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Virulence et spécificité d’hôte de leptospires pathogènes endémiques de Madagascar et ses îles voisines / Virulence and host-specificity of pathogenic Leptospira endemic to Madagascar and surrounding islandsCordonin, Colette 19 March 2019 (has links)
La leptospirose est une zoonose d’importance médicale majeure dans les îles du Sud-Ouest de l’Océan Indien (SOOI) dont certaines enregistrent des incidences parmi les plus élevées au monde. Durant la dernière décennie, les données épidémiologiques moléculaires obtenues avec une approche « One Health » ont mis en évidence une grande diversité de lignées de leptospires ainsi que différentes chaines de transmission sur les différentes îles de la région. Les données moléculaires montrent la présence de leptospires pathogènes et de réservoirs animaux introduits ou endémiques de cette région. La distribution de ces différentes lignées de leptospires est associée à (i) un contraste épidémiologique incluant des différences dans la sévérité des cas humains et (ii) des niveaux de spécificité d’hôtes différents selon les leptospires considérés. Plus particulièrement, les leptospires endémiques du SOOI semblent être moins pathogènes chez les humains et montrent une plus forte affinité pour leur réservoir que les leptospires cosmopolites. Pour compléter nos connaissances sur l’histoire évolutive des leptospires du SOOI, nous avons produit des données provenant de chauves-souris de l’Afrique de l’Est. Ces données confirment la spécificité de certaines lignées de leptospires envers leurs hôtes chiroptères et suggèrent que les chauves-souris d’Afrique ont colonisé Madagascar tout en étant infectées par leurs leptospires. Afin de mieux comprendre le rôle des différents leptospires dans l’épidémiologie régionale de la leptospirose, nous avons mesuré la pathogénicité de trois souches de leptospires retrouvées dans cette région à l’aide d’un modèle hamster. Des souches de Leptospira mayottensis et Leptospira borgpetersenii ont été isolés respectivement de Tenrec ecaudatus (tenrec) et Triaenops menamena (chauve-souris), deux mammifères endémiques du SOOI. Une souche de Leptospira interrogans, dont le génotype est retrouvé dans la majorité des cas humains graves à la Réunion, a été isolée de Rattus rattus (rat). En cohérence avec les données épidémiologiques humaines de Mayotte et de La Réunion, les leptospires endémiques se sont révélées être significativement moins pathogènes que la souche L. interrogans. La spécificité d’hôte des deux souches isolées de mammifères endémiques a été mise à l’épreuve par des infections expérimentales de Rattus norvegicus, connu comme un réservoir important de leptospires. Les rats ont été infectés avec les trois isolats précédemment utilisés. Les rats infectés par les souches endémiques n’ont pas développé d’infection rénale chronique contrairement à la souche cosmopolite. Ces résultats montrent que la spécificité d’hôte des leptospires endémiques observée in natura est probablement due à des facteurs génétiques plutôt qu’à des facteurs écologiques, comme un manque de contacts physiques entre les réservoirs animaux endémiques et introduits. Enfin, le séquençage complet de souches de leptospires du SOOI a été réalisé afin d’identifier des caractéristiques génétiques pouvant être associées à la pathogénicité et la spécificité d’hôte des leptospires pathogènes. Une classification précise de souches de leptospires du SOOI a pu être réalisée sur la base des génomes complets. La comparaison de ces génomes a permis d’identifier des gènes spécifiques à un groupe ou une espèce de leptospires. Cependant des modifications génomiques complexes rendent difficiles l’identification de caractéristiques génomiques responsables d’un phénotype particulier tel que la virulence ou la spécificité d’hôte. / Leptospirosis is a zoonosis of main medical concern on several islands of southwestern Indian Ocean (SWIO), some of which recording among the highest human incidence worldwide. Over the last decade, molecular epidemiology investigations carried out under a One Health framework have revealed a wide variety of Leptospira lineages and distinct transmission chains throughout the islands of the region. These islands are home to pathogenic Leptospira lineages and animal reservoirs that are either introduced or endemic to the SWIO region. Interestingly, the regional distribution of Leptospira diversity is associated with (i) a contrasted severity of human cases and (ii) distinct levels of specificity of Leptospira towards their mammalian hosts. Specifically, endemic Leptospira appear less pathogenic in humans and display higher specificity towards their animal reservoirs than their cosmopolitan counterparts. To complete the dataset of Leptospira diversity in the SWIO region, we produced data from bats of eastern Africa. Results support the previously observed pattern of host specificity of Leptospira towards their bats hosts and, overlaid upon the biogeographic history of Malagasy bats, suggest that these volant mammals have colonized Madagascar from continental Africa while hosting pathogenic Leptospira. To better understand the role of distinct Leptospira lineages in the contrasted epidemiology observed in the SWIO, we investigated the pathogenicity of three Leptospira isolates from this region using a hamster model. Leptospira mayottensis and Leptospira borgpetersenii isolates were obtained from Tenrec ecaudatus (tenrec) on Mayotte and Triaenops menamena (bat) in Madagascar, respectively, both mammals endemic to the SWIO region. A Leptospira interrogans strain, which genotype has been reported in the majority of human acute cases on La Réunion, was isolated from the introduced Rattus rattus (rat). In keeping with a distinct severity of the disease on Mayotte and La Réunion, endemic bat-borne and tenrec-borne Leptospira were significantly less pathogenic than the control cosmopolitan rat-borne isolate. The host specificity of the isolates obtained from endemic hosts was addressed using experimental infection of Rattus norvegicus, a known reservoir of pathogenic Leptospira. This animal model was challenged with all three isolates and mostly failed in supporting chronic infection with bat-borne and tenrec-borne Leptospira. Hence, the strong host-specificity of endemic Leptospira toward their hosts observed in the wild likely results from genetic determinants shaped by long-term co-evolutionary processes rather than from ecological constraints such as a lack of physical contact between introduced and endemic animal reservoirs. Finally, we undertook full genome sequencing of regional strains in order to highlight genomic features that may be associated with virulence and host specificity. Whole genome sequencing allowed the accurate classification of Leptospira isolates obtained on SWIO islands. Comparative genomics allowed to identify genes specific to a group or species of Leptospira but complex changes in Leptospira genome make difficult the identification of genomic elements responsible for specific traits such as virulence and host specificity.
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Genômica comparativa de Xylella fastidiosa: diversidade do pangenoma e análise de genes de patogenicidade / Comparative genomics of Xylella fastidiosa: pan-genome diversity and analysis of patogenicity genesSantana, Wesley Oliveira de 04 February 2013 (has links)
O gênero Xylella é composto de uma única espécie, Xylella fastidiosa, bactéria Gram-negativa, não flagelada, que coloniza o xilema de uma diversidade de plantas cultivadas e silvestres em várias partes do mundo. Em algumas dessas plantas, a bactéria é considerada agente causal de doenças, como a Clorose Variegada do Citros em laranjeiras, a Doença de Pierce das videiras e escaldadura da folha de cafeeiro. Onze diferentes cepas de X. fastidiosa, isoladas de distintos hospedeiros, já tiveram seus genomas sequenciados, entre essas, as cepas 9a5c, isolada de laranjeira, e Temecula 1, isolada de videira. Análises desses genomas indicam uma razoável variabilidade entre suas respectivas sequências e evidenciam vários genes associados a mecanismos de virulência e patogenicidade desta bactéria. No presente trabalho descrevemos o sequenciamento, a montagem e a anotação dos genomas das cepas U24d e Fb7, isoladas de laranjeiras, e da cepa 3124 isolada de cafeeiro, os quais apresentam, respectivamente 2.681.334 pb, 2.733.974 pb e 2.748.594 pb. Destas, apenas a cepa U24d apresenta um plasmídeo, o qual é idêntico ao pXF51 previamente identificado na cepa 9a5c. O genoma da cepa U24d é praticamente colinear ao genoma da cepa 9a5c enquanto que os genomas das cepas Fb7 e 3124 apresentaram maior colinearidade com a cepa Temecula1. Entre as diversas alterações encontradas nas análises comparativas destes genomas, destacamos a inserção no gene pilQ verificada no genoma da cepa U24d. Essa mutação causa ausência do pilus do tipo IV com consequente deficiência na motilidade twitching, sendo que plantas infectadas com a cepa U24d apresentam sintomas localizados restritos ao ponto de inoculação. Na cepa Fb7, detectamos a ausência de formação de biofilme no cultivo in vitro possivelmente devido ausência da expressão dos transcritos de mrkD e pspA, que codificam respectivamente adesina do pilus curto e adesina similar à hemaglutinina. Postulamos que estes genes não são expressos em decorrência de um defeito na via de sinalização de DSF (Fator de Sinalização Difusível) reflexo de uma mutação em rpfC no genoma de Fb7. Assim como as demais cepas de X. fastidiosa, também os genomas de U24d, Fb7 e 3124 apresentaram elevado conteúdo de Elementos Genéticos Móveis (EGM), que aparecem em maior número nas cepas sul-americanas. Os estudos do pangenoma de X. fastidiosa mostraram que essa espécie tem um genoma aberto e grande parte dos genes de EGMs correspondem a genes acessórios. A grande quantidade de EGMs em X. fastidiosa pode estar relacionada a falta do sistema CRISPR/cas completo, um provável resultado de eventos de erosão do genoma desta espécie. A inferência filogenética por MSLA mostrou uma clara distinção dos grupos de cepas da América do Norte em relação às do Sul, sugerindo a ocorrência de mais eventos de recombinações genéticas nas cepas sul-americanas, provavelmente pela falta de isolamento geográfico. Assim, é possível que as cepas norte e sul-americanas sofreram divergência alopátrica e simpátrica, respectivamente. / The genus Xylella consists of a single species, Xylella fastidiosa, a Gram-negative and non-flagellated bacterium that colonizes the xylem of a diversity of cultivated and wild plants in several parts of the world. In some of these plants, this bacterium is considered causal agent of diseases such as the Citrus Variegated Cholorosis in orange trees, Pierce\'s Disease of grapevines and coffee leaf scald. Eleven different strains of X. fastidiosa isolated from different hosts had their genomes sequenced, including 9a5c and Temecula1 strains, respectively isolated from orange tree and grapevine. Analyses of these genomes indicate a reasonable variability in their sequences and showed several genes associated with pathogenicity and virulence mechanisms of this bacterium. In this work we describe the genome sequencing, assembly and annotation of the strains U24d and Fb7, isolated from orange trees, and 3124 isolated from coffee, which have, respectively, 2,681,334 bp, 2,733,974 bp and 2,748,594 bp. Of these, only strain U24d has a plasmid, identical to pXF51 from strain 9a5c. The genome of U24d strain is almost collinear to the genome of strain 9a5c while the genomes of strains Fb7 and 3124 had higher collinearity to Temecula1 strain. Among many changes found in the comparative analysis of these genomes, we highlight an on insertion in pilQ gene that was found in U24d strain genome. This mutation causes lack of type IV pilus with a consequent deficiency in twitching motility. Moreover orange trees infected with U24d strain showed localized symptoms near to the inoculation point. We verified that Fb7 strain does not form biofilm in vitro possibly due to the absence of expression of mrkD and pspA transcripts, which encode, respectively, a short pilus adhesin and a hemagglutinin-like adhesin. We postulate that these genes are not expressed due to a defect in the signaling pathway of DSF (Diffusible Signal Factor) reflecting a mutation on rpfC in the Fb7 genome. Similarly to other X. fastidiosa strains, the genomes of U24d, Fb7 and 3124 also showed high content of mobile genetic elements (MGE), which appear in larger numbers in South American strains. Pan genome studies of X. fastidiosa showed that this species has a open genome and that most of MGE genes correspond to accessory genes. The large number of MGE in X. fastidiosa may be related to the lack of a complete system CRISPR/cas, likely a result of erosion events of the genome of this species. The phylogenetic reconstruction by MLSA showed a clear distinction between groups of strains from North and South America, suggesting the occurrence of more recombination events in South American strains, probably due to lack of geographical isolation. Thus it is possible that North and South American strains underwent allopatric and sympatric divergence, respectively.
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The evolution and transmission of HA-MRSA ST239 through hospitals in Turkey and intercontinental spreadAldeljawi, Mona January 2015 (has links)
Next-generation sequencing technology provides high-resolution data for epidemiological surveillance of bacterial pathogens on local and global scales. This approach has been used for many species including Methicillin Resistant Staphylococcus aureus (MRSA). In this thesis I demonstrate the utility of these data for understanding the spread of the globally disseminated clone MRSA ST239. I focus both on local and national-level epidemiology through sequence data of 71 isolates recovered from four hospitals representing three cities in Turkey; Istanbul (x2). Ankara and Izmir. I analyse whole genome sequence data from a further 33 ST239 isolates from global sources. These data were combined with previously published data for phylogenetic analysis based only on the core genome. I demonstrate how transmission events can be inferred from this approach on multiple levels; within hospital, between hospitals and between countries. The data pointed to a European origin of ST239, and independent introductions from Europe to Turkey, South America and East Asia. I also demonstrate how whole genome sequence data can be used to develop bespoke PCR assays, based on phage variation, for rapid local epidemiology. Finally, I consider how the sequence data might be used to explain variation in virulence potential, and describe the distribution and transfer of an important phage-borne virulence determinant, sasX, within Europe. Finally, I identified a single isolate with very strong biofilm forming ability likely due to the over-expression of the important adhesion SasG.
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Génomique épidémiologique de Salmonella / Genomic and epidemiology of SalmonellaTran Dien, Alicia 11 January 2018 (has links)
Découverte il y a plus d’un siècle, Salmonella n’a cessé d’intriguer les chercheurs. Sa capacité à résister à de nombreux antibiotiques est de plus en plus préoccupante. La surveillance de ce pathogène repose sur un typage rapide et discriminant de façon à identifier le plus précocement possible les sources alimentaires contaminées. Les méthodes classiques sont longues, lourdes et non automatisables. Comprendre l’émergence et l’évolution des Salmonella est la clé pour éradiquer ce pathogène resté l’une des premières causes de diarrhées bactériennes d’origine alimentaire dans le monde. Au cours des dernières décennies, des progrès spectaculaires ont été menés dans le monde de la microbiologie avec l’arrivée des séquenceurs de paillasse, passant du traitement d’une dizaine à des centaines de millions de séquences. L’accès facilité aux séquences génomiques et aux outils qui leurs sont dédiés sont devenus une nécessité. Les outils actuellement disponibles ne sont pas assez discriminants pour sous-typer S. enterica sérotype Typhimurium (STM), sérotype prédominant de Salmonella. Nous avons voulu lors de ce travail, montrer l’intérêt du séquençage entier du génome, pour l’étude génomique de Salmonella. (1) Après avoir séquencé plus de 300 génomes de STM, nous avons mis au point un outil de sous-typage in silico de ce sérotype, basé sur le polymorphisme des CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats). La surveillance à haut débit des salmonelloses a été validée en routine sur plus de 800 génomes. L’étude de la coévolution entre le chromosome (SNPs) et les régions CRISPR ont permis d’établir une nomenclature définissant les différentes populations de STM. (2) L’analyse génomique de 280 souches historiques de STM a montré que les gènes de bêta-lactamase conférant une résistance à l’ampicilline et portés par des plasmides étaient répandus chez STM à la fin des années 1950, bien avant l’utilisation de cet antibiotique. La présence de la pénicilline G dans le milieu agricole où ces composés ont été utilisés en tant que promoteurs de croissance ont pu conduire à la sélection des premières souches résistantes à l’ampicilline. (3) L’étude phylogénétique d’un génome issu du cadavre d’une femme décédée il y a plus de 800 ans, probablement à cause de la fièvre entérique et de 219 génomes historiques et récents des sérotypes Paratyphi C, Choleraesuis et Typhisuis ont montré que leurs génomes étaient très similaires au cours des 4000 dernières années. Ainsi, la combinaison des approches génotypique et phylogénétique ont accru nos connaissances sur l’évolution de ce pathogène.Mots clés : Séquençage entier du génome, surveillance épidémiologique, CRISPR, SNP, résistance antibiotique, phylogénie, évolution / Over a century has passed since the discovery of Salmonella and yet, this pathogen still intrigues researchers. Its ability to withstand many antibiotics is of increasing concern. The monitoring of this pathogen is based on a rapid and discriminatory typing to identify the sources of contaminated food as early as possible. The conventional methods are long, heavy and non-automatable. Understanding the emergence and evolution of Salmonella is the key to eradicate this pathogen, which has remained one of the leading causes of foodborne bacterial diarrhea in the world. During the last decades, spectacular progress has been made in the world of microbiology with the arrival of workbench sequencers, passing from a dozen to hundreds of millions of sequences processed. Facilitated access to numerous genome sequences and dedicated tools are mandatory. Tools currently available are not sufficiently discriminating for the subtype of S. enterica serotype Typhimurium, a predominant serotype of Salmonella. Throughout this study, we showed the interest of whole genome sequencing, a multidisciplinary tool, for the genomic study of Salmonella. (1) After sequencing over 300 S. enterica serotype Typhimurium genomes, we have developed an in silico subtyping tool for this serotype, based on the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) polymorphism. High-throughput microbiological monitoring of salmonellosis has been routinely validated on over 800 genomes. The study of coevolution between the chromosome (SNPs of the core genome) and the two CRISPR regions made it possible to establish a nomenclature defining the different populations of this serotype. (2) Genomic analysis of 280 historical strains of S. enterica serotype Typhimurium showed that plasmids carrying beta-lactamase genes, which confer resistance to ampicillin, were widespread within this serotype in the late 1950s, years before ampicillin was first used for clinical purposes. The presence of penicillin G in the farming environment where these compounds were used as growth promoters, may have led to the selection of the first ampicillin-resistant strains. (3) The phylogenetic study of a genome from the corpse of a young woman who died over 800 years ago, probably due to enteric fever, and 219 historical and recent genomes of the serotypes Paratyphi C, Choleraesuis and Typhisuis have shown, despite the differences in host specificity, that their genomes were very similar over the past 4000 years. Thus, the combination of genotypic and phylogenetic approaches has increased our knowledge of the evolution of this pathogen.Key words: Whole genome sequencing, epidemiological monitoring, CRISPR, SNP, antibiotic resistance, phylogeny, evolution
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Caracterização molecular do Vírus da Anemia Infecciosa Equina do Pantanal e padronização de qPCR para diagnósticoMalossi, Camila Dantas January 2019 (has links)
Orientador: João Pessoa Araujo Junior / Resumo: O Vírus da Anemia Infecciosa Equina (VAIE) é um lentivírus que infecta equídeos causando a Anemia Infecciosa Equina. A doença possui distribuição mundial e o principal método de controle empregado é a eutanásia dos animais positivos, uma vez que não existe cura ou vacina disponível. No Brasil, a região do Pantanal é endêmica para a doença e, somente nessas áreas, a eutanásia não é obrigatória. A alta variação genética de lentivírus durante uma infecção prolongada é bem conhecida em várias espécies e também descrita em sequências de VAIE de outros países. Não há sequência genômica brasileira completa descrita ou mesmo um estudo da variação genética do vírus em um ambiente endêmico como o Pantanal Brasileiro. Com isso, este trabalho teve como objetivos a caracterização molecular do VAIE em equinos da região do Pantanal Brasileiro, e a padronização da PCR quantitativa (qPCR) para detecção do DNA proviral de VAIE. Duas amostras de plasma equino positivas para VAIE foram submetidas ao sequenciamento do genoma completo utilizando uma combinação de técnicas tradicionais e de nova geração. O genoma a partir do RNA viral foi obtido, anotado e comparado com sequências virais obtidas de animais naturalmente infectados em outros países. As sequências provenientes do Pantanal apresentaram identidade entre 75 e 77 % com sequências de outros países e 88,54 % entre si, classificando-as em um clado individual na árvore filogenética dos vírus já descritos, exibindo a maior variação genética de... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Equine infectious anemia virus (EIAV) is a lentivirus that infects equids and causes equine infectious anemia (EIA). This disease presents worldwide distribution and the main control method is the euthanasia of positive animals, since there is no cure or vaccine available. The Pantanal region in Brazil is endemic for the disease, and euthanasia is not mandatory in this area. The high lentivirus genetic variation during a prolonged infection is well known in several species and also described in EIAV sequences in other countries. There are no Brazilian viral genomic sequences available or even a study of genetic variation in an environment such as Brazilian Pantanal. This work aimed to characterize the EIAV complete genome in equines from Brazilian Pantanal and to standardize a quantitative real time PCR (qPCR) for detection of EIAV proviral DNA. Two plasma samples of EIAV positive horses were submitted to massive sequencing by combination of Sanger and NGS sequencing. The complete genome from viral RNA was obtained, annotated and compared with viral sequences of naturally infected animals from other countries. Sequences from Pantanal showed identity between 75% and 77% with other countries sequences and 88.54% with each other, classifying them into an individual cluster in EIAV cladogram and exhibiting the greatest genetic variation between sequences from the same country. With viral envelope gene analysis, both Brazilian sequences were classified as two quasispecies. A qPCR ... (Complete abstract click electronic access below) / Doutor
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Leishmanie podrodu Mundinia: genetická analýza a experimentální infekce hlodavců a přenašečů. / Leishmania of the subgenus Mundinia: genetical analysis and experimental infections of rodents and vectors.Bečvář, Tomáš January 2019 (has links)
Leishmaniasis is a human and animal disease caused by digenetic parasites of the genus Leishmania, which is now divided into 4 subgenera - L. (Leishmania), L. (Viannia), L. (Sauroleishmania) and L. (Mundinia). Subgenus Mundinia was established in 2016 and consists of 5 species - L. enriettii and L. macropodum are parasites of wild mammals and L. martiniquensis, L. orientalis and unnamed L. sp. from Ghana are infectious to humans. Mundinia are geographically widely dispersed, their distribution covers all continents, except of Antarctica. Despite phlebotomine sand flies (Diptera: Psychodidae) also biting midges (Diptera: Ceratopogonidae) are supposed to be involved in transmission of these species, which is a unique feature for this subgenus. But there is little to no current information on natural reservoir hosts and vector species for any Mundinia species. In this thesis we tested possible vectors and potential model organisms (Guinea-pigs) and reservoir hosts of Mundinia species by experimental infections. We used 3 sand fly species sharing geographical distribution with respective Mundinia species and available in our laboratory for experimental infections. Sand flies from Australia had never been colonised so we used the permissive vector Lu. migonei for testing development of L. macropodum....
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Population genetics of Colletotrichum truncatum associated with soybean anthracnose / Genética populacional de Colletotrichum truncatum associado à antracnose da sojaRogério, Flávia 05 July 2019 (has links)
The soybean crop is one of the main agricultural crops, with high global economic relevance. The large area under soybean cultivation in Brazil, including the incorporation of new areas in the northern and midwestern regions, mostly under monoculture and non-tillage system, has been affected the prevalence and the intensity of diseases. Among these, one of most prominent is anthracnose, mainly associated with the fungal species Colletotrichum truncatum. Knowledge of the genetic structure of plant pathogen populations can be used to infer their life histories and the evolutionary processes that shape populations in the agroecosystems, which can help to implement effective disease management strategies. However, the genetic structure of C. truncatum populations associated with soybean remains unknown. We collected C. truncatum isolates from 10 sites representing two of main areas of soybean producing in Brazil and used microsatellite markers and whole-genome sequencing to investigate the population biology and evolutionary history of this important pathogen. The multilocus microsatellite typing of 237 isolates revealed high gene and haplotypic diversity within populations, as well low genetic differentiation and sharing of multilocus haplotypes among populations and regions. In addition, three distinct genetic clusters were detected, coexisting in syntopy in the soybean fields, without evidence of admixture between them. Such finding suggesting that Brazilian C. truncatum populations resulted from at least three founder events, which led to three genetic lineages that spread throughout the country. However, the genetic makeup of these lineages remains unknow, and their extreme geographic proximity raises the question of the maintenance of their genetic integrity in the face of admixture. In order to gain insights into the evolutionary history of C. truncatum lineages and to investigate in more details the possibility of a lack of genetic exchanges between them, we employed a population genomic approach. For that, we produced a draft genome sequence of a typical strain of the species associated with soybean anthracnose, which was used as the reference genome. Eighteen representative C. truncatum isolates from the three lineages were submitted to whole genome sequencing, aligned against the reference genome, and variants were identified. Our population genomic analyzes revealed that the genetic structure of C. truncatum pathogen causing soybean anthracnose is formed by three deeply divergent lineages with levels of genetic diversity consistent with repeated introduction events for each lineage. We also found evidence for sexual recombination within and between lineages, with multiples isolates displaying signatures of admixture. Our findings support a scenario in which the three lineages initially diverged in allopatry before experiencing hybridization following secondary contact. Monitoring of the pathogen\'s diversity over time is needed to reveal whether these lineages maintain or fuse, which can impact the disease control methods currently employed. / A soja é uma das principais culturas agrícolas, com alta relevância econômica global. A grande área sob cultivo de soja no Brasil, incluindo a incorporação de novas áreas nas regiões norte e centro-oeste, principalmente sob monocultura e plantio direto, tem afetado a prevalência e a intensidade das doenças. Entre elas, uma das mais proeminentes é a antracnose, principalmente associada à espécie fúngica Colletotrichum truncatum. O conhecimento da estrutura genética das populações de patógenos de plantas pode ser usado para inferir suas histórias de vida e os processos evolutivos que moldam as populações nos agroecossistemas, o que pode ajudar a implementar estratégias eficazes de manejo da doença. No entanto, a estrutura genética das populações de C. truncatum associadas à soja permanece desconhecida. Coletamos isolados de C. truncatum em 10 áreas, representando duas principais regiões produtoras de soja no Brasil. Utilizamos marcadores microssatélites e sequenciamento do genoma completo para investigar a biologia populacional e a história evolutiva desse importante patógeno. A tipagem de microssatélites multilocus de 237 isolados revelou alta diversidade genética e haplotípica nas populações, bem como baixa diferenciação genética e compartilhamento de haplótipos entre populações e regiões. Além disso, foram detectados três grupos genéticos distintos, coexistindo nas mesmas áreas, sem evidência de mistura entre eles. Isto sugere que as populações C. truncatum no Brasil resultaram de pelo menos três eventos fundadores, o que levou á formação das três linhagens genéticas que se espalharam pelo país. No entanto, a composição genética dessas linhagens permanece desconhecida, e sua extrema proximidade geográfica levanta a questão sobre a manutenção de sua integridade genética em face a mistura entre elas. A fim de analisar a história evolutiva das linhagens de C. truncatum e investigar a possibilidade de ausência de trocas genéticas entre elas, empregamos uma abordagem genômica populacional. Para isso, produzimos uma versão preliminar do genoma completo de um isolado típico da espécie, o qual foi utilizado como genoma de referência. Dezoito isolados representativos das três linhagens foram submetidos ao sequenciamento completo, alinhados ao genoma de referência, e variantes foram identificados. Nossas análises genômicas populacionais revelaram que a estrutura genética do patógeno é formada por três linhagens profundamente divergentes, com níveis de diversidade consistentes com repetidos eventos de introdução para cada linhagem. Também encontramos evidências de recombinação sexual dentro e entre linhagens, com múltiplos isolados apresentando assinaturas de mistura. Nossas descobertas sugerem um cenário no qual as três linhagens divergiram inicialmente em alopatria antes de experimentar hibridação, após contato secundário. O monitoramento da diversidade do patógeno ao longo do tempo é necessário para revelar se essas linhagens se mantêm geneticamente separadas ou se fundem, o que pode afetar os métodos de controle da doença atualmente empregados.
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Structural Variation in the Human GenomePang, Wing Chun Andy 09 August 2013 (has links)
The study of variation found in DNA is fundamental in human genetic studies. Single nucleotide polymorphisms (SNPs) are simple to document because they can be captured in single DNA sequence reads. Larger structural variation including duplications, insertions, deletions, termed as copy number variation (CNV), inversions and translocations are more challenging to discover. Recent studies using microarray and sequencing technologies have demonstrated the prevalence of structural variation in humans. They can disrupt genic and regulatory sequences, be associated with disease, and fuel evolution. Therefore, it is important to identify and characterize both SNPs and structural variants to fully understand their impact.
This thesis presents the analysis of structural variation in the human genome. The primary DNA sample used for my experiments is the DNA of J. Craig Venter, also termed HuRef. It was the first personal human genome sequenced. I combined computational re-analysis of sequence data with microarray-based analysis, and detected 12,178 structural variants covering 40.6 Mb that were not reported in the initial sequencing study. The results indicated that the genomes of two individuals differed 1.3% by CNV, 0.3% by inversion and 0.1% by SNP. Structural variation discovery is dependent on the strategy used. No single approach can readily capture all types of variation, and a combination of strategies is required.
I analyzed the formation mechanisms of all HuRef structural variants. The results showed that the relative proportion of mutational processes changed across size range: the majority of small variants (<1kb) were associated with nonhomologous processes and microsatellite events; median size variants (<10kb) were commonly related to minisatellites and retrotransposons; and large variants were associated with nonallelic homologous recombination.
Eight new breakpoint-resolved HuRef inversions were genotyped in populations to elucidate these understudied variants. I discovered that the structures of inversion could be complex, could create conjoined genes, and their frequencies could exhibit population differentiation.
The data here contributes to our understanding of structural variation in humans. It shows the need to use multiple strategies to identify variants, and it emphasizes the importance to examine the full complement of variation in all biomedical studies.
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Development of genetic tools for metabolic engineering of Clostridium pasteurianumPyne, Michael E 21 April 2015 (has links)
Reducing the production cost of industrial biofuels will greatly facilitate their proliferation and co-integration with fossil fuels. The cost of feedstock is the largest cost in most fermentation bioprocesses and therefore represents an important target for cost reduction. Meanwhile, the biorefinery concept advocates revenue growth through complete utilization of by-products generated during biofuel production. Taken together, the production of biofuels from low-cost crude glycerol, available in oversupply as a by-product of bioethanol production, in the form of thin stillage, and biodiesel production, embodies a remarkable opportunity to advance affordable biofuel development. However, few bacterial species possess the natural capacity to convert glycerol as a sole source of carbon and energy into value-added bioproducts. Of particular interest is the anaerobe Clostridium pasteurianum, the only microorganism known to convert glycerol alone directly into butanol, which currently holds immense promise as a high-energy biofuel and bulk chemical. Unfortunately, genetic and metabolic engineering of C. pasteurianum has been fundamentally impeded due to a complete lack of genetic tools and techniques available for the manipulation of this promising bacterium. This thesis encompasses the development of fundamental genetic tools and techniques that will permit extensive genetic and metabolic engineering of C. pasteurianum.
We initiated our genetic work with the development of an electrotransformation protocol permitting high-level DNA transfer to C. pasteurianum together with accompanying selection markers and vector components. The CpaAI restriction-modification system was found to be a major barrier to DNA delivery into C. pasteurianum which we overcame by in vivo methylation of the recognition site (5’-CGCG-3’) using the M.FnuDII methyltransferase. Systematic investigation of various parameters involved in the cell growth, washing and pulse delivery, and outgrowth phases of the electrotransformation procedure significantly elevated the electrotransformation efficiency up to 7.5 × 104 transformants µg-1 DNA, an increase of approximately three orders of magnitude. Key factors affecting the electrotransformation efficiency include cell-wall-weakening using glycine, ethanol-mediated membrane solubilization, field strength of the electric pulse, and sucrose osmoprotection.
Following development of a gene transfer methodology, we next aimed to sequence the entire genome of C. pasteurianum. Using a hybrid approach involving 454 pyrosequencing, Illumina dye sequencing, and single molecule real-time sequencing platforms, we obtained a near-complete genome sequence comprised of 12 contigs, 4,420,100 bp, and 4,056 candidate protein-coding genes with a GC content of 30.0%. No extrachromosomal elements were detected. We provide an overview of the genes and pathways involved in the organism’s central fermentative metabolism.
We used our developed electrotransformation procedure to investigate the use of established clostridial group II intron biology for constructing chromosomal gene knockout mutants of C. pasteurianum. Through methylome analysis of C. pasteurianum genome sequencing data and transformation assays of various vector deletion constructs, we identified a new Type I restriction-modification system that inhibits transfer of vectors harboring group II intron gene knockout machinery. We designated the new restriction system CpaAII and proposed a recognition sequence of 5’-AAGNNNNNCTCC-3’. Overcoming restriction by CpaAII, in addition to low intron retrohoming efficiency, allowed the isolation of a gene knockout mutant of C. pasteurianum with a disrupted CpaAI Type II restriction system. The resulting mutant strain should be efficienty transformed with plasmid DNA lacking M.FnuDII methylation.
Lastly, we investigated the use of plasmid-based gene overexpression and chromosomal gene downregulation to alter gene expression in C. pasteurianum. Using a β-galactosidase reporter gene, we characterized promoters corresponding to the ferredoxin and thiolase genes of C. pasteurianum and show that both promoters permitted high-level, constitutive gene expression. The thiolase promoter was then utilized to drive transcription of an antisense RNA molecule possessing complementarity to mRNA of our β-galactosidase reporter gene. Our antisense RNA system demonstrated 52-58% downregulation of plasmid encoded β-galactosidase activity throughout the duration of growth. In an attempt to perturb the central fermentative metabolism of C. pasteurianum and enhance butanol titers, we prepared several antisense RNA constructs for downregulation of 1,3-propanediol, butyrate, and hydrogen production pathways. The resulting downregulation strains are expected to exhibit drastically altered central fermentative metabolism and product distribution.
Taken together, we have demonstrated that C. pasteurianum is amendable to genetic manipulation through the development of methods for plasmid DNA transfer and gene overexpression, knockdown, and knockout. Further, our genome sequence should provide valuable nucleotide sequence information for the application of our genetic tools. Thus, the genome sequence, electrotransformation method, and associated genetic tools and techniques reported here should promote extensive genetic manipulation and metabolic engineering of this biotechnologically important bacterium.
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Structural Variation in the Human GenomePang, Wing Chun Andy 09 August 2013 (has links)
The study of variation found in DNA is fundamental in human genetic studies. Single nucleotide polymorphisms (SNPs) are simple to document because they can be captured in single DNA sequence reads. Larger structural variation including duplications, insertions, deletions, termed as copy number variation (CNV), inversions and translocations are more challenging to discover. Recent studies using microarray and sequencing technologies have demonstrated the prevalence of structural variation in humans. They can disrupt genic and regulatory sequences, be associated with disease, and fuel evolution. Therefore, it is important to identify and characterize both SNPs and structural variants to fully understand their impact.
This thesis presents the analysis of structural variation in the human genome. The primary DNA sample used for my experiments is the DNA of J. Craig Venter, also termed HuRef. It was the first personal human genome sequenced. I combined computational re-analysis of sequence data with microarray-based analysis, and detected 12,178 structural variants covering 40.6 Mb that were not reported in the initial sequencing study. The results indicated that the genomes of two individuals differed 1.3% by CNV, 0.3% by inversion and 0.1% by SNP. Structural variation discovery is dependent on the strategy used. No single approach can readily capture all types of variation, and a combination of strategies is required.
I analyzed the formation mechanisms of all HuRef structural variants. The results showed that the relative proportion of mutational processes changed across size range: the majority of small variants (<1kb) were associated with nonhomologous processes and microsatellite events; median size variants (<10kb) were commonly related to minisatellites and retrotransposons; and large variants were associated with nonallelic homologous recombination.
Eight new breakpoint-resolved HuRef inversions were genotyped in populations to elucidate these understudied variants. I discovered that the structures of inversion could be complex, could create conjoined genes, and their frequencies could exhibit population differentiation.
The data here contributes to our understanding of structural variation in humans. It shows the need to use multiple strategies to identify variants, and it emphasizes the importance to examine the full complement of variation in all biomedical studies.
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