INVESTIGADORES
MIRANDA Maria Victoria
congresos y reuniones científicas
Título:
Purification of a recombinant functionally active Streptococcus pneumoniae UDP-glucose pyrophosphorylase and optimization of an assay to screen for inhibitors.
Autor/es:
BOBOFIGLIO; ZABALA; KOVACEC; GUSTAVO LEVIN; ALBERTINA MOGLIONI; MIRANDA MAR√ćA VICTORIA; ERNESTO GARCIA; MOLLERACH
Lugar:
Madrid
Reunión:
Congreso; Europneumo 2013; 2013
Resumen:
The UDP-glucose pyrophosphorylase (GalU) of Streptococcus pneumoniae is absolutely required for the biosynthesis of capsular polysaccharide, the sine qua non virulence factor of pneumococcus. Although GalU is widely distributed, the eukaryotic enzymes are completely unrelated to their prokaryotic counterparts; therefore, we proposed that pneumococcal GalU is an important target to expand our knowledge of the mechanisms underlying capsule formation. This could contribute to the development of new therapeutic strategies to fight the pneumococcus. A recombinant form of the pneumococcal GalU was purified from Escherichia coli and found to be stable and catalytically active. An average of 0.6 g of active rGalU was obtained from 100 ml of culture. We describe a GalU assay that is rapid, sensitive, and easy to perform in 96-well plates. The purified enzyme was tested in the presence of several drugs with structural similarity to natural substrates of the GalU enzyme. Our results document that this colorimetric test is appropriate for screening of chemical libraries for UDP-glucose pyrophosphorylase inhibitors. This work represents a fundamental step in the search of novel antipneumococcal drugs. Keywords: GalU, antipneumococcal inhibitors, high scale purification.