Identification and Characterization of a Novel Shigella flexneri Serotype Yv in China

Shigella flexneri is the major cause of bacterial shigellosis in developing countries. S. flexneri is divided into at least 19 serotypes, the majority of which are modifications of the same basic O-antigen by glucosylation and/or O-acetylation of its sugar residues by phage encoded serotype-converting genes. Recently, a plasmid encoded phosphoethanolamine (PEtN) modification of the O-antigen has been reported, which is responsible for the presence of the MASF IV-1 determinant and results in conversion of traditional serotypes X, 4a and Y to novel serotypes Xv, 4av and Yv, respectively. In this study, we characterized 19 serotype Yv strains isolated in China. A variant of the O-antigen phosphoethanolamine transferase gene opt (formerly called lpt-O) carried by a pSFxv_2-like plasmid was found in serotype Yv strains, which specifies the phosphorylation pattern on the O-antigen of this serotype. For the majority of the O-antigen units, the PEtN modification occurs on Rha^III, while for a minority, modifications occur on both Rha^II and Rha^III. Serotype-specific gene detection and PFGE analysis suggested that these serotype Yv isolates were originated from serotypes Y, Xv and 2a by acquisition of an opt-carrying plasmid and/or inactivation of serotype-specific gene gtrII or gtrX. These data, combined with those of serotypes Xv and 4av reported earlier, demonstrate that the plasmid-encoded PEtN modification is an important serotype conversion mechanism in S. flexneri, in addition to glucosylation and O-acetylation.     

Identification of an O-Acyltransferase Gene (oacB) That Mediates 3- and 4-O-Acetylation of Rhamnose III in Shigella flexneri O Antigens

O antigen (O polysaccharide) is an important and highly variable cell component present on the surface of cells which defines the serospecificity of Gram-negative bacteria. Most O antigens of Shigella flexneri, a cause of shigellosis, share a backbone composed of →2)-α-l-Rhap^III-(1→2)-α-l-Rhap^II-(1→3)-α-l-Rhap^I-(1→3)-β-d-GlcpNAc-(1→ repeats, which can be modified by adding various substituents, giving rise to 19 serotypes. The known modifications include glucosylation on various sugar residues, O-acetylation on Rha^I, and phosphorylation with phosphoethanolamine on Rha^II or/and Rha^III. Recently, two new O-antigen modifications, namely, O-acetylation at position 3 or 4 of Rha^III and position 6 of GlcNAc, have been identified in several S. flexneri serotypes. In this work, the genetic basis for the 3/4-O-acetylation on Rha^III was elucidated. Bioinformatic analysis of the genome of S. flexneri serotype 2a strain Sf301, which carries 3/4-O-acetylation on Rha^III, revealed an O-acyltransferase gene designated oacB. Genetic studies combined with O-antigen structure analysis demonstrated that this gene is responsible for the 3/4-O-acetylation in serotypes 1a, 1b, 2a, 5a, and Y but not serotype 6, which has a different O-antigen backbone structure. The oacB gene is carried by a transposon-like structure located in the proA-adrA region on the chromosome, which represents a novel mechanism of mobilization of O-antigen modification factors in S. flexneri. These findings enhance our knowledge of S. flexneri O-antigen modifications and shed light on the origin of new O-antigen variants.     

Serotype-Converting Bacteriophage SfII Encodes an Acyltransferase Protein That Mediates 6-O-Acetylation of GlcNAc in Shigella flexneri O-Antigens,Conferring on the Host a Novel O-Antigen Epitope

Shigella flexneri O-antigen is an important and highly variable cell component presented on the outer leaflet of the outer membrane. Most Shigella flexneri bacteria share an O-antigen backbone composed of →2)-α-l-Rhap^III-(1→2)-α-l-Rhap^II-(1→3)-α-l-Rhap^I-(1→3)-β-d-GlcpNAc-(1→ repeats, which can be modified by adding various chemical groups to different sugars, giving rise to diverse O-antigen structures and, correspondingly, to various serotypes. The known modifications include glucosylation on various sugar residues, O-acetylation on Rha^I or/and Rha^III, and phosphorylation with phosphoethanolamine on Rha^II or/and Rha^III. Recently, a new O-antigen modification, namely, O-acetylation at position 6 of N-acetylglucosamine (GlcNAc), has been identified in S. flexneri serotypes 2a, 3a, Y, and Yv. In this study, the genetic basis of the 6-O-acetylation of GlcNAc in S. flexneri was elucidated. An O-acyltransferase gene designated oacD was found to be responsible for this modification. The oacD gene is carried on serotype-converting bacteriophage SfII, which is integrated into the host chromosome by lysogeny to form a prophage responsible for the evolvement of serotype 2 of S. flexneri. The OacD-mediated 6-O-acetylation also occurs in some other S. flexneri serotypes that carry a cryptic SfII prophage with a dysfunctional gtr locus for type II glucosylation. The 6-O-acetylation on GlcNAc confers to the host a novel O-antigen epitope, provisionally named O-factor 10. These findings enhance our understanding of the mechanisms of the O-antigen variation and enable further studies to understand the contribution of the O-acetylation to the antigenicity and pathogenicity of S. flexneri.     

A Novel Glucosyltransferase Involved in O-Antigen Modification of Shigella flexneri Serotype 1c

The O antigen of serotype 1c differs from the unmodified O antigen of serotype Y by the addition of a disaccharide (two glucosyl groups) to the tetrasaccharide repeating unit. It was shown here that addition of the first glucosyl group is mediated by the previously characterized gtrI cluster, which is found within a cryptic prophage at the proA locus in the bacterial chromosome. Transposon mutagenesis was performed to disrupt the gene responsible for addition of the second glucosyl group, causing reversion to serotype 1a. Colony immunoblotting was used to identify the desired revertants, and subsequent sequencing, cloning, and functional expression successfully identified the gene encoding serotype 1c-specific O-antigen modification. This gene (designated gtrIC) was present as part of a three-gene cluster, similar to other S. flexneri glucosyltransferase genes. Relative to the other S. flexneri gtr clusters, the gtrIC cluster is more distantly related and appears to have arrived in S. flexneri from outside the species. Analysis of surrounding sequence suggests that the gtrIC cluster arrived via a novel bacteriophage that was subsequently rendered nonfunctional by a series of insertion events.Shigella flexneri is a pathovar of Escherichia coli that is the main causative agent of endemic bacillary dysentery (shigellosis). It is estimated that S. flexneri is responsible for approximately 100 million shigellosis cases annually, resulting in hundreds of thousands of deaths, predominantly in young children (11). Currently no vaccine is available, although there is evidence to suggest that serotype-specific immunity occurs following infection and that induction of immunity can be replicated with vaccines (9). Shigella serotype diversity arises due to differences in the chemical structure of the O-antigen repeating unit in the lipopolysaccharide, which is the main target of the adaptive host immune response following infection.Because immunity to S. flexneri can be conferred by the induction of antibodies directed against the O antigen, an understanding of the prevalence of different serotypes and the underlying basis of serotype diversity can inform appropriate vaccine design. All S. flexneri serotypes (with the exception of serotype 6) share a common O-antigen backbone, consisting of a repeating tetrasaccharide unit that is comprised of one N-acetylglucosamine residue (GlcNAc) and three rhamnose residues (RhaI, RhaII, and RhaIII) (14). The 12 traditionally recognized S. flexneri serotypes differ by the presence or absence of just six different chemical modifications (glucosylations or O acetylations) of the O antigen. The genes responsible for these O-antigen modifications are introduced into the bacterial genome via bacteriophages (3). Glucosylation of the S. flexneri O antigen is mediated by three genes [gtrA, gtrB, and gtr(type)] that are arranged in a single operon known as a gtr cluster. gtrA and gtrB are highly conserved between different gtr clusters and encode proteins involved in transferring the glucosyl group from the cytoplasm into the periplasm, where O-antigen modification is thought to take place. gtr(type) is unique to each gtr cluster and encodes a glucosyltransferase that is responsible for attaching the glucosyl group to a specific sugar unit of the O antigen via a specific linkage (3).Investigations of S. flexneri have typically focused on serotypes for which commercially available typing sera are available. More recently, it has become clear that other serotypes are also epidemiologically important. In Bangladesh in the late 1980s, two novel S. flexneri strains that did not agglutinate with antibodies specific for the traditionally recognized serotypes were isolated (4). Chemical analysis of the O antigen revealed that these strains belonged to a new serotype, which was named serotype 1c due to the similarity its O antigen shares with the O antigens of serotype 1a and 1b strains (19). Serotype 1c has since been isolated in Egypt, Indonesia, Pakistan, and Vietnam (6, 15, 18). Serotype 1c was shown to be the most prevalent S. flexneri serotype in a northern province of Vietnam, accounting for more than a third of all S. flexneri strains isolated from 1998 to 1999 (15). Identification of serotype 1c currently relies on agglutination testing using monoclonal antibody MASF Ic (19).The O antigen of serotype 1c is distinguished by the presence of a disaccharide (two glucosyl groups) linked to the GlcNAc in the tetrasaccharide repeating unit of the O antigen. The first glucosyl group is joined to GlcNAc via an α1→4 linkage, as occurs in the O antigen of serotype 1a and serotype 1b strains (type I modification). The O antigen of serotype 1c is distinguished by the presence of a second glucosyl group that is linked to the first via an α1→2 linkage (Fig. ​(Fig.1).1). Type Ia modification is prerequisite to type Ic modification.Open in a separate windowFIG. 1.Chemical structure of the tetrasaccharide repeat units in the O antigens of S. flexneri serotypes 1a and 1c. Note that the O antigen of serotype 1b (not shown) differs from that of serotype 1a by the O acetylation of l-RhaIII.In this study, the genetic basis of O-antigen modification in serotype 1c was elucidated. Serotype 1c strains isolated from different locations and times were compared to gain insight into the evolution of this serotype. This is the first report of the identification of a glucosyltransferase gene that is responsible for addition of the second glucosyl group, causing serotype conversion from serotype 1a to serotype 1c.     

Serotype-conversion in Shigella flexneri: identification of a novel bacteriophage,Sf101, from a serotype 7a strain

Background

Shigella flexneri is the major cause of bacillary dysentery in the developing countries. The lipopolysaccharide (LPS) O-antigen of S. flexneri plays an important role in its pathogenesis and also divides S. flexneri into 19 serotypes. All the serotypes with an exception for serotype 6 share a common O-antigen backbone comprising of N-acetylglucosamine and three rhamnose residues. Different serotypes result from modification of the basic backbone conferred by phage-encoded glucosyltransferase and/or acetyltransferase genes, or plasmid-encoded phosphoethanolamine transferase. Recently, a new site for O-acetylation at positions 3 and 4 of Rha^III, in serotypes 1a, 1b, 2a, 5a and Y was shown to be mediated by the oacB gene. Additionally, this gene was shown to be carried by a transposon-like structure inserted upstream of the adrA region on the chromosome.

Results

In this study, a novel bacteriophage Sf101, encoding the oacB gene was isolated and characterised from a serotype 7a strain. The complete sequence of its 38,742 bp genome encoding 66 open reading frames (orfs) was determined. Comparative analysis revealed that phage Sf101 has a mosaic genome, and most of its proteins were >90% identical to the proteins from 12 previously characterised lambdoid phages. In addition, the organisation of Sf101 genes was found to be highly similar to bacteriophage Sf6. Analysis of the Sf101 OacB identified two amino acid substitutions in the protein; however, results obtained by NMR spectroscopy confirmed that Sf101-OacB was functional. Inspection of the chromosomal integration site of Sf101 phage revealed that this phage integrates in the sbcB locus, thus unveiling a new site for integration of serotype-converting phages of S. flexneri, and determining an alternative location of oacB gene in the chromosome. Furthermore, this study identified oacB gene in several serotype 7a isolates from various regions providing evidence of O-acetyl modification in serotype 7a.

Conclusions

This is the first report on the isolation of bacteriophage Sf101 which contains the S. flexneri O-antigen modification gene oacB. Sf101 has a highly mosaic genome and was found to integrate in the sbcB locus. These findings contribute an advance in our current knowledge of serotype converting phages of S. flexneri.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-742) contains supplementary material, which is available to authorized users.     

Cloning and analysis of the glucosyl transferase gene encoding type I antigen in Shigella flexneri

The O-antigen of most Shigella flexneri serotypes contains an identical tetrasaccharide repeating unit. Apart from serotype Y, the O-antigen is modified by addition of a glucosyl and/or O-acetyl residue to a specific position in the O-unit. In this study the glucosyl transferase gene from a serotype 1a has been cloned and identified. The bacteriophage SfV integrase (int) gene was used to probe a S. flexneri Y53 (serotype 1a) cosmid library and 18 unique clones were identified. Southern hybridisation of these clones indicated two unlinked regions of the chromosome contained the int homologue. When expressed in a live candidate vaccine strain of S. flexneri serotype Y (SFL124), clones with one region produced type I antigen, whereas clones containing the other region produced mainly type Y antigen. One of the cosmid clones positive for type I antigen by agglutination and Western blotting was selected for further study. Genes involved in O-antigen glucosyl modification were mapped on a 5.8 kb fragment and subclones were produced which fully or partially expressed the type I antigen, depending on the extent of the clone. Fully and partially expressing clones may be useful vaccine candidate strains for protection against disease caused by two serotypes of S. flexneri.     

Structural and Genetic Basis for the Serological Differentiation of Pasteurella multocida Heddleston Serotypes 2 and 5

Pasteurella multocida is classified into 16 serotypes according to the Heddleston typing scheme. As part of a comprehensive study to define the structural and genetic basis of this scheme, we have determined the structure of the lipopolysaccharide (LPS) produced by P. multocida strains M1404 (B:2) and P1702 (E:5), the type strains for serotypes 2 and 5, respectively. The only difference between the LPS structures made by these two strains was the absence of a phosphoethanolamine (PEtn) moiety at the 3 position of the second heptose (Hep II) in M1404. Analysis of the lpt-3 gene, required for the addition of this PEtn residue, revealed that the gene was intact in P1702 but contained a nonsense mutation in M1404. Expression of an intact copy of lpt-3 in M1404 resulted in the attachment of a PEtn residue to the 3 position of the Hep II residue, generating an LPS structure identical to that produced by P1702. We identified and characterized each of the glycosyltransferase genes required for assembly of the serotype 2 and 5 LPS outer core. Monoclonal antibodies raised against serotype 2 LPS recognized the serotype 2/5-specific outer core LPS structure, but recognition of this structure was inhibited by the PEtn residue on Hep II. These data indicate that the serological classification of strains into Heddleston serotypes 2 and 5 is dependent on the presence or absence of PEtn on Hep II.Pasteurella multocida is a gram-negative pathogen that causes serious diseases in animals and humans. It is the causative agent of fowl cholera (7), hemorrhagic septicemia in cattle (9), atrophic rhinitis in pigs (6), and dog and cat bite infections in humans (28).P. multocida isolates may be grouped serologically based on capsular antigens into five serogroups—A, B, D, E, and F—using a passive hemagglutination test with erythrocytes sensitized with capsular antigen. Structural information is available for the capsular polysaccharides synthesized by serogroups A (hyaluronic acid) (22), D (heparin) (10), and F (chondroitin) (10). The genes involved in biosynthesis of the capsules have been identified for all five serogroups (27), and capsule is a critical virulence factor for serogroups A (8) and B (3).Lipopolysaccharide (LPS) is also an important virulence factor in P. multocida (13) and can be used for the identification of strains, with two main somatic typing systems reported (14, 17). The Namioka system is based on a tube agglutination test and is able to recognize 11 serotypes (17), whereas the Heddleston system uses a gel diffusion precipitation test and can recognize 16 serotypes; the Heddleston system is currently the preferred method (14). Current classification of P. multocida strains combines capsular typing with Heddleston somatic typing. Strains are given a designation in which the first letter indicates the capsular group and the number designates the Heddleston LPS serotype (e.g., A:1 indicates a strain that is capsular group A and LPS serotype 1). LPS produced by each of the 16 Heddleston serotype strains has been examined previously for sugar content and reactivity with LPS antisera (21). The LPS isolated from serotype 2 and 5 strains was virtually identical in sodium dodecyl sulfate-polyacrylamide gel electrophoresis migration profile (19), sugar composition, and serological reactivity with anti-LPS antibodies (21). Interestingly, serotypes 2 and 5 were the only serotypes found to elaborate two isomers of heptose in their LPS, namely l-glycero-d-manno-heptose (ld-Hep) and d-glycero-d-manno-heptose (dd-Hep) (21). The aims of this study were to determine whether the LPS molecules made by these two serotypes were structurally distinct and to compare the LPS structures with those previously determined for P. multocida serotypes 1 and 3 (24-26). Furthermore, we identified the transferase genes responsible for the assembly of the outer core LPS structure in each of these strains and characterized the function of each glycosyltransferase.     

Characterization of WbpB, WbpE, and WbpD and Reconstitution of a Pathway for the Biosynthesis of UDP-2,3-diacetamido-2,3-dideoxy-d-mannuronic Acid in Pseudomonas aeruginosa

The lipopolysaccharide of Pseudomonas aeruginosa PAO1 contains an unusual sugar, 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid (d-ManNAc3NAcA). wbpB, wbpE, and wbpD are thought to encode oxidase, transaminase, and N-acetyltransferase enzymes. To characterize their functions, recombinant proteins were overexpressed and purified from heterologous hosts. Activities of His₆-WbpB and His₆-WbpE were detected only when both proteins were combined in the same reaction. Using a direct MALDI-TOF mass spectrometry approach, we identified ions that corresponded to the predicted products of WbpB (UDP-3-keto-d-GlcNAcA) and WbpE (UDP-d-GlcNAc3NA) in the coupled enzyme-substrate reaction. Additionally, in reactions involving WbpB, WbpE, and WbpD, an ion consistent with the expected product of WbpD (UDP-d-GlcNAc3NAcA) was identified. Preparative quantities of UDP-d-GlcNAc3NA and UDP-d-GlcNAc3NAcA were enzymatically synthesized. These compounds were purified by high-performance liquid chromatography, and their structures were elucidated by NMR spectroscopy. This is the first report of the functional characterization of these proteins, and the enzymatic synthesis of UDP-d-GlcNAc3NA and UDP-d-GlcNAc3NAcA.Gram-negative organisms such as Pseudomonas aeruginosa produce lipopolysaccharide (LPS)⁴ as an essential component of the outer leaflet of the outer membrane. LPS can be conceptually divided into three parts: lipid A, which anchors LPS into the membrane; core oligosaccharide, which contributes to membrane stability; and the O-antigen, which is a polysaccharide that extends away from the cell surface. In P. aeruginosa, two types of O-antigen are observed: A-band O-antigen, which is common to most strains, and B-band O-antigen, which is variable and therefore used as the basis of the International Antigenic Typing Scheme (1). P. aeruginosa serotypes O2, O5, O16, O18, and O20 collectively belong to serogroup O2, because they all share common backbone sugar structures in their O-antigen repeat units consisting of two di-N-acetylated uronic acids and one 2-acetamido-2,6-dideoxy-d-galactose (N-acetyl-d-fucosamine). The minor structural variations in the O-antigen repeat units that differentiate this serogroup into five serotypes are: the type of glycosidic linkage between O-units (alpha versus beta) that is formed by the O-antigen polymerase (Wzy), isomers present (d-mannuronic or l-guluronic acid), and acetyl group substituents (2–4). The B-band O-antigen of P. aeruginosa PAO1 (serotype O5) contains a repeating trisaccharide of 2-acetamido-3-acetamidino-2,3-dideoxy-d-mannuronic acid (d-ManNAc3NAmA), 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid (d-ManNAc3NAcA), and 2-acetamido-2,6-dideoxy-d-galactose (3).The biosynthesis of the two mannuronic acid derivatives has yet to be fully understood and has been the subject of investigation by our group. To produce UDP-d-ManNAc3NAcA, a five-step pathway has been proposed (Fig. 1) that requires the products of five genes localized to the B-band O-antigen biosynthesis cluster (5). The O-antigen biosynthesis cluster was shown to be identical for all serotypes within serogroup O2, which further underscores the high similarity between these serotypes (5). The five genes, including wbpA, wbpB, wbpE, wbpD, and wbpI, have been shown to be essential for B-band LPS biosynthesis, because knockout mutants of each of these genes are deficient in B-band O-antigen (6–8). Homologs of all five of the proteins required for the UDP-d-ManNAc3NAcA biosynthesis pathway are conserved in other bacterial pathogens, including Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica. Cross-complementation of P. aeruginosa knockout mutants lacking wbpA, wbpB, wbpE, wbpD, or wbpI with the homologues from B. pertussis could fully restore LPS production in the P. aeruginosa LPS mutants, suggesting that the genes from B. pertussis are functional homologs of the wbp genes (7). Homologs of these genes could be identified in diverse bacterial species, demonstrating the importance of UDP-d-ManNAc3NAcA biosynthesis beyond its role in P. aeruginosa (7).Open in a separate windowFIGURE 1.Proposed pathway for the biosynthesis of UDP-d-ManNAc3NAcA in P. aeruginosa PAO1. The full names of the sugars are as follows: GlcNAc, 2-acetamido-2-deoxy-d-glucose; GlcNAcA, 2-acetamido-2-deoxy-d-glucuronic acid; 3-keto-d-GlcNAcA, 2-acetamido-2-deoxy-d-ribo-hex-3-uluronic acid; GlcNAc3NA, 2-acetamido-3-amino-2,3-dideoxy-d-glucuronic acid; GlcNAc3NAcA, 2,3-diacetamido-2,3-dideoxy-d-glucuronic acid; ManNAc3NAcA, 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid. Adapted from Ref. 8.The first enzyme of the UDP-d-ManNAc3NAcA biosynthesis pathway, WbpA, is a 6-dehydrogenase that converts UDP-2-acetamido-2-deoxy-d-glucose (N-acetyl-d-glucosamine; UDP-d-GlcNAc) to UDP-2-acetamido-2-deoxy-d-glucuronic acid (N-acetyl-d-glucosaminuronic acid, UDP-d-GlcNAcA) using NAD⁺ as a coenzyme (9) (Fig. 1). Following this, the second step in UDP-d-ManNAc3NAcA biosynthesis is proposed to be an oxidation reaction catalyzed by WbpB, forming UDP-2-acetamido-2-deoxy-d-ribo-hex-3-uluronic acid (3-keto-d-GlcNAcA), which in turn is used as the substrate for transamination by WbpE, creating UDP-2-acetamido-3-amino-2,3-dideoxy-d-glucuronic acid (d-GlcNAc3NA).This residue is thought to be the substrate for WbpD, a putative N-acetyltransferase of the hexapeptide acyltransferase superfamily (10) that requires acetyl-CoA as a co-substrate (8). WbpD has been proposed to synthesize UDP-2,3-diacetamido-2,3-dideoxy-d-glucuronic acid (UDP-d-GlcNAc-3NAcA), which is utilized in the B-band O-antigen of P. aeruginosa serotype O1. In P. aeruginosa serogroup O2, the UDP-d-GlcNAc3NAcA is then epimerized by WbpI to create the UDP-d-ManNAc3NAcA required for incorporation into B-band LPS (11). A derivative of UDP-d-ManNAc3NAcA is also used in the synthesis of B-band O-antigen of P. aeruginosa serogroup O2. UDP-d-ManNAc3NAmA is thought to be produced through additional modification of UDP-d-ManNAc3NAcA via the action of WbpG, an amidotransferase, which has also been demonstrated to be essential for the production of B-band O-antigen (12, 13).In the current study, our aim was to define the function of WbpB, WbpE, and WbpD, because only genetic evidence has previously been given for the involvement of wbpB and wbpE (7), and the reaction catalyzed by WbpD could not be demonstrated due to the unavailability of its presumed substrate, UDP-d-GlcNAc3NA (8). The functional characterization of these proteins is also important for understanding LPS biosynthesis in B. pertussis, because the genes in the LPS locus of this species, wlbA, wlbC, and wlbB, could cross-complement knockouts of wbpB, wbpE, and wbpD, respectively, when expressed in P. aeruginosa PAO1 (7). Furthermore, these three proteins form a cassette for the generation of C-3 N-acetylated hexoses and may be important for the biosynthesis of a variety of other sugars. Capillary electrophoresis and MALDI-TOF mass spectrometry were used to analyze reaction mixtures of WbpB and WbpE and showed that the expected products were produced only when both enzymes were present together. Achieving the enzymatic synthesis of the product of both enzymes, which was demonstrated to be UDP-d-GlcNAc3NA by ¹H NMR spectroscopy, was a key breakthrough, because this rare sugar has never before been produced by any means. UDP-d-GlcNAc3NA was also essential for use as the substrate of WbpD, which not only allowed us to determine the enzymatic activity of this protein but also allowed the enzymatic synthesis of UDP-d-GlcNAc3NAcA to be achieved as well. Although this sugar had previously been produced through a 17-step chemical synthesis (11, 14), the 4-step concurrent enzymatic reaction demonstrates the advantage of linking chemistry with biology and represents a significant saving of both time and reagents as compared with chemical synthesis. Finally, our data also showed the success in reconstituting in vitro the 5-step pathway for the biosynthesis of UDP-d-ManNAc3NAcA in P. aeruginosa.     

Genesis of a novel Shigella flexneri serotype by sequential infection of serotype-converting bacteriophages SfX and SfI

Background

Shigella flexneri is the major pathogen causing bacillary dysentery. Fifteen serotypes have been recognized up to now. The genesis of new S. flexneri serotypes is commonly mediated by serotype-converting bacteriophages. Untypeable or novel serotypes from natural infections had been reported worldwide but have not been generated in laboratory.

Results

A new S. flexneri serotype-serotype 1 d was generated when a S. flexneri serotype Y strain (native LPS) was sequentially infected with 2 serotype-converting bacteriophages, SfX first and then SfI. The new serotype 1 d strain agglutinated with both serotype X-specific anti-7;8 grouping serum and serotype 1a-specific anti- I typing serum, and differed from subserotypes 1a, 1b and 1c. Twenty four S. flexneri clinical isolates of serotype X were all converted to serotype 1 d by infection with phage SfI. PCR and sequencing revealed that SfI and SfX were integrated in tandem into the proA-yaiC region of the host chromosome.

Conclusions

These findings suggest a new S. flexneri serotype could be created in nature. Such a conversion may be constrained by susceptibility of a strain to infection by a given serotype-converting bacteriophage. This finding has significant implications in the emergence of new S. flexneri serotypes in nature.     

Lipopolysaccharide with an altered O-antigen produced in Escherichia coli K-12 harbouring mutated, cloned Shigella flexneri rfb genes

Cloning of the rfb genes of Shigella flexneri 2a into Escherichia coli K-12 strain DH1 results in the synthesis of lipopolysaccharides (LPS) with an O-antigen chain having type antigen IV and group antigens 3,4. During genetic studies of these rfb genes in E. coli K-12, we observed that strains harbouring plasmids with certain mutations (inversion and transposon insertions) which should have blocked O-antigen synthesis nevertheless still produced LPS with O-antigen chains. These LPS migrated differently on silver-stained SDS—polyacrylamide gels, compared with the LPS produced by wild-type rfb genes, and the group 3,4 antigens were barely detectable, suggesting that the O-antigen was altered. Investigation of the genetic determinants for production of the altered O-antigen/LPS indicated that: (i) these LPS are produced as a result of mutations which are either polar on rfbF or inactivate rfbF; (ii) the rfbX gene product (or a similar protein in the E. coli K-12 rfb region) is needed for production of the altered O-antigen in the form of LPS; (iii) the rfbG gene product is required for the production of both the parental and altered LPS; (iv) the dTDP-rhamnose biosynthesis genes are required. Additionally, an E. coli K-12 gene product(s) encoded outside the rfb region also contributes to production of the O-antigen of the altered LPS. An antiserum raised to the altered LPS from strain DH1(pPM2217 (rfbX::Tn1725)) was found to cross-react with nearly all S. flexneri serotypes, and with the altered LPS produced by other DH1 strains harbouring plasmids with different rfb mutations, as described above. The reactivity of the altered LPS with a panel of monoclonal antibodies specific for various S. flexneri O-antigen type and group antigens demonstrated that their O-antigen components were closely related to that of S. flexneri serotype 4. The RfbF and RfbG proteins were shown to have similarity to rhamnose transferases, and we identified a motif common to the N-termini of 6-deoxy-hexose nucleotide sugar transferases. We propose that the E. coli K-12 strains harbouring the mutated S. flexneri rfb genes produce LPS with a hybrid O-antigen as a consequence of inactivation of RfbF and complementation by an E. coli K-12 gene product. Analysis of the genetic and immunochemical data suggested a possible structure for the O-antigen component of the altered LPS.     

Rationalizing the design of a broad coverage Shigella vaccine based on evaluation of immunological cross-reactivity among S. flexneri serotypes

No vaccine to protect against an estimated 238,000 shigellosis deaths per year is widely available. S. sonnei is the most prevalent Shigella, and multiple serotypes of S. flexneri, which change regionally and globally, also cause significant disease. The leading Shigella vaccine strategies are based on the delivery of serotype specific O-antigens. A strategy to minimize the complexity of a broadly-protective Shigella vaccine is to combine components from S. sonnei with S. flexneri serotypes that induce antibodies with maximum cross-reactivity between different serotypes. We used the GMMA-technology to immunize animal models and generate antisera against 14 S. flexneri subtypes from 8 different serotypes that were tested for binding to and bactericidal activity against a panel of 11 S. flexneri bacteria lines. Some immunogens induced broadly cross-reactive antibodies that interacted with most of the S. flexneri in the panel, while others induced antibodies with narrower specificity. Most cross-reactivity could not be assigned to modifications of the O-antigen, by glucose, acetate or phosphoethanolamine, common to several of the S. flexneri serotypes. This allowed us to revisit the current dogma of cross-reactivity among S. flexneri serotypes suggesting that a broadly protective vaccine is feasible with limited number of appropriately selected components. Thus, we rationally designed a 4-component vaccine selecting GMMA from S. sonnei and S. flexneri 1b, 2a and 3a. The resulting formulation was broadly cross-reactive in mice and rabbits, inducing antibodies that killed all S. flexneri serotypes tested. This study provides the framework for a broadly-protective Shigella vaccine which needs to be verified in human trials.     

Identification and Molecular Characterisation of a Novel Mu-Like Bacteriophage,SfMu, of Shigella flexneri

S. flexneri is the leading cause of bacillary dysentery in the developing countries. Several temperate phages originating from this host have been characterised. However, all S. flexneri phages known to date are lambdoid phages, which have the ability to confer the O-antigen modification of their host. In this study, we report the isolation and characterisation of a novel Mu-like phage from a serotype 4a strain of S. flexneri. The genome of phage SfMu is composed of 37,146 bp and is predicted to contain 55 open reading frames (orfs). Comparative genome analysis of phage SfMu with Mu and other Mu-like phages revealed that SfMu is closely related to phage Mu, sharing >90% identity with majority of its proteins. Moreover, investigation of phage SfMu receptor on the surface of the host cell revealed that the O-antigen of the host serves as the receptor for the adsorption of phage SfMu. This study also demonstrates pervasiveness of SfMu phage in S. flexneri, by identifying complete SfMu prophage strains of serotype X and Y, and remnants of SfMu in strains belonging to 4 other serotypes, thereby indicating that transposable phages in S. flexneri are not uncommon. The findings of this study contribute an advance in our current knowledge of S. flexneri phages and will also play a key role in understanding the evolution of S. flexneri.     

Structural Analysis of the Core Region of O-Lipopolysaccharide of Porphyromonas gingivalis from Mutants Defective in O-Antigen Ligase and O-Antigen Polymerase

Porphyromonas gingivalis synthesizes two lipopolysaccharides (LPSs), O-LPS and A-LPS. Here, we elucidate the structure of the core oligosaccharide (OS) of O-LPS from two mutants of P. gingivalis W50, ΔPG1051 (WaaL, O-antigen ligase) and ΔPG1142 (O-antigen polymerase), which synthesize R-type LPS (core devoid of O antigen) and SR-type LPS (core plus one repeating unit of O antigen), respectively. Structural analyses were performed using one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy in combination with composition and methylation analysis. The outer core OS of O-LPS occurs in two glycoforms: an “uncapped core,” which is devoid of O polysaccharide (O-PS), and a “capped core,” which contains the site of O-PS attachment. The inner core region lacks l(d)-glycero-d(l)-manno-heptosyl residues and is linked to the outer core via 3-deoxy-d-manno-octulosonic acid, which is attached to a glycerol residue in the outer core via a monophosphodiester bridge. The outer region of the “uncapped core” is attached to the glycerol and is composed of a linear α-(1→3)-linked d-Man OS containing four or five mannopyranosyl residues, one-half of which are modified by phosphoethanolamine at position 6. An amino sugar, α-d-allosamine, is attached to the glycerol at position 3. In the “capped core,” there is a three- to five-residue extension of α-(1→3)-linked Man residues glycosylating the outer core at the nonreducing terminal residue. β-d-GalNAc from the O-PS repeating unit is attached to the nonreducing terminal Man at position 3. The core OS of P. gingivalis O-LPS is therefore a highly unusual structure, and it is the basis for further investigation of the mechanism of assembly of the outer membrane of this important periodontal bacterium.Porphyromonas gingivalis is a gram-negative anaerobe which is strongly implicated in the etiology of periodontal disease. Several putative virulence factors are produced by this organism. These virulence factors include the cysteine proteases Arg-gingipains (Rgps) and Lys-gingipain (Kgp) specific for Arg-X and Lys-X peptide bonds, respectively, which are capable of degrading several host proteins (56), and lipopolysaccharide (LPS), which has the potential to cause an inflammatory response in the periodontal tissues of the host. These factors are important antigens in patients with periodontal disease and may account for a considerable proportion of the immune response directed against P. gingivalis (58).LPS is a major constituent of the outer membrane of gram-negative bacteria and facilitates interactions with the external environment. It consists of three regions: a hydrophobic lipid A embedded in the outer leaflet of the outer membrane, a core oligosaccharide (OS), and the O-polysaccharide (O-PS) side chain composed of several repeating units. The hydrophobic lipid A serves as an anchor for the LPS and consists of β-1,6-linked d-glucosamine disaccharide, which is usually phosphorylated at the 1 and/or 4′ positions and N and/or O acylated at positions 2, 3, 2′, and 3′ with various amounts of fatty acids. The rest of the LPS molecule projects from the surface. The core region is attached to lipid A and is composed of ∼10 sugars in most bacteria studied to date and can be further subdivided into an inner core and an outer core. The inner core usually contains l(d)-glycero-d-(l)-manno-heptose and 3-deoxy-d-manno-octulosonic acid (Kdo) residues, whereas the outer core is usually composed of hexoses. Attached to the outer core are the repeating units of O antigen (O-PS), which vary in composition, stereochemistry, and the sequence of O-glycosidic linkages between bacterial strains and thereby give rise to O-serotype specificity within bacterial species. Attachment of O antigen to core lipid A results in “smooth” LPS (S-type LPS), whereas LPS lacking O antigen is “rough” LPS (R-type LPS). Attachment of one repeating unit of O-PS to core lipid A results in SR-LPS (core-plus-one repeating unit) (41, 47, 48). In addition, the outer core OS region can be either “uncapped” or “capped.” The “uncapped” core OS is devoid of O-PS repeating units, whereas the “capped” core OS contains attached O-PS repeating units (47, 53) due to modifications in the outer core region.P. gingivalis W50 was originally thought to synthesize a single LPS composed of a tetrasaccharide repeating unit in the O-PS, [→6)-α-d-Glcp-(1→4)-α-l-Rhap-(1→3)-β-d-GalNAc-(1→3)-α-d-Galp-(1→], which is modified by phosphoethanolamine (PEA) at position 2 of Rha in a nonstoichiometric manner (43). However, a second LPS in this organism, namely A-LPS (49), which has a phosphorylated mannan-containing anionic polysaccharide (A-PS), was identified in our laboratory. The A-PS repeating unit is built up of a phosphorylated branched d-Man-containing oligomer composed of an α1→6-linked d-mannose backbone to which α1→2-linked d-Man side chains of different lengths (one or two residues) are attached at position 2. One of the side chains contains Manα1→2-Manα-1-phosphate linked via phosphorus to a backbone Man residue at position O-2. Although A-LPS is predominantly composed of α-d-mannose residues, it cannot be referred to as a homopolymer due to the presence of Manα1→2Manα1-phosphate-containing OS side chains forming a nonglycosidic linkage between the backbone α-mannose and side chains. Hence, it is likely that the synthesis of A-PS (A-LPS) occurs via a “wzy-dependent” pathway in which repeating units formed on the cytoplasmic face of the inner membrane are polymerized at the periplasmic face following transport or flipping across the cytoplasmic membrane. A-LPS cross-reacts with monoclonal antibody (MAb) 1B5 raised against one of the isoforms of Arg-gingipains, a family of differentially glycosylated cysteine proteases (14, 19). Deglycosylation of the cross-reacting Rgps with anhydrous trifluoromethane sulfonic acid abolishes their immunoreactivity to MAb 1B5, indicating that this antibody recognizes a carbohydrate-containing epitope also present in A-LPS (14, 44). Hence, there appear to be common elements in the biosynthesis of A-LPS and the Arg-gingipains of this organism.Inactivation of P. gingivalis waaL (PG1051, O-antigen ligase) abolishes the synthesis of both O-LPS and A-LPS (49). Hence, the WaaL O-antigen ligase appears to have dual specificity and is capable of ligating both O-PS and A-PS chains to core lipid A. The dual specificity of WaaL in the final step of LPS biosynthesis has also been demonstrated in the synthesis of Escherichia coli O-LPS and M_LPS (38) and for Pseudomonas aeruginosa A-band and B-band LPSs (1).However, the linkage between O-PS and A-PS and core OS has not been identified in P. gingivalis. In this paper, we describe a structural investigation of the core OS of O-LPS in which we used R-LPS prepared from ΔPG1051 (49) and ΔPG1142 (putative O-antigen polymerase), which we hypothesized would synthesize an SR-LPS (core plus one repeating unit) (60). The putative O-antigen polymerase encoded at PG1142 (42) is a phenylalanine-rich membrane protein consisting of 347 amino acids which shows 46% similarity over 297 amino acids to EpsK of Lactobacillus delbrueckii subsp. bulgaricus. EpsK is proposed to be a polymerase on the basis of homology and topological similarity to the O-antigen polymerase (Wzy) of E. coli and is required for the synthesis of an exopolysaccharide composed of Gal, Glc, and Rha (5:1:1) containing repeating units in L. delbrueckii (32). Application of one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy and methylation and monosaccharide analyses using gas chromatography-mass spectrometry (GC-MS) to purified core-containing OSs isolated from LPS from ΔPG1051 and ΔPG1142 mutants enabled us to solve the LPS core structure of an oral gram-negative bacterium for the first time.     

NMR-based Structural Analysis of the Complete Rough-type Lipopolysaccharide Isolated from Capnocytophaga canimorsus

We here describe the NMR analysis of an intact lipopolysaccharide (LPS, endotoxin) in water with 1,2-dihexanoyl-sn-glycero-3-phosphocholine as detergent. When HPLC-purified rough-type LPS of Capnocytophaga canimorsus was prepared, ¹³C,¹⁵N labeling could be avoided. The intact LPS was analyzed by homonuclear (¹H) and heteronuclear (¹H,¹³C, and ¹H,³¹P) correlated one- and two-dimensional NMR techniques as well as by mass spectrometry. It consists of a penta-acylated lipid A with an α-linked phosphoethanolamine attached to C-1 of GlcN (I) in the hybrid backbone, lacking the 4′-phosphate. The hydrophilic core oligosaccharide was found to be a complex hexasaccharide with two mannose (Man) and one each of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo), Gal, GalN, and l-rhamnose residues. Position 4 of Kdo is substituted by phosphoethanolamine, also present in position 6 of the branched Man^I residue. This rough-type LPS is exceptional in that all three negative phosphate residues are “masked” by positively charged ethanolamine substituents, leading to an overall zero net charge, which has so far not been observed for any other LPS. In biological assays, the corresponding isolated lipid A was found to be endotoxically almost inactive. By contrast, the intact rough-type LPS described here expressed a 20,000-fold increased endotoxicity, indicating that the core oligosaccharide significantly contributes to the endotoxic potency of the whole rough-type C. canimorsus LPS molecule. Based on these findings, the strict view that lipid A alone represents the toxic center of LPS needs to be reassessed.     

Structural elucidation of the O-antigen of the Shigella flexneri provisional serotype 88-893: structural and serological similarities with S. flexneri provisional serotype Y394 (1c)

The structure of the repeating unit of the O-antigen polysaccharide from Shigella flexneri provisional serotype 88-893 has been determined. ¹H and ¹³C NMR spectroscopy as well as 2D NMR experiments were employed to elucidate the structure. The carbohydrate part of the hexasaccharide repeating unit is identical to the previously elucidated structure of the O-polysaccharide from S. flexneri prov. serotype Y394. The O-antigen of S. flexneri prov. serotype 88-893 carries 0.7 mol O-acetyl group per repeating unit located at O-2 of the 3-substituted rhamnosyl residue, as identified by H2BC and BS-CT-HMBC NMR experiments. The O-antigen polysaccharide is composed of hexasaccharide repeating units with the following structure: →2)-α-l-Rhap-(1→2)-α-l-Rhap-(1→3)-α-l-Rhap2Ac-(1→3)[α-d-Glcp-(1→2)-α-d-Glcp-(1→4)]-β-d-GlcpNAc-(1→. Serological studies showed that type antigens for the two provisional serotypes are identical; in addition 88-893 expresses S. flexneri group factor 6 antigen. We propose that provisional serotypes Y394 and 88-893 be designated as two new serotypes 7a and 7b, respectively, in the S. flexneri typing scheme.     

Topology and identification of critical residues of the O-acetyltransferase of serotype-converting bacteriophage, SF6, of Shigella flexneri

The modification of the LPS O-antigen, seen in the diverse serotypes of Shigella flexneri is brought about by the glucosyltransferases (Gtr) and the O-acetyltransferase (Oac). In this study, we establish the membrane topology of Oac using the dual reporter PhoA-LacZα. We have determined that Oac is an integral membrane protein with 10 transmembrane regions. The hydrophilic N- and C-termini are oriented in the cytoplasm. Functionally important cytoplasmic and periplasmic loops have also been identified. Furthermore, cytoplasmic residues R73 and R75R76 were found to be critical to Oac function.     

The Role of O-antigen in P1 Transduction of Shigella flexneri and Escherichia coli with its Alternative S' Tail Fibre

Enterobacteria phage P1 expresses two types of tail fibre, S and S'. Despite the wide usage of phage P1 for transduction, the host range and the receptor for its alternative S' tail fibre was never determined. Here, a ΔS-cin Δpac E. coli P1 lysogenic strain was generated to allow packaging of phagemid DNA into P1 phage having either S or S' tail fibre. P1(S') could transduce phagemid DNA into Shigella flexneri 2a 2457O, Shigella flexneri 5a M90T and Escherichia coli O3 efficiently. Mutational analysis of the O-antigen assembly genes and LPS inhibition assays indicated that P1(S') transduction requires at least one O-antigen unit. E. coli O111:B4 LPS produced a high neutralising effect against P1(S') transduction, indicating that this E. coli strain could be susceptible to P1(S')-mediated transduction. Mutations in the O-antigen modification genes of S. flexneri 2a 2457O and S. flexneri 5a M90T did not cause significant changes to P1(S’) transduction efficiency. A higher transduction efficiency of P1(S') improved the delivery of a cas9 antimicrobial phagemid into both S. flexneri 2457O and M90T. These findings provide novel insights into P1 tropism-switching, by identifying the bacterial strains which are susceptible to P1(S')-mediated transduction, as well as demonstrating its potential for delivering a DNA sequence-specific Cas9 antimicrobial into clinically relevant S. flexneri.     

A Bifunctional Enzyme in a Single Gene Catalyzes the Incorporation of GlcN into the Aeromonas Core Lipopolysaccharide

The core lipopolysaccharide (LPS) of Aeromonas hydrophila AH-3 and Aeromonas salmonicida A450 is characterized by the presence of the pentasaccharide α-d-GlcN-(1→7)-l-α-d-Hep-(1→2)-l-α-d-Hep-(1→3)-l-α-d-Hep-(1→5)-α-Kdo. Previously it has been suggested that the WahA protein is involved in the incorporation of GlcN residue to outer core LPS. The WahA protein contains two domains: a glycosyltransferase and a carbohydrate esterase. In this work we demonstrate that the independent expression of the WahA glycosyltransferase domain catalyzes the incorporation of GlcNAc from UDP-GlcNAc to the outer core LPS. Independent expression of the carbohydrate esterase domain leads to the deacetylation of the GlcNAc residue to GlcN. Thus, the WahA is the first described bifunctional glycosyltransferase enzyme involved in the biosynthesis of core LPS. By contrast in Enterobacteriaceae containing GlcN in their outer core LPS the two reactions are performed by two different enzymes.     

Identification of essential loops and residues of glucosyltransferase V (GtrV) of Shigella flexneri

Lipopolysaccharide (LPS), particularly the O-antigen component, is one of many virulence determinants necessary for Shigella flexneri pathogenesis. O-antigen modification is mediated by glucosyltransferase (gtr) genes encoded by temperate serotype-converting bacteriophages. The gtrV and gtrX genes encode the GtrV and GtrX glucosyltransferases, respectively. These are integral membrane proteins, which catalyze the transfer of a glucosyl residue via an α1,3 linkage to rhamnose II and rhamnose I of the O-antigen unit. This mediates conversion of S. flexneri serotype Y to serotype 5a and X, respectively. Essential regions in the topology of GtrV protein were identified by in vivo recombination and a PCR-mediated approach. A series of GtrX-GtrV and GtrV-GtrX chimeric proteins were constructed based on the fact that GtrV and GtrX share sequence similarity. Analysis of their respective serotype conversion abilities led to the identification of two important periplasmic loops: loops No 2 and No 10 located in the N- and C-termini, respectively. Within these two loops, three conserved motifs were identified; two in loop No 2 and one in loop No 10. These conserved motifs contain acidic residues which were shown to be critical for GtrV function.     

Mapping of chromosomal loci associated with lipopolysaccharide synthesis and serotype specificity in Vibrio cholerae 01 by transposon mutagenesis using Tn5 and Tn2680

Summary Vibrio cholerae strains of the 01 serotype have been classified into three subclasses, Ogawa, Inaba and Hikojima, which are associated with the O-antigen of the lipopolysaccharide (LPS). The DNA encoding the biosynthesis of the O-antigen, the rfb locus, has been cloned and analysed (Manning et al. 1986; Ward et al. 1987). Transposon mutagenesis of the Inaba and Ogawa strains of V. cholerae, using Tn5 or Tn2680 allowed the isolation of a series of independent mutants in each of these serotypes. Some of the insertions were mapped to the rfb region by Southern hybridization using the cloned rfb DNA as a probe, confirming this location to be responsible for both O-antigen production and serotype specificity. The other insertions allowed a second region to be identified which is involved in V. cholerae LPS biosynthesis.