Yes / Whole genome sequencing (WGS) is increasingly used for Mycobacterium tuberculosis (Mtb) research. Countries with the highest tuberculosis (TB) burden face important challenges to integrate WGS into surveillance and research. We assessed the global status of Mtb WGS and developed a 3-week training course coupled with long-term mentoring and WGS infrastructure building. Training focused on genome sequencing, bioinformatics and development of a locally relevant WGS research project. The aim of the long-term mentoring was to support trainees in project implementation and funding acquisition. The focus of WGS infrastructure building was on the DNA extraction process and bioinformatics. Compared to their TB burden, Asia and Africa are grossly underrepresented in Mtb WGS research. Challenges faced resulted in adaptations to the training, mentoring and infrastructure building. Out-of-date laptop hardware and operating systems were overcome by using online tools and a Galaxy WGS analysis pipeline. A case studies approach created a safe atmosphere for students to formulate and defend opinions. Because quality DNA extraction is paramount for WGS, a biosafety level 3 and general laboratory skill training session were added, use of commercial DNA extraction kits was introduced and a 2-week training in a highly equipped laboratory was combined with a 1-week training in the local setting. By developing and sharing the components of and experiences with a sequencing and bioinformatics training program, we hope to stimulate capacity building programs for Mtb WGS and empower high-burden countries to play an important role in WGS-based TB surveillance and research. / Vlaamse Interuniversitaire Raad-secretariaat voor universitaire ontwikkelingssamenwerking (ET2018JOI008A10); the Research Foundation Flanders under FWO Odysseus (grant G0F8316N); the South African Research Chairs Initiative of the Department of Science and Technology and National Research Foundation of South Africa (64751); the South African Medical Research Council.

Yes / The human- and animal-adapted lineages of the Mycobacterium tuberculosis complex (MTBC) are thought to have expanded from a common progenitor in Africa. However, the molecular events that accompanied this emergence remain largely unknown. Here, we describe two MTBC strains isolated from patients with multidrug resistant tuberculosis, representing an as-yet-unknown lineage, named Lineage 8 (L8), seemingly restricted to the African Great Lakes region. Using genome-based phylogenetic reconstruction, we show that L8 is a sister clade to the known MTBC lineages. Comparison with other complete mycobacterial genomes indicate that the divergence of L8 preceded the loss of the cobF genome region - involved in the cobalamin/vitamin B12 synthesis - and gene interruptions in a subsequent common ancestor shared by all other known MTBC lineages. This discovery further supports an East African origin for the MTBC and provides additional molecular clues on the ancestral genome reduction associated with adaptation to a pathogenic lifestyle. / This work was supported by EDCTP2 grant DRIA2014-326—DIAMA of the European Union, the Belgian General Directorate for Development Cooperation (PhD fellowship to J.C.S.N.), Grant ANR-16-CE35-0009 from Agence Nationale de la Recherche, the Swiss National Science Foundation (Grants 310030_188888, IZRJZ3_164171, IZLSZ3_170834 and CRSII5_177163), and the European Research Council (309540-EVODRTB). The views and opinions of authors expressed herein do not necessarily state or reflect those of EDCTP. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

No / The role of mutations in genes associated with phenotypic resistance to bedaquiline (BDQ) and delamanid (DLM) in Mycobacterium tuberculosis complex (MTBc) strains is poorly characterized. A clear understanding of the genetic variants' role is crucial to guide the development of molecular-based drug susceptibility testing (DST). In this work, we analyzed all mutations in candidate genomic regions associated with BDQ- and DLM-resistant phenotypes using a whole-genome sequencing (WGS) data set from a collection of 4,795 MTBc clinical isolates from six countries with a high burden of tuberculosis (TB). From WGS analysis, we identified 61 and 163 unique mutations in genomic regions potentially involved in BDQ- and DLM-resistant phenotypes, respectively. Importantly, all strains were isolated from patients who likely have never been exposed to these medicines. To characterize the role of mutations, we calculated the free energy variation upon mutations in the available protein structures of Ddn (DLM), Fgd1 (DLM), and Rv0678 (BDQ) and performed MIC assays on a subset of MTBc strains carrying mutations to assess their phenotypic effect. The combination of structural and phenotypic data allowed for cataloguing the mutations clearly associated with resistance to BDQ (n = 4) and DLM (n = 35), only two of which were previously described, as well as about a hundred genetic variants without any correlation with resistance. Significantly, these results show that both BDQ and DLM resistance-related mutations are diverse and distributed across the entire region of each gene target, which is of critical importance for the development of comprehensive molecular diagnostic tools.

Yes / Mycobacterium africanum lineage (L) 6 is an important pathogen in West Africa, causing up to 40% of pulmonary tuberculosis (TB). The biology underlying the clinical differences between M. africanum and M. tuberculosis sensu stricto remains poorly understood. We performed ex vivo expression of 2179 genes of the most geographically dispersed cause of human TB, M. tuberculosis L4 and the geographically restricted, M. africanum L6 directly from sputa of 11 HIV-negative TB patients from The Gambia who had not started treatment. The DosR regulon was the most significantly decreased category in L6 relative to L4. Further, we identified nonsynonymous mutations in major DosR regulon genes of 44 L6 genomes of TB patients from The Gambia and Ghana. Using Lebek's test, we assessed differences in oxygen requirements for growth. L4 grew only at the aerobic surface while L6 grew throughout the medium. In the host, the DosR regulon is critical for M. tuberculosis in adaptation to oxygen limitation. However, M. africanum L6 appears to have adapted to growth under hypoxic conditions or to different biological niches. The observed under expression of DosR in L6 fits with the genomic changes in DosR genes, microaerobic growth and the association with extrapulmonary disease. / European Research Council-INTERRUPTB starting grant nr.311725 (to BdJ, BO, FG, MA, CM).

No description available.

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.

Cette étude vise à approfondir les connaissances sur les interactions de trHbN, une hémoglobine tronquée de Mycobacterium tuberculosis (Mtb), avec des membranes lipidiques modèles. Cette protéine est responsable de la détoxification du NO en milieu granulomique chez Mtb et serait vitale à la survie en état stationnaire de la bactérie. Le dichroïsme circulaire, la spectroscopie infrarouge ainsi que la RMN à l’état solide du 31P ont été utilisés pour observer la conformation de trHbN dans les différents milieux ainsi que son effet sur les lipides. Dû à la controverse entourant la conformation et le rôle du segment N-terminal de trHbN, la protéine sauvage ainsi qu’une protéine mutante où le segment n’a pas été exprimé ont été étudiées. Les résultats suggèrent que la protéine serait située à l’interface des bicouches lipidiques et que le segment pré-A pourrait servir « d’ancre », empêchant la protéine sauvage de pénétrer trop profondément dans les membranes.

Thesis (MScMed (Biomedical Sciences. Molecular Biology and Human Genetics))--University of Stellenbosch, 2007. / The expansion of the Mycobacterium tuberculosis PE and PPE gene families seems to be linked to that of the immunologically-important ESAT-6 (esx) gene clusters secretion system, as the ancestral members of these families are found only within the ESAT-6 gene cluster regions. These ancestral members are also the only copies in the earlier mycobacteria like M. smegmatis. The later duplications of the PE and PPE families belonging to the PGRS and MPTR subgroups, have been implicated in virulence and are only found within the genomes of the pathogenic mycobacteria closely related to the M. tuberculosis complex. The aim of this study was to compare the subcellular localization of the later duplications of the PE and PPE gene families belonging to the PGRS and MPTR subgroups with that of the ancestral PE and PPE proteins found in M. smegmatis and to investigate whether the ESX secretion apparatus is involved in the trafficking of these proteins. The PE (Rv3872) and PPE (Rv3873) genes from M. smegmatis were PCR amplified with a C-terminal HA tag using M. smegmatis genomic DNA as template. Two PPE-MPTR genes, Rv0442c and Rv0878c, and one PE_PGRS gene, Rv2615c, were also PCR amplified with a C-terminal HA tag using M. tuberculosis genomic DNA as template. All genes were cloned into the mycobacterial expression vector p19Kpro. Expression and localization was investigated using SDS-PAGE and Western blotting. The PE and PPE genes expressed in M. smegmatis were found to be present within the cell wall, membrane, and cytosol fractions, but not in the culture filtrate, indicating no secretion. The PPE-MPTR and PE_PGRS genes expressed in M. smegmatis, were also found to be present within the cell wall, membrane and cytosol fractions, but not in the culture filtrate, indicating that they are also not secreted. We hypothesize that their secretion is dependent on ESAT-6 gene cluster region 5, which is absent from the genome of M. smegmatis. Ancestral PE and PPE proteins are secreted efficiently in M. tuberculosis. The ESAT-6 gene cluster Region 3 and Region 4 of M. smegmatis were knocked out, and these knockout mutants could be used in future studies to investigate if the ESAT-6 gene cluster region 1 is involved in the secretion of the ancestral and recent PE and PPE proteins.

Comparou-se a eficácia da baciloscopia in natura e pós concentração (Coloração Ziehl Neelsen) e cultura tradicional e automatizada MA de amostras de escarro ou saliva/muco para a detecção do Complexo M. tuberculosis (MTB) em Centro de Referencia Ambulatorial para Tuberculose na Cidade de São Paulo. A identificação do grupo MTB baseou-se no crescimento em meio com Ácido para nitrobenzóico, e visualização do fator corda. Do total de 374 amostras, 228 eram de pacientes sob diagnóstico inicial e 146 em tratamento. 83 amostras eram saliva/muco. Destas, sete foram positivas à baciloscopia, 03 (3,6%) no material concentrado e uma (1,2%) in natura (p=0.5 McNemar Test). Cinco amostras (6%) positivas na cultura pelo método tradicional e 07 (8,4%) p=0.5 pelo MA. As amostras de escarro eram 74 foram positivas na baciloscopia, sendo 34 (11,7%) in natura e 45 (15,5%) no material concentrado, p=0.001. No método tradicional 56 (19,2%) e 67 (23%) pelo MA p=0.001. Independente da característica da amostra a baciloscopia pós concentração e a cultura pelo MA foram mais sensíveis. / Effectiveness of bacilloscopy were compared in natura and pos-concentrated (Ziehl Neelsen staining), and tradicional and automated culture-AM of sputum or saliva/mucus samples for detection of M. tuberculosis complex (MTB) in Ambulatory Reference Center in the city of São Paulo. Identification of MTB group was based on growth in medium with para nitrobenzoic acid, and cord factor visualization. From the total 374 samples, 228 were from patients under initial diagnostic and 146 in treatment. 83 samples were saliva/mucus. Seven of these were bacilloscopy positive, 03 (3.6%) in concentrated material and one (1.2%) in natura (p=.500ª McNemar Test). Five samples (6%) positive for culture by traditional method and 07 (8.4%) p=0.5 for AM. For sputum 74 were bacilloscopy positive, being 34 (11.7%) in in natura and 45 in concentrated material, p=0.001. In traditional method, 56 (19.2%) and 67 (23%) for AM, p=0.001. Independently of sample characteristics, pos-concentrated bacilloscopy and culture by AM were more effectiveness.

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.