PknB is a putative transmembrane eukaryotic-type serine/threonine protein kinase (STPK) in the cariogenic bacterium Streptococcus mutans that affects biofilm formation, genetic competence and acid tolerance. PknB contains extracellular penicillin-binding and serine/threonine kinase associated (PASTA) domains predicted to bind the D-alanyl-D-alanine (D-ala-D-ala) dipeptide of unlinked peptidoglycan. D-ala-D-ala elicits responses dependent and independent of the presence of pknB. Biofilm-derived cells of a pknB-deficient mutant (PKNB) exhibited concentration-dependent growth enhancement with D-ala-D-ala, which was not a nutrient response as addition of L-alanine or D-alanine did not give the same results. A total of 77 genes were differentially expressed in PKNB, including 7 with putative functions in fatty acid biosynthesis. PKNB was more sensitive to cell wall- and membrane-targeting antibiotics compared to wild-type. Based on these results, PknB in S. mutans appears to play an important role in cell wall biosynthesis, response to membrane stress and/or regulation of cell membrane composition.

PknB is a putative transmembrane eukaryotic-type serine/threonine protein kinase (STPK) in the cariogenic bacterium Streptococcus mutans that affects biofilm formation, genetic competence and acid tolerance. PknB contains extracellular penicillin-binding and serine/threonine kinase associated (PASTA) domains predicted to bind the D-alanyl-D-alanine (D-ala-D-ala) dipeptide of unlinked peptidoglycan. D-ala-D-ala elicits responses dependent and independent of the presence of pknB. Biofilm-derived cells of a pknB-deficient mutant (PKNB) exhibited concentration-dependent growth enhancement with D-ala-D-ala, which was not a nutrient response as addition of L-alanine or D-alanine did not give the same results. A total of 77 genes were differentially expressed in PKNB, including 7 with putative functions in fatty acid biosynthesis. PKNB was more sensitive to cell wall- and membrane-targeting antibiotics compared to wild-type. Based on these results, PknB in S. mutans appears to play an important role in cell wall biosynthesis, response to membrane stress and/or regulation of cell membrane composition.

Spr0334, new protein of cell division in Streptococcus pneumoniae Streptococcus pneumoniae is an important human pathogen. The geonome of this bacteria encodes a single gene for eukaryotic-like serine / threonine protein kinase called StkP. StkP regulates many physiological processes such as pathogenesis, competence for genetic transformation, resistance to various stresses and resistance to antibiotics. It also affects the transcription of many genes involved in cell wall biosynthesis, pyrimidine metabolism, DNA repair and iron uptake. Recent studies have shown that StkP is located in the cell division septum and significantly regulates cell division and morphology. Its substrates include, among others, cell division protein DivIVA, FtsZ and FtsA. Analysis of phosphoproteome maps of wild type and ΔstkP mutant strain of S. pneumoniae showed that in vivo StkP phosphorylates several putative substrates including the protein Spr0334. Mass spectrometry analysis identified phosphorylation sites of the protein Spr0334: threonine 67 and threonine 78. Furthermore, it was found that the protein Spr0334 is located in the cell division septum, which led to the hypothesis that it could be newly identified cell division protein in S. pneumoniae. The main aim of this thesis was to describe the function of the...

La capacidad de Mycobacterium tuberculosis para sobrevivir dentro del macrófago contribuye grandemente a su patogenicidad, latencia y persistencia durante la infección. Este bacilo induce alteraciones en el ambiente intrafagosomal e inhibe la maduración del fagosoma, favoreciendo su supervivencia intracelular. M. tuberculosis PknG secuestra al macrófago precisamente al evitar la fusión fagosoma-lisosoma. En este sentido, PknG representa una familia de dianas novedosas para enfrentar la necesidad de nuevos antimicrobianos para la tuberculosis latente. Aquí, apuntamos a: (i) elucidar la base estructural-molecular del ATP y Mg2+ como cofactores de PknG; (ii) caracterizar los parámetros cinéticos que gobiernan la formación del complejo PknG:ATP; e, (iii) identificar péptidos capaces de unirse a PknG para investigar experimentalmente su interactoma usando enfoques combinatorios como “Phage Display”. Nuestros resultados confirman que PknG se une exclusivamente al ATP con una constante de disociación (KD) de 108.8  22.9 µM. El Mg2+ estabiliza térmicamente a PknG de forma ATP-dependiente. Análisis de estado pre-estacionario muestran que la unión y disociación del ATP es rápida en el complejo PknG:ATP. Usando PknGN-Ext, TPR resolvimos la estructura cristalina en el estado unido al ADP mientras que demostramos que el ATP imposibilita la cristalización. Los análisis bioinformáticos de las librerías enriquecidas por Phage Display identificaron 57 potenciales peptidos que interactuarían con PknG. Una comparación cercana con el proteoma de M. tuberculosis proporcionó un subconjunto de 20 proteínas que podrían interactuar con PknG. Nuestros resultados confirmaron cinco proteínas asociadas a PknG previamente reportadas: PknG, DnaK chaperona, transportador ABC Rv1747, Proteína Ribosomal L23 y Factor de Elongación Tu, resaltando la validez de nuestra plataforma para descubrir el interactoma de PknG. Así, nuestros resultados revelan interacciones proteína-proteína putativas que podrían participar en la supervivencia micobacteriana, mientras que también proporcionan bases sólidas para desarrollar drogas antituberculosas al interrumpir estas interacciones o explotar estos peptidos tipo compuesto líder. / The ability of Mycobacterium tuberculosis to survive inside the macrophage greatly contributes to its pathogenicity, latency and persistence during infection. This bacillus induces alterations in the intraphagosomal environment and inhibits phagosome maturation, thus promoting mycobacterial survival. M. tuberculosis PknG hijacks the macrophage precisely by avoiding phagosome-lysosome fusion. In this sense, PknG represents a family of novel targets to cope with the need for new antimicrobials for latent tuberculosis. Here, we aimed to: (i) elucidate the structural-molecular basis of ATP and Mg2+ as PknG cofactors; (ii) characterize the kinetic parameters governing PknG:ATP complex formation; and, (iii) identify PknG-binding peptides to experimentally query PknG’s interactome using combinatorial approach such as Phage Display. Our results confirm that PknG exclusively binds to ATP with a dissociation constant (KD) of 108.8  22.9 µM. Mg2+ thermally stabilizes PknG in an ATP-dependent manner. Pre-steady-state analyses show that ATP binding and dissociation are rapid in the PknG:ATP complex. Using PknGN-Ext, TPR we solved the ADP-state crystal structure while showing that ATP precludes crystallization. Phage Display and bioinformatic analyses identified 57 potential PknG binders. A close comparison to the M. tuberculosis proteome provided a subset of 20 proteins that may interact with PknG. Our results confirmed five previously reported PknG-associated proteins: PknG, DnaK chaperone, ABC transporter Rv1747, Ribosomal Protein L23 and Elongation Factor Tu, highlighting our platform’s validity to uncover the PknG interactome. Altogether, our results reveal putative protein-protein interactions that may play a role in mycobacterial survival, while also providing solid bases for the development of anti-tuberculosis drugs by disrupting these interactions or exploiting these lead-like peptide molecules. / Tesis

Analysis of signaling cascade of protein kinase StkP in Streptococcus pneumoniae Streptococcus pneumoniae is not only an important human pathogen but also an appropriate model organism to investigate cell division in ovoid bacteria. This bacterium lacks both, NO and Min systems for selection of cell division site. Thus, the mechanism which determines the site of cell division is unknown. Additionally, the genome of S. pneumoniae encodes a single gene for eukaryotic-like serine/threonine protein kinase StkP and a single gene for eukaryotic-like serine/threonine protein phosphatase of PP2C type called PhpP. StkP is one of the main regulators of cell division. Cell division is probably affected by the phosphorylation of its substrates, which include, among others, cell division proteins FtsZ, FtsA, DivIVA, MacP, Jag/KhpB/EloR, and LocZ/MapZ. The aim of the first project of this dissertation thesis is determination of the function of protein LocZ in the cell division. In summary, locZ is not essential, however, it is involved in proper septum placement in S. pneumoniae and our data suggest that it is a positive regulator of Z-ring placement. Cells lacking LocZ are able to form Z-ring, but the Z-ring is spatially misplaced resulting in cell division defects, shape deformation, and generation of unequally sized,...

Streptococcus pneumoniae (ou pneumocoque) est un agent pathogène humain responsable de maladies invasives et potentiellement mortelles. Les mécanismes impliqués dans le processus d'invasion restent largement inconnus, mais plusieurs observations suggèrent que les processus de signalisation impliquant la phosphorylation des protéines participeraient au caractère invasif du pneumocoque. Le génome de S. pneumoniae code pour une seule tyrosine-kinase (CpsD) et une seule sérine/thréonine-kinase (StkP). Cette dernière serait notamment impliquée dans la virulence, la compétence et la division cellulaire. Elle représente donc une cible thérapeutique potentielle intéressante pour lutter contre les infections liées au pneumocoque. L'objectif de cette thèse a donc été de caractériser le rôle de cette sérine/thréonine-kinase StkP dans la division cellulaire du pneumocoque. StkP est une protéine transmembranaire qui se caractérise par la présence de motifs structuraux conservés dans son domaine catalytique appelés motifs de Hanks. De plus, StkP possède un domaine extracellulaire composé de la répétition de quatre domaines PASTA (Penicillin-binding protein And Serine/Threonine kinase Associated). Le modèle actuel suggère que ces domaines PASTA seraient capables de fixer des fragments de la paroi cellulaire afin de permettre l'activation de StkP qui se comporterait donc comme un récepteur membranaire permettant de réguler la division cellulaire du pneumocoque. Mes travaux de thèse ont permis de revisiter ce modèle en démontrant que les domaines PASTA ne servent pas uniquement à contrôler l'activité protéine-kinase de StkP mais également à contrôler l'épaisseur de la paroi cellulaire et ainsi permettre la constriction de la cellule. Plus précisément, j'ai démontré que le domaine PASTA distal est spécialisé dans l'interaction avec une hydrolase de la paroi cellulaire alors que les trois autres domaines PASTA sont nécessaires à l'activité kinase de StkP mais également au positionnement du domaine PASTA distal. Ainsi, le domaine extracellulaire de StkP se comporterait comme une règle permettant de définir l'épaisseur de la paroi cellulaire de la bactérie. Ces travaux permettent donc de proposer un nouveau modèle d'activation et de régulation de la division cellulaire par la sérine/thréonine-kinase StkP / Streptococcus pneumoniae (the pneumococcus) is one of the most important human pathogens that causes potentially fatal invasive diseases. Mechanisms required for the pneumococcal invasion process remain largely unknown, but several observations suggest that phosphorylation-based signaling processes will be at play in the invasiveness of the pneumococcus. S. pneumoniae encodes only one tyrosine-kinase (CpsD) and one serine/threonine-kinase (StkP). The latter would be involved in virulence, competence, and cell division. StkP represent therefore a promising target to combat pneumococcal infections. My aims were to better understand the role of StkP in pneumococcal cell division. StkP is a transmembrane protein characterized by the presence of a series of conserved structural motifs called Hanks motifs in its catalytic domain. In addition, StkP possesses an extracellular domain composed of the repetition of four PASTA domains (Penicillin-binding protein And Serine/Threonine kinase Associated). The current model proposes that PASTA domains are able to bind cell wall fragments resulting in StkP kinase activation. StkP would thus behave as an authentic kinase receptor regulating cell division. My thesis works has allowed to revisit this model by showing that PASTA domains do not only serve StkP kinase activation. Rather, they contribute to determine the cell wall thickness and govern cell constriction. More precisely, I demonstrated that the distal PASTA domain possesses unique features for the binding of a cell wall hydrolase whereas the other three contributes to StkP kinase activation and the positioning of the distal PASTA domain. Thus, the extracellular domain of StkP acts as a ruler determining the cell wall thickness. This work allows to propose an alternative model of activation and regulation of cell division by the serine/threonine-kinase StkP

Malgré les modèles établis pour certaines bactéries, la morphogenèse de bactéries de formes atypiques est peu comprise. C'est le cas de la bactérie pathogène pour l'homme Streptococcus pneumoniae, ou pneumocoque, qui possède une forme ovo-diplococcale. Cependant, à mon arrivé au laboratoire, il avait été démontré qu'une sérine/thréonine protéine-kinase membranaire appelée StkP était indispensable à la division cellulaire et à la morphogenèse du pneumocoque. L'objectif de ma thèse a ainsi été de caractériser certains substrats de StkP et d'étudier leur rôle, ainsi que l'impact de leur phosphorylation, au cours du processus de division cellulaire. Dans ce contexte, j'ai montré que le substrat DivIVA et son paralogue GpsB coordonnent l'élongation et la division cellulaire du pneumocoque. Ces travaux permettent de proposer un nouveau modèle de morphogenèse du pneumocoque dans lequel la triade StkP/DivIVA/GpsB organise la synthèse de la paroi cellulaire nécessaire à l'élongation et à la division de la cellule. J'ai également mis en évidence que la protéine MapZ interagit avec la paroi cellulaire lors de l'élongation cellulaire afin de marquer de manière permanente le site de division, où elle recrute la protéine FtsZ. Ces travaux ont ainsi permis d'identifier un système inédit de régulation positive du positionnement du site de division chez les bactéries. Enfin, j'ai caractérisé les déterminants moléculaires du positionnement de MapZ au centre de la cellule. S. pneumoniae étant un pathogène humain important, nous pouvons anticiper que nos données pourraient servir de base fondamentale à des projets plus appliqués de lutte contre les infections bactériennes / Despite the established models for some bacteria, the morphogenesis of bacteria with atypical shapes is poorly understood. This is the case of the human pathogen Streptococcus pneumoniae, or pneumococcus, that displays an ovo-diplococcal shape. However, when I joined the lab, it had just been shown that a membrane serine/threonine kinase named StkP was crucial for the cell division and the morphogenesis of the pneumococcus. The goal of my thesis was to characterize the substrates of StkP and to study their function as well as the impact of their phosphorylation in the cell division process. First, I have shown that the substrate DivIVA together with its paralog GpsB coordinate cell elongation and division of the pneumococcus. Based on these observations, we propose a new model of pneumococcal morphogenesis in which the triad StkP/DivIVA/GpsB organizes cell wall synthesis involved in cell elongation and division. In a second part of my work, I have studied another substrate of StkP that was of unknown function and that we named MapZ. I have shown that MapZ interacts with the cell wall during the cell elongation to position at midcell. Then MapZ recruits the cell division protein FtsZ and controls the closure of the Z-ring. This work has uncovered a new mechanism of positive regulation for the positioning of the division site in bacteria. Finally, I characterized the molecular determinants of MapZ positioning at the division site. S. pneumoniae is an important human pathogen, we can thus anticipate that our work will represent a fundamental base for applied projects in order to develop new strategies against bacterial infections