The O-antigen of Plesiomonas shigelloides serotype O36 containing pseudaminic acid.

The structure of the repeating unit of O-antigen of Plesiomonas shigelloides serotype O36 has been investigated by 1H and 13C NMR spectroscopy, matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry and chemical methods. The new structure of trisaccharide has been established: [Formula: see text] These trisaccharide O-antigen units substitute the core undecasaccharide at C-4 of the ß-D-GlcpNAc residue. The core oligosaccharide and lipid A are identical with these of the serotype O17 (PCM 2231) (Maciejewska, A., Lukasiewicz, J., Kaszowska, M., Jachymek, W., Man-Kupisinska, A.; Lugowski, C. Mar. Drugs.2013, 11 (2), 440-454; Lukasiewicz, J., Dzieciatkowska, M., Niedziela, T., Jachymek, W., Augustyniuk, A., Kenne, L., Lugowski, C. Biochemistry, 2006, 45, 10434-10447).

Identification of Plesiomonas spp.: serological and MALDI-TOF MS methods.

Biochemical and serological profiles of isolates of Plesiomonas shigelloides were assayed using standard procedures in isolates from various clinical samples. Seventy-four isolates, including P. shigelloides type strain, were further characterized by MALDI-TOF MS using 3-methoxy-4-hydroxycinnamic acid as matrix. Multiple ions in the 3- to 12-kDa mass range were found in the spectra of each strain, from which the "species-identifying" unique biomarker ions were identified. After creating the species-specific patterns, a spectral database was generated for reliable, rapid, reproducible and accurate identification of Plesiomonas strains. The classical strain description (biochemical and serological) was thus complemented with the metabolic (proteomic) characterization.

Inhibition of mammalian cathepsins by Plesiomonas shigelloides.

To study molecular mechanisms underlying self-defense of the bacterial pathogen Plesiomonas shigelloides against host inflammatory and immune responses, we evaluated its interactions with mammalian papain-like cathepsins that are essential for host immunity. When grown under anaerobic, but not aerobic, conditions, P. shigelloides was shown to bind and inhibit papain, a model representative of the papain family of cysteine proteinases. This points to mammalian cathepsins as likely physiological targets of a novel cysteine-proteinase inhibitor expressed on bacterial cell surface. Both papain and mammalian cathepsins L and B were inhibited by periplasmic extracts of aerobically and anaerobically grown bacteria, the inhibitory activity being higher in the latter. Inhibition by both intact cells and periplasmic samples was rapid and efficient. The results suggest a possible defensive role of bacterial inhibitors of cathepsins during invasion of a mammalian host. The bacteria thus may modulate host protective responses through inhibiting cathepsins involved in antigen processing and presentation.

Comparison of serological specificity of anti-endotoxin sera directed against whole bacterial cells and core oligosaccharide of Escherichia coli J5-tetanus toxoid conjugate.

The rough mutants of Gram-negative bacteria are widely used to induce protective antisera but the nature of the target epitope for such antibodies is not precisely defined. Endotoxin is one of several antigens present on the surface of bacterial cells, which are able to elicit specific antibodies. We studied the specificity of antibodies produced against a conjugate of E. coli J5 endotoxin core oligosaccharide with tetanus toxoid. The use of chemically defined antigen for immunisation excludes the possibility of production of antibodies against other cell surface antigens. A comparison of this monospecific anti-endotoxin serum with antiserum against E. coli J5 whole cells was performed in order to distinguish the role that endotoxin core oligosaccharide plays in the interaction with humoral host defences from that of other potentially important Gram-negative bacterial surface antigens. The reactivity of both sera with smooth and rough lipopolysaccharides was determined in ELISA, immunoblotting and by flow cytometry. Both antisera reacted with similar specificity with most lipopolysaccharides of identical or related core type. Less distinct reactions with endotoxins of the antibacterial serum in comparison with the anti-conjugate serum were found in all serological tests. LPS of E. coli O100 that showed the strongest reactions with both sera was used to stimulate IL-6, TNFalpha and nitric oxide production by the J-774A.1 cell line. Both sera were used to inhibit that stimulation and no inhibitory effects of the examined sera in comparison with non-immune serum were observed.

Conformational investigation of a cyclic enterobacterial common antigen employing NMR spectroscopy and molecular dynamics simulations.

The three-dimensional structure of a cyclic enterobacterial common antigen (ECA) having four trisaccharide repeating units has been investigated by NMR spectroscopy and molecular dynamics simulations. Three different NMR parameters were determined: (a) (1)H,(1)H cross-relaxation rates from NOE experiments were used for determination of proton-proton distances; (b) trans-glycosidic (3)J(C,H) scalar coupling constants analyzed via a Karplus-type relationship provided information on torsion angles; and (c) (1)H,(13)C one-bond dipolar couplings obtained in a dilute liquid-crystalline medium were interpreted in terms of the orientational order and molecular conformations. The molecular dynamics simulations of the dodecasaccharide were performed with explicit water and counterions, which are important factors that strongly influence molecular conformation. Subsequently, the results from computer simulation were used to generate a three-dimensional structure of the cyclic ECA which is consistent with the experimental NMR parameters.

Comparison of O-antigen gene clusters of Escherichia coli (Shigella) sonnei and Plesiomonas shigelloides O17: sonnei gained its current plasmid-borne O-antigen genes from P. shigelloides in a recent event.

Escherichia coli Sonnei has an O antigen identical to that of Plesiomonas shigelloides O17, and its O-antigen gene cluster is located on a plasmid. By sequencing the chromosomal O-antigen gene cluster of P. shigelloides O17 and comparing it with that of Sonnei, we showed that Sonnei gained its O-antigen genes recently.

New serovars of Plesiomonas shigelloides--1992-1998.

Most of the 26 new O (O77-O102) and 10 new H (H42-H50) and H1a1d antigens were found in various P. shigelloides strains isolated from man and other mammals, birds, fish, and water and water insects, not only in the Czech Republic but also in 12 foreign countries.

The complete DNA sequence of the O antigen gene region of Plesiomonas shigelloides serotype O17 which is identical to Shigella sonnei form I antigen.

We cloned and determined the sequence of a DNA region of approximately 15-kb containing the cluster of genes required for O17 antigen expression in the Escherichia coli K-12 strain from the chromosome of Plesiomonas shigelloides serotype O17:H2 strain. The sequencing analysis revealed that the minimum essential region of the P. shigelloides O17 antigen gene cluster had a size of approximately 11.5-kb and contained 9 contiguous open reading frames (ORFs), which were almost identical to the corresponding ORFs of Shigella sonnei form I antigen gene region, except for IS630 sequence, at the DNA as well as amino acid levels. The putative function of most of the ORFs could be determined on the basis of amino acid sequence similarities and characteristics. In addition, the G+C content of the P. shigelloides O17 antigen genes was lower than that of the chromosomal DNA of P. shigelloides and S. sonnei, suggesting that both P. shigelloides O17 and S. sonnei form I antigen genes had been derived from the same origin with a low G+C content.

New O and H antigens of the international antigenic scheme for Plesiomonas shigelloides.

A revised update of the International antigenic scheme for Plesiomonas shigelloides is presented. Twenty-six new O (O77-O102) and 10 new H (H42-H50 and H1a1d) antigens have been described since 1994. The sources of antigens are mostly human clinical strains, isolates from warmblooded animals and a few environmental cultures.

[An antigenic analysis and the protective properties of the R-form lipopolysaccharides of gram-negative bacteria]. / Antigennyi analiz i protektivnye svoistva lipopolisakharidov R-form nekotorykh gramotritsatel'nykh bakterii.

The antigenic and immunogenic properties of the R-form lipopolysaccharides (R-LPS) of Pseudomonas aeruginosa, Salmonella minnesota, Escherichia coli and Shigella were studied. The results of the study revealed the existence of antigenic relationship between P.aeruginosa R-LPS and R-LPS Escherichia and Shigella. In serological tests no antigenic relationship between P. aeruginosa R-LPS and Salmonella R-LPS was revealed, but as shown in earlier experiments of the protection of mice, Re-glycolipid stimulated protective immunity against Pseudomonas infection in the animals. On the basis of R-LPS obtained from selected P. aeruginosa and Salmonella strains a vaccine was prepared which proved to be effective against infection caused by P. aeruginosa S-strain in experiments on mice. The vaccine induced protection in 40-100% of immunized mice, depending on the scheme of immunization. The vaccine may probably be effective against infections caused by other gram-negative bacteria.

New serovars of plesiomonas shigelloides--1996.

Eight new 0 (91-98) and H (46-49) antigens are described. Their reference strains come from Czech Republic, Cuba and USA. The majority of reference strains are of human origin. Some of the new antigens have been found in other strains coming mostly from water.

Serological characterisation of anti-endotoxin sera directed against the conjugates of oligosaccharide core of Escherichia coli type R1, R2, R3, J5 and Salmonella Ra with tetanus toxoid.

The covalent conjugates of oligosaccharide core: Escherichia coli type R1, R2, R3, J5 and Salmonella Ra with tetanus toxoid have been prepared using reaction of reductive amination. The neoglycoconjugates were good immunogens in rabbits yielding a high level of anti-lipopolysaccharide antibodies of IgG class. The antibodies were used to examine the possibility of their reactions with smooth lipopolysaccharides. We have found that all antisera were able to react with the lipopolysaccharide molecules of identical or related core type, possessing core oligosaccharides substituted with O-specific chains. These reactions were shown in both the ELISA assay and the immunoblotting test.

Anti-Plesiomonas shigelloides agglutinating and complement-fixing antibody titres in normal individuals and diarrhoeal patients in Edo State, Nigeria.

One hundred patients with diarrhoea and 50 asymptomatic individuals attending various hospitals in Edo State, Nigeria, were screened for serum complement-fixing and agglutinating antibodies to Plesiomonas shigelloides using the complement-fixation and agglutination tests. Seventy (70%) of the 100 patients and 20 (40%) of the 50 asymptomatic individuals had detectable complement-fixing antibodies at titres ranging from 1:32 to 1:128 and 1:8 to 1:32 respectively. Results suggest that cases of diarrhoea in this environment may be due to P. shigelloides, but the demonstration of antibodies in asymptomatic individuals show that they also have serum antibodies against P. shigelloides. The exclusive use of antibody responses in the diagnosis of P. shigelloides infections should, therefore, be interpreted with caution.

Structural elucidation of the O-antigen lipopolysaccharide from two strains of Plesiomonas shigelloides that share a type-specific antigen with Shigella flexneri 6, and the common group 1 antigen with Shigella flexneri spp and Shigella dysenteriae 1.

Sugar and methylation analyses of native polysaccharides together with one-dimensional 1H- and 13C-NMR spectroscopy revealed that the two polysaccharides from strains 22074 and 12254 of Plesiomonas shigelloides are identical. The structure of the polysaccharide from strain 22074 was deduced from a uronic acid degradation and by NMR spectroscopy where heteronuclear multiple bond connectivity and two-dimensional nuclear Overhauser effect spectroscopy experiments established the pentasaccharide repeating unit as-->4)-alpha-D-GalpA-(1-->3)-alpha-D-GlcpNAc-(1-->3)-alpha-L- Rhap-(1-->2)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->.

New serovars of Plesiomonas shigelloides 1992.

Fourteen new O (O77-O9O) and 4 new H (H42-H45) antigens were described. Seven O and 2 new H antigens were revealed among strains not agglutinating with antisera against 76 O and 41 H officially recognized serovars. The most frequent was serovar O80:H38 which was isolated in 5 countries (Sweden, Czechoslovakia, Bulgaria, Yugoslavia and Canada) from human material and from sewage and surface water.

Is protection against shigellosis induced by natural infection with Plesiomonas shigelloides?

Shigellosis due to Shigella sonnei is rare among people growing up and living in developing countries; however, infections due to S sonnei becomes more common than those due to S flexneri as societies develop economically. The relation between risk of S sonnei infection and economic development may be explained by the exposure of developing-country populations to Plesiomonas shigelloides. P shigelloides is often found in surface water, and one serotype (serotype 17) possesses a cell-wall lipopolysaccharide identical to that of S sonnei. Thus, exposure to P shigelloides by drinking contaminated water may immunise populations to S sonnei. As economic development occurs, water quality improves and populations become susceptible to S sonnei. Although drinking water has many advantages, immunisation against S sonnei may be one benefit of traditional water sources.

Characterisation of Plesiomonas shigelloides strains that share type-specific antigen with Shigella flexneri 6 and common group 1 antigen with Shigella flexneri spp. and Shigella dysenteriae 1.

Three strains of Plesiomonas shigelloides isolated from patients with diarrhoea were agglutinated with Shigella flexneri 6 antiserum in slide and tube tests. All the strains were also agglutinated with a monoclonal antibody to the common group 1 antigen shared between S. flexneri serotypes and S. dysenteriae type 1. Further studies with one strain also showed sharing of antigenicity in an enzyme-linked immunosorbent assay. The results suggest that the strains share type-specific antigen with S. flexneri 6 and the common group 1 antigen with S. flexneri serotypes and S. dysenteriae 1. The sharing of antigens may have implications for cross-protection. One strain adhered to HEp-2 cell monolayers. None of the strains contained high mol. wt plasmids and there was no sequence homology with the invasiveness plasmid of Shigella spp. in DNA probe hybridisation. They were susceptible to the commonly used antibiotics. However, they possessed four other virulence-associated properties of Shigella spp. that included Congo-red binding, hydrophobicity, toxicity to HeLa cells and HEp-2 cell invasiveness (although they gave negative results in the Sereny test for invasiveness). These data suggest that the three unique strains might be considered pathogenic. Studies in animal models and human volunteers would be necessary to establish their pathogenic potential.

Protection from Shigella sonnei infection by immunisation of rabbits with Plesiomonas shigelloides (SVC 01).

Plesiomonas shigelloides, an organism commonly found in water, is only rarely associated with diarrhoea in man. P. shigelloides serotype O:17 (SVC O1), which is antigenically similar to Shigella sonnei, was found to be neither virulent nor toxic for rabbits. Rabbits immunised by feeding with P. shigelloides (SVC O1) were completely protected against an oral challenge with 10(10) cells of S. sonnei but non-immunised rabbits were not. P. shigelloides (SVC O1) may be a useful vaccine strain for shigellosis.

Age-specific prevalence of serum antibodies to the invasion plasmid and lipopolysaccharide antigens of Shigella species in Chilean and North American populations.

Shigella species have virulence plasmids that encode outer membrane proteins (invasion plasmid antigens, Ipa) associated with pathogenicity. Western blots were used to detect antibodies to Ipa in sera from 390 Chilean children, and these responses were compared with those of a US population of infants and adults. Antibodies to lipopolysaccharide (LPS) of Plesiomonas shigelloides and Shigella flexneri 2a were measured by ELISA. Among the Chileans, there was an age-related acquisition of Ipa antibodies, with 28% of 1-year-olds and 100% of children greater than or equal to 10 years showing positive responses. In contrast, none of the US infants and only 38% of the adults had antibodies to Ipa. Levels of LPS antibodies were also found to increase in an age-related manner among the Chileans. These results corroborate findings of previous epidemiologic studies which show that Shigella infections are endemic in Chile, as in other developing countries. The measurement of Ipa and LPS antibodies is a useful seroepidemiologic tool for investigating previous exposure to Shigella species in populations.