Transcriptional analysis of pneumococcal galU gene and biochemical characterization of GalU protein essential for capsule biosynthesis in Streptococcus pneumoniae

BONOFIGLIO L; GARCÍA E; MOLLERACH M

Buenos Aires, Argentina

Simposio; International Symposium Advances in Biomedical Sciences.; 2008

Universidad de Friburgo - Universidad de Buenos Aires

<!-- /* Font Definitions */ @font-face {font-family:Calibri; panose-1:2 15 5 2 2 2 4 3 2 4; mso-font-charset:0; mso-generic-font-family:swiss; mso-font-pitch:variable; mso-font-signature:-1610611985 1073750139 0 0 159 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin-top:0cm; margin-right:0cm; margin-bottom:10.0pt; margin-left:0cm; line-height:115%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:Calibri; mso-fareast-font-family:Calibri; mso-bidi-font-family:"Times New Roman"; mso-ansi-language:ES-TRAD; mso-fareast-language:EN-US;} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> The pneumococcal UDP-glucose uridyltransferase (UDPG:PP, GalU) is absolutely required for the biosynthesis of capsular polysaccharide, the main virulence factor in this microorganism and the GalU enzyme is present in the 91 serotypes of pneumococci.  Since eukaryotic GalU appear to be completely unrelated to their prokaryotic counterparts, we postulate that GalU may be an appropriate target for the search of new drugs to control pathogenicity of S. pneumoniae. The aim of this study was a) purified and biochemical characterized of GalU, b) characterized the galU promoter region, c) study the transcriptional analysis in different growth curve phases and d) the construction of galU mutants by mariner mutagenesis and evaluation of capsule production. Results: The pneumococcal GalU protein was overexpressed in Escherichia coli, and purified. GalU showed a pI of 4.23, and catalyzed the reversible formation of UDP-glucose and pyrophosphate from UTP and glucose 1-phosphate with KM values of 0.4 mM for UDP-glucose, 0.26 mM for pyrophosphate, 0.19 mM for glucose 1-phosphate, and 0.24 mM for UTP. GalU has a pH optimum of 8−8.5, and requires Mg2+ for activity. Neither ADP-glucose nor TDP-glucose are utilized as substrates in vitro. To study the promoter region we cloned four DNA fragments (F1 a F4) in pLSE4, a broad-host range promoter-probe vector that contains a promoterless lytA gene which codes the main autolytic enzyme of pneumococcus. The purified plasmids were used to transform S. pneumoniae M31 ΔlytA strain and the transformants that showed LytA phenotype proved the presence of a functional promoter in the cloned fragment. Relative quantification of galU expression level was done by Real Time RT-PCR, showing that the expression of galU gene occurs mainly during the exponential growth phase. Mutagenesis mariner was done and galU gene was interrupted in the last 101 nucelotides.  Conclusions: Recombinant GalU was purified. This enzyme showed classical Michaelis-Menten kinetic and kM values were calculated. Sequence analysis upstream of galU gene reveals a putative extended -10 promoter. A functional promoter was detected in a fragment (F1) that contains this putative promoter.  galU gene is expressed mainly in exponential growth phases and 14 times less in stationary phase. galU mutants produce less capsule. Decreased production of polysaccharide may be explained by a change in the conformational stability of the tetrameric enzyme. These results contribute to the knowledge of GalU enzyme, which is considered a target for the search of new antipneumococcal drugs.