UV-MALDI-TOF analysis of the lipopolysaccharide isolated from

A. C. CASABUONO; F. SISTI; D. HOZBOR; A. S. COUTO

Buenos Aires- Argentina

Congreso; 1st Annual Iberoamerican Proteomics Congress-LAHUPO 2007; 2007

LAHUPO

UV-MALDI-TOF analysis of the lipopolysaccharide isolated from Bordetella bronchiseptica. Adriana C. Casabuono1, Federico Sisti2, Daniela Hozbor2 and Alicia S. Couto1 1CIHIDECAR, Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Bs As, 1428, Argentina. 2 Instituto de Bioquímica y Biología Molecular, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina.               Lipopolysaccharides (LPSs) are polysaccharides attached to the bacteria membrane by a lipidic part, the saccharidic part being oriented to the exterior. The general structure of this compound consists in an anchor named lipid A associated with a core polysaccharide, which can bear an O-antigen domain. Bordetella bronchiseptica is a gram-negative bacteria that cause respiratory tract infections characterized by a chronic period. In this period the bacteria present some phenotypic variations that include the LPS structure. While in the first acute period the LPS has a smooth structure, in the chronic period a deep rough LPS is present. This change in the LPS structure could be essential for bacterial survival within the host and, more specifically within the host cell. We have performed a structural analysis by UV-MALDI TOF of the lipid A present in different B. bronchiseptica strains with altered LPS structures. In particular, strains with smooth LPS (as in acute period of the disease) and deep rough LPS (as in chronic period of the disease, named hereafter LPSwaaC).          In all cases LPS was extracted with phenol-water and purified by polymyxin-column chromatography. Further treatment with 1% acetic acid released the Lipid A. It was separated by ultracentrifugation and purified by extraction with Clo:Me.          Analysis by UV-MALDI-TOF using different matrices in the positive and negative modes was performed. Regarding LPSwaaC showed a signal at m/e1587 corresponding to Lipid A structure bearing two hexosamines acylated with C14:0h, C12:0h fatty acids and two phosphate groups. Another signal at m/e1810 corresponding to the Lipid A linked to a KDO unit in accordance with the deep rough structure obtained by SDS-PAGE was observed. As expected, the corresponding Lipid A spectrum showed a signal at m/e 1507 due to the loss of a phosphate unit.