Publication: Caracterización estructural y funcional de la glucosaminidasa LytB de "Streptococcus penumoniae"
Loading...
Official URL
Full text at PDC
Publication Date
2016-03-08
Authors
Advisors (or tutors)
Editors
Journal Title
Journal ISSN
Volume Title
Publisher
Universidad Complutense de Madrid
Citations
Exportar
Abstract
LytB, una enzima codificada por la bacteria Streptococcus pneumoniae, es una murein hidrolasa no lítica, localizada en los polos de la célula, responsable de la separación de las células hijas durante el proceso de división celular (García et al., 1999; De Las Rivas et al., 2002). Consta de un módulo de unión a colina (CBM) (LytBCBM), situado en el extemo N-terminal, formado por 18 repeticiones de secuencia dispuestas en tándem que reconocen de forma específica los motivos de fosforilcolina (P-col) presentes en los ácidos teicoicos de la envuelta celular de neumococo y que es esencial para la catálisis. Además contiene un módulo SH3b (LytBSH3b) y uno de tipo WW (LytBWW), ambos importantes para el reconocimiento del sustrato y la adhesión a las células del hospedador (Bai et al., 2014), y un módulo perteneciente a la familia GH73 de las glicosil hidrolasas (LytBGH73) en la región C-terminal, responsable de su actividad catalítica. A pesar de que LytB es un factor de virulencia implicado en la colonización de la nasofaringe, la evasión de la respuesta inmune (Gosink et al., 2000; Ramos-Sevillano et al., 2011) y la formación de biofilms (Moscoso et al., 2006), y que por tanto podría considerarse como una diana potencial en el desarrollo de nuevas vacunas o antibióticos, su actividad catalítica o su especificidad por el sustrato no han sido hasta ahora estudiadas. De este modo, el objetivo de esta tesis ha sido la purificación de formas estructuralmente homogéneas de LytB y la caracterización de su estabilidad y de su organización estructural, en presencia y ausencia de posibles ligandos, así como analizar los determinantes estructurales de la especificidad de sustrato de la proteína e identificar los residuos relevantes para la catálisis. La caracterización de las muestras de LytB purificadas por cromatografía de afinidad y ultracentrifugación analítica puso de manifiesto la coexistencia de monómeros y formas de mayor tamaño molecular que se separaron por cromatografía de exclusión molecular y, mediante ensayos de dispersión de células de neumococo de la cepa R6B (en la que el gen lytB está inactivado y por tanto crece formando largas cadenas de células), se verificó que ambas formas eran funcionales...
LytB, encoded by Streptococcus pneumoniae, is a non-lytic murein hydrolase, located at the cell poles, and responsible for daughter cell separation during division (García et al., 1999; De Las Rivas et al., 2002). It comprises an N-terminal choline-binding module (CBM) (LytBCBM) made of 18 tandemly-arranged repeats that specifically recognize the phosphocholine (P-col) moieties present in pneumococcal teichoic acids that is essential for catalysis. Besides it contains an SH3b module (LytBSH3b) an a WW-like domain (LytBWW), both relevant for substrate recognition and LytB-mediated host-cell adhesion (Bai et al., 2014), and a C-terminal module (LytBGH73) belonging to the GH73 family of glycoside hydrolases, responsible for its glucosaminidase activity. LytB is a virulence factor involved in nasopharyngeal colonization, evasion of host immunity (Gosink et al., 2000; Ramos-Sevillano et al., 2011) and biofilm formation (Moscoso et al., 2006), therefore, LytB is a putative vaccine/drug target. But none of its catalytic activity neither its substrate specificity have been studied yet. So the aim of this thesis is the purification of structurally homogeneous forms of LytB in order to characterize its stability and structural organization in presence and absence of possible ligands, to analyze the structural determinants of LytB substrate specifity, and to identify the relevant residues for the catalysis. Purified samples characterization by using affinity chromatography and analytic ultracentrifugation, showed the coexistence of monomeric and multimeric forms of the protein. These forms were purified by size exclusion chromatography and revealed both active in cell-dispersing activity analysis using cultures of R6B (a LytB-deficient strain that forms long chains of cells). Thermic stability studies (differential scanning calorimetry (DSC) and circular dichroism (DC)) showed that the moderate stability of free LytB drastically increases upon choline recognition by the CBM module and interestingly this stabilization effect is transmitted to the catalytic module. These results in combination with the analysis of LytB interaction with choline analogs by using isothermal titration calorimetry (ITC), allowed us to discard the participation of the phosphate group and the galactose ring from GalNAc-6-fosfate residues in the cell wall-CBM union, and the quantification introduced by the negative charge of the phosphate group. Besides, LytB doesn’t recognize any of the teichoic acids components, so the polar localization of the enzymes may be due to the peptidoglycan (PG) recognition or surface proteins of the bacteria...
LytB, encoded by Streptococcus pneumoniae, is a non-lytic murein hydrolase, located at the cell poles, and responsible for daughter cell separation during division (García et al., 1999; De Las Rivas et al., 2002). It comprises an N-terminal choline-binding module (CBM) (LytBCBM) made of 18 tandemly-arranged repeats that specifically recognize the phosphocholine (P-col) moieties present in pneumococcal teichoic acids that is essential for catalysis. Besides it contains an SH3b module (LytBSH3b) an a WW-like domain (LytBWW), both relevant for substrate recognition and LytB-mediated host-cell adhesion (Bai et al., 2014), and a C-terminal module (LytBGH73) belonging to the GH73 family of glycoside hydrolases, responsible for its glucosaminidase activity. LytB is a virulence factor involved in nasopharyngeal colonization, evasion of host immunity (Gosink et al., 2000; Ramos-Sevillano et al., 2011) and biofilm formation (Moscoso et al., 2006), therefore, LytB is a putative vaccine/drug target. But none of its catalytic activity neither its substrate specificity have been studied yet. So the aim of this thesis is the purification of structurally homogeneous forms of LytB in order to characterize its stability and structural organization in presence and absence of possible ligands, to analyze the structural determinants of LytB substrate specifity, and to identify the relevant residues for the catalysis. Purified samples characterization by using affinity chromatography and analytic ultracentrifugation, showed the coexistence of monomeric and multimeric forms of the protein. These forms were purified by size exclusion chromatography and revealed both active in cell-dispersing activity analysis using cultures of R6B (a LytB-deficient strain that forms long chains of cells). Thermic stability studies (differential scanning calorimetry (DSC) and circular dichroism (DC)) showed that the moderate stability of free LytB drastically increases upon choline recognition by the CBM module and interestingly this stabilization effect is transmitted to the catalytic module. These results in combination with the analysis of LytB interaction with choline analogs by using isothermal titration calorimetry (ITC), allowed us to discard the participation of the phosphate group and the galactose ring from GalNAc-6-fosfate residues in the cell wall-CBM union, and the quantification introduced by the negative charge of the phosphate group. Besides, LytB doesn’t recognize any of the teichoic acids components, so the polar localization of the enzymes may be due to the peptidoglycan (PG) recognition or surface proteins of the bacteria...
Description
Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Biológicas, Departamento de Bioquímica y Biología Molecular, leída el 04-11-2015