Structural analysis of the lipopolysaccharide core of a rough, cystic fibrosis isolate of Pseudomonas aeruginosa

Abstract

Lipopolysaccharide (LPS) expressed by isolates of Pseudomonas aeruginosa from cystic fibrosis patients lacks the O-polysaccharide chain but the degree to which the rest of the molecule changes has not been determined. We analyzed, for the first time, the core structure of an LPS from a rough, cystic fibrosis isolate of P. aeruginosa. The products of mild acid hydrolysis and strong alkaline degradation of the LPS were studied by ESI MS, MALDI MS, and NMR spectroscopy. The following structure was determined for the highest-phosphorylated core-lipid A backbone oligosaccharide isolated after alkaline deacylation of the LPS:where Kdo and Hep are 3-deoxy-D-manno-octulosonic acid and L-glycero-D-manno-heptose, respectively; all sugars are in the pyranose form and have the D configuration unless stated otherwise. The outer core region occurs as two isomeric glycoforms differing in the position of rhamnose (Rha). The inner core region carries four phosphorylation sites at two Hep residues, Hep I being predominantly bisphosphorylated and Hep II monophosphorylated. In the intact LPS, both Hep residues carry monophosphate and diphosphate groups in nonstoichiometric quantities, GalN is N-acylated by an L-alanyl group, Hep II is 7-O-carbamoylated, and the outer core region is nonstoichiometrically O-acetylated at four sites. Therefore, the switch to the LPSrough phenotype in cystic fibrosis isolates of P. aeruginosa is not accompanied by losses of core monosaccharide, phosphate or acyl components. The exact positions of the O-acetyl groups and the role of the previously undescribed O-acetylation in the LPS core of P. aeruginosa remain to be determined.

Full Structure of the Lipopolysaccharide of Pseudomonas aeruginosa Immunotype 5

“…3). The occurrence of two isomeric core glyco-forms has been reported earlier in P. aeruginosa immunotype 1 (serogroup O6) [6] and in a rough, clinical isolate from a cystic fibrosis patient, P. aeruginosa 2192 [11], whereas a core glycoform that differs also in the number of glucose residues has been found in P. aeruginosa PAO1 (serogroup O5) [5]. Whether the fourth glucose residue is present or not, one of the core glycoforms is not substituted (1), whereas the other glyco-form is substituted with the Opolysaccharide (3) or one O-polysaccharide repeat (2).…”

“…The ESI mass spectrum of OS-IV (Fig. 2a) showed a mixture of core oligosaccharides with the expected monosaccharide composition (RhaGlc 4 GalNHep 2 Kdo) [12] and non-sugar substituents including N-alanyl and O-carbamoyl groups, phosphate, and phosphoethanolamine (PEtn) [9,14], but no O-acetyl groups, which have been detected earlier in the core of some other P. aeruginosa strains [6,11]. Ion peaks for the major compounds with Kdo in an anhydroform having the mono-isotopic molecular masses 1833.…”

“…Another conserved feature is the presence of a carbamoyl group at O7 of Hep II [14], whereas the N-alanyl group that is present on GalN in most P. aeruginosa strains studied [6,8,9,11], may be replaced with an N-acetyl group [10]. Up to four O-acetyl groups at undefined positions have been reported in the core of a rough clinical isolate P. aeruginosa 2192 [11] and several smooth strains [6] but no significant O-acetylation was observed in the core of P. aeruginosa immunotype 5 studied in this work.…”

“…In the Wzy-dependent pathway of LPS biosynthesis, the Opolysaccharide is synthesized by polymerization of the pre-assembled oligosaccharide, the so- [2,4], and in a few of them, the structure of the biological repeating unit was defined [5][6][7]. The core structure was elucidated in type strains of several serogroups [6,8,9] and in a number of rough strains with an O-polysaccharide-lacking LPS [8][9][10][11].…”

“…In the Wzy-dependent pathway of LPS biosynthesis, the Opolysaccharide is synthesized by polymerization of the pre-assembled oligosaccharide, the so- [2,4], and in a few of them, the structure of the biological repeating unit was defined [5][6][7]. The core structure was elucidated in type strains of several serogroups [6,8,9] and in a number of rough strains with an O-polysaccharide-lacking LPS [8][9][10][11].Recently, we have determined the structure of the biological repeating unit of the Opolysaccharide and the core-lipid A carbohydrate backbone of P. aeruginosa immunotype 5 (serogroup O10) and established the mode of the linkage between the O-polysaccharide and the core [12]. It was found that the LPS core of one type is substituted with the O-polysaccharide or one repeating unit of the O-polysaccharide, and the core of the other type is not substituted.…”