RdoA is a bacterial protein kinase from Salmonella enterica serovar Typhimurium first noted for its regulation of dsbA expression in this organism. The crystal structure of RdoA’s homologue, YihE from Escherichia coli, revealed a basic bi-lobal kinase domain that is a hallmark of the eukaryotic Ser/Thr, Tyr protein kinase superfamily. YihE however, bears the greatest structural similarity to choline kinase and aminoglycoside 3’-phosphotransferase [APH(3’)]-IIIa which are both atypical kinases. RdoA and YihE have demonstrated the capacity for autophosphorylation in vitro and the ability to phosphorylate myelin basic protein, however, the native kinase target protein has not been identified. Based on structural alignment with APH(3’)-IIIa, predictions were made of key residues involved in ATP binding and catalysis and five YihE mutants were generated. Both the wildtype and YihE mutants were cloned for expression as N-terminal histidine-tagged proteins. In the work presented here, these proteins have been overexpressed and purified for further study. Mutational analyses revealed that four of the five mutants had decreased kinase activity in comparison to the wildtype protein, thereby establishing the mutated residues as important for enzymatic activity. Several attempts were made to elucidate the substrate of RdoA/YihE, however, it remains unknown. Further investigation is necessary to identify its substrate(s) and to pinpoint its physiological significance. RdoA is a member of the Cpx regulon and its absence stimulates Cpx activation. Since the Cpx system is involved in regulating expression of cell surface appendages and is one of three envelope stress response systems, it is hypothesized that RdoA serves to relay Cpx activation signals. This is supported by studies on the effect of pH on Cpx activity in wildtype and rdoA- cells presented here. RdoA homologues are present in at least 85 different genera. This level of conservation is indicative of an important biological role for this previously uncharacterized bacterial protein kinase. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2007-12-04 18:19:29.574

La protéine kinase AMPc-dépendante (PKA) joue un rôle crucial dans la régulation neurohormonale de la fonction cardiaque. L’activation aiguë de la PKA est bénéfique car elle conduit à une augmentation de la contraction cardiaque en phosphorylant les acteurs clés du couplage excitation-contraction. En revanche, son activation chronique est délétère et ces effets semblent faire intervenir la régulation de protéines nucléaires pouvant conduire au remodelage hypertrophique et à l'insuffisance cardiaque. La localisation subcellulaire de la PKA, assurée par des protéines d’ancrage (AKAPs), est importante pour la rapidité et la spécificité d’action des hormones mettant en jeu la voie de l’AMPc. Les niveaux d’AMPc sont régulés par l’activité des adénylate cyclases et des phosphodiestérases (PDEs), et l’état de phosphorylation des protéines cibles de la PKA dépend de l’activité des Ser/Thr phosphatases (PPs). Dans le cœur, les PDEs les plus importantes dégradant l’AMPc sont les PDE3 et les PDE4. Les principales PPs cardiaques sont PP1, PP2A et PP2B. Dans une première partie de mon travail, j’ai mis au point, dans les cardiomyocytes de rats adultes, une mesure de l’activité de la PKA en temps réel dans les compartiments cytoplasmiques et nucléaires. J’ai utilisé pour cela des sondes de type AKAR (A-kinase activity reporters) basées sur le transfert d’énergie de fluorescence (FRET) et localisées spécifiquement dans le noyau ou dans le cytoplasme par des séquences d’adressage ou d’exclusion nucléaires. J’ai ainsi pu montrer qu’une stimulation maintenue des récepteurs β-adrénergiques active la PKA de façon plus importante dans le cytoplasme que dans le noyau, et que cette activation se développe lentement au niveau nucléaire que dans le cytoplasme. De ce fait, une stimulation brève des récepteurs β-adrénergiques active maximalement la PKA dans le cytoplasme, mais de façon marginale dans le noyau. Dans une seconde partie de l’étude, je me suis intéressée au rôle des PDE3 et PDE4 ainsi qu’à celui de PP1, PP2A et PP2B dans la régulation de l’activité PKA cytoplasmique et nucléaire, en réponse à une stimulation β-adrénergique. J’ai montré que la PDE4, mais pas la PDE3, régule l’activité de la PKA cytoplasmique et nucléaire. L’utilisation de souris invalidées pour les gènes Pde4b et Pde4d a révélé que l’isoforme PDE4B est prédominante pour la modulation de l’activité PKA cytoplasmique, alors que les deux isoformes PDE4B et PDE4D contribuent à la régulation de l’activité PKA nucléaire. Finalement, j’ai montré que la PP1 et la PP2A, mais pas la PP2B, participent à la terminaison des réponses β-adrénergiques dans le cytoplasme, alors qu’au niveau nucléaire, la PP1 semble jouer un rôle majeur. En conclusion, ce travail a mis en évidence le rôle des phosphodiestérases et des phosphatases dans l’intégration différentielle des réponses PKA à une stimulation β-adrénergique dans le cytoplasme et le noyau de cardiomyocytes adultes. / The cAMP-dependent protein kinase (PKA) exerts short term beneficial effects on cardiac function by phosphorylating several key excitation-contraction coupling (ECC) proteins. However, its chronic activation is deleterious on the long term, and this may involve regulation of nuclear effectors ultimately leading to hypertrophic remodelling and heart failure. The subcellular localization of PKA, mediated by anchoring proteins (AKAPs), is important for the speed and specificity of hormones that activate the cAMP pathway. The levels of cAMP are regulated by adenylyl cyclase and phosphodiesterases (PDEs), and PKA activity is counterbalanced by Ser/Thr phosphatases (PPs). In heart, the most important PDEs that degrade cAMP belong to the PDE3 and PDE4 famillies, whereas the major cardiac PPs are PP1, PP2A and PP2B. In a first part, I developed, in adult rat cardiomyocytes, a technique to measure PKA activity in real time specifically in the cytoplasm and the nucleus. For this I used genetically-encoded fluorescence resonance energy transfer (FRET) sensors called AKAR (A-kinase activity reporters) that can be targeted specifically to the nucleus or the cytoplasm by nuclear localization or exclusion sequences, respectively. Using this approach, I showed that maintained β-adrenergic stimulation activates PKA more efficiently and more potently in the cytoplasm than in the nucleus, and that the kinetics of PKA activation was much slower in the nucleus than in the cytoplasm. Accordingly, a short β-adrenergic stimulation maximally activated PKA in the cytoplasm but marginally activated PKA in the nucleus. In a second part, I characterized the respective contribution of PDE3, PDE4, and PP1, PP2A and PP2B families in the regulation of cytoplasmic and nuclear PKA activity in response to β-adrenergic stimulation. PDE4, but not PDE3, regulates PKA activity in the cytoplasm and in the nucleus. The use of knock out mice for Pde4b and Pde4d genes revealed that PDE4B plays a predominant role to modulate β-AR stimulation of cytoplasmic PKA, whereas in the nucleus both PDE4B and PDE4D isoforms contribute. Finally, I showed that both PP1 and PP2A, but not PP2B, participate to the termination of β-adrenergic PKA responses in the cytoplasm, whereas PP1 appears to play a major role in the nuclei. In conclusion, this work highlights the role of phosphodiesterases and phosphatases in the differential integration of PKA responses to β-adrenergic stimulation in the cytoplasm and the nucleus of adult cardiomyocytes.

Tuberculosis, caused by the intracellular pathogen Mycobacterium tuberculosis, is one of the most prevalent infectious diseases in our world today. In order to survive within the host the bacteria need to sense and respond to changes in the environment; however, signal transduction in this bacterium is poorly understood. PknB is a serine/threonine kinase essential for the in vitro survival of M. tuberculosis and therefore a potential drug target against the bacteria. It is the goal of the current study to elucidate downstream substrates of PknB. We have found that PknB shares in vitro substrates with another serine/threonine kinase, PknH, implying the potential complexity of the signaling pathways in the bacteria. We have also provided the first description of the coupling between serine/threonine kinases PknB and PknH with a two-component system response regulator DevR, and further proposed Ser/Thr phosphorylation as the negative regulator of DevR transcription activator activity based on LC-MS/MS analysis. Finally, we have identified a previously unknown phosphoprotein glyceraldehyde 3-phosphate dehydrogenase encoded by the ORF Rv1436, which demonstrates autophosphorylation activity and which phosphorylation is independent of PknB. Overall, the current study has contributed to advance our understanding of the signal transduction pathways and phosphoproteome in Mycobacterium tuberculosis.

Continuous sensation and reaction to environmental fluctuations is especially critical to the survival of unicellular organisms. Stress response mechanisms are essential for cells during the vegetative and sexual life cycles and quiescence. The Schizosaccharomyces pombe mitotic activator and stress response serine/threonine kinase Ssp1 acts independent of the major fission yeast Spc1 MAP kinase stress response cascade. Ssp1 is required at high temperatures in the presence of other stressors, ensures long-term viability in quiescent cells and allows efficient cell division in low-glucose conditions. Ssp1 is cytoplasmic but briefly localizes to the cell membrane after exposure to extracellular stress. It plays a role in actin depolymerization and is required for the change of growth polarity after cell division. After identifying 14-3-3 proteins Rad24 and Rad25 as putative Ssp1 binding partners, we confirmed the interaction with co-immunoprecipitation. Association of Ssp1 with Rad24 diminishes after 15 minutes of hyperosmotic stress, however Rad25 binding is retained. Loss of the rad24 gene product rescues both ssp1- mitotic delay at elevated temperatures and sensitivity to 0. 6M KCl. Conversely, overexpression of rad24 exacerbates ssp1 stress sensitivity and mitotic delay. Diffuse actin polarity and spheroid morphology in rad24- cells improves in an ssp1- background. Ssp1 localization to the cell membrane is negatively regulated by Rad24. Ssp1 does not co-localize with Arp3C (actin-related protein 3 homologue C) after osmotic stress, but instead appears to form a ring around the cell, suggesting localization to fission scars. Ssp1 is basally phosphorylated and hyperphosphorylated after glucose deprivation. Ssp1 is shuttled in and out of the nucleus and accumulates in the nucleus in an exportin Cmr1 dependent manner. Ssp1-GFP levels are constant in all stages of the vegetative cell cycle and Ssp1-GFP is present in both the sexual life cycle and quiescence. C-terminal and N-terminal truncation of ssp1 alters its subcellular localization. The C-terminal region is the site of hyperphosphorylation following glucose deprivation and is also necessary for membrane localization following osmotic stress. / Thesis (Ph.D, Biology) -- Queen's University, 2012-01-24 09:49:58.225

Tuberculosis, caused by the intracellular pathogen Mycobacterium tuberculosis, is one of the most prevalent infectious diseases in our world today. In order to survive within the host the bacteria need to sense and respond to changes in the environment; however, signal transduction in this bacterium is poorly understood. PknB is a serine/threonine kinase essential for the in vitro survival of M. tuberculosis and therefore a potential drug target against the bacteria. It is the goal of the current study to elucidate downstream substrates of PknB. We have found that PknB shares in vitro substrates with another serine/threonine kinase, PknH, implying the potential complexity of the signaling pathways in the bacteria. We have also provided the first description of the coupling between serine/threonine kinases PknB and PknH with a two-component system response regulator DevR, and further proposed Ser/Thr phosphorylation as the negative regulator of DevR transcription activator activity based on LC-MS/MS analysis. Finally, we have identified a previously unknown phosphoprotein glyceraldehyde 3-phosphate dehydrogenase encoded by the ORF Rv1436, which demonstrates autophosphorylation activity and which phosphorylation is independent of PknB. Overall, the current study has contributed to advance our understanding of the signal transduction pathways and phosphoproteome in Mycobacterium tuberculosis.

Tuberculosis, caused by the intracellular pathogen Mycobacterium tuberculosis, is one of the most prevalent infectious diseases in our world today. In order to survive within the host the bacteria need to sense and respond to changes in the environment; however, signal transduction in this bacterium is poorly understood. PknB is a serine/threonine kinase essential for the in vitro survival of M. tuberculosis and therefore a potential drug target against the bacteria. It is the goal of the current study to elucidate downstream substrates of PknB. We have found that PknB shares in vitro substrates with another serine/threonine kinase, PknH, implying the potential complexity of the signaling pathways in the bacteria. We have also provided the first description of the coupling between serine/threonine kinases PknB and PknH with a two-component system response regulator DevR, and further proposed Ser/Thr phosphorylation as the negative regulator of DevR transcription activator activity based on LC-MS/MS analysis. Finally, we have identified a previously unknown phosphoprotein glyceraldehyde 3-phosphate dehydrogenase encoded by the ORF Rv1436, which demonstrates autophosphorylation activity and which phosphorylation is independent of PknB. Overall, the current study has contributed to advance our understanding of the signal transduction pathways and phosphoproteome in Mycobacterium tuberculosis. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

La photosynthèse oxygénique peut être le lieu de formation des Formes Réactives de l’Oxygéne (FROs). Les FROs altèrent toutes les macromolécules de la cellule, générant ainsi un stress oxydant. Toute perturbation du métabolisme cellulaire peut conduire à ce type de stress. Les cyanobactérie sétant les premiers organismes à avoir émis de l’oxygène sur terre, elles ont du développer très tôt au cours de l’évolution des mécanismes de perception et de défense pour lutter contre ce stress. Nous nous sommes intéressés à l’étude des mécanismes qui permettent à la cyanobactérie filamentuse et diazotrophe Anabaena PCC 7120 de s’adapter à diverses conditions de stress et de carence : stress oxydant, carence en fer et en azote combiné. En réponse à une carence en azote combiné, elle différencie en 24 h des hétérocystes : cellules spécialisées dans la fixation de l’azote atmosphérique. Nous avons étudié la réponse transcriptomique globale de cette bactérie à la fois au stress oxydant et à la carence en fer et nous avons établit la connection existant entre ces deux stress. Nous avons pu identifier le régulateur transcriptionnel pleiotrope impliqué dans la perception et la signalisation du stress peroxyde et nous en avons élucidé le mécanisme d’action. Nous avons également étudié une Ser/Thr kinase qui joue un rôle important à la fois dans la réponse au stress oxydant et à la carence en azote combiné. Notre étude a montré que cette kinase pourrait être le lien moléculaire entre ces deux conditions, puisque une cible potentielle de cette kinase semble être la protéine HetR qui est le régulateur clé du processus de différenciation cellulaire. / Oxygenic photosynthesis may generate Reactive forms of Oxygéne (ROS). These reactive oxygen species can damage all the macromolecules of the cell, inducing oxidative stress. Any disruption of cellular metabolism can lead to oxidant damage. Cyanobacteria were the first organisms producing oxygen on Earth, they therefore had to develop very early during evolution the mechanisms of perception and defence to cope with this tstress. We are interested in studying the mechanisms that allow the diazotrophic filamentous cyanobacterium Anabaena PCC 7120 to adapt to various conditions of stress and stravations: oxidative stress, iron and combined nitrogen starvations. Anabaena PCC 7120 is a simple model for the study of cell differentiation. In response to combined nitrogen stravation it can differentiate heterocysts, cell specialized in molecular nitrogen fixation. We studied the global transcriptomic response of this bacterium to both oxidative stress and iron deficiency and we establish the crosstalk between these two stresses. We were able to identify the global transcriptional regulator involved in the perception and in the signaling of a peroxide stress. Its mechanism of action was elucidated. We also studied a Ser / Thr kinase that plays an important role both in the response to oxidative stress and combined nitrogen stravation. Our study showed that this kinase may be the molecular link between these two conditions, as a potential target of this kinase appears to be the HetR protein which is the key regulator of cellular differentiation process.

Afin de déjouer les défenses immunitaires de l’hôte et créer les niches nécessaires à leur survie, les bactéries pathogènes mettent on œuvre de nombreux mécanismes ciblant les voies de signalisation de la cellule hôte. L’un de ces mécanismes repose sur la sécrétion de protéines bactériennes dans les cellules cibles afin de moduler directement leurs réseaux de signalisation. Cependant, les signaux, les senseurs et les effecteurs impliqués dans ces régulations sont encore peu ou mal connus. La détection de l’environnement dans la cellule hôte lors de l'infection est l’élément clé d’une réponse adaptée, et les systèmes de signalisation basés sur les mécanismes de phosphorylation sont indispensables à l'adaptation hôte-pathogène. L’aspect innovant de ce projet repose sur l’étude du rôle des Ser/Thr kinases et phosphatases sécrétées lors des interactions hôte-pathogène, modifiant ainsi la réponse globale de l’hôte durant l’infection. Pendant ma thèse, j’ai tout d’abord étudié le rôle d’une nouvelle protéine kinase bactérienne identifiée chez Coxiella burnetii, nommée CstK (Coxiella serine threonine Kinase). C. burnetii, l’agent étiologique de la zoonose appelée fièvre Q, modifie les défenses de la cellule hôte, permettant sa réplication dans des vacuoles spécifiques à l’intérieur de la cellule hôte. Par ailleurs, la sécrétion d’un grand nombre d’effecteurs bactériens est indispensable au détournement du phagosome par Coxiella. Nous ainsi avons démontré que cette potentielle protéine kinase, identifiée in silico dans le génome de C. burnetii, est capable de s’autophosphoryler et par conséquent possède une activité kinase. De plus, nous avons identifié différentes protéines spécifiques de la cellule hôte interagissant avec CstK à l'aide du modèle amibe Dictyostelium discoideum, un phagocyte professionnel eucaryote, permettant des études génétiques et biochimiques. Dans la deuxième partie de mon projet, je me suis intéressée au rôle d’une probable protéine sécrétée, la tyrosine phosphatase PtpA, durant l’infection par Staphylococcus aureus. Bien connue dans les hôpitaux, où elle est responsable de nombreuses maladies nosocomiales, cette bactérie possède un grand nombre de facteurs de virulence, responsables d’infections variées, et l’apparition exponentielle de souches multi-résistantes en font un problème majeur. Ce pathogène est capable d’envahir et de persister dans un grand nombre de types cellulaires différents chez l’Homme, en sécrétant des protéines effectrices qui vont moduler les réponses cellulaires. Nous avons démontré que PtpA était sécrétée durant la phase de croissance bactérienne, et pu déterminer que PtpA possédait une activité tyrosine phosphatase, régulée par la tyrosine kinase CapA1B2 de S. aureus. Enfin, en utilisant le modèle D. discoideum, nous avons pu identifier des protéines de l’hôte qui interagissent avec PtpA, mais leur rôle dans l’infection n’est pas encore connu. / Bacterial pathogens have developed diverse strategies towards host signalling pathways, in order to subvert the immune response and/or create permissive niches for their survival. One such strategy is based on the secretion of bacterial signalling proteins into the target host cells, thereby directly modulating the status of host signalling networks. Because the mechanisms involved are largely intractable to most in vivo analyses, very little is known about the signals, sensors, and effectors mediating these adaptations. Sensing the host environment is a key component to execute appropriate developmental programs, and the eukaryotic-like phosphosignaling systems in prokaryotes are emerging as equally important regulatory systems as the well-known eukaryotic systems, but the study of their functions is still in its infancy. The innovative aspect of this project resides in the study of the emerging role of secreted Ser/Thr kinases and phosphatases in the control of host-pathogen interactions thus modifying the global host response during infection. During my thesis, I first investigated the role of a novel bacterial protein kinase identified in Coxiella burnetii that we named CstK (Coxiella serine threonine Kinase). C. burnetii, the etiological agent of the emerging zoonosis Q fever, subverts host cell defenses, permitting its intracellular replication in specialized vacuoles within host cells. Secretion of a large number of bacterial effectors into host cell is absolutely required for rerouting the Coxiella phagosome. We demonstrated that this putative protein kinase identified by in silico analysis of the C. burnetii genome is able to autophosphorylate and undergoes in vitro phosphorylation. Moreover, we identified specific host cell proteins interacting with CstK, by the use of the model amoeba Dictyostelium discoideum, an eukaryotic professional phagocyte amenable to genetic and biochemical studies. In the second part of my project, I was interested in the role of a putative secreted protein tyrosine phosphatase (PtpA) during Staphylococcus aureus infection. Well-known in hospital-acquired diseases, this bacteria produces multiple virulence factors that lead to various severe diseases, and the increase of multi-resistant strains is a major concern. This pathogen has the ability to invade and persist in a number of different human host cell types, secreting effector proteins to modulate cellular responses. Here we demonstrated that PtpA is secreted during the bacterial growth. We also determined that PtpA presents a tyrosine phosphatase activity that is regulated by the tyrosine protein kinase CapA1B2 of S. aureus. At last, using the D. discoideum model, we identified some host proteins that interact with PtpA, but their link with infection still remain to be studied.

Reversible protein phosphorylation is considered the universal language for intracellular communication in all living organisms. This process, catalysed by protein kinases and phosphatases, enables the translation of extracellular signals into cellular responses and also allows for adaptation to a constantly changing environment. In recent years, a number of bacterial eukaryotic-type Ser/Thr protein kinases and phosphatases have been identified. However, their precise functions and substrates are not yet well defined. The genome of opportunistic human pathogen Pseudomonas aeruginosa contains at least five genes encoding putative eukaryotic-type Ser/Thr protein kinases and phosphatases. In the first part of this study, we have attempted to establish the role of Ser/Thr protein kinase PpkA and phosphatase PppA, which belong to type VI secretion system H1-T6SS. Double mutant strain ∆pppA-ppkA was prepared in P. aeruginosa PAO1 background. Phenotypic studies revealed that the mutant grew slower than the wild-type strain in minimal media and exhibited reduced secretion of pigment pyocyanin. In addition, the mutant had altered sensitivity to oxidative and hyperosmotic stress conditions. Consequently, mutant cells had an impaired ability to survive in murine macrophages and an attenuated virulence in the...

La recrudescence de la tuberculose et l'apparition des souches de Mycobacterium tuberculosis (Mtb) résistantes aux antibiotiques souligne la nécessité de rechercher de nouveaux antituberculeux avec de nouveaux mécanismes d'action. Les mycobactéries possèdent une membrane externe (appelée aussi mycomembrane), très riche en lipides qui leur confère une imperméabilité remarquable. Les constituants majeurs de la mycomembrane sont des acides gras originaux et à très longue chaine, appelés acides mycoliques (AMs). La biosynthèse de ces AMs est essentielle à la croissance mycobactérienne et comporte des enzymes-clefs, dont nombreuses ont été validées comme des cibles prometteuses pour la recherche de nouveaux antituberculeux. Parmi elles, FadD32, une Fatty-acyl AMP Ligase impliquée dans l'étape ultime de cette voie de biosynthèse. Si les propriétés physico-chimiques de l'enzyme ont été caractérisées, la régulation de l'activité de cette enzyme essentielle reste encore à découvrir. La première partie de cette thèse porte sur la mise en évidence de la régulation post-traductionnelle par phosphorylation de FadD32 par des Sérine/Thréonine Protéine Kinases (STPKs) de type eucaryote. Nous avons montré que FadD32 pouvait être phosphorylée in vitro par plusieurs STPKs de Mtb. Cette phosphorylation réversible module négativement l'activité de la protéine, comme démontré pour d'autres protéines de la voie de biosynthèse des AMs. Afin de mieux comprendre l'impact de la phosphorylation par STPK sur la production des AMs et sur la synthèse de la mycomembrane, nous avons surexprimé une STPK de Mtb chez M. smegmatis, espèce modèle des mycobactéries. Nous avons pu observer un changement significatif de la quantité des AMs. Dans la deuxième partie de la thèse, nous avons optimisé et réalisé le criblage biochimique d'une chimiothèque sur l'activité de FadD32 et avons identifié des touches qui devraient servir dans la recherche de nouveaux agents antituberculeux. / The resurgence of tuberculosis (TB), together with the emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb), highlights the need for new anti-TB drugs with novel mechanisms of action. Mycobacteria possess an outer membrane (also called mycomembrane), which confers to them a remarkably impermeable cell envelope. The major constituents of the mycomembrane are very long-chain fatty acids with original structures, the so-called mycolic acids (MAs). The biosynthesis of MAs is essential for mycobacterial growth and involves several key enzymes, many of which have been validated as promising anti-TB drug targets. Among them, FadD32, a Fatty-acyl AMP Ligase implicated in the last step of the biosynthesis pathway, has been characterized biochemically and its protein structure has been solved recently. However, the regulation aspect of the enzyme is still to be deciphered. The first part of the thesis focuses on the post-translational regulation by protein phosphorylation of FadD32 by eukaryotic-like Serine/Threonine Protein Kinases (STPKs). We showed that FadD32 is the substrate of several Mtb STPKs. The reversible phosphorylation negatively modulates the adenylation activity of the protein. In order to determine the impact of the post-translational regulation on MA production, we over-expressed a STPK of Mtb in M. smegmatis, a validated surrogate of Mtb. We observed a significant change in the amounts of MAs. In the second part of the thesis, we screened a chemical library against FadD32 and identified several hits that should help in the development of new anti-TB agents.