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Submitted by Carla Ferreira (carlaferreira66@gmail.com) on 2014-07-31T12:23:26Z No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) HerminioSobrinho-2008 (1).PDF: 811492 bytes, checksum: 63a0aabcbb6458adb1051bc054066d08 (MD5) / Made available in DSpace on 2014-07-31T12:23:26Z (GMT). No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) HerminioSobrinho-2008 (1).PDF: 811492 bytes, checksum: 63a0aabcbb6458adb1051bc054066d08 (MD5) Previous issue date: 2008 / Rheumatoid Arthritis (RA) and Systemic Lupus Erythematosus (SLE) are autoimmune diseases of unknown etiology. Some species of mycoplasmas cause arthritis in animals and humans, and their lipid-associated membrane proteins (LAMPs) and Mycoplasma arthritidis mitogen (MAM superantigen) are potent stimulators of the immune system. Thus, it has been proposed that mycoplasma can be involved in autoimmune-disease etiology. The objective of the present work was to detect antibodies to MAM and LAMPs of M. hominis and M. fermentans in the patient sera, and to characterize the profile of IgG antibodies reactivity with LAMPs in order to identify the major immunogenic mycoplasmal lipoproteins that could be involved in the etiopathogenesis of these autoimmune diseases. Serum samples were obtained from peripheral blood of female patients at the same age of healthy controls. Recombinant MAM (from M. arthrititidis), LAMPs of M. hominis PG21 and M. fermentans PG18 were used in Western blotting assays. Antibodies to MAM were detected in the patient and control sera (RA: 27.5% vs 18.8%; SLE: 21.7% vs 20.0%). At least 23 LAMPs were found in the preparations of M. hominis PG21 and of M. fermentans PG18 with molecular masses between 20 and 192 KDa. The sera of RA patients recognized a larger number of LAMPs of M. hominis PG21 and M. fermentans PG18 than the control sera (RA: 11 ± 4 vs controls: 7 ± 3, n = 35; p < 0,05). Most of the sera of RA patients presented strong reactivity with LAMPs of M. hominis PG21 (RA: 65.7% vs controls: 20%, p < 0.05). LAMPs of M. hominis PG21 with molecular masses < 49 and ? 20 KDa and LAMPs of M. fermentans PG18 < 102 and ? 58 were mainly recognized by IgG antibodies of RA patients. When comparing sera from SLE patients and controls there was detected no significant differences between the profiles of IgG reactivity. Therefore, M. hominis PG21 LAMPs (< 49 and ? 20 KDa) and M. fermentans PG18 LAMPs (< 102 and ? 58 KDa) are high immunogenic mycoplasmal antigens that can induce antibody cross reactivity with self antigen, contributing with the RA pathogenesis. / A artrite reumatóide (AR) e o lúpus eritematoso sistêmico (LES) são doenças autoimunes de etiologia desconhecida. Algumas espécies de micoplasmas causam artrite séptica em seres humanos, sendo estas bactérias fortes candidatos à etiopatogênese destas doenças. O superantígeno MAM é uma proteína secretada por Mycoplasma arthritidis, que juntamente com lipoproteínas (LAMPs) de M. hominis e M. fermentans, ativam as células do sistema imune e podem estar envolvidos na etiopatogenia da AR e do LES. O objetivo do presente trabalho foi detectar e caracterizar a resposta de anticorpos IgG contra superantígeno MAM e LAMPs de M. fermentans e M. hominis em soros de pacientes com AR ou LES, a fim de detectar as LAMPs mais imunogênicas candidatas a antígenos envolvidos na etiopatogenia destas doenças. Os pacientes com AR ou LES e os controles saudáveis eram indivíduos do sexo feminino e da mesma faixa etária. Foi usado MAM recombinante e LAMPs de M. hominis PG21 e M. fermentans PG18 extraídas com detergente Triton X-114, para avaliar o perfil de anticorpos IgG por meio da técnica de Western blotting. Anticorpos IgG anti-MAM foram detectados tanto nos soros de pacientes quanto nos dos controles (AR: 27,5% vs 18,8%; LES: 21,7% vs 20,0%). Foram detectadas pelo menos 23 LAMPs nas preparações de M. hominis PG21 e de M. fermentans PG18 com massas moleculares entre 20 e 192 KDa. Os soros de pacientes com AR reconheceram um maior número de LAMPs de M. hominis PG21 e de M. fermentans PG18 do que os soros controles (AR: 11 ± 4 vs controles: 7 ± 3, n = 35; p < 0,05). A maioria dos soros dos pacientes com AR apresentou forte reatividade com LAMPs de M. hominis PG21 (AR: 65,7% vs controles: 20%, p < 0,05). As LAMPs de M. hominis PG21 com massas moleculares <49 e ³ 20 KDa e de M. fermentans PG18 < 102 e ? 58 foram mais frequentemente reconhecidas por anticorpos IgG de soros de pacientes com AR do que por anticorpos dos soros controles. Não foram atestadas diferenças significantes entre os perfis de reatividade dos soros de pacientes com LES e controles, nem com relação ao número de LAMPs reconhecidas, nem com as diferentes faixas de massas moleculares das LAMPs. Portanto, as LAMPs de M. hominis (<49 e ³ 20 KDa) e M. fermentans (< 102 e ? 58) podem ser antígenos que induzem a produção de anticorpos que reagem cruzadamente com antígenos próprios, contribuindo para o processo da patogênese da AR.

Diss. (sammanfattning) Linköping : Univ., 2004. / Härtill 5 uppsatser.

BACKGROUND:Mycoplasma hyopneumoniae causes respiratory disease in swine and contributes to the porcine respiratory disease complex, a major disease problem in the swine industry. The M. hyopneumoniae strain 232 genome is one of the smallest and best annotated microbial genomes, containing only 728 annotated genes and 691 known proteins. Standard protein databases for mass spectrometry only allow for the identification of known and predicted proteins, which if incorrect can limit our understanding of the biological processes at work. Proteogenomic mapping is a methodology which allows the entire 6-frame genome translation of an organism to be used as a mass spectrometry database to help identify unknown proteins as well as correct and confirm existing annotations. This methodology will be employed to perform an in-depth analysis of the M. hyopneumoniae proteome.RESULTS:Proteomic analysis indicates 483 of 691 (70%) known M. hyopneumoniae strain 232 proteins are expressed under the culture conditions given in this study. Furthermore, 171 of 328 (52%) hypothetical proteins have been confirmed. Proteogenomic mapping resulted in the identification of previously unannotated genes gatC and rpmF and 5-prime extensions to genes mhp063, mhp073, and mhp451, all conserved and annotated in other M. hyopneumoniae strains and Mycoplasma species. Gene prediction with Prodigal, a prokaryotic gene predicting program, completely supports the new genomic coordinates calculated using proteogenomic mapping.CONCLUSIONS:Proteogenomic mapping showed that the protein coding genes of the M. hyopneumoniae strain 232 identified in this study are well annotated. Only 1.8% of mapped peptides did not correspond to genes defined by the current genome annotation. This study also illustrates how proteogenomic mapping can be an important tool to help confirm, correct and append known gene models when using a genome sequence as search space for peptide mass spectra. Using a gene prediction program which scans for a wide variety of promoters can help ensure genes are accurately predicted or not missed completely. Furthermore, protein extraction using differential detergent fractionation effectively increases the number of membrane and cytoplasmic proteins identifiable my mass spectrometry.

Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Mycoplasma nasistruthionis sp. nov. str. Ms03 (Ms03) is one of three Mycoplasma species that were identified from ostriches. Mycoplasmas infections have been implicated in ostrich chick mortalities, growth retardation and downgrading of ostrich carcasses. Currently there is no vaccine available for the treatment of mycoplasmosis in ostriches. This study investigated the development of DNA vaccines against Ms03 infections in ostriches. To this end, the Ms03 genome was sequenced and annotated. The vaccine candidate gene, oppA, was identified within the genome sequence and characterized before DNA vaccines containing the oppA were developed and tested. The genome of Ms03 was sequenced and the resulting 172 contigs were annotated. This dissertation presents the first Ms03 draft genome and annotation which contributed to the understanding of Ms03 as a miniature genetically independent organism. In Ms03, genome replication, cell division, RNA transcription, protein translation and glycolysis resemble that of the closely related Mycoplasma synoviae 53. Purine and pyrimidine metabolism was incomplete and de novo synthesis thereof was not possible. Amino acid synthesis in Ms03 was mostly absent and only the genes that convert aspartate to asparagine and glycine to serine were found. More importers than exporters were annotated owing to the lack of synthesis pathways in Ms03, which is typical for mycoplasmas that have parasitic life styles. Two oligopeptide permease (opp) operons were annotated within the Ms03 genome. The potential of the oppA as a vaccine candidate gene was evaluated by investigating the need for a substrate-binding domain (OppA) as part of the OppBCDF transporter within Mycoplasma species. An oppA homologue could be identified for each oppBCDF operon in all species and therefore must play an essential role in oligopeptide transport. All mycoplasmas (except for hemoplasma) had one, two or three opp operons that could be divided into three types (Type A, B and C). Each type had unique InterPro and MEME domains and motifs which together with the phylogenetic analysis suggest unique roles in their survival under different conditions. Ms03 had a Type A and a Type B opp operon, the Type A oppA was used as vaccine candidate gene. The Type A oppA was cloned and site-directed mutagenesis was used for codon correction before the mutated gene was sub-cloned into three DNA vaccine vectors. The three DNA vaccines (pCI-neo_oppA, VR1012_oppA and VR1020_oppA) were used to vaccinate ostriches and the OppA-antibody response was analysed by ELISA. The VR1020_oppA and pCI-neo_oppA constructs elicited a primary immune response in ostriches, indicating that the OppA protein was expressed in vivo and was immunogenic. This can therefore be viewed as the first step in the development of a DNA vaccine for the control of mycoplasma infections in ostriches. / AFRIKAANSE OPSOMMING: Mycoplasma nasistruthionis sp. nov. str. Ms03 (Ms03) is een van drie mikoplasma spesies wat volstruise infekteer. Mikoplasma-infeksies in volstruise veroorsaak kuiken vrektes, vertraagde groei en afgradering van volstruis karkasse. Daar is tans geen geregistreerde mikoplasma entstof beskikbaar vir gebruik in volstruise nie. Hierdie studie het die ontwikkeling van DNS-entstowwe teen Ms03-infeksies in volstruise ondersoek. Vir hierdie doel was die Ms03-genoomvolgorde bepaal en geannoteer. Die entstofkandidaat-geen, oppA, was geïdentifiseer in die genoomvolgorde en gekarakteriseer voordat DNS-entstowwe (wat die oppA-geen bevat) ontwikkel en getoets is. Die Ms03-genoomvolgorde was bepaal en die gegenereerde 172 aaneenlopende volgordes was geannoteer. Hierdie proefskrif bied die eerste voorlopige volgorde en annotering van die Ms03-genoom wat bygedra het tot die kennis van Ms03 as 'n miniatuur geneties onafhanklike organisme. Genoom-replikasie, seldeling, RNS-transkripsie, proteïen-translasie en glikolise in Ms03 stem ooreen met dié prosesse in die naverwante Mycoplasma synoviae 53. Die purien en pirimidien metabolisme was onvolledig en de novo sintese daarvan was nie moontlik in Ms03 nie. Aminosuursintese in Ms03 was meestal afwesig en net die gene wat aspartaat omskep na asparagien en glisien na serien was gevind in die annoteerde genoom. Meer invoerders as uitvoerders was geannoteer, wat dui op die gebrek aan sintesepadweë in Ms03. Dit is tipies van mikoplasmas wat ‘n parasitiese lewensstyle het. Twee oligopeptied-permeases (opp) operons was gevind in die Ms03-genoom. Die potensiaal van die oppA-geen as 'n entstofkandidaat-geen was geëvalueer deur die behoefte van 'n substraatbindingsdomein (OppA) as deel van die OppBCDF-vervoerder binne mikoplasma spesies te ondersoek. 'n Homoloog van die oppA-geen kon geïdentifiseer word vir elke oppBCDF-operon in al die spesies en behoort daarom 'n noodsaaklike rol te speel in die vervoer van oligopeptiede. Alle mikoplasmas (behalwe vir hemoplasmas) het een, twee of drie opp-operons, wat verdeel kan word in drie tipes (Tipe A, B en C). Elke tipe het unieke InterPro en MEME domeine en motiewe wat saam met die filogenetiese ontleding daarop dui dat hulle unieke rolle in oorlewing onder verskillende omstandighede speel. Ms03 het 'n Tipe A en Tipe B opp-operon, die Tipe A oppA is gebruik as entstofkandidaat-geen. Die Tipe A oppA-geen was gekloneer en teikengerigte-mutagenese was gebruik vir kodonregstellings voordat die gemuteerde geen in drie DNS-entstof vektore gesubkloneer was. Die drie DNS-entstowwe (pCI-neo_oppA, VR1012_oppA en VR1020_oppA) was gebruik om volstruise in te ent en die OppA-teenliggaamsreaksie was geanaliseer deur ELISA. Inenting met die VR1020_oppA en pCI-neo_oppA entstowwe het tot 'n primêre immuniteitsreaksie in volstruise gelei. Dit dui daarop dat die OppA proteïen in vivo uitgedruk en immunogenies was. Dit kan beskou word as die eerste stap in die ontwikkeling van 'n DNS-entstof vir die beheer van mikoplasma-infeksies in volstruise.

A Mycoplasma pneumoniae se le atribuye entre un 15 a 20% de las neumonías adquiridas en la comunidad en los adultos mayores. Este estudio, el primero realizado en Chile para adultos mayores, se hizo para conocer la frecuencia, mediante la técnica de Reacción de la Polimerasa en Cadena, de Mycoplasma pneumoniae en este grupo etario y para probar la sensibilidad y especificidad analítica de ésta usando los partidores para el gen de la adhesina P1 y el gen del rRNA de 16S en el diagnóstico de dicho microorganismo, datos que no han sido estandarizados, como sí lo son las pruebas diagnósticas de Serología. Sin embargo, las pruebas de Serología no cuentan con una sensibilidad lo suficientemente buena, por lo que es necesario aplicar técnicas más sensibles y rápidas para la detección de Mycoplasma pneumoniae como lo es la Reacción de la Polimerasa en Cadena. Para llevar a cabo el estudio se tomaron muestras de expectoración y de sangre a 84 adultos mayores con neumonía adquirida en la comunidad, pertenecientes al Hospital Clínico de la Universidad de Chile, entre Agosto de 2002 a Septiembre de 2004, a los cuales se les realizó la técnica de Reacción de la Polimerasa en Cadena para ambos genes y la técnica de Serología Inmunofluorescencia Indirecta para IgM e IgG. A partir de los datos obtenidos se determinó que el porcentaje de Mycoplasma pneumoniae presente en los pacientes con neumonía fue de 8.3%, cifra inferior a la citada por la literatura y a la obtenida mediante la complementación de las técnicas de Reacción de la Polimerasa en Cadena y serología. Entre ambos partidores no existen diferencias significativas en cuanto a su sensibilidad y especificidad analítica. No obstante, se requiere de más estudios que verifiquen la calidad de estas técnicas para que lleguen a estandarizarse y constituir así técnicas de diagnóstico rápido de Mycoplasma pneumoniae, lo cual contribuiría importantemente a la clínica.

Les mycoplasmes sont des bactéries atypiques, dépourvues de paroi et souvent considérées comme des cellules minimales du fait de la taille réduite de leur génome. De nombreuses espèces sont pathogènes et ont un impact économique important dans les filières d'élevage, notamment pour les ruminants. Les mycoplasmes n'échappent pas au phénomène mondial de la résistance aux antibiotiques. Contrairement à la plupart des autres bactéries, les mycoplasmes ne contiennent pas de plasmides conjugatifs souvent incriminés dans la dissémination horizontale de gènes de résistance, la base moléculaire principalement décrite étant la mutation chromosomique des gènes cibles. De façon générale, le transfert horizontal de gènes (HGT) chez les mycoplasmes a longtemps été sous-estimé. Récemment, deux mécanismes de HGT ont été décrits chez Mycoplasma agalactiae : le transfert d'élément conjugatif et intégratif (ICE), et le transfert non-conventionnel de régions chromosomiques par conjugaison appelé MCT (Mycoplasma Chromosomal Transfer). Nos travaux se sont attachés à explorer ce dernier mécanisme et à évaluer son impact sur l'acquisition de la résistance aux antibiotiques. Une analyse de génomique comparative a été conduite à partir du séquençage de nombreux mutants spontanément résistants et de transconjugants générés par des expériences de mating et sélectionnés pour leur résistance. Nos résultats montrent que le MCT conduit de façon distributive au transfert simultané de nombreux fragments. En une seule étape de conjugaison impliquant deux souches, ce phénomène génère une population variée de génomes hautement mosaïques. Il accélère la dissémination de l'antibiorésistance, permettant l'acquisition de plusieurs mutations distantes associées à la résistance en un seul événement. De par les multiples possibilités de réassemblage génomique qu'il produit, le MCT pourrait avoir des conséquences importantes sur d'autres processus adaptatifs comme la virulence ou la spécificité d'hôte. Enfin, les modalités distributives et l’ampleur du MCT expliquent l'origine des transferts de gènes précédemment détectés in silico entre de nombreux mycoplasmes. Ce phénomène pourrait donc avoir eu des répercussions importantes sur l'évolution de ces bactéries minimales et être un facteur clé de leur persistance et virulence actuelles.

A partir de meados da década de 80, com a expansão da cultura do milho para além das épocas tradicionais de cultivo, quer pela prática da safrinha ou por plantios irrigados, vem ocorrendo um aumento na incidência de doenças a secundária. Dentro deste contexto, o enfezamento vermelho do milho, relatado no país primeiramente em 1970, vem ocorrendo de forma freqüente, apresentando altos índices de ocorrência, muitas vezes com comprometimento total da produção. As plantas infectadas apresentam uma sintomatologia complexa facilmente confundida com viroses. O sintoma mais característico é o avermelhamento foliar. Além do avermelhamento as plantas apresentam redução na altura, perfilhamento basal e axilar, espigas extranumerárias e colmos afinados. Essa doença é causada por um procarioto não cultivável em meio de cultura, habitante exclusivo do floema, denominado fitoplasma, veiculado de forma persistente e propagativa pela cigarrinha Dalbulus maidis. Devido às características do patógeno, a única forma de controle promissora é a utilização de variedades tolerantes/resistentes. Para eficiência na obtenção destas variedades é necessário um diagnóstico correto e conhecimento sobre a variabilidade metodologia mais eficiente tanto para diagnose correta como para investigar essa variabilidade tem sido o PCR. O PCR, utilizando oligonucleotídeos baseados no gene 16SrDNA seguido da análise de RFLP, proporciona uma identificação mente a classificação de fitoplasmas é fundamentada no perfil molecular obtido por análise de RFLP de fragmentos do gene 16SrDNA amplificados. Com base nestas considerações, o trabalho teve como objetivo caracterizar molecularmente isolados do fitoplasma associado ao enfezamento vermelho do milho, coletados em quatro regiões produtoras de milho do estado de São Paulo. A sintomatologia para cada amostra de milho foi anotada. Foram usados dois pares de oligonucleotídeos universais para fitoplasmas em duplo PCR, um par de oligonucleotídeo especificamente desenvolvido para detecção do fitoplasma do enfezamento vermelho do milho, além de oligonucleotídeos para detecção de fitoplasmas pertencentes a grupos específicos. Após amplificação e eletroforese, 29 isolados foram selecionados para a identificação através de RFLP. Fragmentos de DNA foram submetidos à digestão com diferentes enzimas de restrição com o objetivo de identificar/classificar o fitoplasma. Os padrões de bandas obtidos após a eletroforese em gel de poliacrilamida foram comparados com os padrões atuais para classificação dos fitoplasmas. Todos os isolados analisados apresentaram idênticos padrões de bandas, para cada enzima de restrição, considerada individualmente. Não houve diferenciação de acordo com a região geográfica de coleta ou de acordo com intensidade de sintomas apresentados. Todos os isolados foram identificados como pertencentes ao grupo I e subgrupo B da classificação molecular atualmente adotada para estes microorganismos. / Since the middle 80s, an increase in year round cropping of maize resulted in a spread of secondary diseases in the crop’s major production areas. In this context, maize busy stunt, firstly appointed in Brazil in 1970, is occurring more frequently, often with total damage of production. Infected plants show a complex symptomatology, easily confounded with virus-caused diseases. The most characteristic symptom is leaf reddening. Besides the reddening diseased plants show stunting, often developing tillering. This disease is caused by a phytoplasma, a wall- less prokaryote, uncultivable, phloem inhabitant. This pathogen is transmitted by the leafhopper Dalbulus maidis, a in persistent and propagative manner. Due to the pathogen’s characteristics, the best control measure is the use of tolerant/resistant plants. For efficiency in breeding, accurate procedures of detection and an investigation of the pathogen’s genetic variability are necessary. The more accurate manner is using PCR. PCR, using 16SrDNA based primers pairs and followed by RFLP analysis, offers a safe identification of the pathogen. Today the phytoplasma classification is based in molecular patterns obtained by RFLP analysis of amplified 16SrDNA gene fragments. This work’s objective was the molecular characterization of maize bushy stunt phytoplasma strains collected in four corn production areas in São Paulo state, Brazil. The simptomatology to every maize sample was saved. Two primer pairs in nested PCR and a specific primer pair developed to MBS detection were used, besides group specific phytoplasma primers pairs. After amplification and electrophoresis, 29 samples were selected. These selected samples were digested with different restriction enzymes to identify/classify the phytoplasma. The fragment’s sizes obtained by electrophoresis through 4,5% polyacrilamide gel were compared with the reference’s classification patterns. All analyzed samples showed identical fragment-size patterns, for each restriction enzyme considered individually. There was no difference between these samples according to geographic collect region or according to symptoms. All strains were identified as belonging to group I and subgroup B of molecular classification.