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

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

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.