Das hochadaptierte, zellwandlose Bakterium Mycoplasma pneumoniae gehört zu den häufigsten Erregern von ambulant erworbenen Pneumonien. Während den alle 3-7 Jahre auftretenden Epidemien könnten gefährdete Personengruppen bei Verfügbarkeit einer effektiven Vakzinierung vor einer Infektion geschützt werden. Ziel dieser Arbeit war die Erarbeitung von Grundlagen zum Aufbau einer schützenden Immunantwort durch Immunisierung mit rekombinant hergestellten Teilbereichen verschiedener Adhärenzproteine von M. pneumoniae. Da die bereits bekannten Adhäsine des Erregers einerseits unverzichtbare Virulenzfaktoren darstellen und andererseits als dominante Immunogene des Bakteriums beschrieben wurden, war die Feincharakterisierung dieser Proteine im Hinblick auf ihre Eignung als Vakzinekandidat Ausgangspunkt der Untersuchungen. Definierte Teilbereiche des Hauptadhäsins P1 und weiterer adhärenzassoziierter Proteine wurden rekombinant hergestellt und auf ihre Antigenität qualitativ und quantitativ mit Seren experimentell infizierter Tiere und Seren von Patienten mit bestätigter M. pneumoniae-Infektion geprüft. Dabei gingen der C-terminale Bereich des P1-Adhäsins (RP14; AS 1287-1518) und das Protein P30 (RP30; AS 17-274) als hoch antigene Protein(regionen) hervor. Die Oberflächenexposition beider Proteinbereiche wurde experimentell bestätigt. Parallel dazu sollte der Einfluss der monospezifischen Seren gegen die rekombinanten Proteine auf die Adhärenz von M. pneumoniae an verschiedene Zelllinien bewertet werden. Nach Entwicklung eines quantitativen und schnell durchzuführenden in vitro Adhärenzhemmtests wurde gezeigt, dass Antikörper sowohl gegen RP14 als auch gegen RP30 die Adhärenz des Erregers an humane Zellen signifikant reduzieren. Im Hinblick auf die Beeinflussung von mehr als einer an der Adhärenz beteiligten Struktur wurden beide Adhäsinregionen in einem Hybridprotein vereinigt, wodurch die adhärenzhemmende Wirkung des entsprechenden Serums in vitro mehr als additiv verstärkt werden konnte und dem Effekt des polyspezifischen Serums gegen M. pneumoniae entspricht. In Immunisierungs- und Infektionsversuchen mit dem Hybridprotein konnte über eine neu etablierte sehr sensitive real-time PCR basierend auf einer Multicopy-Targetsequenz in M. pneumoniae jedoch nur in einem Teil der Versuchstiere eine signifikante Hemmung der Kolonisierung des Respirationstraktes mit dem Erreger nachgewiesen werden. Eine Steigerung der humoralen Immunantwort durch Zugabe des mukosalen Adjuvans MALP-2 konnte nicht ausreichend belegt werden. Dagegen wurde durch Immunisierung auf intranasalem Wege eine signifikante Erhöhung des sekretorischen IgA-Titers in der BAL auf das Vierfache erreicht. Um einen stabil hohen sekretorischen Antikörperspiegel im Tiermodell zu erreichen, sind weitere Optimierungen des Immunisierungsschemas notwendig. Der Einsatz des Hybridproteins HP14/30, bestehend aus mehreren immunogenen und funktionalen Adhäsinbereichen, hat sich als sehr vielsprechend zur Entwicklung einer effektiven Vakzinierung von Risikopopulationen zur Prophylaxe von Infektionen mit M. pneumoniae erwiesen.

Thèse d'exercice : Médecine. Médecine générale : Paris 12 : 2006. / Titre provenant de l'écran-titre. Bibliogr. f. 83-88.

Les mycoplasmes sont des bactéries pathogènes, dotées de petits génomes d’environ 1Mbp, avec une faible teneur en G+C. L'intérêt de la communauté scientifique pour ces bactéries a été récemment renouvelé par des avancées dans les domaines de la synthèse et de la transplantation de génomes. Ces nouvelles approches ont ouvert la voie à l'ingénierie génomique à grande échelle des mycoplasmes. Les systèmes CRISPR/Cas sont des systèmes de défense adaptatifs procaryotes contre les acides nucléiques invasifs. Le système CRISPR de Streptococcus pyogenes est composé d’une endonucléase (SpCas9) et de deux CRISPR ARNs (crRNA et tracrRNA) qui dirigent Cas9 vers sa séquence d’ADN cible. La reconnaissance de l’ADN cible se fait par appariement du crRNA et de la présence en aval d’une séquence nommée protospacer adjacent motif (PAM). Apres cette reconnaissance, Cas9 coupe l’ADN cible. A partir de ce système, un outil génétique simplifié composé de Cas9 et d’un ARN guide (gRNA) a été développé pour de nombreux organismes. Le premier objectif de ma thèse était de combiner les méthodes de biologie synthétique de clonage et de la transplantation de génomes avec les outils CRISPR/Cas9 pour l’ingénierie des génomes de mycoplasmes clonés dans la levure. Nous avons réussi à utiliser cette approche pour enlever des gènes et des régions génomiques dans trois espèces: Mycoplasma mycoides subsp. capri (Mmc), M. capricolum subsp. capricolum et M. pneumoniae. Afin de développer un système plus adapté aux mycoplasmes, nous avons ensuite caractérisé le système CRISPR/Cas9 de Mycoplasma gallisepticum (Mg). En utilisant une combinaison d'approches in silico et in vivo, la séquence PAM de MgCas9 a été caractérisée comme NNNAAAA. Nous avons alors entrepris de développer un système CRISPR/Cas minimal de M. gallisepticum pour une utilisation directe dans les cellules de mollicutes: le gène codant MgCas9 a été introduit dans le génome de Mmc, mais son activation avec un gRNA chimère entre le crRNA et le tracrRNA de M. gallisepticum n’a pas été obtenue pour le moment. / Mycoplasmas are small pathogenic bacteria that are characterized by reduced genomes of about 1 Mbp with a low G+C content. The interest of the scientific community towards these species has been recently renewed by successful synthesis of their genome and transplantation experiments. These new genetic tools opened the way to further applications and developments for large-scale genome engineering programmes. CRISPR/Cas systems are natural systems that provide bacteria and archaea with an adaptive defense mechanism against invading nucleic acids. The CRISPR system from Streptococcus pyogenes includes an endonuclease (SpCas9) and two CRISPR RNAs (crRNA et tracrRNA) which role are to drive Cas9 to a target sequence. Target recognition depends on a specific pairing of the crRNA and the presence of a motif named protospacer adjacent motif (PAM). After recognition, Cas9 cleaves the targeted DNA. From the natural S. pyogenes system, a simplified genetic tool including Cas9 and a guide RNA (gRNA) was developed for many organisms . The first goal of my thesis was to combine the synthetic biology methods of genome cloning in yeast and back transplantation into recipient cells with a CRISPR/Cas9 tool for efficient engineering of mycoplasma genomes cloned in yeast. We succeeded in removing genes and genomic regions in three different species, Mycoplasma mycoides subsp. capri (Mmc), M. capricolum subsp. capricolum and M. pneumoniae. Then, in order to develop a system optimized for mycoplasma genome editing, we characterized a natural CRISPR/Cas9 system derived from Mycoplasma gallisepticum (Mg). Using a combination of in silico and in vivo approaches, MgCas9 PAM sequence was characterized as NNNAAAA. We then started to develop a minimal CRISPR/Cas system from M. gallisepticum for direct genome editing in mollicutes. Thus we introduced MgCas9 encoding gene in Mmc and tried to activate it with a newly designed gRNA, a chimeric molecule between the crRNA and the tracrRNA of M. gallisepticum, without success yet.

Memoria para optar al Título Profesional de Médico Veterinario. / Se recolectaron 453 muestras de suero de gallinas ponedoras pertenecientes a una empresa productora de huevos ubicada en la Región de Valparaíso, y se determinó la seroprevalencia de Mycoplasma gallisepticum (MG) y Mycoplasma synoviae (MS) a través de la detección de anticuerpos utilizando la prueba de ensayo por inmunoabsorción ligado a enzimas (ELISA). Las muestras se obtuvieron desde los 10 sectores con aves de diferentes edades pertenecientes a las 4 granjas que componen la empresa, y se evidenció que todos éstos presentaron individuos positivos a MG y/o MS. Del total de las 453 muestras de sueros, se observó que 239 (52,8%) eran positivas a MG, mientras que 322 (71,1%) eran positivas a MS. Los rangos de positividad fluctuaron entre un 6,7% y un 100% tanto para MG como para MS en los distintos sectores evaluados. Para ambos agentes se evaluaron diferencias en los factores grupo etario, tipo de galpón, granja y sector productivo. En el caso de MG, la granja Los Ceibos, los sectores Ceibo 1, Ceibo 2 y Ceibo 3, y los galpones abiertos, fueron los que mostraron una mayor cantidad de muestras positivas. Mientras que para MS, la granja Los Ceibos, los sectores Ceibo 1, Ceibo 3, Los Boldos y Los Espinos, los galpones abiertos y los grupos etarios de mediana y avanzada edad fueron los que mostraron una mayor cantidad de muestras positivas. / 453 serum samples from laying hens belonging to an egg producer located in the Valparaíso region were collected to determine the seroprevalence of Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) by using the enzyme-linked immunosorbent assay (ELISA). The samples were obtained from 10 sectors of the company, which had birds of different ages belonging to the company 4 farms. It was shown that all these sectors have positive MG and / or MS birds. Of the total of 453 serum samples 239 (52.8%) were positive to MG, while 322 (71.1%) were positive to MS. The ranges of positivity ranged between 6.7% and 100% for both MG and MS in the different sectors evaluated. Differences in certain factors were evaluated for both agents: age, type of barn, farm and production sector. For MG, the farm Los Ceibos, the Ceibo 1 sector, Ceibo 2 sector, Ceibo 3 sector and open sheds showed a greater number of positive samples. For MS, the farm Los Ceibos, the Ceibo 1 sector, Ceibo 3 sector, Los Boldos sector, Los Espinos sector, open sheds and the age groups of middle and old age were those who had a higher number of positive samples.

Alicerçado em trabalhos contidos na literatura internacional sobre a implicação dos micoplasmas nas aIterações do trato geniturinário e sobretudo nas chamadas \"falhas da reprodução\", o presente estudo teve como objetivo, além da introdução de uma tecnologia de pesquisa desses microrganismos acessível ao laboratório clínico de rotina diagnóstica, também determinar as taxas de colonização de Mycoplasma hominis e de Ureaplasma urealyticum em gestantes pertencentes a uma classe de baixo nível sócio-econômico. Estas gestantes, em número de 74 (setenta e quatro), foram divididas em dois grupos: um grupo de 37 (trinta e sete) gestantes que possuía histórico anterior de alterações perigestacionais como: abortos, natimortos, prematuros, etc., e que constituíram o Grupo de Risco; e o outro constituído de 37 (trinta e sete) gestantes com gestações anteriores normais, Grupo Controle. Utilizando-se os materiais e métodos adaptados às condições de nosso meio, foram encontrados os seguintes resultados: a proporção de micoplasmas isolada foi de 94,6% no grupo de risco e de 81,2% no grupo controle. Do grupo de risco, Ureaplasma urealyticum foi isolado de vlnte e um pacientes (56,8%), Mycoplasma hominis de dois (5,4%) e ambos foram encontrados em seis pacientes (16,2%). No grupo controle, Ureaplasma urealyticum foi encontrado em 18 pacientes (48,6%), Mycoplasma hominis não foi isolado de nehuma (0,0%), enquanto que a associação Ureaplasma urealyticum e Mycoplasma hominis foi observado em seis casos (16,2%). Foi ainda verificado que onze (30,5%) gestações terminaram anormalmente no grupo de risco, sendo quatro (11,1%) de pacientes que não possuíam quaisquer alterações clínicas que as justificassem. No grupo controle, nenhuma alteração foi verificada entre os casos notificados. A análise estatística, dos resultados, permitiu verificar que não houve diferenças significantes entre os grupos estudados. / Abstracts Not Available

Les mycoplasmes sont des bactéries à petit génome dérivées d’ancêtres à Gram positif par une succession de pertes de matériel génétique. Il a longtemps été considéré que la réduction génétique était la seule force régissant l’évolution de ces bactéries, cependant, des transferts horizontaux de grandes régions chromosomiques au sein et entre les espèces de mycoplasmes ont été récemment mis en évidence. Des éléments conjugatifs et intégratifs (ICE) découverts chez certaines espèces de mycoplasme pourraient être à l’origine de ces transferts. Ces ICEs codent les systèmes nécessaires pour leur excision, leur transfert conjugatif et leur intégration dans la cellule receveuse.Mycoplasma hominis est un mycoplasme commensal des voies génitales qui peut être responsable d’infections gynécologiques, d’infections néonatales et d’infections extragénitales. L’analyse préliminaire de génomes de M. hominis avait montré la présence de régions codantes caractéristiques des ICEs. Les objectifs de cette thèse étaient de rechercher et caractériser les ICEs chez 12 isolats cliniques de M. hominis entièrement séquencés et de déterminer la prévalence de ces ICEs au sein de l’espèce M. hominis. Pour cela, une étude rétrospective sur une période de 6 ans a été menée sur des isolats cliniques obtenus au CHU de Bordeaux. Les concentrations minimales inhibitrices des tétracyclines et des fluoroquinolones ainsi que les mécanismes de résistance ont été déterminés, permettant de disposer d’une collection d’isolats cliniques caractérisés pour l’étude des ICEs.Des ICEs de près de 30 kpb ont été trouvés en une ou plusieurs copies dans sept des 12 souches de M. hominis séquencées. Seulement cinq de ces ICEs semblaient fonctionnels puisqu’une forme circulaire a pu être détectée. Tous les ICEs de M. hominis présentaient une structure similaire avec un module spécifique de M. hominis d’environ 4-kpb, codant des protéines ayant des caractéristiques structurelles similaires à des effecteurs TAL (transcription activator-like), impliqués dans la reconnaissance de nucléotides et dans la transduction de signaux chez les bactéries symbiotiques. La caractérisation des mécanismes de résistance aux antibiotiques des isolats cliniques de M. hominis collectés au CHU de Bordeaux nous a permis de disposer d’une collection de 183 isolats isolés entre 2010 et 2015, parmi lesquels 14,8% étaient porteur du gène tet(M) responsable de la résistance aux tétracyclines, 2,7% étaient résistant à la lévofloxacine et 1,6% étaient résistants à la moxifloxacine par mutation des gènes de la topoisomérase IV et de l’ADN gyrase. Le screening de 120 de ces isolats cliniques a révélé une prévalence élevée des ICEs dans l’espèce M. hominis, mesurée à 45%. Il n’y avait pas de prédominance des ICEs dans les isolats portant le gène tet(M), suggérant que les ICEs n’étaient pas responsables de la dissémination de la résistance à la tétracycline.Des expériences complémentaires de conjugaison seront nécessaires pour confirmer la fonctionnalité des ICEs retrouvés dans l’espèce M. hominis. Cependant, la forte prévalence et le caractère très conservé des ICEs chez M. hominis suggèrent que ces ICEs pourraient conférer un avantage sélectif pour la physiologie ou la physiopathologie de la bactérie. Ce travail ouvre ainsi la voie à de futures études qui permettront une meilleure compréhension des transferts horizontaux de gènes et des facteurs de virulence chez M. hominis. / Mycoplasmas are small-genome bacteria derived from Gram-positive ancestors by a succession of genetic material losses. It has long been considered that genetic reduction was the only force governing the evolution of these bacteria, however, horizontal transfers of large chromosomal regions within and between mycoplasma species have recently been reported. Conjugative and integrative elements (ICE) found in some species of mycoplasma may be responsible for these transfers. These ICEs encode the systems necessary for excision, conjugative transfer and integration into a recipient cell.Mycoplasma hominis is a commensal genital mycoplasma that can be responsible for gynecological infections, neonatal infections and extragenital infections. Preliminary analysis of M. hominis genomes had showed the presence of coding regions characteristic of ICEs. The objectives of this thesis were to search for and characterize ICEs in one reference strain and 11 fully sequenced M. hominis clinical isolates and to determine the prevalence of these ICEs in the M. hominis species. To do so, a retrospective study over a period of 6 years was conducted on clinical isolates collected at the Bordeaux University Hospital. The minimum inhibitory concentrations of tetracyclines and fluoroquinolones as well as resistance mechanisms were determined, providing a collection of clinical isolates characterized for the study of ICEs.ICEs of 27-30 kpb were found in one or two copies in seven of the 12 M. hominis sequenced strains. Only five of these ICEs seemed functional since circular forms of extrachromosomal ICE were detected. All M. hominis ICEs exhibited a similar structure consisting of a 4.0-5.1 kb module composed of five to six juxtaposed CDSs, encoding proteins that share common structural features with transcription activator-like (TAL) effectors, involved in polynucleotide recognition and signal transduction in symbiotic bacteria. The characterization of antibiotic resistance mechanisms in M. hominis clinical isolates collected at Bordeaux University Hospital enabled us to obtain a collection of 183 isolates isolated between 2010 and 2015, of which 14.8% harbored the tet(M) gene responsible for tetracycline resistance, 2.7% were resistant to levofloxacin and 1.6% were resistant to moxifloxacin by mutation in topoisomerase IV and DNA gyrase genes. Screening of 120 of these clinical isolates revealed a high prevalence of ICEs in M. hominis, measured to be 45%. The proportion of ICEs was not higher in isolates carrying the tet (M) gene, suggesting that ICEs were not responsible for the spread of tetracycline resistance.Additional mating experiments will be necessary to confirm the functionality of the ICEs found in the M. hominis species. However, the conserved and specific structure of M. hominis ICEs and the high prevalence in clinical strains suggest that these ICEs may confer a selective advantage for the physiology or pathogenicity of the bacteria. This work opens the way for future studies that will provide a better understanding of horizontal gene transfers and virulence factors in M. hominis.

No description available.

Mycoplasma hyopneumoniae é o agente etiológico da pneumonia enzoótica suína (PES), uma doença crônica, caracterizada pela alta morbidade e baixa mortalidade, e que infecta 200 milhões de suínos anualmente, gerando milhões de dólares de perdas em todo o mundo. M. hyopneumoniae adere-se aos cílios das células do epitélio traqueal, causando a redução do movimento ciliar, predispondo o animal a patógenos secundários. É uma das menores bactérias presentes na natureza, apresentando um genoma reduzido, sem parede celular e com alto conteúdo de guanina+citosina. Atualmente, estão disponíveis as sequências dos genomas de três linhagens de M. hyopneumoniae, duas patogênicas (7448 e 232) e uma não patogênica (J), porém pouco se sabe a respeito das sequências que regulam e controlam a expressão gênica em espécies de Mycoplasma ou das proteínas que ligam ao DNA, que desempenham um papel fundamental em todos os aspectos genéticos do organismo, como transcrição, replicação e reparo. Tipicamente, 2-3% de um genoma procariótico e 6-7% de um eucariótico codificam proteínas que ligam ao DNA. A interação mais comum entre proteína e DNA ocorre entre uma alfa-hélice da proteína com a cavidade maior do DNA, sendo que aproximadamente 84% dos fatores de transcrição de um componente utilizam o motivo hélice-volta-hélice para a ligação ao DNA Sendo assim, a hélice-volta-hélice assume um papel central na regulação da trancrição e, portanto, torna-se um excelente alvo para análises in silico. Não há relatos de trabalhos de identificação de proteínas que ligam ao DNA de M. hyopneumoniae utilizando métodos experimentais e predições in silico. Neste trabalho foi empregado um método de cromatografia de afinidade DNA-celulose para obter amostras enriquecidas com proteínas de superfície, onde foram identificas 32 proteínas, sendo que muitas delas são conhecidas e estão realmente envolvidas em processos que requerem a ligação ao DNA. Além da análise experimental, uma análise utilizando o genoma de M. hyopneumoniae 7448 foi realizada, utilizando vários algoritmos designados a encontrar genes que codificam proteínas que ligam ao DNA, sendo que 59 proteínas foram preditas como ligantes a DNA. Da sobreposição dos dados obtidos in silico e experimentalmente foram identificadas sete proteínas, sendo duas hipotéticas. A identificação destas proteínas disponibiliza um grupo de proteínas-alvo para futuros estudos de regulação e controle da expressão gênica de M. hyopneumoniae. / Mycoplasma hyopneumoniae is the etiological agent of Enzootic Pneumonia (EP), a chronical disease, characterized by high morbidity and low mortality that infects 200 million pigs every year causing hundreds of millions of dollars of loss for swine farmers worldwide. M. hyopneumoniae attaches to the cilia of the tracheal epithelial cells, causing a reduction in the ciliary action, predisposing the swine to secondary pathogens. M. hyopneumoniae is one of the smallest bacteria present in nature with a small genome, no cell wall and high guanine/cytosine content. Published genome sequences of three M. hyopneumoniae strains, two pathogenic (232 and 7448) and one nonpathogenic (J) are available, but little is understood about the sequences that regulates and control gene expression in Mycoplasma species and its DNA-binding proteins (DBPs), that plays a central role in all aspects of genetic activity within an organism, such transcription, replication and repair. Typically 2-3% of a prokaryotic genome and 6-7% of a eukaryotic genome encodes genes that codify DBPs proteins. The common protein-DNA interaction region in bacteria occurs between an alpha-helix present in the protein and the major groove of DNA. Among the bacterial one-component transcription factors (TFs), up to 84% of the output domains comprise a DNA-binding helix–turn–helix (HTH) region. Therefore, the HTH motif assumes a central role in the transcription regulation and becomes a main target to in silico predictions. There have been no reports of the in silico and experimental identification of DBPs in M. hyopneumoniae. In this work we employed a method of DNA-cellulose column chromatography to obtain a DBPs enriched sample, for later mass espectrometry analysis, were we identify 32 proteins, many of which are involved in biological processes that require DNA binding. In addition to the proteomic approach, genomic analysis of the M. hyopneumoniae 7448 genome was performed in silico with several algorithms designed to identify genes that encoded DBPs and we have characterized 59 proteins predict as DBPs. The overlap analyzes between bioinformatics and proteomics has resulted in the identification of seven proteins, two of which are hypothetical proteins. The identification of these proteins has provided a valuable set of proteins that may be used in the studies to analyze the regulation and control of the gene expression in M. hyopneumoniae.

Thesis (Ph. D.)--Mississippi State University. Department of Poultry Science. / Title from title screen. Includes bibliographical references.

Introduction: Mycoplasma pneumoniae(M. pneumoniae) causes 10% to 30% of community-acquired pneumonia (CAP). The commonly used first-line antibiotic macrolide (ML) against respiratory tract infection may lead to the increase of ML-resistant M. pneumoniaeinfection. To resolve the problem, a rapid and accurate method for detection of ML-resistant M. pneumoniaeis necessary for treatment adjustment. Aims: The study aims to (1) develop a rapid method for diagnosis of ML-resistance of M. pneumoniaedirectly from clinical specimens; and (2) investigate the prevalence of M. pneumoniaeand ML-resistant M. pneumoniae. Methods: The M. pneumoniaeqPCR results of 689 respiratory tract samples from Queen Mary Hospital collected during April 2010 to May of 2012 were analyzed. Positive nucleic acid from M. pneumoniaeqPCR samples were tested with SimpleProbe real-time PCR coupled to melting curve analysis (SimpleProbe PCR), pyrosequencing and 23S rRNA gene sequencing(23S sequencing) for detection of ML-resistance. Results: A total of 111 samples (16.11%) in 689respiratory tract samples were found M. pneumoniaepositive by qPCR. Of 111, 96 positive nucleic acids were available for this study. Overall, 29 (30.21%, n=96) of ML-resistant M. pneumoniaewere found. 23S sequencing identified 28 mutants (29.17%) and 62 wild–type (64.58%), while 6 (6.25%) of them are failed to be identified. Pyrosequencing identified 28 mutants (29.17%) and 63 wild–type (65.63%), while 5 (5.21%) of them are failed to be identified. The SimpleProbe PCR identified 29 mutants (30.21%) and 65 wild–type (67.71%), while 2 (2.08%) of them are failed to be identified. All ML-resistant M. pneumoniaepositives were found to have A2063G mutation either by 23S sequencing or pyrosequencing. Conclusion: From this study, SimpleProbe PCR is the most sensitive and simple to perform. Therefore, it is highly recommended to be included in the routine testing with positive M. pneumoniaesamples for diagnosis of ML-resistant strain. 23S sequencing or pyrosequencing is recommended to use as a confirmatory test if necessary. / published_or_final_version / Microbiology / Master / Master of Medical Sciences