Immunochemical analysis of O-specific polysaccharides from the soil nitrogen-fixing bacteria Azospirillum brasilense

Immunochemical reactivity of O-specific polysaccharide and the monosaccharide composition of O-antigenic determinants of the lipopolysaccharide isolated from the type strain Sp7 of Azospirillum brasilense were studied. An original modification of the method of spectroturbidimetry for disperse biological systems and a nonstandard procedure for the preparation of monospecific antibodies against cell surface antigens were used. The polysaccharide fraction, which contained residues of galactose, rhamnose, and galacturonic acid, was able to bind about 50% of the antibodies raised against whole bacterial cells. Twelve immunodeterminant groups were shown to be present in its molecule. Galactose and, less effectively, rhamnose but not galacturonic acid inhibited the antigen-antibody reactions. It is concluded that the serotype of the strain studied is determined by galactose residues.     

Conformational differences among mono- and oligosaccharide fragments of O-specific polysaccharides of Vibrio cholerae O1 revealed by circular dichroism

The circular dichroism (CD) of synthetic mono- and oligosaccharides that represent the terminal, non-reducing group of O-antigens of Vibrio cholerae O1 from the subtypes Ogawa and Inaba was measured in various solvents. We found differences in the CD of the monosaccharides of these subtypes that decrease with increasing chain lengths of the oligosaccharides. The differences can be explained by different orientations of the N-acyl side chain of the terminal monosaccharides. The linear relationship of ellipticity versus the number of residues in an oligosaccharide chain follows the principle of optical superposition. This, together with a similar contribution by internal units to the overall ellipticity, suggests an identical, regular conformation of oligosaccharide fragments of both Ogawa and Inaba series.     

O-Acetylation in the O-specific polysaccharide isolated from Shigella flexneri serotype 2a

Shigella flexneri causes diarrheal diseases especially in infants and children in developing countries. Modifications of the lipopolysaccharide (LPS) molecule, like bacteriophage-mediated glucosylation and acetylation of the O-specific chain (O-SP), are important for the LPS antigenicity and consequently for the immunogenicity of the polysaccharide-based vaccines against shigellosis. Here, we report the degree of O-acetylation and the localisation of O-acetyl groups and side-chain glucose substitution in the O-SP (scheme) in different preparations of S. flexneri type 2a LPS. [structure: see text]     

Basic structure of the oligosaccharide repeating-unit of the Shigella flexneri O-antigens.

The reaction of sodium D-glucuronate with a synthetic peptide, AcTyrLysGlyNH₂ acetate, under physiological conditions, gave as major product the sodium salt of AcTyr-N-(D-arabino-5-carboxy-2,3,4,5-tetrahydroxy-1-pentenyl)-N-(D-arabino- 5-carboxy-3,4,5-trihydroxy-2-oxopentylidene)LysGlyNH₂ (2). The structure was elucidated on the basis of p.m.r., ¹³C-n.m.r., i.r., and u.v. spectra, and pH titration. Compound 2 is the product of oxidation of the sodium salt of AcTyr-N,N-bis(D- arabino-5-carboxy-2,3,4,5-tetrahydroxy-1-pentenyl)LysGlyNH₂, the bis-enol form of the di-D-fructuronic acid peptide obtained through the Amadori rearrangement. A new type of condensation that gives a product having a conjugated enol-keto-immonium group might take place when D-glucuronic acid reacts with peptides or proteins containing a lysine residue.     

Conformational studies of the O-specific polysaccharide of Shigella flexneri 5a and of four related synthetic pentasaccharide fragments using NMR and molecular modeling

As part of a program for the development of synthetic vaccines against the pathogen Shigella flexneri, we used a combination of NMR and molecular modeling methods to study the conformations of the O-specific polysaccharide (O-SP) of S. flexneri 5a and of four related synthetic pentasaccharide fragments. The NMR study, based on the analysis of 1H and 13C chemical shifts, the evaluation of inter-residue distances, and the measurement of one- and three-bond heteronuclear coupling constants, showed that the conformation of one of the four pentasaccharides is similar to that of the native O-SP in solution. Interestingly, inhibition enzyme-linked immunosorbent assay demonstrated that a protective monoclonal antibody specific for S. flexneri 5a has a greater affinity for this pentasaccharide than for the others. We carried out a complete conformational search on the pentasaccharides using the CICADA algorithm interfaced with MM3 force field. We calculated Boltzmann-averaged inter-residue distances and 3JC,H coupling constants for the different conformational families and compared the results with NMR data for all pentasaccharides. Our experimental data are consistent with only one conformational family. We also used molecular modeling data to build models of the O-SP with the molecular builder program POLYS. The models that are in agreement with NMR data adopt right-handed 3-fold helical structures in which the branched glucosyl residue points outwards.     

Acid tolerance of Shigella sonnei and Shigella flexneri

AIMS: Determination of the behaviour of Shigella sonnei and Sh. flexneri under acid conditions. METHODS AND RESULTS: The growth and survival of Shigella spp. (9 isolates) in acidified Brain Heart Infusion (BHI) (pH 5.0-3.25 with pH intervals of 0.25) was determined after 6, 24 and 30 h incubation at 37 degrees C. Subsequently, survival of shigellae was studied in apple juice and tomato juice stored at 7 degrees C and 22 degrees C for up to 14 days and in strawberries and a fresh fruit salad, kept at 4 degrees C for 4 and 48 h. CONCLUSIONS: The minimum pH for growth in acidified BHI for Sh. flexneri and Sh. sonnei was, respectively, pH 4.75 and pH 4.50. Survival in fruit juices and fresh fruits depended upon their pH, the type of strain and the incubation temperature. Shigella spp. Survived for up to 14 days in tomato juice and apple juice stored at 7 degrees C. The shortest survival time (2-8 d) was observed in apple juice at 22 degrees C. Sh. sonnei but not Sh. flexneri was recovered after 48 h from strawberries and fruit salad kept at 4 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: Acid foods, especially if kept at refrigeration temperatures, support survival of Shigella spp. and may cause Shigella food poisoning.     

Quantitative analysis of Shigella flexneri protein expression under acid stress

As an important foodborne pathogen, Shigella flexneri can cause widespread enteric infection with bacteria as few as hundreds. This is, at least in part, attributed to its robust anti‐acid strategies because passage through the highly acidic human digestive tract is a prerequisite for successful bacterial infection. Nevertheless, our understanding of these mechanisms and the impact of acid stress on Shigella protein expression still remains largely incomplete. Herein we conducted a proteomic survey of Shigella spp. under acid stress. Out of 1754 protein identifications, we found 131 altered proteins, most of which were down‐regulated, including virulence factors and cell envelope proteins. Rather, many metabolic enzymes and pyrimidine/amino acid biosynthesis proteins were up‐regulated. In addition to induction of many known anti‐acid systems, we also found marked increase of 2‐oxoglutarate dehydrogenase (SucAB), a metabolic enzyme in the tricarboxylic acid cycle. Importantly, overproduction of this enzyme significantly enhanced Shigella acid resistance and hence SucAB‐mediated metabolic pathways may represent novel anti‐acid strategies.     

Comprehensive proteomic analysis of Shigella flexneri 2a membrane proteins

Shigella flexneri is the causative agent of most shigellosis cases in developing countries. We used different proteolytic enzymes to selectively shave the protruding proteins on the surface of purified bacterial membrane sheets or vesicles, and recovered peptides were subsequently identified using 2-D LC-MS/MS. As a result, a total of 666 proteins were unambiguously assigned, including 159 integral membrane proteins, 35 outer membrane proteins and 114 proteins previously annotated as hypothetical. The former had an average grand average hydrophobicity score of 0.362 and were predicted to separate within a pH range of 4.1-10.6 with molecular mass 8-148 kDa, which represents the largest validated set of integral membrane proteins in this organism to date. A functional classification revealed that a large proportion of the identified proteins were involved in cell envelope biogenesis and energy production and conversion. For the first time, this work provides a global view of the S. flexneri 2a membrane subproteome.     

Complete genome sequence of Shigella flexneri 5b and comparison with Shigella flexneri 2a

Background

On most common microarray platforms many genes are represented by multiple probes. Although this is quite common no one has systematically explored the concordance between probes mapped to the same gene.

Results

Here we present an analysis of all the cases of multiple probe sets measuring the same gene on the Affymetrix U133a GeneChip and found that although in the majority of cases both measurements tend to agree there are a significant number of cases in which the two measurements differ from each other. In these cases the measurements can not be simply averaged but rather should be handled individually.

Conclusion

Our analysis allows us to provide a comprehensive list of the correlation between all pairs of probe sets that are mapped to the same gene and thus allows microarray users to sort out the cases that deserve further analysis. Comparison between the set of highly correlated pairs and the set of pairs that tend to differ from each other reveals potential factors that may affect it.     

Synthesis of oligosaccharide fragments of O-specific Shigella flexneri polysaccharides. II. Synthesis of trisaccharide Glc alpha1----3RHa alpha1----2Rha alpha1----OMe and tetrasaccharide GlcNAc beta1----2(Llc alpha1----3)Rha alpha1----2Rh alpha1----OMe

Methyl glycosides of the title linear trisaccharide and branched tetrasaccharide were synthesized by stepwise glycosylation. These oligosaccharides represent the fragments of O-antigenic polysaccharides of Shigella flexneri serotypes 2b, 3a, 5b, and X.