Browsing by Subject "growth conditions"

Campylobacter species, particularly C. jejuni and C. coli, are the most common cause of human gastroenteritis worldwide. Lipooligosaccharides (LOS) are heat-stable amphiphilic glycolipids essential for viability and outer membrane stability of Gram-negative bacteria. LOS trigger the activation of both innate and adaptive immune systems. This project aimed to investigate variability of C. coli LOS expression under different growth conditions and its impact on immunogenicity. Moreover, it aims to highlight possible method-based biases in investigating LOS-host interaction. In this study the LOS of eight C. coli strains were extracted by three different methods. Despite differences in growth conditions, the electrophoretic mobility of analyzed LOS was consistent within strains. SDS-PAGE analysis and TLR4 activation assay revealed a significant overestimation of the LOS concentration in the samples by the commonly used LOS quantitation method – purpald assay. Furthermore, it was shown that standardization of lyophilized LOS samples by weight lead to an incorrect estimation of LOS concentration. In conclusion, growth conditions did not cause a visible change in the electrophoretic mobility, suggesting no significant changes in LOS expression. Crude LPS extraction was suitable for LOS profiling and immune response evaluation in HEK-TLR4 cells. LOS quantitation by weight and/or by the purpald assay may result in incorrect estimation of LOS concentration and it might significantly affect downstream immunological analyses. The results of this thesis suggest that the validity of data produced using LOS quantified with either purpald assay or weight of lyophilized material should be critically reassessed.