Full structural characterization of Shigella flexneri M90T serotype 5 wild-type R-LPS and its ΔgalU mutant:: glycine residue location in the inner core of the lipopolysaccharide

Shigella flexneri is a Gram-negative bacterium responsible for serious enteric infections that occur mainly in the terminal ileum and colon. High interest in Shigella, as a human pathogen, is driven by its antibiotic resistance and the necessity to develop a vaccine against its infections. Vaccines of the last generation use carbohydrate moieties of the lipopolysaccharide as probable candidates. For this reason, the primary structure of the core oligosaccharide from the R-LPS produced by S. flexneri M90T serotype 5 using chemical analysis, nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MALDI), is herein reported. This is the first time that the core oligosaccharide primary structure by S. flexneri M90T is established in an unambiguous multi-disciplinary approach. Chemical and spectroscopical investigation of the de-acetylated LPS showed that the inner core structure is characterized by a L,D-Hep-(1 -> 7)-L,D-Hep-(1 -> 3)-L,D-Hep-(1 -> 5)-[Kdo-(2 -> 4)]-Kdo sequence that is the common structural theme identified in Enterobacteriaceae. In particular, in S. flexneri M90T serotype 5 LPS, a glucosamine residue is additionally sitting at O-7 of the last heptose whereas the outer core is characterized by glucose and galactose residues. Also, in order to exactly define the position of glycine that is an integral constituent of the core region of the LPS, we created a S. flexneri M90T Delta galU mutant and studied its LOS. In this way it was possible to establish that glycine is sitting at O-6 of the second heptose in the inner core.