénomique des procaryotes sur des échelles de temps humaines qui sont considérées comme étant courtes du point de vue évolutif, par exemple de l’ordre de quelques années. La plupart des études sur ce sujet impliquent des espèces relativement distantes qui ont divergé depuis des millions d’années. De plus, l'équilibre des forces évolutives majeures impliquées, telles que le THG, la sélection, la dérive génétique et les mutations, n’est pas clairement défini et est au cœur d’un débat dans la littérature. Ce projet de maîtrise permet donc d’élargir le portrait évolutif des pangénomes de procaryotes en s’intéressant à l’évolution des gènes transférés horizontalement, aussi appelés gènes mobiles, sur de courtes échelles de temps. Pour ce faire, nous allons d’abord passer en revue la littérature pertinente en lien avec ce sujet, notamment les méthodes employées pour détecter les gènes mobiles et les modèles d’évolution pangénomique. Nous allons ensuite analyser l’évolution d’une collection de 37 853 gènes mobiles impliqués dans des THG récents détectés dans le microbiote intestinal d’individus provenant d’Amérique du Nord ou des îles Fidji. Pour détecter des signatures évolutives des forces en action, nous estimerons divers paramètres de génétique des populations à partir de l’alignement entre les lectures de séquençage métagénomique de 176 microbiotes fidjiens et cette collection de gènes mobiles. Nous expliquerons aussi l’outil de simulations évolutives que nous avons développé afin de valider et expliquer certaines de nos observations. Sans exclure la présence de pressions de sélection pour des gènes mobiles ayant des fonctions spécifiques, les données réelles et les simulations nous amènent à conclure que l’évolution des gènes mobiles sur de courtes échelles de temps peut être expliquée par un modèle d’évolution où les gènes mobiles ne sont pas largement adaptifs à leurs hôtes humains ou microbiens, contrairement à ce qui est parfois observé sur de longues échelles de temps évolutif. / The pangenome is the collection of unique genes found in a species. For prokaryotes, especially those present in the human gut microbiota, variation in gene content is characterized by gene gain through horizontal gene transfer (HGT) and gene loss. In human gut, gene content variations can occur at faster rates than mutation, which allow microbes to adapt rapidly to environmental changes. This justifies the study of the prokaryotes pangenome evolution on human time scales which are considered evolutionarily short, e.g. in the order of few years. Most studies about the evolution of prokaryotic pangenomes involve relatively distant species that have diverged since millions of years. In addition, the balance of major evolutionary forces involved, such as horizontal transfer, selection, genetic drift, and mutations, is not clearly defined and is debated in literature. This master's project therefore aims to broaden the evolutionary portrait of prokaryotic pangenome evolution by focusing on near-term evolution. To do this, we will first review the relevant literature related to this topic, including the methods used to detect mobile genes and the pangenome evolution models. We will then analyze the evolution of a pre-existing collection of 37 853 mobile genes involved in recent HGT events detected in the gut microbiota of individuals from North America and Fiji Islands. To detect evolutionary signatures of the forces in action, we will estimate various population genetics parameters from the alignment between metagenomic sequencing reads of 176 Fijian microbiomes and this collection of mobile genes. We will also explain the evolutionary simulation tool that we have developed in order to validate and explain some of our observations. While we don’t exclude the importance of selection for specific cellular functions for pangenome evolution, we found that the near-term evolution of mobile genes can be explained by a model in which mobile genes can spread selfishly without being largely adaptive to their human or microbial hosts, contrarily to what is often observed over longer evolutionary time scales.
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Développement de méthodes de fouille de données basées sur les modèles de Markov cachés du second ordre pour l'identification d'hétérogénéités dans les génomes bactériens / Data Mining methods based on second-order Hidden Markov Models to identify heterogeneities into bacteria genomesEng, Catherine 15 June 2010 (has links)
Les modèles de Markov d’ordre 2 (HMM2) sont des modèles stochastiques qui ont démontré leur efficacité dans l’exploration de séquences génomiques. Cette thèse explore l’intérêt de modèles de différents types (M1M2, M2M2, M2M0) ainsi que leur couplage à des méthodes combinatoires pour segmenter les génomes bactériens sans connaissances a priori du contenu génétique. Ces approches ont été appliquées à deux modèles bactériens afin d’en valider la robustesse : Streptomyces coelicolor et Streptococcus thermophilus. Ces espèces bactériennes présentent des caractéristiques génomiques très distinctes (composition, taille du génome) en lien avec leur écosystème spécifique : le sol pour les S. coelicolor et le milieu lait pour S. thermophilus / Second-order Hidden Markov Models (HMM2) are stochastic processes with a high efficiency in exploring bacterial genome sequences. Different types of HMM2 (M1M2, M2M2, M2M0) combined to combinatorial methods were developed in a new approach to discriminate genomic regions without a priori knowledge on their genetic content. This approach was applied on two bacterial models in order to validate its achievements: Streptomyces coelicolor and Streptococcus thermophilus. These bacterial species exhibit distinct genomic traits (base composition, global genome size) in relation with their ecological niche: soil for S. coelicolor and dairy products for S. thermophilus. In S. coelicolor, a first HMM2 architecture allowed the detection of short discrete DNA heterogeneities (5-16 nucleotides in size), mostly localized in intergenic regions. The application of the method on a biologically known gene set, the SigR regulon (involved in oxidative stress response), proved the efficiency in identifying bacterial promoters. S. coelicolor shows a complex regulatory network (up to 12% of the genes may be involved in gene regulation) with more than 60 sigma factors, involved in initiation of transcription. A classification method coupled to a searching algorithm (i.e. R’MES) was developed to automatically extract the box1-spacer-box2 composite DNA motifs, structure corresponding to the typical bacterial promoter -35/-10 boxes. Among the 814 DNA motifs described for the whole S. coelicolor genome, those of sigma factors (B, WhiG) could be retrieved from the crude data. We could show that this method could be generalized by applying it successfully in a preliminary attempt to the genome of Bacillus subtilis
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The evolution of RNA interference system, blue light sensing mechanism and circadian clock in Rhizophagus irregularis give insight on Arbuscular mycorrhizal symbiosisLee, Soon-Jae 08 1900 (has links)
No description available.
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A Biased Urn Model for Taxonomic Identification / Ein gewichtetes Urnenmodell zur taxonomischen IdentifikationSurovcik, Katharina 26 June 2008 (has links)
No description available.
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Genomic and transcriptomic characterization of novel iron oxidizing bacteria of the genus “Ferrovum“ / Charakterisierung von neuartigen eisenoxidierenden Bakterien der Gattung „Ferrovum” auf Genom- und TranskriptomebeneUllrich, Sophie 30 June 2016 (has links) (PDF)
Acidophilic iron oxidizing bacteria of the betaproteobacterial genus “Ferrovum” are ubiquitously distributed in acid mine drainage (AMD) habitats worldwide. Since their isolation and maintenance in the laboratory has proved to be extremely difficult, members of this genus are not accessible to a “classical” microbiological characterization with exception of the designated type strain “Ferrovum myxofaciens” P3G.
The present study reports the characterization of “Ferrovum” strains at genome and transcriptome level. “Ferrovum” sp. JA12, “Ferrovum” sp. PN-J185 and “F. myxofaciens” Z-31 represent the iron oxidizers of the mixed cultures JA12, PN-J185 and Z-31. The mixed cultures were derived from the mine water treatment plant Tzschelln close to the lignite mining site in Nochten (Lusatia, Germany). The mixed cultures also contain a heterotrophic strain of the genus Acidiphilium. The genome analysis of Acidiphilium sp. JA12-A1, the heterotrophic contamination of the mixed culture JA12, indicates an interspecies carbon and phosphate transfer between Acidiphilium and “Ferrovum” in the mixed culture, and possibly also in their natural habitat. The comparison of the inferred metabolic potentials of four “Ferrovum” strains and the analysis of their phylogenetic relationships suggest the existence of two subgroups within the genus “Ferrovum” (i.e. the operational taxonomic units OTU-1 and OUT-2) harboring characteristic metabolic profiles. OTU-1 includes the “F. myxofaciens” strains P3G and Z-31, which are predicted to be motile and diazotrophic, and to have a higher acid tolerance than OTU-2. The latter includes two closely related proposed species represented by the strains JA12 and PN-J185, which appear to lack the abilities of motility, chemotaxis and molecular nitrogen fixation. Instead, both OTU-2 strains harbor the potential to use urea as alternative nitrogen source to ammonium, and even nitrate in case of the JA12-like species. The analysis of the genome architectures of the four “Ferrovum” strains suggests that horizontal gene transfer and loss of metabolic genes, accompanied by genome reduction, have contributed to the evolution of the OTUs.
A trial transcriptome study of “Ferrovum” sp. JA12 supports the ferrous iron oxidation model inferred from its genome sequence, and reveals the potential relevance of several hypothetical proteins in ferrous iron oxidation. Although the inferred models in “Ferrovum” spp. share common features with the acidophilic iron oxidizers of the Acidithiobacillia, it appears to be more similar to the neutrophilic iron oxidizers Mariprofundus ferrooxydans (“Zetaproteobacteria”) and Sideroxydans lithotrophicus (Betaproteobacteria). These findings suggest a common origin of ferrous iron oxidation in the Beta- and “Zetaproteobacteria”, while the acidophilic lifestyle of “Ferrovum” spp. may have been acquired later, allowing them to also colonize acid mine drainage habitats.
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Genomic and transcriptomic characterization of novel iron oxidizing bacteria of the genus “Ferrovum“Ullrich, Sophie 30 May 2016 (has links)
Acidophilic iron oxidizing bacteria of the betaproteobacterial genus “Ferrovum” are ubiquitously distributed in acid mine drainage (AMD) habitats worldwide. Since their isolation and maintenance in the laboratory has proved to be extremely difficult, members of this genus are not accessible to a “classical” microbiological characterization with exception of the designated type strain “Ferrovum myxofaciens” P3G.
The present study reports the characterization of “Ferrovum” strains at genome and transcriptome level. “Ferrovum” sp. JA12, “Ferrovum” sp. PN-J185 and “F. myxofaciens” Z-31 represent the iron oxidizers of the mixed cultures JA12, PN-J185 and Z-31. The mixed cultures were derived from the mine water treatment plant Tzschelln close to the lignite mining site in Nochten (Lusatia, Germany). The mixed cultures also contain a heterotrophic strain of the genus Acidiphilium. The genome analysis of Acidiphilium sp. JA12-A1, the heterotrophic contamination of the mixed culture JA12, indicates an interspecies carbon and phosphate transfer between Acidiphilium and “Ferrovum” in the mixed culture, and possibly also in their natural habitat. The comparison of the inferred metabolic potentials of four “Ferrovum” strains and the analysis of their phylogenetic relationships suggest the existence of two subgroups within the genus “Ferrovum” (i.e. the operational taxonomic units OTU-1 and OUT-2) harboring characteristic metabolic profiles. OTU-1 includes the “F. myxofaciens” strains P3G and Z-31, which are predicted to be motile and diazotrophic, and to have a higher acid tolerance than OTU-2. The latter includes two closely related proposed species represented by the strains JA12 and PN-J185, which appear to lack the abilities of motility, chemotaxis and molecular nitrogen fixation. Instead, both OTU-2 strains harbor the potential to use urea as alternative nitrogen source to ammonium, and even nitrate in case of the JA12-like species. The analysis of the genome architectures of the four “Ferrovum” strains suggests that horizontal gene transfer and loss of metabolic genes, accompanied by genome reduction, have contributed to the evolution of the OTUs.
A trial transcriptome study of “Ferrovum” sp. JA12 supports the ferrous iron oxidation model inferred from its genome sequence, and reveals the potential relevance of several hypothetical proteins in ferrous iron oxidation. Although the inferred models in “Ferrovum” spp. share common features with the acidophilic iron oxidizers of the Acidithiobacillia, it appears to be more similar to the neutrophilic iron oxidizers Mariprofundus ferrooxydans (“Zetaproteobacteria”) and Sideroxydans lithotrophicus (Betaproteobacteria). These findings suggest a common origin of ferrous iron oxidation in the Beta- and “Zetaproteobacteria”, while the acidophilic lifestyle of “Ferrovum” spp. may have been acquired later, allowing them to also colonize acid mine drainage habitats.:EIDESSTATTLICHE ERKLÄRUNG ... 2
CONTENT ... 4
SUMMARY ... 9
CHAPTER I ... 11
ORIGIN AND MICROBIOLOGY OF ACID MINE DRAINAGE ... 11
ACIDOPHILIC IRON OXIDIZING BACTERIA OF THE GENUS “FERROVUM” ... 12
APPLICATION OF OMICS-BASED APPROACHES TO CHARACTERIZE ACIDOPHILES ... 14
AIMS OF THE PRESENT WORK ... 15
CHAPTER II ... 17
ABSTRACT ... 18
INTRODUCTION ... 18
METHODS ... 19
GENOME PROJECT HISTORY ... 19
GROWTH CONDITIONS AND GENOMIC DNA PREPARATION ... 20
GENOME SEQUENCING AND ASSEMBLY ... 20
GENOME ANNOTATION ... 21
RESULTS ... 21
CLASSIFICATION AND FEATURES ... 21
GENOME PROPERTIES ... 24
INSIGHTS FROM THE GENOME SEQUENCE ... 24
COMPARATIVE GENOMICS ... 28
CONCLUSIONS ... 30
ACKNOWLEDGMENTS ... 32
AUTHOR CONTRIBUTIONS ... 32
CHAPTER III ... 33
ABSTRACT ... 34
INTRODUCTION ... 34
METHODS ... 36
ORIGIN AND CULTIVATION OF “FERROVUM” STRAIN JA12 ... 36
GENOME SEQUENCING, ASSEMBLY AND ANNOTATION ... 37
VISUALIZATION OF THE NEARLY COMPLETE GENOME ... 38
PHYLOGENETIC ANALYSIS ... 39
PREDICTION OF MOBILE GENETIC ELEMENTS ... 39
NUCLEOTIDE SEQUENCE ACCESSION NUMBER ... 39
RESULTS AND DISCUSSION ... 39
PHYLOGENETIC CLASSIFICATION OF “FERROVUM” STRAIN JA12 ... 39
GENOME PROPERTIES ... 40
NUTRIENT ASSIMILATION AND BIOMASS PRODUCTION ... 44
Carbon dioxide fixation ... 44
Central carbon metabolism ... 45
Nitrogen ... 47
Phosphate ... 49
Sulfate ... 50
ENERGY METABOLISM ... 50
Ferrous iron oxidation ... 50
Other redox reactions connected to the quinol pool ... 54
Predicted formate dehydrogenase ... 55
STRATEGIES TO ADAPT TO ACIDIC ENVIRONMENTS, HIGH METAL LOADS AND OXIDATIVE STRESS ... 55
Acidic environment ... 55
Strategies to cope with high metal and metalloid loads ... 58
Oxidative stress ... 59
HORIZONTAL GENE TRANSFER ... 60
CONCLUSIONS ... 61
ACKNOWLEDGMENTS ... 62
AUTHORS\' CONTRIBUTIONS ... 62
CHAPTER IV ... 63
ABSTRACT ... 64
INTRODUCTION ... 64
METHODS ... 66
ORIGIN AND CULTIVATION OF “FERROVUM” STRAINS PN-J185 AND Z-31 ... 66
GENOME SEQUENCING, ASSEMBLY AND ANNOTATION ... 66
PREDICTION OF MOBILE GENETIC ELEMENTS ... 67
COMPARATIVE GENOMICS ... 68
Phylogenomic analysis ... 68
Assignment of protein-coding genes to the COG classification ... 68
Identification of orthologous proteins ... 68
Comparison and analysis of genome architectures ... 69
RESULTS ... 69
GENERAL GENOME FEATURES AND PHYLOGENETIC RELATIONSHIP OF THE FOUR “FERROVUM” STRAINS ... 69
COMPARISON OF INFERRED METABOLIC TRAITS ... 71
Identification of core genes and flexible genes ... 71
Comparison of the central metabolism ... 74
Central carbon metabolism ... 74
Nitrogen metabolism ... 77
Energy metabolism ... 78
Cell mobility and chemotaxis ... 78
Diversity of predicted stress tolerance mechanisms ... 78
Maintaining the intracellular pH homeostasis ... 78
Coping with high metal loads ... 79
Oxidative stress management ... 79
IDENTIFICATION OF POTENTIAL DRIVING FORCES OF GENOME EVOLUTION ... 80
Prediction of mobile genetic elements ... 81
Linking the differences in the predicted metabolic profiles to the genome architectures ... 82
Gene cluster associated with flagella formation and chemotaxis in “F. myxofaciens” ... 84
Gene clusters associated with the utilization of alternative nitrogen sources ... 86
Gene cluster associated with carboxysome formation in “F. myxofaciens” and OTU-2 strain JA12 ... 87
Putative genomic islands in the OTU-strain JA12 ... 89
CRISPR/Cas in “F. myxofaciens” Z-31: a defense mechanism against foreign DNA ... 91
DISCUSSION ... 92
THE COMPARISON OF THEIR METABOLIC PROFILES INDICATES THE EXISTENCE OF OTU- AND STRAIN-SPECIFIC FEATURES ... 92
GENOME EVOLUTION OF THE “FERROVUM” STRAINS APPEARS TO BE DRIVEN BY HORIZONTAL GENE TRANSFER AND GENOME REDUCTION ... 94
Horizontal gene transfer ... 94
Mechanisms of genome reduction ... 95
CONCLUDING REMARKS ... 98
ACKNOWLEDGMENTS ... 98
AUTHOR CONTRIBUTIONS ... 98
CHAPTER V ... 99
ABSTRACT ... 100
INTRODUCTION ... 100
METHODS ... 102
CULTIVATION OF THE “FERROVUM”-CONTAINING MIXED CULTURE JA12 ... 102
Up-scaling of pre-cultures for the transcriptome study ... 103
Experimental setup of the transcriptome study ... 103
Cell harvest from large culture volumes ... 106
EXTRACTION OF TOTAL RNA ... 106
LIBRARY CONSTRUCTION AND SEQUENCING ... 107
DATA ANALYSIS ... 107
Processing of raw data ... 107
Quantification of gene expression levels ... 108
Functional analysis ... 108
RESULTS ... 108
CULTIVATION OF THE MIXED CULTURE JA12 IN THE MULTIPLE BIOREACTOR SYSTEM ... 108
Growth monitoring ... 108
Microbial composition ... 111
RNA SEQUENCING (RNA-SEQ) ... 112
FUNCTIONAL CATEGORIZATION OF EXPRESSED GENES ... 113
Functional assignment of highly expressed genes ... 117
Functional assignment of poorly expressed genes ... 121
COMPARISON OF EXPRESSION LEVELS OF GENES PREDICTED TO BE INVOLVED IN OXIDATIVE STRESS MANAGEMENT ... 122
DISCUSSION ... 124
METABOLIC PATHWAYS RELEVANT UNDER CULTURE CONDITIONS MIMICKING THE NATURAL CONDITIONS IN THE MINE WATER TREATMENT PLANT ... 125
Novel insights into the energy metabolism of “Ferrovum” sp. JA12 ... 125
Insights from poorly expressed genes ... 126
VARIATION OF GENE EXPRESSION PATTERNS UNDER THE DIFFERENT CONDITIONS ... 128
EVALUATION OF THE EXPERIMENTAL SET-UP INVOLVING THE MULTIPLE BIOREACTOR SYSTEM ... 129
CONCLUDING REMARKS: SIGNIFICANCE OF THE PRESENT TRANSCRIPTOME STUDY ... 130
ACKNOWLEDGMENTS ... 131
AUTHOR CONTRIBUTIONS ... 131
CHAPTER VI ... 133
ABSTRACT ... 133
EXTENDED INSIGHTS INTO THE FERROUS IRON OXIDATION IN BETAPROTEOBACTERIA ... 133
MECHANISMS OF PHYLOGENETIC AND METABOLIC DIVERSIFICATION WITHIN THE GENUS “FERROVUM” ... 136
INFERRED ROLES OF “FERROVUM” SPP. IN THE MICROBIAL NETWORK OF THE MINE WATER TREATMENT PLANT ... 138
PERSPECTIVES ... 143
REFERENCES ... 145
SUPPLEMENTARY MATERIAL ... 170
DATA DVD ... 170
SUPPLEMENTARY MATERIAL FOR CHAPTER III ... 171
NUCLEOTIDE ACCESSION NUMBERS ... 171
PHYLOGENETIC ANALYSIS ... 171
GENOME PROPERTIES ... 173
NUTRIENT ASSIMILATION ... 174
Carbon metabolism ... 174
FERROUS IRON OXIDATION ... 176
HORIZONTAL GENE TRANSFER ... 179
SUPPLEMENTARY MATERIAL FOR CHAPTER IV ... 180
PHYLOGENETIC ANALYSIS ... 180
ASSIGNMENT OF PROTEIN-CODING GENES TO THE COG CLASSIFICATION ... 180
COMPARISON OF THE CENTRAL METABOLISM ... 181
Predicted metabolic potential of the four “Ferrovum” strains ... 181
Genes predicted to be involved in the central metabolism, energy metabolism, cell motility and stress management in the four “Ferrovum” strains ... 183
PREDICTED MOBILE GENETIC ELEMENTS IN THE GENOMES OF THE FOUR “FERROVUM” STRAINS ... 184
THE FLAGELLA AND CHEMOTAXIS GENE CLUSTER ... 184
THE UREASE GENE CLUSTER ... 185
THE CARBOXYSOME GENE CLUSTER ... 186
PUTATIVE GENOMIC ISLANDS IN “FERROVUM” SP. JA12 ... 187
Gene content of the genomic islands ... 187
Flanking sites of the putative genomic islands 1 and 2 ... 188
SUPPLEMENTARY MATERIAL FOR CHAPTER V ... 189
ORGANIZATION AND OPERATION OF THE LABFORS 5 MULTIPLE BIOREACTOR SYSTEM ... 189
INVESTIGATION OF THE MICROBIAL COMPOSITION IN THE IRON OXIDIZING MIXED CULTURE JA12 ... 192
SUPPLEMENTARY DATA OF THE TRANSCRIPTOME DATA ANALYSIS ... 193
RNA-Seq statistics ... 193
Expression strength of protein-coding genes ... 194
Expression of genes involved in carboxysome formation ... 197
Expression of a ribosomal proteins-encoding gene cluster ... 199
Expression of a gene cluster presumably involved in ferrous iron oxidation ... 202
Lowest expressed genes ... 205
Expression of genes predicted to be involved in oxidative stress response ... 206
ACKNOWLEDGMENTS ... 208
COLLEAGUES ... 208
ERFOLGSTEAM “JUNGE FRAUEN AN DIE SPITZE” (“YOUNG WOMEN TO THE TOP“) ... 208
FAMILY AND FRIENDS ... 209
FUNDING ... 209
CURRICULUM VITAE ... 210
LIST OF PUBLICATIONS ... 212
RESEARCH ARTICLES ... 212
CONFERENCE PROCEEDINGS ... 212
ORAL PRESENTATIONS AND POSTERS ... 213
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Étude des vésicules extracellulaires du parasite Leishmania et de leur rôle dans le phénomène de la résistance aux antimicrobiensDouanne, Noélie 02 1900 (has links)
Bien que l’évolution de la résistance aux médicaments soit l'un des plus grands défis dans la lutte contre les maladies infectieuses, beaucoup de phénomènes restent inexpliqués. Dans le cas de la leishmaniose, la résistance est un problème majeur depuis plusieurs années. Cette maladie zoonotique négligée est causée par le parasite protozoaire Leishmania, transmis par des phlébotomes lors du repas sanguin. Chez l’humain, elle se décline en trois formes principales : viscérale, cutanée et muco-cutanée. La leishmaniose peut aussi se développer chez les chiens infectés, qui constituent un réservoir majeur de transmission. Sans vaccin efficace, le contrôle de la maladie repose principalement sur la chimiothérapie, mais peu de molécules homologuées sont disponibles. En outre, les mêmes produits sont utilisés chez les chiens et les humains, ce qui favorise l'émergence et la propagation de souches résistantes aux médicaments. Leishmania est en effet connu pour détenir d’incroyables particularités génomiques (telles que la formation d’amplicons circulaires contenant des gènes de résistance) qui lui permettent de survivre dans des conditions de stress, telles que la pression médicamenteuse. Par ailleurs, Leishmania est un eucaryote qui a conservé la capacité de produire des vésicules extracellulaires (EVs) au cours de l’évolution. Ces particules de taille nanométrique sont produites naturellement par la majorité des cellules biologiques et ont un contenu riche en protéines, lipides et acides nucléiques. Bien que des caractéristiques clés des EVs du protozoaire aient été découvertes, aucune étude n’a encore été réalisée sur les EVs de souches résistantes. Ainsi, cet aspect constitue le cœur de mon projet de recherche de doctorat : l’étude des EVs de Leishmania et leur rôle dans le phénomène de la résistance aux médicaments. Nos travaux sont les premiers à démontrer que les mécanismes de résistance aux médicaments peuvent induire des changements dans la morphologie, la taille et la distribution des EVs de Leishmania. Nous avons identifié le protéome de base des EVs du parasite et nous avons mis en évidence des protéines enrichies dans les EVs libérées par des parasites résistants à l'antimoine, à la miltéfosine et à l'amphotéricine B. Nous avons également étudié le contenu en ADN des EVs de parasites résistants et confirmé l'enrichissement des amplicons porteurs de gènes de résistance aux médicaments, associés aux EVs. En complément, nos tests de transferts d’EVs ont prouvé que ces vésicules permettent le transfert horizontal de gènes de résistance : un mécanisme alternatif de résistance aux médicaments. Finalement, nous avons montré que les EVs des parasites résistants améliorent la croissance des promastigotes et réduisent l'accumulation de ROS, ce qui favorise la survie et la propagation des populations résistantes aux médicaments. En conclusion, ces découvertes permettront le développement de nouveaux tests diagnostiques et de nouvelles approches thérapeutiques pour certaines maladies infectieuses zoonotiques, basés sur les profils des EVs. De plus, elles ont une importance majeure dans la compréhension de la résistance médicamenteuse et prouve que les EVs fonctionnent comme des médiateurs efficaces dans le transfert horizontal de gène. Ce nouveau mécanisme facilite ainsi la transmission des gènes de résistance aux médicaments entre les parasites et favorise leur survie lorsqu'ils sont confrontés à des environnements stressants. / Although the evolution of drug resistance is one of the greatest challenges in the fight against infectious diseases, many phenomena remain unexplained. In the case of leishmaniasis, resistance has been a major problem for several years. This neglected zoonotic disease is caused by the protozoan parasite Leishmania, transmitted by sandflies during the blood meal. In humans, it comes in three main forms: visceral, cutaneous, and mucocutaneous. Leishmaniasis can also develop in infected dogs, which constitute a major reservoir of transmission. Without an effective vaccine, the control of the disease relies mainly on chemotherapy, but few approved molecules are available. Furthermore, the same products are used in dogs and humans, which promotes the emergence and spread of drug-resistant strains. Leishmania is indeed known to possess incredible genomic peculiarities (such as the formation of circular amplicons containing resistance genes) which allow it to survive under stressful conditions, such as drug pressure. Furthermore, Leishmania is a eukaryote that has retained the ability to produce extracellular vesicles (EVs) during evolution. These nano-sized particles are produced naturally by most biological cells and have a rich content of proteins, lipids, and nucleic acids. Although key characteristics of the protozoan EVs have been discovered, no studies have yet been performed on the EVs of resistant strains. Thus, this aspect constitutes the heart of my doctoral research project: the study of Leishmania EVs and their role in the phenomenon of drug resistance. Our work is the first to demonstrate that drug resistance mechanisms can induce changes in the morphology, size, and distribution of Leishmania EVs. We have identified the basic proteome of parasite EVs, and we have demonstrated enriched proteins in EVs released by parasites resistant to antimony, miltefosine and amphotericin B. We also studied the DNA content of EVs from resistant parasites and confirmed the enrichment of amplicons carrying drug resistance genes associated with EVs. In addition, our EV transfer tests have proven that these vesicles allow the horizontal transfer of resistance genes: an alternative mechanism of drug resistance. Finally, we showed that EVs from resistant parasites enhance promastigote growth and reduce ROS accumulation, which promotes the survival and spread of drug-resistant populations. In conclusion, these discoveries will allow the development of new diagnostic tests and new therapeutic approaches for certain zoonotic infectious diseases, based on the profiles of EVs. Moreover, these findings are of major importance in the understanding of drug resistance and prove that EVs function as effective mediators in horizontal gene transfer. This new mechanism thus facilitates the transmission of drug resistance genes between parasites and promotes their survival when confronted with stressful environments.
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La génomique évolutive mitochondriale révèle des échanges génétiques et la ségrégation chez les GloméromycètesBeaudet, Denis 06 1900 (has links)
Les champignons mycorhiziens à arbuscules (CMA) sont des organismes microscopiques du sol qui jouent un rôle crucial dans les écosystèmes naturels et que l’on retrouve dans tous les habitats de la planète. Ils vivent en relation symbiotique avec la vaste majorité des plantes terrestres. Ils sont des biotrophes obligatoires, c'est-à-dire qu'ils ne peuvent croître qu'en présence d'une plante hôte. Cette symbiose permet entre autres à la plante d'acquérir des nutriments supplémentaires, en particulier du phosphore et du nitrate. Malgré le fait que cette symbiose apporte des services importants aux écosystèmes, la richesse des espèces, la structure des communautés, ainsi que la diversité fonctionnelle des CMA sont mal connues et l'approfondissement des connaissances dans ces domaines dépend d’outils de diagnostic moléculaire. Cependant, la présence de polymorphisme nucléaire intra-isolat combiné à un manque de données génomiques dans différents groupes phylogénétique de ces champignons complique le développement de marqueurs moléculaires et la détermination de l'affiliation évolutive à hauts niveaux de résolution (c.a.d. entre espèces génétiquement similaires et/ou isolats de la même espèce).
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Pour ces raisons, il semble une bonne alternative d’utiliser un système génétique différent en ciblant le génome mitochondrial, qui a été démontré homogène au sein d'un même isolat de CMA. Cependant, étant donné le mode de vie particulier de ces organismes, une meilleure compréhension des processus évolutifs mitochondriaux est nécessaire afin de valoriser l'utilisation de tels marqueurs dans des études de diversité et en génétique des populations. En ce sens, mon projet de doctorat consistait à investiguerétudier: i) les vecteurs de divergences inter-isolats et -espèces génétiquement rapprochéesphylogénétiquement apparentées, ii) la plasticité des génomes mitochondriaux, iii) l'héritabilité mitochondriale et les mécanismes potentiels de ségrégation, ainsi que iv) la diversité mitochondriale intra-isolat in situ.
À l'aide de la génomique mitochondriale comparative, en utilisant le séquençage nouvelle génération, on a démontré la présence de variation génétique substantielle inter-isolats et -espèces, engendrées par l'invasion d'éléments mobiles dans les génomes mitochondriaux des CMA, donnant lieu à une évolution moléculaire rapide des régions intergéniques. Cette variation permettait de développer des marqueurs spécifiques à des isolats de la même espèce. Ensuite, à l'aide d'une approche analytique par réseaux de gènes sur des éléments mobiles, on a été en mesure de démontrer des évènements de recombinaisons homologues entre des haplotypes mitochondriaux distincts, menant à des réarrangements génomiques. Cela a permis d'ouvrir les perspectives sur la dynamique mitochondriale et l'hétéroplasmie dans un même isolatsuggère une coexistence de différents haplotypes mitochondriaux dans les populations naturelles et que les cultures monosporales pourraient induirent une sous-estimation de la diversité allélique mitochondriale. Cette apparente contradiction avec l'homogénéité mitochondriale intra-isolat généralement observée, a amené à investiguer étudier les échanges génétiques à l'aide de croisements d'isolats génétiquement distincts. Malgré l'observation de quelques spores filles hétéroplasmiques, l'homoplasmie était le statut par défaut dans toutes les cultures monosporales, avec un biais en faveur de l'un des haplotypes parentaux. Ces résultats suggèrent que la ségrégation opère durant la formation de la spore et/ou le développement de la coloniedu mycélium. De plus, ils supportent la présence d'une machinerie protéique de ségrégation mitochondriale chez les CMAAMF, où l'ensemble des gènes impliqués dans ce mécanisme ont été retrouvé et sont orthologues aux autres champignons. Finalement, on est revenue aux sources avecon a étudié le polymorphisme mitochondrial intra-isolat à l'aide d'une approche conventionnelle de PCR en utilisant une Taq polymérase de haute fidélité, suivie de clonage et de séquençage Sanger, sur deux isolats de R. irregularis. Cela a permis l'observation d'hétéroplasmie in situ, ainsi que la co-expression de variantes de variantes de protéines'ARNm dans une souche in vitro. Les résultats suggèrent que d'autres études basées sur le séquençage nouvelle génération aurait potentiellement ignorée cette variation, offrant ainsi plusieurs nouveaux arguments permettant de considérer les CMA comme des organismes possédant une population de génomes mitochondriaux et nucléaires distincts. / The association between arbuscular mycorrhizal fungi (AMF) and plant roots is one of the most widespread symbioses involving plants, and thus has an important role in terrestrial ecosystems. In exchange for carbohydrates, AMF improve plant fitness by enhancing mineral nutrient uptake, especially in particular phosphate and nitrate. Although this symbiosisDespite the fact that these symbioses contribute provides to important services toin ecosystems, the species richness, community structure and functional diversity of AMF is not well understood due to a lack of reliable molecular tools. The intra-isolate genetic polymorphism of nuclear DNA observed in AMF, combined with a lack of genomic data in a broad range of phylogenetic groups, has made it difficult to develop molecular markers and to determine evolutionary relatedness at high levels of resolution (i.e. between genetically-similar species and/or isolates).
For these reasons, it seems a good alternative to use a different genetic system by targeting the mitochondrial genome, which have been shown to be homogeneous within AMF isolates. However, given the peculiar lifestyle of these organisms, a better understanding of the mitochondrial evolutionary processes and dynamics were is necessary in order to validate the usefulness of such markers in diversity and population genetics studies. In that regard, the objectives of my PhD project were to investigate: i) the divergence between closely related species and isolates, ii) mitochondrial genomes plasticity, iii) mitochondrial heritability and potential segregation mechanisms and iv) in situ mitochondrial intra-isolate allelic diversity.
With Using comparative mitochondrial genomics using and next generation sequencing (NGS) sequencing, we found substantial sequence variation in intergenic regions caused by the invasion of mobile genetic elements. This variation gives risecontributes to rapid mitochondrial genome evolution among closely related isolates and species, which makes it possible to design reliable intra- and inter-specific markers. Also, an extensive gene similarity network-based approach allowed us to provide strong evidence of inter-haplotype recombination in AMF, leading to a reshuffled mitochondrial genome. These findings suggest the coexistence of distinct mtDNA haplotypes in natural populations and raise questions as to whether AMF single spore cultivations artificially underestimates mitochondrial genetic diversity in natural population.. This apparent contradiction with the intra-isolate mtDNA homogeneity usually observed in these fungi, led to the investigation of mitochondrial heritability in the spore progeny resulting from crossed-cultures. Although an heteroplasmic state was observed in some daughter spores, we found that homoplasmy was the dominant state in all monosporal cultures, with an apparent bias towards one of the parental haplotypes. These results strongly support the presence of a putative mitochondrial segregation proteic machinery in AMF, whose complete set of genes were orthologous with those found in other fungi. Our findings suggest that segregation takes place either during spore formation or colony mycelium development. Finally, we performed a conventional PCR based approach with a high fidelity Taq polymerase, followed by downstream cloning and Sanger sequencing using the model organism Rhizophagus irregularis. We found in situ heteroplasmy along with substantial intra-isolate allelic variation within the mtDNA that persists in the transcriptome. Our study also suggest that genetic variation in Glomeromycota is higher than meets the eye and might be critically underestimated in most NGS based-AMF studies both in nuclei and mitochondria.
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Χαρακτηρισμός των συμβιωτικών σχέσεων του βακτηρίου Wolbachia με έντομα αγροτικής, δασικής και ιατρικής σημασίαςΝτουντούμης, Ευάγγελος 01 August 2014 (has links)
Το βακτήριο Wolbachia είναι ένα ενδοκυττάριο και μητρικά κληρονομούμενο συμβιωτικό βακτήριο. Ανήκει στην ομοταξία των Alphaproteobacteria και την τάξη των Rickettsiales. Αποτελεί ίσως τον πιο διαδεδομένο ενδοκυττάριο συμβιωτικό οργανισμό στον πλανήτη, καθώς έχει εντοπιστεί μέχρι στιγμής σε πληθώρα αρθροπόδων και νηματωδών της φιλαρίασης. Πρόσφατες μελέτες εκτιμούν ότι πάνω από το 40% των ειδών αρθροπόδων είναι μολυσμένα με το βακτήριο Wolbachia. Το συμβιωτικό αυτό βακτήριο επηρεάζει τις βιολογικές λειτουργίες και ιδιότητες των ξενιστών του και είναι υπεύθυνο για μια σειρά αναπαραγωγικών ανωμαλιών, όπως η κυτταροπλασματική ασυμβατότητα, η παρθενογένεση, η θανάτωση των αρσενικών εμβρύων και η θηλυκοποίηση. Τα μοναδικά αυτά βιολογικά χαρακτηριστικά του βακτηρίου Wolbachia προσελκύουν όλο και περισσότερο το ενδιαφέρον διαφόρων ερευνητών τόσο για το ρόλο του βακτηρίου σε εξελικτικές διαδικασίες (κυρίως ειδογένεση) όσο και για τη χρησιμοποίησή του σε περιβαλλοντικά φιλικές εφαρμογές καταπολέμησης οργανισμών που είναι επιβλαβείς στους τομείς του γεωργικού και δασικού περιβάλλοντος, και της υγείας.
Τα είδη του γένους Glossina (Diptera: Glossinidae), γνωστά και ως μύγες τσε-τσε, αποτελούν ξενιστές του βακτηρίου Wolbachia. Η μύγα τσε-τσε είναι ο σημαντικότερος φορέας των παθογόνων τρυπανοσωμάτων στην τροπική Αφρική, τα οποία προκαλούν την ασθένεια του ύπνου (sleeping sickness) στον άνθρωπο και την αντίστοιχη τρυπανοσωμίαση, γνωστή ως nagana, στα ζώα. Η χρησιμοποίηση του βακτηρίου Wolbachia σε μεθόδους βιολογικής καταπολέμησης της μύγας τσε-τσε προαπαιτεί την πλήρη γνώση της γενετικής του ταυτότητας και των αλληλεπιδράσεων του με το ξενιστή.
Προς την κατεύθυνση αυτή, και στα πλαίσια της παρούσας διατριβής, πραγματοποιήθηκε η ανίχνευση του συμβιωτικού βακτηρίου Wolbachia σε περισσότερα από 5300 άτομα από φυσικούς και εργαστηριακούς πληθυσμούς 11 διαφορετικών ειδών μύγας τσε-τσε από 13 Αφρικανικές χώρες. Τα αποτελέσματα έδειξαν τεράστια απόκλιση της παρουσίας του βακτηρίου τόσο μεταξύ ειδών όσο και μεταξύ πληθυσμών του ίδιου είδους. Επίσης, πραγματοποιήθηκε ο γενετικός χαρακτηρισμός των στελεχών Wolbachia από συνολικά 29 αντιπροσωπευτικά δείγματα διαφόρων πληθυσμών και ειδών μύγας τσε-τσε, ενώ σε αρκετά από αυτά παρατηρήθηκαν πολλαπλά στελέχη του βακτηρίου. Διαπιστώθηκε εντυπωσιακή γενετική ποικιλότητα στελεχών Wolbachia που απαντούν στα διάφορα είδη μύγας τσε-τσε καθώς και ασυμφωνία μεταξύ των φυλογενειών των στελεχών Wolbachia και των μυγών τσε-τσε ξενιστών της, γεγονός που σημαίνει οριζόντια μετακίνηση του συμβιωτικού βακτηρίου κατά την εξέλιξη.
Επιπρόσθετα, εντοπίστηκαν για πρώτη φορά εκτεταμένα γεγονότα οριζόντιας μεταφοράς βακτηριακών γονιδίων στο γονιδίωμα τριών ειδών μύγας τσε-τσε: στο Glossina morsitans morsitans, Glossina pallidipes και Glossina austeni. Από εξελικτικής σκοπιάς, κρίσιμα ερωτήματα προκύπτουν από τα παραπάνω ευρήματα, και πιο συγκεκριμένα σχετικά με: την προέλευση-μηχανισμό αυτών των γεγονότων οριζόντιας μεταφοράς, τον χρονικό προσδιορισμό τους, τον πιθανό ρόλο τους σε διαδικασίες ειδογένεσης και την επιλεκτική εμφάνισή τους σε ορισμένα μόνο είδη Glossina π.χ. στo υποείδos Glossina morsitans centralis που είναι πολύ συγγενικό του Glossina morsitans morsitans δεν παρατηρήθηκε το φαινόμενο. Εξίσου σημαντική και επιβεβλημένη κρίνεται η διεξοδική διερεύνηση του ενδεχομένου τα βακτηριακά γονίδια που ενσωματώθηκαν στο ευκαρυωτικό γονιδίωμα της μύγας τσε-τσε να ευθύνονται για την έκφραση νέων λειτουργιών-ιδιοτήτων (ή να μεταβάλλουν τις ήδη υπάρχουσες), ιδίως μάλιστα εάν αυτές συνδέονται με την αποδοτικότητα μετάδοσης της νόσου της τρυπανοσωμίασης μέσω του φορέα της, δηλαδή της μύγας τσε-τσε. Τέλος, διαπιστώθηκε πιθανή αρνητική συσχέτιση της παρουσίας του βακτηρίου Wolbachia με τον παθογόνο ιό Salivary Gland hypertrophy Virus (SGHV), γεγονός που συζητείται στα πλαίσια βιολογικών εφαρμογών καταπολέμησης του εντόμου-φορέα και της τρυπανοσωμίασης.
Παράλληλα, μεγάλο ενδιαφέρον παρουσιάζει η προοπτική χρησιμοποίησης του βακτηρίου Wolbachia για τη βιολογική καταπολέμηση εντόμων αγροτικής ή /και περιβαλλοντικής σημασίας, όπως είναι οι αφίδες και η καρπόκαψα καστανιάς. Το γεγονός αυτό προϋποθέτει την ανίχνευση και τη γενετική ταυτοποίηση του βακτηρίου σε φυσικούς πληθυσμούς εντόμων.
Στα πλαίσια της παρούσας διατριβής πραγματοποιήθηκε ανίχνευση και χαρακτηρισμός του βακτηρίου Wolbachia σε 78 συνολικά άτομα από 22 είδη αφίδων, από 26 φυσικούς πληθυσμούς από την Ελλάδα. Από αυτούς τους 26 πληθυσμούς, μόλις οι 4 βρέθηκαν να είναι μολυσμένοι με το βακτήριο Wolbachia και συγκεκριμένα πληθυσμοί των ειδών: Aphis fabae, Aphis hederae, Metopolophium dirhodum και Baizongia pistaciae. Τα αποτελέσματα αυτά δείχνουν για πρώτη φορά ότι η παρουσία του βακτηρίου Wolbachia στις αφίδες είναι πιθανά πιο διαδεδομένη από ότι προέκυπτε από προηγούμενες μελέτες. Επίσης, μελετήθηκε η ανίχνευση και ο χαρακτηρισμός του βακτηρίου Wolbachia στα είδη Cydia splendana, Cydia fagiglandana και Pammene fasciana. Το βακτήριο Wolbachia ανιχνεύθηκε για πρώτη φορά στα συγκεκριμένα είδη και μάλιστα διαπιστώθηκε ότι η συχνότητα εμφάνισής του ποικίλει τόσο μεταξύ των δύο ειδών Cydia όσο και μεταξύ των πληθυσμών του κάθε είδους. Στο είδος Pammene fasciana, το βακτήριο ανιχνεύθηκε σε όλα τα άτομα που μελετήθηκαν.
Τα αποτελέσματα της παρούσας διατριβής συζητούνται από τη σκοπιά τόσο της οικολογικής και εξελικτικής σημασίας τους όσο και της προοπτικής χρησιμοποίησης του συμβιωτικού βακτηρίου Wolbachia για τον πληθυσμιακό έλεγχο επιβλαβών εντόμων όπως οι μύγες τσε-τσε, οι αφίδες και η καρπόκαψα καστανιάς. / Wolbachia is an intracellular and maternally inherited symbiotic bacterium that belongs to the class of Alphaproteobacteria and the order of Rickettsiales. It is the most ubiquitous intracellular symbiotic organism of the planet, since it has been estimated that over 40% of insect species, in addition to filarial nematodes, crustaceans, and arachnids are infected with Wolbachia. In arthropods Wolbachia affects the biological functions and properties of its hosts and it is responsible for a number of reproductive abnormalities, such as cytoplasmic incompatibility (CI), thelytokous parthenogenesis, feminization of genetic males and male killing. These unique biological characteristics of Wolbachia are attracting the interest of various researchers for: (a) decyphering the role of Wolbachia in evolutionary processes (mainly speciation), and (b) for its use in environmentally friendly applications for the control of agricultural pests and disease vectors.
The species of genus Glossina (Diptera: Glossinidae) known as tsetse flies, have been found to be infected with Wolbachia. Tsetse flies are the sole vectors of pathogenic trypanosomes in tropical Africa, causing the “sleeping sickness” in humans and the “nagana” in animals. The potential use of Wolbachia for the control of tsetse flies, prerequisite a thorough knowledge of its genetic identity and the interactions with the host.
To further characterize the prevalence of Wolbachia in tsetse flies an extensive screen of more than 5300 specimens from natural and laboratory populations of 11 different Glossina species originating from 13 African countries was carried out. Our results indicated a huge divergence in the prevalence of Wolbachia, both among the species and among populations of the same species. Further characterization by MLST and wsp genotyping was carried out for the Wolbachia strains of 29 representative populations and species of tsetse flies. An impressive genetic diversity of Wolbachia strains in tsetse flies was revealed. Interestingly, disconcordance between the phylogeny of Wolbachia and that of the tsetse flies was observed, suggesting horizontal transmission of Wolbachia during the evolution.
Moreover, extended horizontal gene transfer events were detected for first time in Glossina morsitans morsitans, Glossina pallidipes και Glossina austeni. These results raise critical questions concerning: (a) the origin/mechanism of these horizontal gene transfer events, (b) their temporal determination, (c) their potential role as agents of speciation and (d) their selective appearance in only some Glossina species e.g in the subspecies Glossina morsitans centralis which is closely related with Glossina morsitans morsitans the phenomenon was not observed. Equally important will be to examine if genes from the chromosomal insertions were potentially expressed and examine if these genes are associated with the vectorial capacity of tsetse flies for the trypanosoma transmission. Finally, a negative correlation between the presence of Wolbachia with the Salivary Gland Hypertrophy Virus (SGHV) was identified. This is further discussed in the context of biological applications for control of tsetse fly-vector and trypanosomiasis.
Finally in this thesis, the detection and characterization of Wolbachia in 78 specimens of 22 aphids species, from 26 natural populations, from Greece was examined. Only 4 out of 26 populations were found to be infected with Wolbachia, and specifically the species: Aphis fabae, Aphis hederae, Metopolophium dirhodum και Baizongia pistaciae. These results indicated that the presence of Wolbachia in aphids is probably more prevalent than it was derived from previous studies. Also, detection and characterization of Wolbachia in the Cydia splendana, Cydia fagiglandana and Pammene fasciana was carried out. Wolbachia was detected for first time in these species, and it was found that the prevalence of Wolbachia varies between the two species of Cydia and among populations of each species, with the infection in Pammene fasciana being fixed.
At the end the ecological and evolutionary importance of Wolbachia, together with the use of the bacterium for the population control of harmful insects like tsetse flies, aphids and moths is further discussed.
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