aminen

• Cioetto-Mazzabò, Laura; Boldrin, Francesca; Beauvineau, Claire; Speth, Martin Tobias; Marina, Alberto & Namouchi, Amine [Vis alle 17 forfattere av denne artikkelen] (2023). SigH stress response mediates killing of Mycobacterium tuberculosis by activating nitronaphthofuran prodrugs via induction of Mrx2 expression. Nucleic Acids Research (NAR). ISSN 0305-1048. 51(1), s. 144–165. doi: 10.1093/nar/gkac1173. Fulltekst i vitenarkiv Vis sammendrag

  Abstract The emergence of drug-resistant Mycobacterium tuberculosis strains highlights the need to discover anti-tuberculosis drugs with novel mechanisms of action. Here we discovered a mycobactericidal strategy based on the prodrug activation of selected chemical derivatives classified as nitronaphthofurans (nNFs) mediated by the coordinated action of the sigH and mrx2 genes. The transcription factor SigH is a key regulator of an extensive transcriptional network that responds to oxidative, nitrosative, and heat stresses in M. tuberculosis. The nNF action induced the SigH stress response which in turn induced the mrx2 overexpression. The nitroreductase Mrx2 was found to activate nNF prodrugs, killing replicating, non-replicating and intracellular forms of M. tuberculosis. Analysis of SigH DNA sequences obtained from spontaneous nNF-resistant M. tuberculosis mutants suggests disruption of SigH binding to the mrx2 promoter site and/or RNA polymerase core, likely promoting the observed loss of transcriptional control over Mrx2. Mutations found in mrx2 lead to structural defects in the thioredoxin fold of the Mrx2 protein, significantly impairing the activity of the Mrx2 enzyme against nNFs. Altogether, our work brings out the SigH/Mrx2 stress response pathway as a promising target for future drug discovery programs.

• Bernard-Raichon, Lucie; Colom, André; Monard, Sarah C.; Namouchi, Amine; Cescato, Margaux & Garnier, Hugo [Vis alle 20 forfattere av denne artikkelen] (2021). A pulmonary lactobacillus murinus strain induces th17 and rorγt+ regulatory t cells and reduces lung inflammation in tuberculosis. Journal of Immunology. ISSN 0022-1767. 207(7), s. 1857–1870. doi: 10.4049/jimmunol.2001044. Fulltekst i vitenarkiv
• Guellil, Meriam; Kersten, Oliver; Namouchi, Amine; Luciani, Stefania; Marota, Isolina & Arcini, Caroline [Vis alle 17 forfattere av denne artikkelen] (2020). A genomic and historical synthesis of plague in 18th century Eurasia. Proceedings of the National Academy of Sciences of the United States of America (PNAS). ISSN 0027-8424. 117(45), s. 28328–28335. doi: 10.1073/pnas.2009677117. Fulltekst i vitenarkiv Vis sammendrag

  Plague continued to afflict Europe for more than five centuries after the Black Death. Yet, by the 17th century, the dynamics of plague had changed, leading to its slow decline in Western Europe over the subsequent 200 y, a period for which only one genome was previously available. Using a multidisciplinary approach, combining genomic and historical data, we assembled Y. pestis genomes from nine individuals covering four Eurasian sites and placed them into an historical context within the established phylogeny. CHE1 (Chechnya, Russia, 18th century) is now the latest Second Plague Pandemic genome and the first non-European sample in the post-Black Death lineage. Its placement in the phylogeny and our synthesis point toward the existence of an extra-European reservoir feeding plague into Western Europe in multiple waves. By considering socioeconomic, ecological, and climatic factors we highlight the importance of a noneurocentric approach for the discussion on Second Plague Pandemic dynamics in Europe.

• Bramanti, Barbara; Namouchi, Amine; Schmid, Boris Valentijn; Dean, Katharine Rose & Stenseth, Nils Christian (2019). Reply to Barbieri et al.: Out of the Land of Darkness: Plague on the fur trade routes. Proceedings of the National Academy of Sciences of the United States of America (PNAS). ISSN 0027-8424. 116(16). doi: 10.1073/pnas.1902274116. Fulltekst i vitenarkiv
• Guellil, Meriam; Kersten, Oliver; Namouchi, Amine; Bauer, Egil Lindhart; Derrick, Michael & Jensen, Anne Østergaard [Vis alle 8 forfattere av denne artikkelen] (2018). Genomic blueprint of a relapsing fever pathogen in 15th century Scandinavia. Proceedings of the National Academy of Sciences of the United States of America (PNAS). ISSN 0027-8424. 115(41), s. 10422–10427. doi: 10.1073/pnas.1807266115. Fulltekst i vitenarkiv Vis sammendrag

  Louse-borne relapsing fever (LBRF) is known to have killed millions of people over the course of European history and remains a major cause of mortality in parts of the world. Its pathogen, Borrelia recurrentis, shares a common vector with global killers such as typhus and plague and is known for its involvement in devastating historical epidemics such as the Irish potato famine. Here, we describe a European and historical genome of B. recurrentis, recovered from a 15th century skeleton from Oslo. Our distinct European lineage has a discrete genomic makeup, displaying an ancestral oppA-1 gene and gene loss in antigenic variation sites. Our results illustrate the potential of ancient DNA research to elucidate dynamics of reductive evolution in a specialized human pathogen and to uncover aspects of human health usually invisible to the archaeological record.

• Namouchi, Amine; Guellil, Meriam; Kersten, Oliver; Hänsch, Stephanie; Ottoni, Claudio & Schmid, Boris Valentijn [Vis alle 16 forfattere av denne artikkelen] (2018). Integrative approach using Yersinia pestis genomes to revisit the historical landscape of plague during the Medieval Period. Proceedings of the National Academy of Sciences of the United States of America (PNAS). ISSN 0027-8424. 115(50), s. E11790–E11797. doi: 10.1073/pnas.1812865115. Fulltekst i vitenarkiv Vis sammendrag

  Over the last few years, genomic studies on Yersinia pestis, the causative agent of all known plague epidemics, have considerably increased in numbers, spanning a period of about 5,000 y. Nonetheless, questions concerning historical reservoirs and routes of transmission remain open. Here, we present and describe five genomes from the second half of the 14th century and reconstruct the evolutionary history of Y. pestis by reanalyzing previously published genomes and by building a comprehensive phylogeny focused on strains attributed to the Second Plague Pandemic (14th to 18th century). Corroborated by historical and ecological evidence, the presented phylogeny, which includes our Y. pestis genomes, could support the hypothesis of an entry of plague into Western European ports through distinct waves of introduction during the Medieval Period, possibly by means of fur trade routes, as well as the recirculation of plague within the human population via trade routes and human movement.

• Frye, Stephan Alfons; Beyene, Getachew Tesfaye; Namouchi, Amine; Munoz, Marta Gomez; Homberset, Håvard & Kalayou, Shewit [Vis alle 9 forfattere av denne artikkelen] (2017). The helicase DinG responds to stress due to DNA double strand breaks. PLOS ONE. 12:e0187900(11), s. 1–23. doi: 10.1371/journal.pone.0187900. Fulltekst i vitenarkiv
• Namouchi, Amine; Cimino, Mena; Favre-Rochex, Sandrine; Charles, Patricia & Gicquel, Brigitte (2017). Phenotypic and genomic comparison of Mycobacterium aurum and surrogate model species to Mycobacterium tuberculosis: Implications for drug discovery. BMC Genomics. 18(1), s. 1–9. doi: 10.1186/s12864-017-3924-y. Fulltekst i vitenarkiv
• Namouchi, Amine; Munoz, Marta Gomez; Frye, Stephan Alfons; Moen, Line Victoria; Rognes, Torbjørn & Tonjum, Tone [Vis alle 7 forfattere av denne artikkelen] (2016). The Mycobacterium tuberculosis transcriptional landscape under genotoxic stress. BMC Genomics. 17:791, s. 1–13. doi: 10.1186/s12864-016-3132-1. Fulltekst i vitenarkiv Vis sammendrag

  Background: As an intracellular human pathogen, Mycobacterium tuberculosis (Mtb) is facing multiple stressful stimuli inside the macrophage and the granuloma. Understanding Mtb responses to stress is essential to identify new virulence factors and pathways that play a role in the survival of the tubercle bacillus. The main goal of this study was to map the regulatory networks of differentially expressed (DE) transcripts in Mtb upon various forms of genotoxic stress. We exposed Mtb cells to oxidative (H2O2 or paraquat), nitrosative (DETA/NO), or alkylation (MNNG) stress or mitomycin C, inducing double-strand breaks in the DNA. Total RNA was isolated from treated and untreated cells and subjected to high-throughput deep sequencing. The data generated was analysed to identify DE genes encoding mRNAs, non-coding RNAs (ncRNAs), and the genes potentially targeted by ncRNAs. Results: The most significant transcriptomic alteration with more than 700 DE genes was seen under nitrosative stress. In addition to genes that belong to the replication, recombination and repair (3R) group, mainly found under mitomycin C stress, we identified DE genes important for bacterial virulence and survival, such as genes of the type VII secretion system (T7SS) and the proline-glutamic acid/proline-proline-glutamic acid (PE/PPE) family. By predicting the structures of hypothetical proteins (HPs) encoded by DE genes, we found that some of these HPs might be involved in mycobacterial genome maintenance. We also applied a state-of-the-art method to predict potential target genes of the identified ncRNAs and found that some of these could regulate several genes that might be directly involved in the response to genotoxic stress. Conclusions: Our study reflects the complexity of the response of Mtb in handling genotoxic stress. In addition to genes involved in genome maintenance, other potential key players, such as the members of the T7SS and PE/PPE gene family, were identified. This plethora of responses is detected not only at the level of DE genes encoding mRNAs but also at the level of ncRNAs and their potential targets.

• Yimer, Solomon Abebe; Namouchi, Amine; Zegeye, Ephrem Debebe; Holm-Hansen, Carol J C; Norheim, Gunnstein & Abebe, Markos [Vis alle 8 forfattere av denne artikkelen] (2016). Deciphering the recent phylogenetic expansion of the originally deeply rooted Mycobacterium tuberculosis lineage 7. BMC Evolutionary Biology. 16:146, s. 1–10. doi: 10.1186/s12862-016-0715-z. Fulltekst i vitenarkiv Vis sammendrag

  Background: A deeply rooted phylogenetic lineage of Mycobacterium tuberculosis (M. tuberculosis) termed lineage 7 was discovered in Ethiopia. Whole genome sequencing of 30 lineage 7 strains from patients in Ethiopia was performed. Intra-lineage genome variation was defined and unique characteristics identified with a focus on genes involved in DNA repair, recombination and replication (3R genes). Results: More than 800 mutations specific to M. tuberculosis lineage 7 strains were identified. The proportion of non-synonymous single nucleotide polymorphisms (nsSNPs) in 3R genes was higher after the recent expansion of M. tuberculosis lineage 7 strain started. The proportion of nsSNPs in genes involved in inorganic ion transport and metabolism was significantly higher before the expansion began. A total of 22346 bp deletions were observed. Lineage 7 strains also exhibited a high number of mutations in genes involved in carbohydrate transport and metabolism, transcription, energy production and conversion. Conclusions: We have identified unique genomic signatures of the lineage 7 strains. The high frequency of nsSNP in 3R genes after the phylogenetic expansion may have contributed to recent variability and adaptation. The abundance of mutations in genes involved in inorganic ion transport and metabolism before the expansion period may indicate an adaptive response of lineage 7 strains to enable survival, potentially under environmental stress exposure. As lineage 7 strains originally were phylogenetically deeply rooted, this may indicate fundamental adaptive genomic pathways affecting the fitness of M. tuberculosis as a species.

• Meftahi, Nedra; Namouchi, Amine; Mhenni, Besma; Brandis, Gerrit; Hughes, Diarmaid & Mardassi, Helmi (2016). Evidence for the critical role of a secondary site rpoB mutation in the compensatory evolution and successful transmission of an MDR tuberculosis outbreak strain. Journal of Antimicrobial Chemotherapy. ISSN 0305-7453. 71(2), s. 324–332. doi: 10.1093/jac/dkv345. Fulltekst i vitenarkiv
• Beyene, Getachew Tesfaye; Balasingham, Seetha; Frye, Stephan Alfons; Namouchi, Amine; Homberset, Håvard & Kalayou, Shewit [Vis alle 8 forfattere av denne artikkelen] (2016). Characterization of the Neisseria meningitidis Helicase RecG. PLOS ONE. 11:e0164588(10), s. 1–31. doi: 10.1371/journal.pone.0164588. Fulltekst i vitenarkiv Vis sammendrag

  Neisseria meningitidis (Nm) is a Gram-negative oral commensal that opportunistically can cause septicaemia and/or meningitis. Here, we overexpressed, purified and characterized the Nm DNA repair/recombination helicase RecG (RecGNm) and examined its role during genotoxic stress. RecGNm possessed ATP-dependent DNA binding and unwinding activities in vitro on a variety of DNA model substrates including a Holliday junction (HJ). Database searching of the Nm genomes identified 49 single nucleotide polymorphisms (SNPs) in the recGNm including 37 non-synonymous SNPs (nsSNPs), and 7 of the nsSNPs were located in the codons for conserved active site residues of RecGNm. A transient reduction in transformation of DNA was observed in the Nm ΔrecG strain as compared to the wildtype. The gene encoding recGNm also contained an unusually high number of the DNA uptake sequence (DUS) that facilitate transformation in neisserial species. The differentially abundant protein profiles of the Nm wildtype and ΔrecG strains suggest that expression of RecGNm might be linked to expression of other proteins involved in DNA repair, recombination and replication, pilus biogenesis, glycan biosynthesis and ribosomal activity. This might explain the growth defect that was observed in the Nm ΔrecG null mutant.

• Thabet, Sara; Namouchi, Amine & Mardassi, Helmi (2015). Evolutionary trends of the transposase-encoding open reading frames A and B (orfA and orfB) of the mycobacterial IS6110 insertion sequence. PLOS ONE. 10:e0130161(6). doi: 10.1371/journal.pone.0130161. Fulltekst i vitenarkiv
• Yimer, Solomon Abebe; Norheim, Gunnstein; Namouchi, Amine; Zegeye, Ephrem Debebe; Kinander, Wibeke & Tonjum, Tone [Vis alle 11 forfattere av denne artikkelen] (2015). Mycobacterium tuberculosis lineage 7 strains are associated with prolonged patient delay in seeking treatment for pulmonary tuberculosis in Amhara region, Ethiopia. Journal of Clinical Microbiology. ISSN 0095-1137. 53(4), s. 1301–1309. doi: 10.1128/JCM.03566-14. Fulltekst i vitenarkiv

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• Namouchi, Amine; Guellil, Meriam; Kersten, Oliver Sven; Hänsch, Stephanie; Ottoni, Claudio & Schmid, Boris Valentijn [Vis alle 16 forfattere av denne artikkelen] (2020). Correction for Namouchi et al., Integrative approach using Yersinia pestis genomes to revisit the historical landscape of plague during the Medieval Period. Proceedings of the National Academy of Sciences of the United States of America (PNAS). ISSN 0027-8424. 17(22). doi: 10.1073/pnas.2007983117. Fulltekst i vitenarkiv
• Namouchi, Amine; Nome, Torfinn; To, Thu-Hien; Grønvold, Lars; Sandve, Simen Rød & Våge, Dag Inge (2019). SalmoBase2.0: An integrative genomic data resource for salmonids. Fulltekst i vitenarkiv
• To, Thu-Hien; Grønvold, Lars; Nome, Torfinn; Namouchi, Amine; Våge, Dag Inge & Sandve, Simen Rød (2019). A salmonid ohnology pipeline. Fulltekst i vitenarkiv
• Bramanti, Barbara; Namouchi, Amine; Schmid, Boris Valentijn; Dean, Katharine Rose & Stenseth, Nils Christian (2019). Out of the land of darkness: Plague on the fur trade routes. Proceedings of the National Academy of Sciences of the United States of America (PNAS). ISSN 0027-8424. 116(16), s. 7622–7623. doi: 10.1073/pnas.1902274116. Fulltekst i vitenarkiv
• Zegeye, Ephrem Debebe; Gómez-Muñoz, Marta; Namouchi, Amine; Balasingham, Seetha; Szpinda, Irena & Förstner, Konrad [Vis alle 8 forfattere av denne artikkelen] (2019). A nutrient supplement promotes the rapid detection and improved sensitivity of mycobacteria in clinical samples via the differential regulation of dormancy genes and noncoding RNAs. Fulltekst i vitenarkiv
• Namouchi, Amine (2017). Revisiting the evolutionary history of Yersinia pestis: Zooming into the second pandemic with new ancient genomes. Fulltekst i vitenarkiv
• Munoz, Marta Gomez; Namouchi, Amine; Balasingham, Seetha & Tonjum, Tone (2016). Identifiying ncRNAs expressed under genotoxic stress and dormancy in Mycobacterium tuberculosis. Fulltekst i vitenarkiv Vis sammendrag

  Mycobacterium tuberculosis (Mtb) is the intracellular pathogen that causes tuberculosis (TB), an infectious disease responsible for 1.5 million deaths worldwide in 2015. This bacterium is inhaled in aerosol droplets and once in the lung, it is engulfed by alveolar macrophages. Inside the activated macrophage, Mtb is exposed to a hostile environment, including reactive oxygen species (ROS) and reactive nitrogen species (RNS) that cause oxidation, deamination and alkylation of macromolecules. This condition is fatal to most bacterial pathogens, but Mtb has the ability to sense the host environment and eventually enter into a non-replicating, persistent state. The tubercle bacilli can remain in this latent stage for a lifetime without causing disease or, under certain conditions, it can awake resulting in TB disease. In this context, the aim of our study is to identify and characterize non-coding RNAs (ncRNAs) transcribed under genotoxic stress as well as during reactivation from the dormant stage. Here, Mtb strain H37Rv was exposed to various forms of genotoxic stress (oxidative, nitrosative or alkylative stress or double strand DNA breaks) and defined culture supplements. Total RNA from treated and untreated H37Rv cells was isolated and subjected to high-throughput RNA-seq. ncRNAs were identified using Rockhopper. A subset of the ncRNAs detected was selected for a more detailed analysis, including validation by northern blot. The secondary structure of the ncRNAs under study was predicted using mfold and their potential gene targets were predicted using intaRNA and CopraRNA. Ahead, we will combine our molecular and bioinformatics results with in vitro studies, including mapping 5’ and 3’ by RACE, establishing their secondary structure with SHAPE, and detecting sRNA-mRNA interactions by performing EMSA. The potential physiological role of the Mtb ncRNAs predicted will be tested by employing mutants or by repression, deletion or over-expression of the ncRNA of interest and measuring the potential phenotypic outcome by next-generation quantitative mass spectrometry.

• Frye, Stephan Alfons; Balasingham, Seetha; Beyene, Getachew Tesfaye; Homberset, Håvard; Namouchi, Amine & Tonjum, Tone (2016). Meningococcal DNA binding and unwinding proteins. Fulltekst i vitenarkiv Vis sammendrag

  Background: DNA helicases are a ubiquitous group of enzymes that use the energy of nucleoside triphosphate (dNTP) hydrolysis to catalyze the separation of double-stranded DNA (dsDNA). Helicases are involved in essentially every step in DNA metabolism, including replication, DNA repair, recombination, transcription, Holliday junction movement, and displacement of proteins from DNA. We investigated the DNA helicases RecG and DinG from Neisseria meningitidis (NmRecG and NmDinG) and their roles during genotoxic stress, including DNA damage. These helicases belong to superfamily 2, are ATP dependent and exert 5′to 3′ directionality. Our aim was to define the potential roles of NmRecG and NmDinG in DNA repair, recombination and replication (3R). Methods and results: Cell lysates from Nm wildtype and recG and dinG null mutants (NmrecG and dinG) were assessed by quantitative mass spectrometry (MS). In the ∆recG mutant, 16 proteins were differentially expressed as compared to the Nm wildtype. RecN, Ssb and DnaX were upregulated in the Nm∆recG mutant, while the type 4 pilus structural subunit protein PilE as well as pilus biogenesis components (PilF, PilT and PilQ) were downregulated. Global proteomics analysis of Nm wildtype and ∆recG mutant strains thus revealed the most abundant differentially expressed proteins and linked RecG to DNA repair, recombination and replication, pilus biogenesis components and glycan biosynthesis. Notably, when NmdinG cells grown under mitomycin C (MMC) stress, 134 proteins were shown to be differentially abundant compared to the unstressed NmdinG. Most of them are involved in metabolic functions like carbon, amino acid and nucleotide synthesis. Among 3R proteins, polymerase III subunits and recombinational repair proteins RuvA, RuvB, RecB and RecD were significantly downregulated while TopA and SSB were upregulated under stress. Genotoxic stress analysis demonstrated that NmdinG was more sensitive to double-strand DNA breaks (DSB) induced MMC than the Nm wildtype, defining the role of neisserial DinG in DSB repair. An unusually high number of DNA uptake sequences (DUS) that facilitate the transformation in neisserial species were identified in the Nm recG gene, signifying its importance. The genes encoding NmRecG and NmDinG were cloned and overexpressed, the recombinant NmRecG and NmDinG proteins were purified to homogeneity and their enzymatic activity was characterized. NmRecG acts through its ability to process Holliday junction (HJ) intermediates and catalyse branch migration of complex DNA structures. NmRecG and NmDinG possess 5′to3′ directionality and prefer DNA substrates containing a 5′overhang. ATPase activity of NmRecG and NmDinG is strictly DNA-dependent, and DNA unwinding activity requires nucleoside triphosphate in addition to divalent metal cations. This study contributes to understand the overall role of RecG in neisserial genome maintenance and evolution. Conclusions: This study gives new insight into the functional roles and interactions of the helicases RecG and DinG, elucidation their roles in meningococcal DNA repair and recombination, with a link of RecG to transformation also through pilus biogenesis.

• Namouchi, Amine (2015). Mycobacterium tuberculosis in the era of next generation sequencing. Fulltekst i vitenarkiv
• Yimer, Solomon Abebe; Namouchi, Amine; Riaz, Tahira; Zegeye, Ephrem Debebe; Godana, Alemayehu & Holm-Hansen, Carol J C [Vis alle 9 forfattere av denne artikkelen] (2015). Genomic and proteomic analysis of Mycobacterium tuberculosis lineage 7 strains from Ethiopia. Oral presentation at the 46th Union World Conference on Lung Health Cape Town, South Africa: 2 December - 6 December 2015. Fulltekst i vitenarkiv Vis sammendrag

  Background: Understanding the various factors driving the tuberculosis (TB) pandemic is essential to achieve the Global Plan for TB control and elimination. Recently, a new phylogenetic M. tuberculosis (Mtb) lineage (lineage 7) has been identified in Ethiopia, which is associated with prolonged delay among patients and grows slowly in vitro compared to other lineages. Given the potential implications for pathogenesis and virulence of the bacteria, genomic and proteomic studies on Mtb are warranted. The aim of this study was to delineate the molecular characteristics of Mtb lineage 7 strains by analysing genomic and proteomic profiles. Methods: The BACTEC MGIT 960 culture system was used to cultivate 30 Mtb lineage 7 isolates. DNA was extracted and whole genome sequencing was performed using the Illumina Hiseq2000. Overall, after aligning the generated reads for each strain to the genome sequence of the Mtb H37Rv strain, the coverage rate was above 300 times. High resolution Q-Exactive mass spectrometer was used to achieve proteome characterization. MaxQuant software was used to define the amounts of the various proteins present, while post-translational modifications (PTMs) were identified by Proteome Discoverer and manual inspection. Results: More than 800 mutations or single nucleotide polymorphisms (SNPs) specific to Mtb lineage 7 strains were identified. Compared to other lineages, Mtb lineage 7 strains exhibited a high number of mutations in genes involved in carbohydrate transport and metabolism, transcription, energy production and conversion. Notably, low frequencies of mutations were observed in genes involved in amino acid, lipid and coenzyme transport and metabolism, post translational modification, protein turnover and chaperone. Several interesting proteins, such as transcription factors, membrane proteins and proteins involved in pathogenesis of the bacteria were identified in lineage 7 strains. We also identified novel site-specific PTMs. Conclusions: This study generated novel knowledge regarding the genomic and proteomic profile of Mtb lineage 7 strains. Further research is essential to understand the consequences of the differential frequencies of mutations observed in the main gene groups of lineage 7 strains. Taken together, these studies have generated significant novel knowledge on a new lineage of Mtb, which can elucidate how these strains can have fitness for survival despite their relatively slow growth.

• Namouchi, Amine; Gómez-Muñoz, Marta; Debebe, Ephrem; Frye, Stephan Alfons; Tonjum, Tone & Balasingham, Seetha (2015). Global transcriptional responses to genotoxic stress in Mycobacterium tuberculosis. Fulltekst i vitenarkiv Vis sammendrag

  The aim of this study is to map the regulatory networks of differentially expressed genes in Mycobacterium tuberculosis (Mtb) and non-coding RNAs (ncRNAs) upon genotoxic stress. Understanding how these networks operate during the adaptive responses in Mtb will shed light on the pathogenesis and virulence of this successful pathogen. In this study, we exposed the Mtb cells to oxidative (H2O2 or paraquat), nitrosative (diethylenetriamine nitric oxide adduct), alkylative (methylnitronitrosoguanidine) or mitomycin–C stress. Thereafter, high-throughput RNA-sequencing was carried out on the isolated total RNA from the treated Mtb cells. The obtained data were subjected to differential expression analyses and ncRNAs discovery. We have identified number coding-genes which, upon genotoxic stress, are more than 50 fold up or down-regulated. Nitrosative and mitomycin-C stress induce pronounced effects on the global transcriptomic profile of Mtb. Under nitrosative stress we observed, more than 200 genes which are down-regulated 10-50 times. These genes are from almost all clusters of orthologous groups. For each genotoxic stress condition, we discovered stress specific small RNAs. Currently, these data are being validated. Novel candidates will be subjected to functional studies. We will also study the expression level of the identified genes and ncRNAs upon phagocytosis of Mtb by human macrophages. The outcome of these studies will give further understanding of the global transcriptomic network in Mtb, increase our basic understanding of the host–pathogen interaction and identify bacterial factors necessary for survival and growth within the human host. The gained knowledge will be used to design improved therapeutic interventions to eradicate TB worldwide.

• Yimer, Solomon Abebe; Norheim, Gunnstein; Namouchi, Amine; Zegeye, Ephrem Debebe; Kinander, Wibeke & Tonjum, Tone [Vis alle 9 forfattere av denne artikkelen] (2015). Lineage 7 Mycobacterium tuberculosis strains are associated with longer patient delay in pulmonary TB patients. Fulltekst i vitenarkiv
• Tonjum, Tone; Balasingham, Seetha; Yimer, Solomon Abebe & Namouchi, Amine (2015). Outbreak of tuberculosis in a Norwegian dance academy delineated by whole genome sequencing and high end mass spectrometry. Fulltekst i vitenarkiv Vis sammendrag

  Objectives: The aim of this study is to delineate the molecular characteristics of the strain of Mycobacterium tuberculosis (Mtb) that caused an outbreak of tuberculosis in a Norwegian dance academy in 2013-2014. Methods: An index case with tuberculosis (TB) teaching at a dance academy initiated an outbreak investigation. Clinical specimens were cultivated in the culture system BACTEC MGIT 960 system and Løwenstein-Jenssen and Middlebrook 7H10 solid media. Mtb was detected directly in clinical specimens by auramin-staining and fluorescence microscopy, PCR (Roche) or GeneXpert (Cepheid) analysis. Clinical Mtb isolates were identified by the BD ID test (Beckton-Dickinson) and/or by mass spectrometry (Bruker). Drug susceptibility testing (DST) was performed by BACTEC MGIT 960 culture and by GeneXpert to detect rifampicin resistance. Whole genome sequencing (WGS) was performed by MiSeq (Illumina). Peptide characterization and quantitation as well as identification of post-translational modifications (PTMs) were performed by electrospray-based high resolution mass spectrometry (Q-Exactive, Thermo-Fischer). Results: Totally, Mtb positive cultures from 11 cases attending the dance academy were isolated and identified in our laboratory. In addition, 10 related cases were identified in other laboratories. DST revealed that the isolates were streptomycin-resistant, otherwise they were drug sensitive. Over time, the isolate from the index case became isoniazid-resistant. Phylogenetic analysis of the WGS showed that the Mtb outbreak strain belongs to the W/Beijing family. The genomes of the Mtb outbreak strains had a massive genomic duplication spanning 350 kb and encompassing >300 genes. In total, this equates to 8% of the genome being present as two copies. The presence of IS6110 elements at both ends of the region of duplication, and in the novel junction region, suggests that it arose through unequal homologous recombination of sister chromatids at the IS6110 sequences. Next-generation mass spectrometry was employed to improve strain resolution, revealing unique PTMs seen for the first time in Mtb. Conclusion: The Mtb dance academy outbreak in 2013-2014 was fuelled by a W/Beijing strain whose evolution resulted in a massive gene duplication of 350 kb. This 350-kb duplication is restricted to the most recently evolved sublineages of the W/Beijing family, yielding a strain particularly fit to combat the specific selective pressure(s) encountered inside the host. Unique PTMs were detected in Mtb. Taken together, the findings from this study has generated novel knowledge on a significant outbreak of TB in a low-incidence country. The presence of the 350 kb duplication and its perpetuation most probably can explain the fitness enabling this particular Mtb W/Beijing strain to cause such an unusual TB outbreak.

• Tonjum, Tone; Balasingham, Seetha; Namouchi, Amine & Yimer, Solomon Abebe (2015). Recent evolution in Mycobacterium tuberculosis: Genome dynamics, small RNAs and vesicles. Fulltekst i vitenarkiv Vis sammendrag

  Mycobacterium tuberculosis (Mtb) is an intracellular pathogen of human macrophages and is the cause of tuberculosis (TB). Inside the macrophage, Mtb is subjected to multiple forms of genotoxic stress. DNA repair is vital for the fitness for survival, adaptation and evolution of Mtb and, hence, in-depth understanding of Mtb DNA metabolic pathways may also reveal novel targets for intervention. We are addressing the nature and role of DNA repair, small RNAs and vesicles in Mtb by convergently employing functional and comparative genomics, RNomics, proteomics and biochemical approaches in the context of structure-function and cellular systems to analyze stress responses in Mtb. Thereby, we test the hypothesis that DNA repair and small non-coding RNA (ncRNA) responses to genotoxic stress and the adaptive potential in Mtb is cradled in its ability to repair, replicate and recombine DNA and RNA. We performed comparative genomics and mapped the regulatory networks of differentially expressed genes upon genotoxic stress in various Mtb lineages. Mtb cells were subjected to oxidative (H2O2 or paraquat), nitrosative (diethylenetriamine nitric oxide adduct), alkylative (methylnitronitrosoguanidine) or mitomycin–C stress. RNA-seq data were subjected to differential expression analyses and ncRNAs discovery. We identified mRNAs and ncRNAs which, upon genotoxic stress, were more than 50 fold up or down-regulated. Nitrosative and mitomycin-C stress induced the most pronounced effects on the Mtb global transcriptomic profile. For example, under nitrosative stress, more than 200 genes were down-regulated 10-50 times. Comparative genomics of the same Mtb strains revealed an accumulation of single nucleotide polymorphisms in genes encoding DNA repair, recombination and replication enzymes. Understanding how these networks operate during the adaptive responses in Mtb will shed light on the pathogenesis and virulence of this successful pathogen. The findings on Mtb damage repair pathways and ncRNAs have implications for the subsequent delineation of mutagenicity and evolution, antigenic variation and drug resistance development.

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