Mycobacterium tuberculosis atteint le tiers de la population mondiale et cause plus de 1.5 millions de décès chaque année. L’augmentation des infections chez les patients immuno-compromis et l’émergence d’infections à de nouvelles souches multirésistantes aux antibiotiques somme la communauté scientifique à la découverte de nouvelles cibles thérapeutiques ainsi qu’au développement de nouveaux antibiotiques, vaccins et thérapies. Parmi les ~ 4000 gènes du génome de Mycobacterium tuberculosis, un d’entre eux, glbN, code pour l’hémoglobine tronquée N. Les hémoglobines sont de petites métalloprotéines qui fixent réversiblement l’oxygène et qui effectuent plusieurs activités catalytiques importantes. Ainsi, nos recherches s’inscrivent dans le cadre d’une approche biochimique qui vise à définir une fonction pour l’hémoglobine tronquée N de Mycobacterium tuberculosis à l’aide de techniques biochimiques modernes et de la spectroscopie à flux-arrêté. Nos recherches nous amènent également à résoudre la structure tridimensionnelle du complexe oxygéné de trHbN, à caractériser les interactions protéines-ligands au site actif de l’hémoglobine et à définir leurs rôles dans l’établissement du potentiel fonctionnel de l’enzyme en utilisant les spectroscopies d’absorption, de résonance Raman et de diffraction des rayons X. Nous avons découvert que l’activité rapide et efficace de détoxication aérobie du •NO de l’hémoglobine tronquée N mesurée chez Mycobacterium bovis BCG pourrait remplir un rôle similaire chez Mycobacterium tuberculosis et permettre la persistance de l’infection tuberculeuse dans l’hôte par la prévention des effets cytotoxiques associés au •NO et à ses dérivés réactifs azotés. De plus, l’architecture et la polarité du site actif de l’hémoglobine tronquée N définissent le potentiel fonctionnel de la protéine et contrôlent la diffusion, la fixation, la stabilisation, l’activation des ligands coordonnées au fer de l’hème et assurent le maintien d’une activité catalytique rapide et efficace. Finalement, nos découvertes suggèrent que l’hémoglobine tronquée N pourrait constituer une nouvelle cible thérapeutique pour le développement éventuel de drogues inhibitrices qui inactiveraient la première ligne de défense du parasite et perturberaient son adaptation métabolique face aux stress oxydatifs. / Mycobacterium tuberculosis infects over one-third of the human population, causing 1.5 millions deaths each year. The increase incidence of infections among immunocompromised patients and the emergence of strains with resistance to multiple antibiotics urge the scientific community to discover new therapeutic targets as well as develop new antibiotics, vaccines and therapies. Among the ~ 4000 genes that compose the genome of Mycobacterium tuberculosis, one of them, glbN, encodes the truncated hemoglobin N. Hemoglobins are small metalloproteins that reversibly bind oxygen and perform a wide array of important catalytic activities. Thus, our research aims at defining a function for Mycobacterium tuberculosis truncated hemoglobin N using modern biochemical techniques and stopped-flow spectroscopy. Our research also leads us to solve the three-dimensional structure of the oxygenated complex of trHbN, characterize the proteins-ligands interactions within the active site of the hemoglobin and define their roles in establishing the functional potential of the enzyme using absorption, resonance Raman and x-rays diffraction spectroscopies. We discovered that the fast and efficient •NO detoxification activity of truncated hemoglobin N measured in Mycobacterium bovis BCG could fulfill a similar role in Mycobacterium tuberculosis and allow the persistence of the tuberculous infection in the host by preventing the cytotoxic effects associated with •NO and its reactive nitrogen derivatives. Moreover, the architecture and polarity of truncated hemoglobin N active site define the functional potential of the protein and control the diffusion, binding, stabilization, and activation of the heme-iron coordinated ligands and ensure the maintenance of a fast and efficient catalytic activity. Finally, our discoveries suggest that truncated hemoglobin N could constitute a new therapeutic target for the development of inhibitors that would inactivate the first line of defence of the parasite and disturb its metabolic adaptation to nitrosative stresses.

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The role of the transposable insertion sequence IS<i>6110 </i>was studied in the evolution of <i>Mycobacterium tuberculosis </i>in 202 isolates from 40 countries. The isolates were analyzed by IS<i>6110 </i>insertion site mapping, spoligotyping, IS<i>6110 </i>RFLP fingerprints and <i>in silico </i>comparisons. Different IS<i>6110 </i>insertion sites exhibited a wide range of variation in the presence or absence of IS<i>6110 </i>in isolates varying between sites with only one isolate identified with the insertion, singletons, to sites where many isolates harboured an IS<i>6110 </i>insertion, common insertions.  95% of isolates were split into ten IS<i>6110 </i>cluster groups or lineages (ICG-1 to 8, ICG-a and ICG-b) based on their common IS<i>6110</i> insertion site patterns.  No <i>M. tuberculosis </i>isolates were found which were intermediate between ICG cluster groups.  A non-random association of IS<i>6110 </i>alleles over loci and also a high correlation between the common IS<i>6110 </i>cluster groups and spoligotype families suggested that common IS<i>6110 </i>insertions are predominantly the result of unique evolutionary event polymorphisms and they are therefore robust and valuable markers for phylogenetic and evolutionary studies of <i>M. tuberculosis.</i> 14 IS<i>6110-</i>assocaited deletions, including nine new regions of deletion were detected in the studied isolates.  Phylogenetic analysis of these genomic deletions demonstrated that they can also be used to classify the majority of MTB lineages including ICG-1/Beijing, ICG-3/CAS, ICG-5/a part of T, ICG-4/S and a major part of the ICG-6/LAM lineage. Published literature and DNA sequence databases were used to establish a global IS<i>6110 </i>database comprising of 524 different IS<i>6110</i> insertion sites across the genome and this database was used to study the role of IS<i>6110</i> in the fitness of <i>M. tuberculosis.  </i>The distribution of these sites showed a significant bias into intergenic regions, non-essential genes, multi-copy genes, other insertion elements and genomic repeat regions.  Common IS<i>6110</i> insertions may well have played an important role in the evolution of <i>M. tuberculosis.</i>

Mvcohacterium spp, the etiological agent of mycobacteriosis, has recently been responsible for serious infections in two economically important fish species cultured in Thailand; snakehead (Channa striata) and Siamese fighting fish (Berta splendens). Attempts to detect and identify the pathogens to species level, in fish tissue and the environment, have so far been unsuccessful, mainly due to the poor levels of sensitivity and specificity of the detection methods used, based on conventional bacteriology and histology. In this study, a variety of novel techniques were developed and used for more effective identification of Mycobacterium spp., including monoclonal antibody-based assays, DNA-based techniques and mass spectrometry. A monoclonal antibody (Mab 8F7) probe was developed against M. marinum, which was successfully used to identify M. marinum in infected fish tissue by immunohistochemistry (IHC), and from pure bacterial cultures by enzyme-liked immunosorbent assay (ELISA). The molecular-based techniques employed to detect the pathogen included in situ hybridisation (ISH), polymerase chain reaction (PCR) and reverse cross blot hybridisation. The PCR was developed using primers available from the literature which amplified mycobacterial 16S rDNA. The products of the reaction were identified to species level by PCR-reverse cross blot hybridisation. M. inarinum, M. fortuitum and M. chelonae were identified using this method. The same primers as those used in the PCR, were used as probes in ISH to identify Mycobacterium spp to genus level in infected fish tissues. spectrometry. A range of Mvcohocterium spp. isolated from fish located in different geographical regions were identified and characterised using Mab 8F7, pyrolysis mass spectrometry (PyMS) and PCR-reverse cross blot hybridisation and PyMS analysis showed that three distinct groups of mycobacteria were involved in mycobacteriosis in Thailand and Israel. The groups were clustered around either type strains M. fortuitum-M. chelonae or M. marinum, or around an unspeciated Mycobacterium spp. The unspeciated isolates were identified as M. marinum by PCR analysis and were mainly isolated from fish cultured in Israel. M. marinum from Israel and Thailand appeared to be different from each other since the isolates from Thailand reacted positively with Mab 8F7, whereas isolates obtained from fish in Israel were negative. PCR-reverse cross blot hybridisation was used to establish the identity of Mycobacterium spp involved in mycobacteriosis outbreaks affecting Siamese fighting fish and snakehead fish in Thailand. PCR was also utilised to analyse environmental samples taken from these farm sites. Siamese fighting fish farmers are known to suffer from skin lesions caused by Mycobacterium spp and therefore biopsies were taken from the farmers for analysis by PCR-reverse cross blot hybridisation. Analysis revealed that two species, M. fortuitum and M. marinum, were involved in the mycobacterial infections observed in both fish species. M. fortuitum and M. marinum were also both found in environmental samples including water, sediment and fish food. However, M. fortuitum was the isolate most frequently found. Skin lesions were only observed amongst the Siamese fighting fish farmers, while the snakehead fish farmers did not seem to be effected. Analysis of the biopsies from the skin lesions by PCR reverse cross blot hybridisation revealed that M. fortuitum was the main etiological agent associated with these lesions.

It was the purpose of this investigation to isolate and characterize the pigments of Mycobacterium rhodochrous by partition behavior, chromatographic data, and absorption spectra. In addition, it was the purpose to determine whether or not the type of pigments found in M. rhodochrous is typical of those found in other mycobacteria.

La tuberculosis en cualquiera de sus manifestaciones, en la actualidad, es sin duda uno de los problemas de salud pública más importantes y la búsqueda de un método de diagnóstico rápido ha sido y es, uno de los principales objetivos en muchas partes del mundo, sobre todo en países como el nuestro que presenta altas tasas de incidencia y cuya población más afectada no cuenta con los medios económicos para acceder a cualquiera de los métodos de diagnóstico rápido existentes. La finalidad del presente estudio fue evaluar el medio de cultivo Löwenstein - Jensen modificado con nitrato de potasio (KNO3) para el diagnóstico de Mycobacterium tuberculosis, compararando la sensibilidad y especificidad de este medio modificado con el medio Löwenstein - Jensen convencional y evaluando los factores importantes que influyen en el proceso como por ejemplo el tiempo. En el presente estudio, se evaluó un total de 120 muestras provenientes de pacientes diagnosticados con tuberculosis pulmonar por baciloscopía, las muestras (esputo, lavado bronquial, aspirado bronquial, aspirado gástrico) se trataron por el método Petroff modificado para su descontaminación y homogenización y se sembraron en los medios Löwenstein - Jensen convencional y modificado con nitrato de potasio al 35%. Luego estos cultivos fueron incubados a 37°C durante 7,10, 14 días, después de los cuales se determinó la reducción de nitratos a nitritos. El nitrito producido por Mycobacterium tuberculosis permitió identificar la presencia de la bacteria empleando el reactivo de Peter Griess, detectado por medio de un viraje de color del medio de cultivo. En el medio Löwenstein - Jensen modificado, se determinó que la sensibilidad era del 41% y la especificidad del 100%, con un valor predictivo positivo de 100%, y valor predictivo negativo de 34%. Al comparar estadísticamente la sensibilidad y especificidad de la prueba en el medio modificado y el cultivo convencional o Estándar de oro, se observó que los factores predominantes en el proceso de evaluación son: tipo (esputo, lavado bronquial, aspirado bronquial, aspirado gástrico) y calidad de la muestra (Carga Bacilar), obtenidas adecuadamente. Los resultados fueron obtenidos en su mayoría a los 10 días, con un promedio ponderado de 12 días para el diagnóstico de Mycobacterium tuberculosis. El estudio realizado justifica el empleo del medio Löwenstein - Jensen modificado conteniendo nitrato de potasio (KNO3), presentándose como una alternativa para el diagnóstico presuntivo de Mycobacterium tuberculosis. / --- The tuberculosis in anyone of his manifestations, one becomes of without a doubt the public- health problems more important at the present time and it has been a diagnostic shoot's quest and it is, one join of the objective main things in many parts worldly, most of all in countries as the our that he presents high incidence rates and whose more population once was affected does not count on the economic means to accede to anyone of the diagnostic- shoot methods existents. The present study's purpose was to evaluate Löwensein-Jensen's midway modified with nitrate reductasa in order to the pulmonary tuberculosis's diagnosis, Mycobacterium tuberculosis's identification, comparing sensibility and modified specificity of this midway with the cultivation conventional Löwenstein-Jensen and evaluating the mains factors that have influence in the process for example theTime. In the present study evaluated 120 samples coming from patients diagnosed with pulmonary tuberculosis for baciloscopy; the samples them tried to him for the method Petroff once was modified in order to his decontamination and homogenization before being sown in the Löwensein-Jensen's midway conventional and modified , with potassium nitrate to 37 per cent.. The cultures were incubates went to 37 ºC for 7, 10, 14 days; then the nitrate reduction was suggesting growth to nitrite. To positive reaction was detected by means of a color turn. In the Löwensein-Jensen's midway conventional obtained a sensibility of 41 per cent and specificity of 100 per cent in the Löwenstein-Jensen midway modificated, with a positive value once of 100 per cent and negative value once of 34 per cent. To the comparing statistically sensibility and specificity of the essay test with nitrate reductasa and the conventional cultivation or Gold Standard heeded that the prevailing factors in the evaluation process are: the type and quality of the sample quality (charges Bacilar), fitting to obtain. The results were obteined aftermaths in the main to the 10 days, with a 12 days average prudent in order to TB'S diagnosis – Pulmonar The study once was accomplished justifies test's job Löwenstein – Jensen modified with potassium of Nitrate (KNO3), encountering as an alternative in order to the Mycobacterium tuberculosis's diagnosis.

A tuberculose é considerada um problema emergente de saúde pública. Este estudo tem como objetivo avaliar a associação da patogenicidade/virulência e propriedades imunomoduladoras de isolados clínicos de Mbv (cepas B2 e MP287/03) e Mtb (cepa Beijing 1471), e da cepa de Mtb H37Rv, como referência de virulência. Os isolados, MP287/03 e Beijing 1471, apresentaram maior virulência em relação às demais cepas, levando os camundongos à morte ainda na fase aguda de infecção. Foi verificada baixa produção de mediadores pró-inflamatórios nos animais infectados com o isolado MP287/03, enquanto nos infectados com o isolado Beijing 1471 os níveis destes mediadores foram exacerbados. O desbalanço na produção destes mediadores pode ter contribuído para morte precoce dos animais. Baseado nesse estudo, nós podemos concluir que as propriedades que conferem hipervirulência aos isolados clínicos de Mbv e Mtb estão principalmente relacionadas à alta capacidade de crescimento intracelular das bactérias, que parece ser pouco alterada pela presença de citocinas pró-inflamatórias. Sendo assim, as infecções por isolados hipervirulentos podem acarretar consequências semelhantes, mesmo quando associadas a diferentes padrões de modulação da resposta imune. / Tuberculosis is an emergent problem of public health. This study aimed to evaluate the association between pathogenicity/virulence and immunemodulatory ability of Mbv (B2 and MP287/03) and Mtb (Beijing 1471) clinical isolates, using H37Rv strain as reference of virulence. The virulence was assessed in C57BL/6 mice infected with a low dose of bacilli (~100 bacteria) via intratracheal route. MP287/03 and Beijing 1471 isolates showed higher virulence than all others strains, leading to mice death during the acute phase. It was verified low production of pro-inflammatory mediators in mice infected by MP287/03 bacteria, whereas in mice infected by Beijing 1471 bacteria were observed exacerbated levels of pro-inflammatory mediators. The disbalance of these mediators may have contributed to the early mouse death. Based on this study, we concluded that the properties that confer hypervirulence to Mbv and Mtb clinical isolates are primarily related to the high intracellular growth capacity of the bacteria, which seems to be marginally affected by the presence of pro-inflammatory cytokines. Therefore, the infection by hypervirulent isolates can lead to similar outcomes, even when associated to different patterns of modulation of the immune response.

A tuberculose é uma doença infectocontagiosa causada por bactérias do complexo Mycobacterium tuberculosis (MTBC) que afeta humanos e/ou animais. Membros desse complexo evoluíram clonalmente e possuem grande similaridade genômica, diferenciando-se por polimorfismos de nucleotídeo único (SNPs) e regiões de diferença (RDs). Dentre os patógenos da tuberculose em animais, Mycobacterium bovis, causador da tuberculose bovina, é o membro do MTBC de maior importância global. Desta maneira, o presente estudo tem por objetivo o sequenciamento, a anotação e a análise da estirpe brasileira SP38 de M. bovis, seguido da genômica comparativa desse com outros genomas de M. bovis depositados no GenBank. Mycobacterium bovis SP38 apresenta um genoma tradicional de micobactéria tuberculosa, sendo esse único, circular com 4.347.646 pb, alto conteúdo de GC (65,6%) e 4.216 genes, incluindo 154 pseudogenes, 3 genes de rRNA (RNA ribossomal), 45 de tRNA (RNA transportador), 2 de ncRNA (RNA não codificante), 1 tmRNA (RNA transferência-mensageiro) e 4.011 sequências de DNA codificante (CDSs) (NZ_CP015773.1). Dentre as CDSs, a maioria (2.805 - 69,93%) foi anotado com função e 1.206 (30,07%) como hipotéticos. Para a genômica comparativa, os 31 genomas completos ou em drafts de M. bovis depositados no GenBank, 32 genomas de Mycobacterium bovis BCG e 23 genomas de Mycobacterium tuberculosis foram selecionados. Análises in silico dos padrões de RD resultaram na exclusão de três genomas anotados equivocadamente como M. bovis virulentos. A análise de genes ortólogos sugere que M. bovis está sob processo de decay genômico. A quantificação de sítios polimórficos indica uma maior variabilidade genética em números totais (8.335 em M. tuberculosis, 3.448 em M. bovis virulentos, e 1.088 em M. bovis BCGs) e comparações par-a-par (p &le;0,05) de M. tuberculosis em relação a M. bovis virulentos e BCGs, indicando uma maior pressão evolutiva sob M. tuberculosis, contrastando com o fato de que M. bovis é capaz de infectar um maior número de espécies hospedeiras que M. tuberculosis. A maioria desses sítios polimórficos estão localizados em CDSs hipotéticos (31,7% - 51,3%), sendo associados a família gênica PE/PPE, e apresentam uma proporção de mutações não sinônimas crescentes pela ordem M. bovis BCG, M. bovis virulentos e M. tuberculosis (48,90%, 51,92% e 59,52%, respectivamente). Essa menor proporção de mutações não sinônimas e a categorização funcional dissimilar entre CDSs contendo sítios polimórficos, indica que M. bovis BCG está sujeito a diferentes pressões seletivas quando comparado a M. bovis virulentos e M. tuberculosis. Por fim, a análise filogenética baseada em sítios polimórficos indica agrupamentos filogenéticos de M. bovis suportados pela classificação dos Complexos Clonais (CCs) e não por hospedeiros de origem dos isolados, confirmando que sítios polimórficos podem ser utilizados para classificação filogenética de linhagens genéticas desta espécie bacteriana. Além do mais, 2/28 (7,14%) genomas de M. bovis não puderam ser classificados nos CCs atualmente descritos, sugerindo a existência de complexos ainda não determinados. Este estudo representa o primeiro genoma de uma estirpe nacional de M. bovis a ser completamente sequenciado e a primeira análise de genômica comparativa de genomas desta espécie bacteriana. / Tuberculosis is an infectious disease caused by bacteria of the Mycobacterium tuberculosisComplex (MTBC) that affects human beings and/or animals. Members of this complex clonally evolved and have high genomic similarity, differentiated by single nucleotide polymorphisms (SNPs) and regions of difference (RDs). Among the animal tuberculosis pathogens, Mycobacterium bovis, the causative agent of bovine tuberculosis, is the MTBC member of greatest global importance. Therefore, the aim of the present study is to sequence, assemble and annotate the genome of the Brazilian strain SP38 of M. bovis, followed by the comparative genomics with other M. bovis genomes available in GenBank. Mycobacterium bovis SP38 has a traditional mycobacteria genome. It has a single and circular chromosome with 4,347,646 bp, high GC content (65.6%), and 4,216 genes, including 154 pseudogenes, 3 rRNA genes (ribosomal RNA), 45 tRNA (transfer RNA), 2 ncRNA (non-coding RNA), 1 tmRNA (transfer-messenger RNA), and 4,011 coding DNA sequences (CDSs) (NZ_CP015773.1). The majority of CDSs (2,805 - 69,93%) was annotated with function and 1,206 (30,07%) are hypothetical. For the comparative genomics analyses, the 31 genomes (complete and drafts) of M. bovis available in GenBank, 32 Mycobacterium bovis BCG and, 23 of Mycobacterium tuberculosis were chosen. In silico analysis of the RDs patterns resulted in the exclusion of three genomes, mistakenly annotated as virulent M. bovis. Orthologous gene analysis suggests that strains of M. bovis are under genomic decay. The quantification of polymorphic sites indicates the greater variability in absolute numbers (8,335 in M. tuberculosis, 3,448 in virulent M. bovis, and 1,088 in M. bovis BCG) and in pairwise comparisons (p&le;0,05) of M. tuberculosis compared to virulent M. bovis and M. bovis BCG, suggesting that M. tuberculosis is under high evolutionary pressure. This is in contrast to the fact that M. bovis is capable of infecting a higher number of host species than M. tuberculosis. Most of these polymorphic sites are located in hypothetical CDSs (31.7% - 52.3%), being associated with PE/PPE family, and demonstrating a nonsynonymous mutations proportion of the following increasing order: M. bovis BCG, virulent M. bovis and M. tuberculosis (48.90%, 51.92% and 59.52%, respectively). This lower proportion of nonsynonymous mutations and the dissimilar functional categorization of CDSs with polymorphic sites indicates that M. bovis BCG is subjected to different selective pressure when compared to virulent M. bovis and M. tuberculosis. Finally, the phylogenetic analysis based on polymorphic sites indicates that the phylogenetic grouping of M. bovis is supported by Clonal Complexes (CCs), and not by the host of M. bovis isolates, confirming that polymorphic sites can be used for phylogenetic classification of genetic lineages of this bacterial species. Furthermore, 2/28 (7.14%) genomes of M. bovis could not be classified in the currently described CCs, suggesting the existence of complexes yet to be determined. This study represents the first genome of a Brazilian strain of M. bovis to be completely sequenced and the first comparative genomic analysis of the genomes of this bacterial species.