Application of two different kinds of sera against the Proteus penneri lipopolysaccharide core region in search of epitopes determining cross-reactions with antibodies

Abstract

Introduction: Proteus penneri lipopolysaccharide (LPS) core regions are characterized by a greater structural variability than that observed in other Enterobacteriaceae. This fact and the small amount of published data concerning the serological activity of this part of P. penneri LPS prompted an examination of which fragment might determine cross-reactions with antibodies. To date, such epitopes have been found in the LPS core regions of P. mirabilis and P. vulgaris strains. Materials and Methods: Proteus sp. LPSs were tested with unabsorbed rabbit antisera by enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot, and once again by ELISA or passive immunohemolysis after the absorption of these antisera with selected LPSs. Results: The serological studies of P. penneri 8 LPS demonstrated antibodies in the tested antisera recognizing a common epitope located in the core regions of six of the LPSs, i.e. P. penneri 8, 34, 133, 7, 14, and 15. Additionally, another type of antibody directed against some fragment of P. penneri 13 and the core regions of other LPSs investigated was observed in one antiserum. Conclusions: A distal, trisaccharide fragment of the P. penneri 8 LPS core region is suggested to determine the cross-reactions of the tested antisera with the six P. penneri LPSs.Key words: Proteus penneri, lipopolysaccharide, core region, anti-conjugate serum, epitope.Abbreviations: LPS -lipopolysaccharide, Glc -glucose, GlcN -glucosamine, IgG -immunoglobulin G, IgM -immunoglobulin M, ELISA -enzyme-linked immunosorbent assay, PIH -passive immunohemolysis.Corresponding author: Agata Palusiak, Department of General Microbiology, Institute of Microbiology and Immunology, University of £ódŸ, Banacha 12/16, 90-237 £ódŸ, Poland, tel.: +48 42 635-44-69, fax: +48 42 665-58-18, e-mail: agatapal @biol.uni.lodz.pl O-polysaccharide-specific antibodies in such antisera can influence the cross-reactivity of core-specific antibodies and may not demonstrate the real serological activity of the core region of the lipopolysaccharide (LPS). In this paper we report on serological studies performed with two different kinds of sera against P. penneri 8 containing only core-specific antibodies. The application of this material allowed a more precise determination of which fragment of the P. penneri 8 LPS core region may react with specific antibodies. MATERIALS AND METHODS Bacterial strainsP. penneri 8 (O67) was a clinical isolate from the urine of a patient with bacteriuria in Missouri (USA). This strain and 26 others, i.e. P. penneri 1 (O72a), 2 (O66), 4 (O72a,b), 7 (O61), 11 (O58), 12 (O58) ,

Defining Function of Lipopolysaccharide O-antigen Ligase WaaL Using Chemoenzymatically Synthesized Substrates

“…Interestingly, however, the band of desired product lipid A-core R3 -GlcNAc was not observed. The previous observation that the distally ligated glycan moiety can block the accessibility of the LPS core epitope(s) to core-specific antibodies has frequently been reported in Western blotting analyses of LPS (30,31). Therefore, we reasoned that newly ligated GlcNAc may prevent core-specific antibodies from binding to the core epitope(s), and furthermore, GlcNAc is itself not able to form the O-antigen epitope(s).…”

Classification of Proteus penneri lipopolysaccharides into core region serotypes

“…Table 2 includes all serotypes of LPS core regions formed on the basis of the results (Table 1; Figs. 2, 3) and previous serological studies [16, 17, 19, 20]. The studies were possible to perform owing to a unique set of anti-core sera obtained by: (1) the adsorption of serum against the bacterial whole cells by the LPS possessing the same O-polysaccharide as the strain homologous to the serum and different core region serotypes, (2) the immunization of rabbit with the conjugate of core oligosaccharide with diphtheria toxoid and (3) the immunization of rabbit with the whole cells of rough strains or of smooth strains but having the majority of LPS molecules unsubstituted with O-polysaccharide chains.…”

“…In previous serological studies, sera specific to appropriate P. penneri strains were tested with a set of 40 P. penneri LPSs and the core region serospecificity for the majority of those antigens was determined [16, 17, 19, 20]. In the present work, the reactivities of the core regions of P. penneri LPSs 2, 11, 12, 13, 16, 17, 18, 19, 24, 26, 28, 31, 35, 36, 38, 60, 63, 75, 100, 103, 107, 112, 114, 115 and 124 with five sera specific to the strains P. penneri 17, 103 (with defined structures of LPS core regions [11]), 28, 60 and 124 were analyzed by ELISA and Western blot and classified into core serotypes.…”

“…The procedure of antiserum adsorption, by LPS molecules coated on SRBC as well as on the bacterial cells, is a proven method for assessing serological similarities between antigens. Even though slight differences in reactivities have been observed for a group of LPSs with unadsorbed serum, the adsorption procedure usually appears to be helpful in explaining disputable results [16, 17]. …”

“…LPSs samples were checked with the appropriate antiserum in the enzyme-linked immunosorbent assay (ELISA), in Western blot after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and/or in PIH according to the previously described procedures [17, 18]. For PIH, sheep red blood cells (SRBCs) were sensitized with alkali-treated LPSs (64–200 μg/0.2 ml of SRBCs); 50 ng of LPS per well was used for coating microtiter plates in ELISA.…”