The poly- and oligosaccharides comprising core OS substituted by ECA repeating units were identified in R1, R2, and R4

The poly- and oligosaccharides comprising core OS substituted by ECA repeating units were identified in R1, R2, and R4. by the O-PS in the case of smooth phase I. Specifically, the distal -d-Glcresidue in the outer core region of the chemotype R1 was substituted at position 3 by ECA [7,8] (Figure 1). Open in a separate window Figure 1 Structures of the core oligosaccharides (OS) and [ECA]-core OS in phase II and R1, R2, R3, and R4 lipooligosaccharides (LOS). The ECA stands for enterobacterial common antigen. The sugar residue of the ligation between core OS and ECA is colored in red. The ECA repeating unit is colored in green. R stands for nonstoichiometric substituents, such as a phosphate group (P), a pyrophosphate group (PP), pyrophosphorylethanolamine (PPEtn). The symbol * indicates heterogeneity of the core OS regarding terminal sugar residues. Symbol ** indicates ECA ligation sites assumed by GW788388 the mass spectrometry only. Symbol *** indicates an inverted anomeric configuration of the D-GlcO14 rabbit serum with various rough and smooth strains. It was further demonstrated that GW788388 only a few strains (serotype O14, O54, O124, and O144) elicited highly cross-reactive anti-ECA antibodies upon immunization of rabbits [6,12]. O14 was proven later on to be rough and synthesize LOS characterized by the R4 core chemotype [13,14]. Further studies demonstrated that ECALPS-dependent immunogenicity generally was limited only to a few rough enterobacteria with complete core OS, such as, for example, R1, R4, and K-12 [15], R2 and R3 [15,16,17], [18], phase II [19], and O:3 and O:9 [20,21,22,23]. Nowadays, LPS (including O-PS) and ECA biosynthesis pathways are, to a great extent, well-described and share some similarities. Lipid A-core GW788388 OS part is assembled on the cytoplasmic side of the inner membrane and translocated across the inner membrane. The O-PS is synthesized in a separate pathway, whereas both O-PS and ECA polysaccharides are produced via the Wzx/Wzy-dependent assembly Rabbit Polyclonal to AIBP pathway [3,24,25,26]. Completed O-PS is finally ligated to the lipid A-core OS by a WaaL ligase to form a mature LPS molecule ready for transport to the outer membrane [3]. Since repeating units of both polymers are assembled on the same lipid carrierundecaprenyl pyrophosphate (Und-PP)and undergone similar processing [25], key features of ECALPS structures are predicted partially on the basis of biosynthesis pathway analyses: (i) ECALPS can only be observed in strains incapable of producing the O-PS [3,25]; (ii) ECA is ligated to the core OS in the position used to be occupied by O-PS, and (iii) an inverted anomeric configuration of the D-Glcphase II ECALPS [7,8]. Successful identification of ECALPS in phase II prompted us to further survey for ECALPS in rough mutants of prototype R1, R2, R3, and R4 strains to correlate serological observations that were made before concerning the presence or absence of ECALPS. O39 strain GW788388 (PCM 209) characterized as the R1 core OS chemotype was selected for the purpose of seeking ECALPS presence in a smooth bacterium. ECALPS was identified in R1, R2, and R4, where it occupied the outer core region in the GW788388 position that used to be substituted by O-PS in smooth LPS. No ECALPS-derived fragments were found in R3. Several new examples of ECALPS presented herein were the preconditions for the further evaluation of biosynthesis and immunogenicity of this form of ECA. Finally, presented herein structural findings regarding ECALPS were discussed in relation to its origin (biosynthesis) and immunogenicity (serology). 2. Results 2.1. Isolation and Purification of E. coli R1, R2, R3, R4, and O39 ECALPS-Derived Poly- and Oligosaccharides Poly- and oligosaccharides of ECALPS were searched for among products of LOS hydrolysis. Lipooligosaccharides (LOS) were extracted from bacterial cells by a hot phenol/water method. Purified LOS preparations were delipidated by mild acid hydrolysis and separated.