A New Look at the Enterobacterial Common Antigen Forms Obtained during Rough Lipopolysaccharides Purification.

Enterobacterial common antigen (ECA) is a conserved antigen expressed by enterobacteria. It is built by trisaccharide repeating units: →3)-α-D-Fucp4NAc-(1→4)-ß-D-ManpNAcA-(1→4)-α-D-GlcpNAc-(1→ and occurs in three forms: as surface-bound linear polysaccharides linked to a phosphoglyceride (ECAPG) or lipopolysaccharide - endotoxin (ECALPS), and cyclic form (ECACYC). ECA maintains, outer membrane integrity, immunogenicity, and viability of enterobacteria. A supernatant obtained after LPS ultracentrifugation was reported as a source for ECA isolation, but it has never been assessed for detailed composition besides ECACYC. We used mild acid hydrolysis and gel filtration, or zwitterionic-hydrophilic interaction liquid (ZIC®HILIC) chromatography combined with mass spectrometry for purification, fractionation, and structural analysis of rough Shigella sonnei and Escherichia coli R1 and K12 crude LPS preparations. Presented work is the first report concerning complex characteristic of all ECA forms present in LPS-derived supernatants. We demonstrated high heterogeneity of the supernatant-derived ECA that contaminate LPS purified by ultracentrifugation. Not only previously reported O-acetylated tetrameric, pentameric, and hexameric ECACYC have been identified, but also devoid of lipid moiety linear ECA built from 7 to 11 repeating units. Described results were common for all selected strains. The origin of linear ECA is discussed against the current knowledge about ECAPG and ECALPS.

NACE-ESI-MS/MS method for separation and characterization of phosphorylation and acylation isomers of lipid A.

Lipid A represents a heterogeneous group of bacterial outer membrane phosphoglycolipids, which play a major role in the pathogenesis of Gram-negative sepsis. The number and position of phosphoryl and acyl groups in lipid A molecules are key structural determinants in their bioactivities. In this study, a NACE-ESI-MS/MS method was developed for the simultaneous analysis of lipid A isomers possessing a different degree of phosphorylation and acylation. Various C4'- and C1-monophosphorylated lipid A isobars, as well as acylation isomers, were baseline separated within 43 min in a separation medium of methanol/dichloromethane/triethylamine/acetic acid 60:40:1.08:0.36 (v/v/v/v). Both normal and reverse CE polarities could be applied for proper detection of the analytes owing to the combination of a suction effect caused by the nebulizer gas at the outlet end of the capillary and external pressure applied on the inlet vial. The separated lipid A species could be identified unequivocally by their characteristic fragmentation patterns through CID performed in both negative- and positive-ionization modes. The uniqueness of the NACE-ESI-MS/MS method lies in its simplicity and reliability for proving the phosphorylation isomerism (C1 or C4') and acylation pattern of native lipid A species or those designed for therapeutic applications.

Superstructure formation by RodZ hexamers of Shigella sonnei maintains the rod shape of bacilli.

The rod shape of bacilli is maintained by bacterial cytoskeletal protein MreB, an actin homolog that acts in concert with the inner membrane protein RodZ. We previously reported RodZ binds RNA to control the posttranscriptional regulation of invE (virB), which controls the type III secretion system essential for the virulence of Shigella. Here, we show that purified RodZ forms "superstructures" of high molecular mass that dissociate into a midsized "basal complex" in the presence of nonionic detergent, or to a monomer in the presence of dithiothreitol. We used mass spectrometry to show that the basal complex was a hexamer. Electrophoresis mobility shift assays combined with gel filtration detected the RNA-binding activity in fractions containing molecules larger than the basal hexamer. The superstructure was consistently detected with MreB in crude cell lysates of S. sonnei that were fractionated using gel filtration. Immunofluorescence microscopy using two different super-resolution settings showed that wild-type RodZ was distributed in cells as separate dots. Consistent with the superstructure comprising homohexamers, majority of the dots distributed among areas of discrete values. In addition, simultaneous immunodetection of MreB provided the first evidence of colocalization with RodZ as larger patch like signals. These findings indicate that native RodZ forms clusters of various sizes, which may correspond to a superstructure comprising multiple hexamers required for the RNA-binding activity.

Detection and discrimination of Shigella sonnei and Shigella flexneri based on vacuolar responses in Saccharomyces cerevisiae.

This study provided a system for bacteria detection based on a lysosome-like-vacuole response in the yeast Saccharomyces cerevisiae. Vacuoles are factors known to activate the immune system in the presence of foreign substances. Here, Shigella sonnei and Shigella flexneri were exposed to yeast to analyze the alteration of vacuolar enzymes. The ability to detect the bacteria was evaluated by confocal microscopy after exposing and staining vacuoles with LysoTracker. Results showed that the treatment of yeast with these bacteria increased the number of red vacuole-like organelles surrounding yeast nuclei. Thus, vacuole alteration can be used as a biomarker for bacteria detection. Next, the expression of vacuolar enzymes under the influence of bacteria was examined using two-dimensional gel electrophoresis (2-DE) method for screening specific biomarkers for each Shigella strain. Finally, the recombinant yeasts that contained biomarkers fused to different fluorescent proteins confirmed the ability of yeast to detect these two Shigella strains at concentrations ranging from 10 to 100 CFU/mL.

Evaluation of toll-like receptor 4 expression in human bone marrow mesenchymal stem cells by lipopolysaccharides from Shigella.

Lipopolysaccharides (LPS) from gram negative bacteria stimulate toll-like receptor 4 (TLR4) expression in immune cells. Recent reports state that bone marrow-derived cells such as mesenchymal stem cells (MSCs) also express TLR proteins. Numerous researches have studied the effect of a number of LPSs on TLR4 expression, but no data exists on the effect of LPSs from different strains of one bacterial genus on TLR4 expression. In this study, we investigate the effects of various concentrations of LPS from different Shigella strains on TLR4 expression in human bone marrow (hBM)-MSCs. At the mRNA level, we have found that untreated hBM-MSCs (control) did not express TLR4 compared to the experimental groups. Cells treated with LPS from Shigella flexneri had the highest expression of TLR4, whereas cells treated with LPS from Shigella sonnei had the lowest expression. We observed that LPSs had a dose-dependent effect on TLR4 expression in all of the treatment groups. ELISA findings for interleukin-6 secretion have confirmed mRNA expression results for all treatment groups. Hence, LPS from S. flexneri can be considered as an optimum LPS to stimulate the immune system for vaccine production against shigellosis. Also, TLR activation in hBM-MSCs can modulate their function such as homing.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis of Escherichia coli categories.

The mass profiles of cell-free extracts of 180 commensal and pathogenic strains of Escherichia coli were determined by MALDI-TOF mass spectrometry (MS). While some peaks were highly conserved in all E. coli, several peaks occurred only in some strains, showing heterogeneity among them. We did not detect strain-specific peaks for any of the E. coli categories tested. However, review of the fully conserved and the variable peaks suggested that MALDI-TOF MS has the potential to distinguish commensal and uropathogenic E. coli strains. Additionally, eight Shigella sonnei isolates were tested and found to be indistinguishable from E. coli by MALDI-TOF MS under the test conditions.

Antagonistic activity expressed by Shigella sonnei: identification of a putative new bacteriocin.

Bacteriocins are antibacterial, proteinaceous substances that mediate microbial dynamics. Bacteriocin production is a highly disseminated property among all major lineages of bacteria, including Shigella. In this paper, we addressed the purification and characterisation of a bacteriocin produced by a Shigella sonnei strain (SS9) isolated from a child with acute diarrhoea. The substance was purified through ammonium-sulphate precipitation and sequential steps of chromatography. The intracellular fraction obtained at 75% ammonium sulphate maintained activity following exposure to pH values from 1-11 and storage at -80ºC for more than two years and was inactivated by high temperatures and proteases. The molecular mass of the purified bacteriocin was determined by mass spectrometry to be 18.56 kDa. The N-terminal sequence of the bacteriocin did not match any other antibacterial proteins described. A putative new bacteriocin produced by S. sonnei has been detected. This bacteriocin may represent a newly described protein or a previously described protein with a newly detected function. Considering that SS9 expresses antagonism against other diarrhoeagenic bacteria, the bacteriocin may contribute to S. sonnei virulence and is potentially applicable to either preventing or controlling diarrhoeal disease.

Antagonistic activity expressed by Shigella sonnei: identification of a putative new bacteriocin

Bacteriocins are antibacterial, proteinaceous substances that mediate microbial dynamics. Bacteriocin production is a highly disseminated property among all major lineages of bacteria, including Shigella. In this paper, we addressed the purification and characterisation of a bacteriocin produced by a Shigella sonnei strain (SS9) isolated from a child with acute diarrhoea. The substance was purified through ammonium-sulphate precipitation and sequential steps of chromatography. The intracellular fraction obtained at 75% ammonium sulphate maintained activity following exposure to pH values from 1-11 and storage at -80ºC for more than two years and was inactivated by high temperatures and proteases. The molecular mass of the purified bacteriocin was determined by mass spectrometry to be 18.56 kDa. The N-terminal sequence of the bacteriocin did not match any other antibacterial proteins described. A putative new bacteriocin produced by S. sonnei has been detected. This bacteriocin may represent a newly described protein or a previously described protein with a newly detected function. Considering that SS9 expresses antagonism against other diarrhoeagenic bacteria, the bacteriocin may contribute to S. sonnei virulence and is potentially applicable to either preventing or controlling diarrhoeal disease.

Structural variability of endotoxins from R-type isogenic mutants of Shigella sonnei.

The structural variations in the rough-type endotoxins [lipopolysaccharides (LPSs)] of Shigella sonnei mutant strains (S. sonnei phase II-4303, R41, 562H and 4350) were investigated by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and tandem MS. A series of S. sonnei mutants had previously been the subject of analytical studies on the biosynthesis of heptose components in the core oligosaccharide region of LPSs. This study gives a complete overview on the structures of the full core and lipid A of S. sonnei mutant strains by MS. We found that the LPSs of the isogenic rough mutants were formed in a step-like manner containing 0:1:2:3 heptose in the deep core region of 4350, 562H, R41 and 4303, respectively, and the longest LPS from the mutant S. sonnei 4303 contained also five hexoses. The structural variations in the lipid A moiety and in the oligosaccharide part of the intact LPS were followed by MALDI-TOF-MS/MS. For the dissolution and the ionization of the samples, 2,5-dihydroxybenzoic acid in citric acid solution was applied as matrix. The detailed evaluation of the mass spectra indicates heterogeneity in the lipid part due to the differences in the phosphate and fatty acid composition.

The elucidation of the structure of the core part of the LPS from Plesiomonas shigelloides serotype O17 expressing O-polysaccharide chain identical to the Shigella sonnei O-chain.

Plesiomonas shigelloides O17 LPS contains the same O-antigenic polysaccharide chain as a causative agent of dysentery, Shigella sonnei. This polysaccharide can be used as a component of a vaccine against dysentery. Core part of the P. shigelloides O17 LPS was studied using NMR and mass spectrometry and the following structure was proposed: [structure : see text]. Significant similarity of the P. shigelloides O17 LPS core with the structure of the P. shigelloides O54 core was observed.

Estudio comparativo de la actividad antibacteriana in vitro de los neumatóforos de Heritiera fomes y Xylocarpus moluccensis / A comparative study on the in vitro antibacterial activity of the pneumatophores of Heritiera fomes and Xylocarpus moluccensis

Se evaluó la actividad antibacteriana in vitro de los extractos de etanol de los neumatóforos de Xylocarpus moluccensis (Familia: Meliaceae) y Heritiera fomes (Familia: Sterculiaceae) frente a diversas cepas bacterianas utilizando el ensayo de difusión en disco. Ambos extractos presentaron perfiles antibacterianos similares, y las zonas de inhibición fueron >10 mm en la mayoría de los casos. Estos extractos presentaron la máxima actividad frente a aerógenos Enterobacter, siendo las zonas de inhibición de 19 y 21 mm, respectivamente. La concentración inhibitoria mínima(CIM) se determinó mediante el método de dilución en caldo de cultivo. El extracto de X. moluccensis fue el más potente frente a Shigella boydii y Shigella sonnie (CIM = 200 y 300 mg/mL, respectivamente). Se puede asumirque X. moluccensis y H. fomes podrían ser fuentes potenciales de nuevos descubrimientos para el desarrollo de fármacos (AU)

The ethanol extracts of the pneumatophores of Xylocarpus moluccensis (Family: Meliaceae) and Heritiera fomes (Family: Sterculiaceae) were assessed for in vitro antibacterial activities against a number of bacterial strains using the disc diffusion assay. Both extracts showed similar antibacterial profiles, and the zones of inhibitions were >10 mm in the most cases. These extracts exhibited the most prominent activity against Enterobacter aerogenes, with the zones of inhibition of 19 and 21 mm, respectively. The minimum inhibitory concentration (MIC) was determined by the broth dilution method. The extract of X. moluccensis was the most potent against Shigella boydii and Shigella sonnie (MIC = 200 and 300 mg/mL, respectively). It can be assumed that that X. moluccensis and H. fomes could be potential sources for novel ‘lead’ discovery for antibacterial drug development (AU)

uspA of Shigella sonnei.

One of the strategies that bacteria utilize to combat environmental stress is to synthesize stress-responding proteins. In Escherichia coli, adverse environmental factors, such as starvation, heat, and the presence of acid, oxidants, heavy metals, and antibiotics, trigger the expression of the universal stress protein (USP). The gene of the USP, uspA, in E. coli K-12 and E. coli O157:H7 has been identified and sequenced. In this study, the nucleotide sequence of uspA in a strain of Shigella sonnei implicated in the 1998 parsley-related outbreak of shigellosis was determined. Within an 800-bp region sequenced, there were 17 bp mismatches between the uspA of S. sonnei and that of E. coli K-12. Among the 17 mismatched nucleotides, 8 were within the structure gene of uspA. A total of 12 bp variations were identified between the uspA of S. sonnei and that of E. coli O157:H7, of which 5 bp were internal to the coding region of uspA. However, unlike the mismatches between the uspA of E. coli K-12 and the same gene of E. coli O157:H7 and S. sonnei that resulted in a single amino acid substitution and changed an alanine to an arginine at position 140, the mismatches between S. sonnei and E. coli O157:H7 were silent and did not result in any amino acid substitution.

CE to monitor endotoxins by protein complexation.

A new CE method for fast and efficient analysis of bacterial endotoxins (lipopolysaccharides) is described. It is based on the strong interaction between proteins and endotoxins. The UV absorption of the protein component in the complex is used for the detection. The electrophoretic mobility of the complex hemoglobin/endotoxin can be employed for qualitative analysis of the endotoxin. For instance, the structural differences between "smooth" and "rough" lipopolysaccharides from Salmonella minnesota (wild-type), Salmonella minnesota R595 and Shigella sonnei R562H are reflected in the electrophoretic mobilities of their hemoglobin complex.

The immunosuppressive activity of chemically modified lipopolysaccharide of Shigella sonnei.

In the present investigations we aimed to study the effect of Shigella sonnei lipopolysaccharide (LPS) on delayed type hypersensitivity (DTH) to non-bacterial antigen in CBA mice. These experiments showed that intraperitoneal injection of phenol-water extracted LPS and avirulent S. sonnei did not affect the level of DTH. However, an injection of avirulent bacteria and LPS treated with 2-mercaptoethanol reduced significantly the levels of DTH. Gel filtration of redox-reactivated LPS through Sephadex G-200 shows that LPS contains three immunosuppressive components: approximately 800 kDa and higher, 150-200 and 50-70 kDa. These components differed by their specificity and heat-sensitivity.

Identification and structural analysis of synthetic oligosaccharides of Shigella sonnei using MALDI-TOF MS.

MALDI-TOF mass spectroscopy was used for the molecular weight determination of protected synthetic oligosaccharides related to a cell surface bacterial polysaccharide. By-products containing chlorinated protecting groups caused isotopic patterns characteristic of the natural isotopic distribution of chlorine, were identified on the basis of isotopic distribution. 2,4,6-Trihydroxyacetophenone (THAP) as a matrix was better than 2,5-dihydroxybenzoic acid (DHB) for compounds containing chlorine, since monoisotopic resolution and no fragmentation were observed. In the post source decay (PSD) mode the identification of the oligosaccharide sequence through cleavage of the interglycosidic linkages was also possible, thus providing a sensitive and accurate tool for the structural verification of synthetic oligosaccharide intermediates.

Protein profile characterization of bacterial lysates by capillary electrophoresis.

A fast and reproducible method was developed to characterize cell lysates by their electrophoretic profiles using capillary electrophoresis (CE). Characteristic and reproducible patterns were recorded for each bacterial strains when "dynamic sieving" CE, using a polymer solution in the capillary, was applied to distinguish four strains of the Enterobacteriaceae family. The electropherograms showed distinct differences when comparing them to the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein profiles. This is certainly a result of the differences in the separation principles and in the detection methods of the two techniques.

Conformational stabilization of the altruronic acid residue in the O-specific polysaccharide of Shigella sonnei/Plesiomonas shigelloides.

Complete assignments for the 1H- and the 13C-NMR spectra of the O-specific polysaccharide of S. sonnei/Plesiomonas shigelloides are reported. Evidence is presented that in this polysaccharide both pyranose residues exist preferentially in the 4C1 chair conformation and that the polysaccharide exists in the zwitterion form.

Chemical analysis of lipopolysaccharides of Shigella sonnei form II strains expressed by cloned form I antigen genes.

A compositional sugar analysis was carried out on lipopolysaccharide (LPS) from Shigella sonnei form II in which a plasmid with cloned form I antigen genes had been introduced. The recipient form II strains contained galactose, glucose, heptose, glucosamine, and 2-keto-3-deoxyoctonic acid (KDO) (2: 3: 1: 2: 2) in its LPS, while the transformant form I LPS contained, besides these sugars, N-acetyl-L-altrosaminouronic acid as an additional sugar constituent, which is known to be one of the antigenic determinants of form I antigen.

[Lipopolysaccharide changes in smooth-type avirulent Shigella in comparison with initial virulent strains]. / Izmeneniia lipopolisakharidov u avirulentnykh shigell gladkogo tipa po sravneniiu s iskhodnymi virulentnymi shtammami.

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.

Use of multiple markers for investigation of an epidemic of Shigella sonnei infections in Monroe County, New York.

Antibiotic susceptibility patterns, plasmid profiles, and endonuclease restriction analysis of plasmid DNA were used in the investigation of an epidemic of Shigella sonnei infections in Monroe County, New York, in 1988 and 1989. The epidemic peaked during the winter, included the simultaneous transmission of the disease from person to person and from common food sources, and especially affected inhabitants of the poor, inner-city neighborhoods, young children of both sexes, and women. Resistance to ampicillin, tetracycline, or trimethoprim-sulfamethoxazole, encoded in a 70-MDa plasmid, was found in most of the examined isolates. Unexpectedly, isolates from patients involved in a food-borne outbreak exhibited three different antibiotic susceptibility patterns, suggesting deletion of antibiotic resistance determinants in some strains. Antibiograms clearly separated food-borne outbreak-related and non-foodborne outbreak-related strains, distinguished more strains than did the plasmid profiles, and were useful in tracing the dissemination of individual isolates in the community. Restriction endonuclease analysis substantially increased the discriminatory value of plasmid profiles and validated the antibiogram results. The present study illustrates the complexity of epidemics of S. sonnei infections and shows the value of combining different biological markers in the investigation.