Chemical and serological properties of lipopolysaccharides from Vibrio parahaemolyticus O-untypeable strains isolated from patients

Chemical and serological studies have been carried out on the O-antigenic lipopolysaccharides (LPS) of six strains, U-6443, W-90144, X-3972, AD-7999, 90A-6611 and KX-V212, of Vibrio parahaemolyticus isolated from patients. The O-serotypes of these strains have not been identified because they were not agglutinated by any diagnostic antisera against known O-serotype strains. A compositional sugar analysis of their LPS revealed that out of the six O-untypeable (OUT) strains, U-6443, W-90144 and AD-7999 strains belonged to chemotype II (chemotype of O2), 90A-6611 and KX-V212 strains to chemotype III (chemotype of O3, O5, O11 and O13) and X-3972 strain to chemotype IV (chemotype of O4). A structural analysis of LPS isolated from KX-V212 revealed that the inner core region of the LPS consisted of only one mole of 2-keto-3-deoxy-D-manno-octonic acid, which carried a phosphate group at position C4 and the outer core at position C5. In passive hemolysis tests performed by using LPS as the antigen to sensitize sheep red blood cells (SRBC), and diagnostic antisera (O1 to O11) or anti-whole-cell rabbit antisera raised against O12, O13 and the six OUT strains, strong cross-reactivity was observed among LPS derived from the strains belonging to chemotype II (U-6443, W-90144, AD-7999 and O2). Strong cross-reactivity was also observed between X-3972 (chemotype IV) and O4 LPS. In contrast, LPS from two of the strains belonging to chemotype III (90A-6611 and KX-V212) did not react with any of the antisera raised against known O-serotypes. Cross-absorption tests showed that the O-antigens of U-6443, W-90144 and AD-7999 were identical to that of O2, and the O-antigen of X-3972 to that of O4. On the other hand, after the absorption of antisera raised against 90A-6611 and KX-V212 with O2 cells, the hemolytic activities against SRBC sensitized with homologous LPS were still retained at a high titer, whereas the hemolytic activities against SRBC sensitized with LPS from other O-serotype strains were completely eliminated. A cross-absorption test revealed that the O-antigens of these two strains were identical to each other. Thus, it was demonstrated that the O-serotype of OUT strains 90A-6611 and KX-V212 was not involved in the known O-serotypes; rather it represented a novel serotype which has not hitherto been reported.     

Serological cross-reaction between Yersinia enterocolitica O9 and non-O1 Vibrio cholerae bio-serogroup Hakata and antigenic analysis of their relationship by their lipopolysaccharides.

Cross-agglutination and cross-agglutinin absorption experiments were carried out on non-O1 Vibrio cholerae bio-serogroup Hakata (Hakata) and Yersinia enterocolitica O9 (O9). It was shown that the O-antigen of Hakata was closely related to that of O9 in an a, b-a, c type of relationship. The antigenic relationship between the O-antigens of the two bacteria was analyzed by passive hemolysis (PH) and passive hemolysis inhibition (PHI) tests by using their lipopolysaccharides (LPS) as antigen for sensitizing sheep red blood cells (SRBC) and, in the case of the latter, as an inhibitor in a PH system consisting of LPS-coated SRBC, guinea-pig complement and anti-Hakata or O9 antiserum, both unabsorbed and absorbed with the heterologous Hakata or O9 antigen. In the PH experiment, unabsorbed anti-Hakata antiserum had hemolytic titers of 126,100 and 2,600 against Hakata- and O9-LPS-coated SRBC, respectively, and anti-O9 antiserum had hemolytic titers of 19,400 and 38,800, respectively, against these SRBC. The PH experiment showed that anti-O9 antiserum contains a hemolysin reacting with the heterologous Hakata antigen at a high titer (19,400), while anti-Hakata antiserum contains a hemolysin reacting with the heterologous O9 antigen at a significant titer (2,600). The former was completely removed from anti-O9 antiserum with the Hakata antigen and the latter from anti-Hakata antiserum with the O9 antigen. Thus, serological cross-reactivity was demonstrated between the Hakata and O9 strains.     

Lipopolysaccharide changes in smooth-type avirulent Shigella in comparison with initial virulent strains]

The comparative study of the lipopolysaccharides (LPS) of virulent and avirulent strains of S. sonnei, phase I (smooth colonies), has been made. Electrophoresis of LPS and subsequent densitometry of electrophoregrams have revealed the increase of the fraction of long 0-chains with a considerable number of recurring elements in 2 out of 3 LPS preparations obtained from avirulent shigellae. In mice immunized with these LPS preparations a considerably greater number of antibody-producing cells can be detected in Jerne's test on sheep red blood cells (SRBC) sensitized with the LPS of a virulent strain than on those sensitized with the above LPS preparations. Long 0-specific chains supposedly inhibit the fixation of individual complement components on the corresponding sensitized SRBC. The LPS of the third avirulent strain of S. sonnei, phase I, with transposon integrated into its genome, which has led to the formation of the avirulent variant of a previously virulent strain, seems to contain fine structural differences from the initial virulent strain. The immunogenicity of the LPS of this avirulent strain is greatly (3-4 times) decreased, which is manifested by the number of antibody-producing cells detected in Jerne's test on SRBC sensitized with LPS preparations obtained from these two strains.     

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]     

Identification of alternative pathway serum complement activity in the blood of the American alligator (Alligator mississippiensis)

Incubation of different dilutions of alligator serum with sheep red blood cells (SRBCs) that had not been sensitized with antibodies resulted in concentration-dependent hemolytic activity. This hemolytic activity was not affected by the presence of ammonium hydroxide and methylamine, known inactivators of the classical complement cascade. However, the hemolytic activities were inhibited by EDTA and salicylaldoxime, indicating that the alternate pathway is primarily responsible for these activities. Immunofixation of electrophoretically-resolved alligator serum proteins with antihuman C3 polyclonal antibodies resulted in detection of a protein antigenically similar to human C3 in alligator serum. SDS-PAGE, followed by Western blot analysis, revealed the presence of two alligator serum proteins with nearly identical molecular weights as human C3alpha and C3beta. SRBC hemolysis and antibacterial activity by alligator serum was significantly reduced in the presence of antihuman C3 antibodies. The hemolytic effect of alligator serum was shown to occur rapidly, with significant activity within 5 min and maximal activity occurring at 15 min. SRBC hemolysis was also temperature-dependent, with reduced activity below 15 degrees C and above 30 degrees C. These data suggest that the antibiotic properties of alligator serum are partially due to the presence of a complement-facilitated humoral immune response analogous to that described in mammalian systems.     

Anti-LPS factor in the horseshoe crab, Tachypleus tridentatus. Its hemolytic activity on the red blood cell sensitized with lipopolysaccharide

Anti-LPS factor, which inhibits the endotoxin mediated coagulation system in the horseshoe crab, Tachypleus tridentatus, was found to lyse red blood cells sensitized with gram-negative bacterial LPS, but not to lyse unsensitized cells. This hemolysis occurred even at 0 degree C and was completed within 1 min. The binding of anti-LPS factor to LPS must be essential for the hemolysis, because free LPS inhibited the hemolytic action of anti-LPS factor.     

Serological cross-reaction between intact and chemically modified lipopolysaccharides of O1 Vibrio cholerae Inaba and non-O1 V. cholerae bio-serogroup Hakata.

Serological cross-reaction of intact as well as chemically modified LPS from O1 Vibrio cholerae 569B (Inaba) with non-O1 V. cholerae Hakata LPS, which contain alpha(1-->2)-linked N-acetyl perosamine-homopolymer constituting their O polysaccharide chain, was studied by passive hemolysis test by using their LPS as antigen for sensitizing sheep red blood cells (SRBC). The N-deacylation of the alpha(1-->2)-linked linear 3-deoxy-tetronyl perosamine-homopolymer constituting the O polysaccharide chain in 569B LPS resulted in virtual elimination of their serological reactivity with both homologous Inaba and heterologous Hakata antisera. Furthermore, when the resultant NH2 groups of the N-deacylated perosamine-homopolymers in 569B LPS were N-acylated with acetyl, propionyl or butanoyl groups, they markedly recovered the serological reactivity to a marked extent, in particular, their pronounced cross-serological reactivity with heterologous Hakata antiserum. These results are believed to be compatible with the interpretation that the Inaba antigen factor C possessed by the two bacteria studied is related to the common occurrence of the N-acyl groups, regardless of what the acyl groups are, residing in the perosamine residues of the perosamine-homopolymers constituting the O polysaccharide chain of their LPS.     

Role of divalent metal ions in serum complement activity of the American alligator (Alligator mississippiensis)

Treatment of alligator serum with different concentrations of EDTA resulted in a concentration-dependent inhibition of serum-mediated sheep red blood cell (SRBC) hemolysis. This inhibition of serum-dependent hemolysis was observed for other chelators of divalent metal ions, such as phosphate and citrate. Treatment of alligator serum with 5 mM EDTA completely inhibited SRBC hemolysis, which could be totally restored by the addition of 5 mM Ca(2+) or Mg(2+), but not Cu(2+) or Ba(2+). These data indicate a specific need for Ca(2+) and/or Mg(2+) in the serum-mediated hemolysis of SRBCs. Kinetic analyses revealed that the addition of 30 mM EDTA 1 min after incubation of SRBCs with serum resulted in only 30% inhibition of hemolytic activity. However, addition of EDTA as early as 3 min post-incubation resulted in complete SRBC hemolysis. Pretreatment of serum with EDTA inhibited the hemolytic activity, but the activity could be restored in a time-dependent manner by the addition of Ca(2+)or Mg(2+). These data indicate that, as in human serum, the need for divalent metal ions occurs early in the alligator serum complement cascade.     

痢疾菌苗研究进展

痢疾是全世界范围内流行的肠道传染病。随着分子生物学技术的发展,迄今已构建了新一代预防痢疾的疫苗,其中包括:具有侵袭力和不具侵袭力的单价口服活菌苗候选株;它们与大肠杆菌、伤寒沙门氏菌和血清型不同的痢疾杆菌组成的双价和三价菌苗候选株;以及以脂多糖和核糖体为基础的不经胃肠的组分菌。猴体和人体试验证明它们安全有效。预计在未来的10年中,将会有一个或几个安全有效的痢疾疫苗投放市场。     

Acetylation of O-specific lipopolysaccharides from Shigella flexneri 3a and 2a occurs in Escherichia coli K-12 carrying cloned S. flexneri 3a and 2a rfb genes.

Most of the Shigella flexneri O-specific serotypes result from O-acetyl and/or glucosyl groups added to a common O-repeating unit of the lipopolysaccharide (LPS) molecule. The genes involved in acetylation and/or glucosylation of S. flexneri LPS are physically located on lysogenic bacteriophages, whereas the rfb cluster contains the biosynthesis genes for the common O-repeating unit (D.A.R. Simmons and E. Romanowska, J. Med. Microbiol. 23:289-302, 1987). Using a cosmid cloning strategy, we have cloned the rfb regions from S. flexneri 3a and 2a. Escherichia coli K-12 containing plasmids pYS1-5 (derived from S. flexneri 3a) and pEY5 (derived from S. flexneri 2a) expressed O-specific LPS which reacted immunologically with S. flexneri polyvalent O antiserum. However, O-specific LPS expressed in E. coli K-12 also reacted with group 6 antiserum, indicating the presence of O-acetyl groups attached to one of the rhamnose components of the O-repeating unit. This was confirmed by measuring the amounts of acetate released from purified LPS samples and also by the chemical removal of O-acetyl groups, which abolished group 6 reactivity. The O-acetylation phenotype was absent in an E. coli strain with an sbcB-his-rfb chromosomal deletion and could be restored upon conjugation of F' 129, which carries sequences corresponding to a portion of the deleted region. Our data demonstrate that E. coli K-12 strains possess a novel locus which directs the O acetylation of LPS and is located in the sbcB-rfb region of the chromosomal map.     

The effect of the potential PhoQ histidine kinase inhibitors on Shigella flexneri virulence

PhoQ/PhoP is an important two-component system that regulates Shigella virulence. We explored whether the PhoQ/PhoP system is a promising target for new antibiotics against S. flexneri infection. By using a high-throughput screen and enzymatic activity coupled assay, four compounds were found as potential PhoQ inhibitors. These compounds not only inhibited the activity of SF-PhoQc autophosphorylation but also displayed high binding affinities to the SF-PhoQc protein in the Surface Plasmon Resonance response. A S. flexneri cell invasion assay showed that three of these potential PhoQ inhibitors inhibit the invasion of HeLa cells by S. flexneri 9380. In a Mouse Sereny test, mice inoculated with S. flexneri 9380 pre-treated with the potential PhoQ inhibitors 1, 2, 3 or 4 displayed no inflammation, whereas mice inoculated with S. flexneri 9380 alone displayed severe keratoconjunctival inflammation. All four potential PhoQ inhibitors showed no significant cytotoxicity or hemolytic activity. These data suggest that the four potential PhoQ inhibitors inhibited the virulence of S. flexneri and that PhoQ/PhoP is a promising target for the development of drugs against S. flexneri infection.     

Molecular cloning and genetic analysis of the rfb region from Shigella flexneri type 6 in Escherichia coli K-12

The rfb gene cluster which determines the biosynthesis of the Shigella flexneri serotype 6 O-antigen specificity has been cloned in pHC79, generating plasmids pPM3115 and pPM3116. These plasmids mediate expression, in Escherichia coli K-12, of lipopolysaccharides (LPS) immunologically similar to the S. flexneri type 6 LPS as judged by SDS-PAGE and Western-immunoblot analysis using S. flexneri type 6 specific antisera. Thus, unlike other S. flexneri serotypes, no additional loci are required for serotype specificity. This expression is independent of E. coli K-12 rfb genes. Southern-hybridization analysis using the 16.2-kb BglII probe from S. flexneri type 6 rfb region detected very little sequence homology in S. flexneri serotypes 1-5, however, some homology was detected with E. coli O2 and O18, but not in E. coli 0101 strains, Salmonella and Vibrio cholerae.     

Regulation of O-antigen chain length is required for Shigella flexneri virulence

It is shown that Shigella flexneri maintains genetic control over the modal chain length of the O-antigen polysaccharide chains of its lipopolysaccharide (LPS) molecules because such a distribution is required for virulence. The effect of altering O-antigen chain length on S. flexneri virulence was investigated by inserting a kanamycin (Km)-resistance cassette into the rol gene (controlling the modal O-antigen chain length distribution), and into the rfbD gene, whose product is needed for synthesis of dTDP-rhamnose (the precursor of rhamnose in the O-antigen). The mutations had the expected effect on LPS structure. The rol ::Km mutation was impaired in the ability to elicit keratoconjunctivitis, as determined by the Serény test. The rol ::Km and rfbD ::Km mutations prevented plaque formation on HeLa cells, but neither mutation affected the ability of S. flexneri to invade and replicate in HeLa cells. Microscopy of bacteria-infected HeLa cells stained with fluorescein isothiocyanate (FITC)-phalloidin demonstrated that both the rol ::Km and rfbD ::Km mutants were defective in F-actin tail formation: the latter mutant showed distorted F-actin tails. Plasma-membrane protrusions were occasionally observed. Investigation of the location of IcsA (required for F-actin tail formation) on the cell surface by immunofluorescence and immunogold electron microscopy showed that while most rol mutant bacteria produced little or no cell-surface IcsA, 10% resembled the parental bacterial cell (which had IcsA at one cell pole; the rfbD mutant had IcsA located over its entire cell surface although it was more concentrated at one end of the cell). That the O-antigen chains of the rol ::Km mutant did not mask the IcsA protein was demonstrated by using the endorhamnosidase activity of Sf6c phage to digest the O-antigen chains, and comparing untreated and Sf6c-treated cells by immunofluorescence with anti-IcsA serum.     

Alcohol-conferred hemolysis in yeast is a consequence of increased respiratory burden

We have previously reported that growth on alcohol vapors confers hemolytic properties on certain yeast species and strains ('microbial alcohol conferred hemolysis'; MACH). Here, a Saccharomyces cerevisiae deletion library consisting of c. 4800 clones was screened for MACH mutants in the presence of n-butanol vapors; 136 mutants were MACH-negative, and 325 exhibited reduced hemolysis and/or growth. Of the MACH-negative mutants, 35.3% were affected in mitochondrial-related genes. The data suggest that intact mitochondrial and respiratory chain functions are critical for the observed MACH phenomenon. We propose that the uncontrolled cellular uptake of alcohol results in yeast 'hyper-respiration', leading to elaboration of hemolytic molecules such as hydrogen peroxide and hemolysis-causing lipids. To support this premise, we showed that: (1) exogenous catalase and glutathione reduce alcohol-conferred hemolysis in S. cerevisiae BY4741 and Candida tropicalis 59445; (2) C. tropicalis produces hydrogen peroxide following growth on ethanol and n-butanol, as shown using xylenol orange; and (3) a lysophospholipid-containing lipid extract from alcohol-grown C. tropicalis specifically causes hemolysis.     

Interactions between earthworm hemolysins and sheep red blood cell membranes

The hemolytic activity exhibited by the coelomic fluid of the Annelid Eisenia fetida andrei is mediated by two lipoproteins of mass 40 and 45 kDa, each of them capable of hemolysis. Such an activity is not inhibited by zymosan, inulin or lipopolysaccharide (LPS), nor by hydrazine or methylamine, suggesting that earthworm hemolysins are not related to C3 or C3b complement components. Among the membrane lipids tested (phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, sphingomyelin and cholesterol) only sphingomyelin inhibited hemolysis. The analysis of E.f. andrei proteins bound to sphingomyelin microvesicles, as well as to sheep red blood cell (SRBC) membranes, revealed a polymerization of E.f. andrei 40 kDa and/or 45 kDa hemolysins. Consequently, sphingomyelin appears a likely candidate for hemolytic complex receptor. Electron microscopy observations suggested that the polymerization causes an open channel through the lipid bilayer. As demonstrated using metal ions, heparin, chondroitin sulfate, poly(L-lysine) and protamine chloride, the mode of action of earthworm hemolytic complex is not analogous to that of C9 or perforine.     

The normal chain length distribution of the O antigen is required for the interaction of Shigella flexneri 2a with polarized Caco-2 cells

Shigella flexneri causes bacillary dysentery in humans. Essential to the establishment of the disease is the invasion of the colonic epithelial cells. Here we investigated the role of the lipopolysaccharide (LPS) O antigen in the ability of S. flexneri to adhere to and invade polarized Caco-2 cells. The S. flexneri 2a O antigen has two preferred chain lengths: a short O antigen (S-OAg) regulated by the WzzB protein and a very long O antigen (VL-OAg) regulated by Wzz pHS2. Mutants with defined deletions of the genes required for O-antigen assembly and polymerization were constructed and assayed for their abilities to adhere to and enter cultured epithelial cells. The results show that both VL- and S-OAg are required for invasion through the basolateral cell membrane. In contrast, the absence of O antigen does not impair adhesion. Purified LPS does not act as a competitor for the invasion of Caco-2 cells by the wild-type strain, suggesting that LPS is not directly involved in the internalization process by epithelial cells.     

Nonspecific Elicitation of Antibody-Forming Cells in the Mouse Spleen by Bacterial Lipopolysaccharide

Mechanisms of nonspecific elicitation of anti-sheep erythrocyte (SRBC) hemolytic antibody plaque-forming cells (PFC) in mouse spleens with an injection of bacterial endotoxin (lipopolysaccharide (LPS)) were studied in comparison with the genesis of naturally occurring ‘background’ PFC in normal mouse spleens and of rapidly arising PFC in mouse spleens after immunization with SRBC. The cytokinetic pattern of anti-SRBC PFC response after an injection of LPS was quite different from that of the response elicited after immunization with SRBC. In addition, even though LPS nonspecifically elicited anti-SRBC PFC response in mice, LPS could not confer any immunological memory on mouse immunocytes for a ‘secondary-type’ anti-SRBC PFC response to restimulation with LPS or SRBC. The administration of rabbit anti-mouse thymocyte immunoglobulin or anti-SRBC antiserum in mice markedly suppressed the PFC response after immunization with SRBC, but did not do so after stimulation with LPS. Neonatally thymectomized mice could still respond to stimulation with LPS, producing anti-SRBC PFC in their spleens. Injections of actinomycin D or cyclophosphamide into mice resulted in obvious reductions of the PFC responses elicited by either LPS or SRBC. However, injections of these immunosuppressive antisera or drugs did not affect the number of anti-SRBC PFC in normal mouse spleens. These results suggest that the geneses of anti-SRBC PFC developed under different conditions, i.e., background PFC, LPS-stimulated PFC, and antigen-stimulated PFC, are quite different from each other, and that the nonspecific elicitation of anti-SRBC PFC by LPS does not require the helper function of T lymphocytes. No obvious difference, however, was observed in the time of ontogenic maturation among these three different anti-SRBC PFC in the mouse spleens judging from when they were first manifested after birth.     

Full structural characterization of Shigella flexneri M90T serotype 5 wild-type R-LPS and its delta galU mutant: glycine residue location in the inner core of the lipopolysaccharide

Shigella flexneri is a gram-negative bacterium responsible for serious enteric infections that occur mainly in the terminal ileum and colon. High interest in Shigella, as a human pathogen, is driven by its antibiotic resistance and the necessity to develop a vaccine against its infections. Vaccines of the last generation use carbohydrate moieties of the lipopolysaccharide as probable candidates. For this reason, the primary structure of the core oligosaccharide from the R-LPS produced by S. flexneri M90T serotype 5 using chemical analysis, nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MALDI), is herein reported. This is the first time that the core oligosaccharide primary structure by S. flexneri M90T is established in an unambiguous multidisciplinary approach. Chemical and spectroscopical investigation of the de-acetylated LPS showed that the inner core structure is characterized by a L,D-Hep-(1 -->7)-L,D-Hep-(1 -->3)-L,D-Hep-(1 -->5)-[Kdo-(2 -->4)]-Kdo sequence that is the common structural theme identified in Enterobacteriaceae. In particular, in S. flexneri M90T serotype 5 LPS, a glucosamine residue is additionally sitting at O-7 of the last heptose whereas the outer core is characterized by glucose and galactose residues. Also, in order to exactly define the position of glycine that is an integral constituent of the core region of the LPS, we created a S. flexneri M90T delta galU mutant and studied its LOS. In this way it was possible to establish that glycine is sitting at O-6 of the second heptose in the inner core.     

Characterization of five Shigella flexneri variant Y-specific monoclonal antibodies using defined saccharides and glycoconjugate antigens

The structural domains of the Shigella flexneri variant Y O-antigen epitopes 3,4 have defied definition, despite knowledge of the structure of the linear polysaccharide chain of the LPS molecule. The dual epitope designation of group antigen 3,4 is based on absorption data using polyvalent rabbit antisera. Five monoclonal antibodies specific for the Y antigen, generated after immunization of BALB/c mice or LOU/C rats, were selected on the basis of ELISA by using well-characterized S. flexneri Y LPS and chemically defined glycoconjugates. Chemically defined LPS from all S. flexneri serogroups, synthetic oligosaccharides, and saccharides obtained by phage Sf6-mediated hydrolysis of the O-polysaccharide were used either as free haptens or glycoconjugates in Farr assays and ELISA titrations. Two different patterns of antibody specificities were seen: two monoclonal antibodies had combining sites recognizing the terminal nonreducing end of the O-polysaccharide complementary to the tetrasaccharide repeating unit; and three antibodies bound to intrachain determinants and had larger combining sites, possibly accommodating at least an octasaccharide. The precise specificity of these two general types of antibodies indicate that variant Y polysaccharide generates more than two O-factors.     

Interaction between bacteriophage Sf6 and Shigella flexner.

The Shigella flexneri phage Sf6 has an isometric head with hexagonal symmetry 53nm in diameter. The noncontractile tails in 16 nm long and terminates with a base plate containing six spikes. Sf6 is typical of the C phages in the morphological classification of Bradley. Phage Sf6 processes alpha-1,3-endorhamnosidase activity as demonstrated by methylation and reducing end group sugar analyses of the products obtained on interaction with the O-polysaccharide chain of S.flexneri strains which have the O-group 3,4 antigen. The major end product was an octasaccharide with the following structure: Rha III-GlcNAc-Rha I-Rha II-Rha III-GlcNAc-Rha I-Rha II. Acetylation of 0-2 of rhamnose III of the O-polysaccharide chain, either brought about by Sf6 lysogenization or found in wild-type S. flexneri (3b) strains, prevented enzymatic hydrolysis. O-deacetylation of the polysaccharide chain again made it susceptible to the S6f endorhamnosidase.