Analysis of genetic variability within the Beijing lineage of «Mycobacterium tuberculosis»
Bibliographic record
Abstract
Previous research has demonstrated that the DosR/DosS two-component signaling system is constitutively overexpressed in modern Beijing strains of Mycobacterium tuberculosis (M.tb).It is hypothesized that constitutive overexpression of this regulatory system is related to the unique pathogenic properties reported for this important strain lineage.Within this thesis we attempted to determine the cause of this overexpression phenotype.We first compared known DosR signaling stimuli between different strains of M. tb, looking specifically at NO (nitric oxide) and redox (reduction-oxidation) balance.We demonstrated that there was no difference in endogenous NO production between strains, but we showed that there was a significantly more reductive NADH/NAD pool in modern Beijing strains.To determine the specific factor responsible, we transformed four independent strains of M. tb with a DosR-dependent XylE reporter and developed a novel colony-screening assay to determine DosR activity.We then performed whole-genome transposon mutagenesis to screen for genes that are potentially able to modify DosR expression and/or activity.After screening ~80,000 transductants, we identified 49 genes that appear to influence reporter activity.Surprisingly, when analyzed by qRT-PCR, we were not able to demonstrate a modulation in dosR expression for any of these putative candidates, which suggested that expression of our reporter was being modified in a DosR-independent manner.Further analysis has revealed that many candidate genes directly affect the activity of the reporter protein by modulating H 2 O 2 levels within the bacteria.Intriguingly, this lead us to the observation that modern Beijing strains have elevated catalase activity compared to other strains of M. tb.This work further characterizes the unique properties of the Beijing lineage of M. tb and identifies a novel phenotype that may be associated with its pathogenesis.RÉSUMÉ Des recherches antérieures ont démontré que le système de signalisation DosR/DosS est surexprimé dans les souches de Beijing moderne, une sous-catégorie du Mycobacterium tuberculosis (M tb).Une hypothèse explique que la surexpression du système règlementaire est la raison des propriétés plus pathogénique de la souche Beijing moderne.Dans cette thèse, nous avons fait une tentative pour trouver la cause de la surexpression.Pour débuter, les stimuli de signalisation DosR connu a été comparé entre différentes souches de M. tb, en regardant spécifiquement les données concernant l'oxyde nitrique (NO) et l'équilibre d'oxydo-réduction (redox).Nous avons démontré que la production NO endogène est semblable aux autres souches.Par contre, nous avons prouvé que les souches Beijing moderne expriment une plus grande proportion de NADH/NAD, ce qui lui donne une propriété plus réduisant que les autres souches.Pour trouver l'unique facteur responsable, nous avons transformé quatre souches de M. tb possédant un reporteur DosR-dépendant et développé une analyse innovatrice : un criblage de colonie.Ceci a pu évaluer l'activité du DosR.Ensuite, nous avons exécuté une mutagénèse utilisant un transposon pour déterminer les gênes capables de modifier l'expression et/ou l'activité du DosR.Après l'analyse d'environ 80,000 colonies, nous avons identifié 49 gênes qui semblaient avoir influencé l'activité du reporteur.À notre surprise, vérifiant l'analyse du qRT-PCR sur nos bactéries, nous étions incapable de démontrer une modulation dans l'expression du dosR.Ceci suggérait donc que l'expression du reporteur était en fait modifiée de manière indépendante du DosR.De plus profondes analyses ont révélé que plusieurs gênes bactériennes, dont nous avons antérieurement identifié, jouent directement un rôle sur l'activité du reporteur en contrôlant les niveaux de H 2 O 2 de la bactérie.Cette découverte a mené à la conclusion que les souches Beijing moderne ont un niveau plus élevé de catalase comparé aux autres.En outre, cette thèse s'interroge plus profondément sur les propriétés uniques de la souche Beijing moderne et identifie un nouveau phénotype qui pourrait être associé au pathogène.perform research in his laboratory.I would like to thank him for the idea for my project and for all the time he dedicated to discussing and helping me to critically analyze my results.I would also like to thank him for being so hands-on, and for caring about my future.I would like to thank Dr. Pilar Domenech for keeping the lab running and supplies available.I would also like to thank Pilar for always being available to discuss results and for help troubleshooting experiments.I would like to thank Ashley Fallow and Gaëlle Kolly for preliminary cDNA and for experimental advice.I would like to thank I would like to thank Fiona McIntosh for supervising the BL3 lab and keeping it organized and maintained.I would like
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.001 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.001 | 0.001 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".