Anikó Kilár

Structural variability of endotoxins from R-type isogenic mutants of Shigella sonnei†

The structural variations in the rough-type endotoxins [lipopolysaccharides (LPSs)] of Shigella sonnei mutant strains (S. sonnei phase II-4303, R41, 562H and 4350) were investigated by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and tandem MS. A series of S. sonnei mutants had previously been the subject of analytical studies on the biosynthesis of heptose components in the core oligosaccharide region of LPSs. This study gives a complete overview on the structures of the full core and lipid A of S. sonnei mutant strains by MS. We found that the LPSs of the isogenic rough mutants were formed in a step-like manner containing 0:1:2:3 heptose in the deep core region of 4350, 562H, R41 and 4303, respectively, and the longest LPS from the mutant S. sonnei 4303 contained also five hexoses. The structural variations in the lipid A moiety and in the oligosaccharide part of the intact LPS were followed by MALDI-TOF-MS/MS. For the dissolution and the ionization of the samples, 2,5-dihydroxybenzoic acid in citric acid solution was applied as matrix. The detailed evaluation of the mass spectra indicates heterogeneity in the lipid part due to the differences in the phosphate and fatty acid composition.

Quantitative microfluidic analysis of S- and R-type endotoxin components with chip capillary electrophoresis

A novel, fast, and sensitive ME method was developed to analyze and differentiate the smooth (S) and rough (R) type bacterial endotoxin components labeled covalently with a fluorescent dye. The quantitative analysis of purified lipopolysaccharides, or partially purified samples from whole‐cell lysates becomes possible with this method. Two groups with three sub‐groups in the first group of S‐type lipopolysaccharides can be classified based on the electrophoretic profiles. The LOD of the endotoxins from S‐ and R‐type Gram‐negative bacteria was found to be 2.6 ng and 6.9 ng, respectively. This method is capable to replace the commonly used SDS‐PAGE combined with silver staining.

Novel quantitative electrophoretic analysis of endotoxins on microchips

A novel microchip electrophoresis method was developed and applied for sensitive detection and quantitative analysis of endotoxins extracted from Gram‐negative bacteria. The method provides a fast and quantitative differentiation of smooth and rough endotoxins based on the solubilization and complexation of the lipopolysaccharides with dodecylsulfate, and then with a fluorescent dye. The migration of the complexes was followed by LIF detection. The novel method is able to replace the SDS‐PAGE with the advantage of high speed and better sensitivity, and by avoiding the laborious gel‐preparation and silver staining.

Capillary electrophoresis chips for screening of endotoxin chemotypes from whole-cell lysates.

A fast microchip electrophoresis method was developed to analyze and differentiate bacterial endotoxins directly from whole-cell lysates after removal of the proteinaceous components with proteinase K digestion and a precipitation of the endotoxin components. The partially purified endotoxin components were visualized by the interaction with dodecyl sulphate and then a fluorescent dye. The lipopolysaccharide (LPS) profiles can be directly evaluated from digested bacterial cells, and the electrophoresis patterns very closely resembled to those of pure LPSs, and the R and S chemotypes can be used to assign the strains. The method has been found to be useful in the screening of a large number of bacterial mutants and the structural characterization of endotoxins extracted only from 1 ml cultures.

Characterization of complex, heterogeneous lipid A samples using HPLC–MS/MS technique II. Structural elucidation of non-phosphorylated lipid A by negative-ion mode tandem mass spectrometry

Non-phosphorylated lipid A species confer reduced inflammatory potential for the bacteria. Knowledge on their chemical structure and presence in bacterial pathogens may contribute to the understanding of bacterial resistance and activation of the host innate immune system. In this study, we report the fragmentation pathways of negatively charged, non-phosphorylated lipid A species under low-energy collision-induced dissociation conditions of an electrospray ionization quadrupole time-of-flight instrument. Charge-promoted consecutive and competitive eliminations of the acyl chains and cross-ring cleavages of the sugar residues were observed. The A-type fragment ion series and the complementary X-type fragment(s) with corresponding deprotonated carboxamide(s) were diagnostic for the distribution of the primary and secondary acyl residues on the non-reducing and the reducing ends, respectively, of the non-phosphorylated lipid A backbone. Reversed-phase liquid chromatography in combination with negative-ion electrospray ionization quadrupole time-of-flight tandem mass spectrometry could provide sufficient information on the primary and secondary acyl residues of a non-phosphorylated lipid A. As a standard, the hexa-acylated ion at m/z 1636 with the Escherichia coli-type acyl distribution (from E. coli O111) was used. The method was tested and refined with the analysis of other non-phosphorylated hexa- and several hepta-, penta-, and tetra-acylated lipid A species detected in crude lipid A fractions from E. coli O111 and Proteus morganii O34 bacteria. Copyright

Capillary electrophoresis chips for fingerprinting endotoxin chemotypes from whole-cell lysates.

Endotoxins (lipopolysaccharides, LPSs) are components of the envelope of Gram-negative bacteria. These molecules, responsible for both advantageous and harmful biological activities of these microorganisms, are highly immunogenic and directly involved in numerous bacterial diseases in humans such as Gram-negative sepsis. The characterization of endotoxins is of importance, since their physiological and pathophysiological effects depend on their chemical structure. The differences among LPSs from different bacterial serotypes and their mutants include variations mainly within the composition and length of their O-specific polysaccharide chains.Proper assignation of the S or R chemotypes of endotoxins is possible by analyzing their electrophoretic profiles. The recent microchip electrophoretic methods provide fast characterizations and differentiations of endotoxins. The methods are applicable for determination directly from whole-cell lysates after destruction of the proteinaceous components by proteinase K digestion and precipitation of the LPS components. The partially purified LPS components are visualized either by interaction with dodecyl sulfate and a fluorescent dye, or by a covalently bound fluorescent dye. These chip electrophoretic methods have advantages of high speed and quantification and replace the sodium dodecyl sulfate-polyacrylamide gel electrophoresis with silver staining.

Structural background for serological cross‐reactivity between bacteria of different enterobacterial serotypes

The structure of the oligosaccharide repeating units of endotoxins from Gram‐negative bacteria is characteristic for the different serogroups and serotypes of bacteria. Detailed examination of the cross‐reactions of three enterobacterial serotypes, Proteus morganii O34, Escherichia coli O111, and Salmonella enterica sv. Adelaide O35, was performed using sensitive tests (ELISA, immunoblotting). Fine differences between the endotoxins of the bacteria were detected using silver staining of SDS‐PAGE gels and chip‐technology for the intact lipopolysaccharides (LPSs). The compositions of the O‐specific polysaccharides of LPSs extracted from the bacteria were studied, and it was proven that the three cross‐reacting bacteria contain O‐antigens built from the same monosaccharides, namely colitoses linked to glucose, galactose, and N‐acetyl‐galactosamine. The NMR and GC‐MS studies revealed that the most probable component for the cross‐reaction is the rare sugar, colitose.

Terminology of bioanalytical methods (IUPAC Recommendations 2018)

Abstract Recommendations are given concerning the terminology of methods of bioanalytical chemistry. With respect to dynamic development particularly in the analysis and investigation of biomacromolecules, terms related to bioanalytical samples, enzymatic methods, immunoanalytical methods, methods used in genomics and nucleic acid analysis, proteomics, metabolomics, glycomics, lipidomics, and biomolecules interaction studies are introduced.

Phosphoglycolipid Profiling of Bacterial Endotoxins

Abstract Much interest is at present focused on bacterial endotoxins, also known as lipopolysaccharides (LPS), as they are responsible for the development of clinical symptoms of Gram-negative sepsis which is the leading cause of death in intensive care units. Endotoxicity is associated with the special phosphoglycolipid part of LPS, termed lipid A. Main challenges in the structural elucidation of lipid A arise from its amphiphilic character and inherent heterogeneity. A mass spectrometrybased de novo method combined with reversed-phase liquid chromatography for the detailed structural characterization of complex lipid A mixtures (obtained by mild acid hydrolysis of LPS) from different bacterial sources has been developed. Tandem mass spectrometric measurements were performed with an electrospray-ionisation quadrupole time-of-flight (ESI-Q-TOF) mass spectrometer in both negative- and positive-ionization modes in order to explore fragmentation pathways. It was found that characteristic product ions in the positive-ion mode could be used for the unambiguous assignment of the phosphorylation site, whereas the use of both ionization modes provided consistent and/or complementary information about the fatty acyl distribution between the two glucosamine moieties of lipid A. Since the immunostimulatory (advantageous) vs. proinflammatory (endotoxic) effect of the lipid A is closely related to the fine chemical structure, our relatively simple structural elucidation strategy could offer great potential in the bioanalysis of native lipid A samples and lipid A-based vaccines

Mass Spectrometry for Profiling LOS and Lipid A Structures from Whole-Cell Lysates: Directly from a Few Bacterial Colonies or from Liquid Broth Cultures

Lipopolysaccharides (LPSs, endotoxins) are components of the outer cell membrane of most Gram-negative bacteria and can play an important role in a number of diseases of bacteria, including Gram-negative sepsis. The hydrophilic carbohydrate part of LPSs consists of a core oligosaccharide (in the case of an R-type LPS or lipooligosaccharide, LOS) linked to an O-polysaccharide chain (in the case of an S-type LPS), which is responsible for O-specific immunogenicity. The hydrophobic lipid A anchor is composed of a phosphorylated diglucosamine backbone to which varying numbers of ester- and amide-linked fatty acids are attached and this part of the LPSs is associated with endotoxicity. The detailed chemical characterization of endotoxins requires long-lasting large-scale isolation procedures, by which high-purity LPSs can be obtained. However, when a large number of bacterial samples and their LPS content are to be compared prompt, small-scale isolation methods are used for the preparation of endotoxins directly from bacterial cell cultures. The purity of the endotoxins extracted by these methods may not be high, but it is sufficient for analysis.Here, we describe a fast and easy micromethod suitable for extracting small quantities of LOS and a slightly modified micromethod for the detection of the lipid A constituents of the LPSs from bacteria grown in different culture media and evaluate the structures with mass spectrometry. The cellular LOS and lipid A were obtained from crude isolates of heat-killed cells, which were then subjected to matrix-assisted laser desorption/ionization mass spectrometry analysis. The observed ions in the 10-colony samples were similar to those detected for purified samples. The total time for the sample preparation and the MS analysis is less than 3 h.