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Shigella is one of the commonest causes of diarrhoea worldwide and a major public health problem. Shigella serotyping is based on a standardized scheme that splits Shigella strains into four serogroups and 60 serotypes on the basis of biochemical tests and O-antigen structures. This conventional serotyping method is laborious, time-consuming, impossible to automate, and requires a high level of expertise. Whole-genome sequencing (WGS) is becoming more affordable and is now used for routine surveillance, opening up possibilities for the development of much-needed accurate rapid typing methods. Here, we describe ShigaPass, a new in silico tool for predicting Shigella serotypes from WGS assemblies on the basis of rfb gene cluster DNA sequences, phage and plasmid-encoded O-antigen modification genes, seven housekeeping genes (EnteroBase’s MLST scheme), fliC alleles and clustered regularly interspaced short palindromic repeats (CRISPR) spacers. Using 4879 genomes, including 4716 reference strains and clinical isolates of Shigella characterized with a panel of biochemical tests and serotyped by slide agglutination, we show here that ShigaPass outperforms all existing in silico tools, particularly for the identification of Shigella boydii and Shigella dysenteriae serotypes, with a correct serotype assignment rate of 98.5 % and a sensitivity rate (i.e. ability to make any prediction) of 100 %.

International audience; Shigella flexneri 3a causes bacillary dysentery. Its O-antigen has the {2)-[α-d-Glcp-(1→3)]-α-l-Rhap-(1→2)-α-l-Rhap-(1→3)-[Ac→2]-α-l-Rhap-(1→3)-[Ac→6]≈40 % -β-d-GlcpNAc-(1→} ([(E)ABAc CAc D]) repeating unit, and the non-O-acetylated equivalent defines S. flexneri X. Propyl hepta-, octa-, and decasaccharides sharing the (E')A'BAc CD(E)A sequence, and their non-O-acetylated analogues were synthesized from a fully protected BAc CD(E)A allyl glycoside. The stepwise introduction of orthogonally protected mono- and disaccharide imidate donors was followed by a two-step deprotection process. Monoclonal antibody binding to twenty-six S. flexneri types 3a and X di- to decasaccharides was studied by an inhibition enzyme-linked immunosorbent assay (ELISA) and STD-NMR spectroscopy. Epitope mapping revealed that the 2C -acetate dominated the recognition by monoclonal IgG and IgM antibodies and that the BAc CD segment was essential for binding. The glucosyl side chain contributed to a lesser extent, albeit increasingly with the chain length. Moreover, tr-NOESY analysis also showed interaction but did not reveal any meaningful conformational change upon antibody binding.

International audience; Bacteriophage research is gaining more importance due to increasing antibiotic resistance. However, for treatment with bacteriophages, diagnostics have to be improved. Bacteriophages carry adhesion proteins, which bind to the bacterial cell surface, for example tailspike proteins (TSP) for specific recognition of bacterial O-antigen polysaccharide. TSP are highly stable proteins and thus might be suitable components for the integration into diagnostic tools. We used the TSP of bacteriophage Sf6 to establish two applications for detecting Shigella flexneri (S. flexneri), a highly contagious pathogen causing dysentery. We found that Sf6TSP not only bound O-antigen of S. flexneri serotype Y, but also the glucosylated O-antigen of serotype 2a. Moreover, mass spectrometry glycan analyses showed that Sf6TSP tolerated various O-acetyl modifications on these O-antigens. We established a microtiter plate-based ELISA like tailspike adsorption assay (ELITA) using a Strep-tag®II modified Sf6TSP. As sensitive screening alternative we produced a fluorescently labeled Sf6TSP via coupling to an environment sensitive dye. Binding of this probe to the S. flexneri O-antigen Y elicited a fluorescence intensity increase of 80% with an emission maximum in the visible light range. The Sf6TSP probes thus offer a promising route to a highly specific and sensitive bacteriophage TSP-based Shigella detection system.

International audience; The bacterium Pseudomonas aeruginosa is well known to have a remarkable adaptive capacity allowing it to colonise many environments. A recent study on environmental isolates from dental unit waterlines (DUWLs) hinted at a genetic clustering into two groups. Isolates from one of these groups, named cluster III, were shown to have unusual phenotypes for environmental isolates, such as an increased biofilm production. To have a better ecological view, more specifically on isolates from cluster III, the complete genomes of 39 isolates including 16 from DUWLs were sequenced. In addition to an investigation of antibiotic resistance and secretion system gene content, a molecular phylogeny allowed confirmation of the split of the 16 environmental isolates in two groups and also sheds light on a correlation between the phylogenetic positions and the serotypes of the isolates. Isolates from cluster III harboured insertion sequences ISPa11 inserted into the O-specific antigen biosynthesis clusters and the gene lasR, encoding for a master regulator of the quorum sensing. Investigation of key regulators revealed another truncated gene, gacS. Alteration in lasR and gacS genes was consistent with phenotypic assays confirming their inactivation. These results bring new perspectives regarding the ecological adaptive potential of P. aeruginosa.

Enteropathogenic Escherichia coli (EPEC) comprise one of the six categories of diarrhoeagenic E. coli (DEC). EPEC is subgrouped into typical (tEPEC) and atypical (aEPEC). The identification of DEC cannot be based only on cultural and biochemical criteria, since they are indistinguishable from the non-pathogenic E. coli commonly found in human feces. Several PCR methods, with both single and multiple target genes, have been reported for detecting the different DEC pathotypes. In the present study five hundred E. coli isolates from children with diarrhea were subjected into multiplex PCR. Furthermore the strains were typed serologically with O antisera and their fliC gene was characterized by PCR-RFLP. The results obtained revealed that overall 41 (8.2%) isolates could be detected as EPEC by this multiplex PCR assay. Of these isolates; 27 (66%) were typical (escv+, bfp+) and 14 (34%) atypical EPEC (escv+, bfp-). None of these 41 isolates contained the Stx1 and Stx2 genes. Among 37 (90%) typeable strains, nine different serogroups were present. The most common serogroups were O111, followed by O86, O55 and O119 and 10 different H types were found among these isolates. The multiplex PCR assay was found to be rapid and reliable in comparison to serological test; especially when screening the large number of isolates. Keywords: diarrhea, enteropathogenic Escherichia coli, polymerase chain reaction

Shigella , the leading cause of bacillary dysentery, uses a type III secretion system (TTSS) to inject proteins into human cells, leading to bacterial invasion and a vigorous inflammatory response. The bacterium is protected against the response by the O antigen of lipopolysaccharide (LPS) on its surface. We show that bacteriophage-encoded glucosylation of Shigella O antigen, the basis of different serotypes, shortens the LPS molecule by around half. This enhances TTSS function without compromising the protective properties of the LPS. Thus, LPS glucosylation promotes bacterial invasion and evasion of innate immunity, which may have contributed to the emergence of serotype diversity in Shigella .

International audience; Combined with chemical synthesis, the use of biocatalysts holds great potential to open the way to novel molecular diversity. We report in vitro chemoenzymatic pathways that, for the first time, take advantage of enzyme engineering to produce complex microbial cell-surface oligosaccharides and circumvent the chemical boundaries of glycochemistry. Glycoenzymes were designed to act on nonnatural conveniently protected substrates to produce intermediates compatible with a programmed chemical elongation. The study was focused on the synthesis of oligosaccharides mimicking the O-antigen motif of Shigella flexneri serotypes 1b and 3a, which could be used for the development of multivalent carbohydrate-based vaccines. A semirational engineering approach was successfully applied to amylosucrase, a transglucosidase that uses a low cost sucrose substrate as a glucosyl donor. The main difficulty was to retain the enzyme specificity toward sucrose, while creating a new catalytic function to render the enzyme able to regiospecifically glucosylate protected nonnatural acceptors. A structurally guided library of 133 mutants was generated from which several mutants with either completely new specificity toward methyl alpha-l-rhamnopyranoside or a tremendously enhanced one toward allyl 2-acetamido-2-deoxy-alpha-d-glucopyranoside acceptors were isolated. The best variants were used to synthesize glucosylated building blocks. They were then converted into acceptors and potential donors compatible with chemical elongation toward oligosaccharide fragments of the O-antigens of the two targeted serotypes. This is the first report of a successful engineering of an alpha-transglycosidase acceptor binding site that led to new specificities. It demonstrates the potential of appropriate combinations of a planned chemoenzymatic pathway and enzyme engineering in glycochemistry.

International audience; The use of carbohydrate-mimicking peptides to induce immune responses against surface polysaccharides of pathogenic bacteria offers a novel approach to vaccine development. Factors governing antigenic and immunogenic mimicry, however, are complex and poorly understood. We have addressed this question using the anti-lipopolysaccharide monoclonal antibody F22-4, which was raised against Shigella flexneri serotype 2a and shown to protect against homologous infection in a mouse model. In a previous crystallographic study, we described F22-4 in complex with two synthetic fragments of the O-antigen, the serotype-specific saccharide moiety of lipopolysaccharide. Here, we present a crystallographic and NMR study of the interaction of F22-4 with a dodecapeptide selected by phage display using the monoclonal antibody. Like the synthetic decasaccharide, the peptide binds to F22-4 with micromolar affinity. Although the peptide and decasaccharide use very similar regions of the antigen-binding site, indicating good antigenic mimicry, immunogenic mimicry by the peptide was not observed. The F22-4-antigen interaction is significantly more hydrophobic with the peptide than with oligosaccharides; nonetheless, all hydrogen bonds formed between the peptide and F22-4 have equivalents in the oligosaccharide complex. Two bridging water molecules are also in common, adding to partial structural mimicry. Whereas the bound peptide is entirely helical, its structure in solution, as shown by NMR, is helical in the central region only. Moreover, docking the NMR structure into the antigen-binding site shows that steric hindrance would occur, revealing poor complementarity between the major solution conformation and the antibody that could contribute to the absence of immunogenic mimicry.

International audience; Six tri- to hexasaccharide fragments of the {2)-[alpha-D-Glcp-(1-->3)]-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->3)-[Ac-->2]-alpha-L-Rhap-(1-->3)-beta-D-GlcpNAc-(1-->}(n) polymer ([(E)AB(Ac)CD](n)) were synthesized as their propyl glycosides. All targets share the (E)AB sequence. Following a thorough investigation on the use of N-trichloroacetylglucosamine- versus N-acetylglucosamine-containing tri- and tetrasaccharide acceptors, the successful strategy was based on an efficient combination of the trichloroacetimidate chemistry, a trichloroacetyl used as permanent N-protection, and an allyl aglycon as temporary and/or permanent anomeric protection of selected building blocks. Use of an EAB intermediate orthogonally protected at 2(A) provided both the trisaccharide target and acceptor 12, the condensation of which with a chain terminator D followed by full deprotection, gave tetrasaccharide D(E)AB. Alternatively, stepwise glycosylation of 12 with a D donor compatible with a selective deblocking at position 3(D) and a 2-O-acetyl C donor following exposure of OH-3(D) led to a pentasaccharide, which was partially and fully deprotected into free (Ac)CD(E)AB and CD(E)AB, respectively. Furthermore, chain elongation of the common D(E)AB acceptor with a 2(B)-O-levulinoyl rhamnobiose donor BC and subsequent partial or total deprotection of the resulting hexasaccharide provided B(Ac)CD(E)AB and BCD(E)AB, respectively. All of the synthesized oligosaccharides are parts of the O-antigen of Shigella flexneri 3a, a prevalent serotype. Moreover, the non-O-acetylated fragments are also parts of the S. flexneri serotype X O-antigen.

The anti-LPS IgG mAb F22-4, raised againstShigella flexneriserotype 2a bacteria, protects against homologous, but not heterologous, challenge in an experimental animal model. We report the crystal structures of complexes formed between Fab F22-4 and two synthetic oligosaccharides, a decasaccharide and a pentadecasaccharide that were previously shown to be both immunogenic and antigenic mimics of theS. flexneriserotype 2a O-antigen. F22-4 binds to an epitope contained within two consecutive 2a serotype pentasaccharide repeat units (RU). Six sugar residues from a contiguous nine-residue segment make direct contacts with the antibody, including the nonreducing rhamnose and both branching glucosyl residues from the two RUs. The glucosyl residue, whose position of attachment to the tetrasaccharide backbone of the RU defines the serotype 2a O-antigen, is critical for recognition by F22-4. Although the complete decasaccharide is visible in the electron density maps, the last four pentadecasaccharide residues from the reducing end, which do not contact the antibody, could not be traced. Although considerable mobility in the free oligosaccharides can thus be expected, the conformational similarity between the individual RUs, both within and between the two complexes, suggests that short-range transient ordering to a helical conformation might occur in solution. Although the observed epitope includes the terminal nonreducing residue, binding to internal epitopes within the polysaccharide chain is not precluded. Our results have implications for vaccine development because they suggest that a minimum of two RUs of synthetic serotype 2a oligosaccharide is required for optimal mimicry of O-Ag epitopes.

Protection against reinfection with noncapsulated Gram-negative bacteria, such as Shigella, an enteroinvasive bacterium responsible for bacillary dysentery, is mainly achieved by Abs specific for the O-Ag, the polysaccharide part of the LPS, the major bacterial surface Ag. The use of chemically defined glycoconjugates encompassing oligosaccharides mimicking the protective determinants carried by the O-Ag, thus expected to induce an efficient anti-LPS Ab response, has been considered an alternative to detoxified LPS-protein conjugate vaccines. The aim of this study was to identify such functional oligosaccharide mimics of the S. flexneri serotype 2a O-Ag. Using protective murine mAbs specific for S. flexneri serotype 2a and synthetic oligosaccharides designed to analyze the contribution of each sugar residue of the branched pentasaccharide repeating unit of the O-Ag, we demonstrated that the O-Ag exhibited an immunodominant serotype-specific determinant. We also showed that elongating the oligosaccharide sequence improved Ab recognition. From these antigenicity data, selected synthetic oligosaccharides were assessed for their potential to mimic the O-Ag by analyzing their immunogenicity in mice when coupled to tetanus toxoid via single point attachment. Our results demonstrated that induction of an efficient serotype 2a-specific anti-O-Ag Ab response was dependent on the length of the oligosaccharide sequence. A pentadecasaccharide representing three biological repeating units was identified as a potential candidate for further development of a chemically defined glycoconjugate vaccine against S. flexneri 2a infection.

The gene wchA (cap8E) belongs to the cps8 locus that is involved in biosynthesis of the capsular polysaccharide (CPS) repeat unit (RU) of the virulent Streptococcus pneumoniae serotype 8. We report here the biochemical characterization of the membrane-associated protein WchA (Cap8E), overexpressed in Escherichia coli BL21(DE3)/pLysS. Our results demonstrate that the recombinant enzyme transfers in vitro a glucosyl-1-phosphate from UDP-glucose to an endogenous phosphoryl-polyprenol, thereby priming the RU biosynthetic pathway of S. pneumoniae CPS 8. We also show that the C-terminal half of WchA is the glycosyltransferase domain as observed for the galactosyl-1-phosphate transferase WbaP from Salmonella enterica, previously described to prime the first step of O-antigen biosynthesis. These results demonstrate that WchA plays a prominent function in the capsule biosynthesis and explain the key role it occupies in the pneumococcal capsule variation.

International audience; 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.

International audience; Synthetic antigens that mimic the terminal hexasaccharide epitope of the O-specific polysaccharide of Vibrio cholerae O1, serotype Ogawa, were conjugated to bovine serum albumin (BSA). Conjugates with carbohydrate-to-carrier molar ratios of 15.5:1, 9.2:1, and 4.6:1 were tested for immunogenicity and efficacy in mice. The role of preimmunity to BSA and the use of adjuvant in the generation of the serologic response to the O-specific polysaccharide and protection against virulent V. cholerae was examined. Preimmunity to BSA did not affect the anti-Ogawa titers but seemed to enhance the protective capacity of antiserum. All 3 conjugates were immunogenic, but adjuvant was effective at inducing higher and earlier antibody responses. In tertiary serum samples, a correlation was found between vibriocidal activity and protection. The protective capacity of antiserum was evident in serum from mice immunized with all conjugates, but it was highest in the groups that received the conjugate with the lowest level of substitution. Further studies are required to increase understanding of the reason for differential protection.