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Abstract
Bordetella pertussis is a gram-negative bacterium that is the causative agent of whooping cough, a highly contagious disease that can be fatal in very young children. The outer membrane of the bacteria consists mainly of lipolysaccharide (LPS), and when LPS is modified with glucosamine, the bacteria can repel cationic antimicrobial peptides (CAMPs) which are currently the last known defense against multi-drug resistant bacteria. The mechanism by which the bacteria can modify its outer membrane has been predicted based on genetic analysis of B. pertussis, but up until now the activity of the enzymes responsible for the pathway has not been individually observed. Using a fluorescently tagged undecaprenyl phosphate analogue (an essential lipid carrier utilized in bacterial glycan synthesis pathways), I was able to monitor each step in the glucosamine modification pathway using fluorescence HPLC. Knowledge of the biochemical steps of this pathway is essential to further research into other ways to treat B. pertussis and other multi-drug resistant bacteria.The glucosamine modification of lipopolysaccharide in B. pertussis is a three-step process thought to be performed by three enzymes: LgmA, LgmC, and LgmB (listed in the order in which they are proposed to act). In the first step, LgmA appends N-acetylglucosamine to undecaprenyl phosphate. In the second step, LgmC deacetlyates the product of LgmA to form undecaprenyl phosphate-glucosamine. Finally, LgmB appends the glucosamine to lipid A, a component of LPS. These three steps involve undecaprenyl phosphate, which is why fluorescently tagged bactoprenyl phosphate was chosen to probe this pathway. The difference in retention time of the fluorescent undecaprenyl phosphate analogue and fluorescent undecaprenyl phosphate-linked sugars was directly observed via HPLC, with supporting data from mass spectrometry, to show the expected products after incubation of the enzymes with their proposed substrates, for the first time producing evidence of the individual enzymatic steps of glucosamine modification.