In vivo versus in vitro protein abundance analysis of Shigella dysenteriae type 1 reveals changes in the expression of proteins involved in virulence, stress and energy metabolism.

BACKGROUND: Shigella dysenteriae serotype 1 (SD1) causes the most severe form of epidemic bacillary dysentery. Quantitative proteome profiling of Shigella dysenteriae serotype 1 (SD1) in vitro (derived from LB cell cultures) and in vivo (derived from gnotobiotic piglets) was performed by 2D-LC-MS/MS and APEX, a label-free computationally modified spectral counting methodology. RESULTS: Overall, 1761 proteins were quantitated at a 5% FDR (false discovery rate), including 1480 and 1505 from in vitro and in vivo samples, respectively. Identification of 350 cytoplasmic membrane and outer membrane (OM) proteins (38% of in silico predicted SD1 membrane proteome) contributed to the most extensive survey of the Shigella membrane proteome reported so far. Differential protein abundance analysis using statistical tests revealed that SD1 cells switched to an anaerobic energy metabolism under in vivo conditions, resulting in an increase in fermentative, propanoate, butanoate and nitrate metabolism. Abundance increases of transcription activators FNR and Nar supported the notion of a switch from aerobic to anaerobic respiration in the host gut environment. High in vivo abundances of proteins involved in acid resistance (GadB, AdiA) and mixed acid fermentation (PflA/PflB) indicated bacterial survival responses to acid stress, while increased abundance of oxidative stress proteins (YfiD/YfiF/SodB) implied that defense mechanisms against oxygen radicals were mobilized. Proteins involved in peptidoglycan turnover (MurB) were increased, while ß-barrel OM proteins (OmpA), OM lipoproteins (NlpD), chaperones involved in OM protein folding pathways (YraP, NlpB) and lipopolysaccharide biosynthesis (Imp) were decreased, suggesting unexpected modulations of the outer membrane/peptidoglycan layers in vivo. Several virulence proteins of the Mxi-Spa type III secretion system and invasion plasmid antigens (Ipa proteins) required for invasion of colonic epithelial cells, and release of bacteria into the host cell cytosol were increased in vivo. CONCLUSIONS: Global proteomic profiling of SD1 comparing in vivo vs. in vitro proteomes revealed differential expression of proteins geared towards survival of the pathogen in the host gut environment, including increased abundance of proteins involved in anaerobic energy respiration, acid resistance and virulence. The immunogenic OspC2, OspC3 and IpgA virulence proteins were detected solely under in vivo conditions, lending credence to their candidacy as potential vaccine targets.

Structural insights into the membrane receptor ShuA in DDM micelles and in a model of gram-negative bacteria outer membrane as seen by SAXS and MD simulations.

Successful crystallization of membrane proteins in detergent micelles depends on key factors such as conformational stability of the protein in micellar assemblies, the protein-detergent complex (PDC) monodispersity and favorable protein crystal contacts by suitable shielding of the protein hydrophobic surface by the detergent belt. With the aim of studying the influence of amphiphilic environment on membrane protein structure, stability and crystallizability, we combine molecular dynamics (MD) simulations with SEC-MALLS and SEC-SAXS (Size Exclusion Chromatography in line with Multi Angle Laser Light Scattering or Small Angle X-ray Scattering) experiments to describe the protein-detergent interactions that could help to rationalize PDC crystallization. In this context, we compare the protein-detergent interactions of ShuA from Shigella dysenteriae in n-Dodecyl-ß-D-Maltopyranoside (DDM) with ShuA inserted in a realistic model of gram-negative bacteria outer membrane (OM) containing a mixture of bacterial lipopolysaccharide and phospholipids. To evaluate the quality of the PDC models, we compute the corresponding SAXS curves from the MD trajectories and compare with the experimental ones. We show that computed SAXS curves obtained from the MD trajectories reproduce better the SAXS obtained from the SEC-SAXS experiments for ShuA surrounded by 268 DDM molecules. The MD results show that the DDM molecules form around ShuA a closed belt whose the hydrophobic thickness appears slightly smaller (~22 Å) than the hydrophobic transmembrane domain of the protein (24.6 Å) suggested by Orientations of Proteins in Membranes (OPM) database. The simulations also show that ShuA transmembrane domain is remarkably stable in all the systems except for the extracellular and periplasmic loops that exhibit larger movements due to specific molecular interactions with lipopolysaccharides (LPS). We finally point out that this detergent behavior may lead to the occlusion of the periplasmic hydrophilic surface and poor crystal contacts leading to difficulties in crystallization of ShuA in DDM.

Structure of the heme/hemoglobin outer membrane receptor ShuA from Shigella dysenteriae: heme binding by an induced fit mechanism.

Shigella dysentriae and other Gram-negative human pathogens are able to use iron from heme bound to hemoglobin for growing. We solved at 2.6 A resolution the 3D structure of the TonB-dependent heme/hemoglobin outer membrane receptor ShuA from S. dysenteriae. ShuA binds to hemoglobin and transports heme across the outer membrane. The structure consists of a C-terminal domain that folds into a 22-stranded transmembrane beta-barrel, which is filled by the N-terminal plug domain. One distal histidine ligand of heme is located at the apex of the plug, exposed to the solvent. His86 is situated 9.86 A apart from His420, the second histidine involved in the heme binding. His420 is in the extracellular loop L7. The heme coordination by His86 and His420 involves conformational changes. The comparisons with the hemophore receptor HasR of Serratia marcescens bound to HasA-Heme suggest an extracellular induced fit mechanism for the heme binding. The loop L7 contains hydrophobic residues which could interact with the hydrophobic porphyring ring of heme. The energy required for the transport by ShuA is derived from the proton motive force after interactions between the periplasmic N-terminal TonB-box of ShuA and the inner membrane protein, TonB. In ShuA, the TonB-box is buried and cannot interact with TonB. The structural comparisons with HasR suggest its conformational change upon the heme binding for interacting with TonB. The signaling of the heme binding could involve a hydrogen bond network going from His86 to the TonB-box.

Glycan encapsulated gold nanoparticles selectively inhibit shiga toxins 1 and 2.

Shiga toxins (Stx) released by Escherichia coli O157:H7 and Shigella dysentriae cause life-threatening conditions that include hemolytic uremic syndrome (HUS), kidney failure, and neurological complications. Cellular entry is mediated by the B-subunit of the AB(5) toxin, which recognizes cell surface glycolipids present in lipid raft-like structures. We developed gold glyconanoparticles that present a multivalent display similar to the cell surface glycolipids to compete for these toxins. These highly soluble glyconanoparticles were nontoxic to the Vero monkey kidney cell line and protected Vero cells from Stx-mediated toxicity in a dose-dependent manner. The inhibition is highly dependent on the structure and density of the glycans; selective inhibition of Stx1 and the more clinically relevant Stx2 was achieved. Interestingly, natural variants of Stx2, Stx2c, and Stx2d possessing minimal amino acid variation in the receptor binding site of the B-subunit or changes in the A-subunit were not neutralized by either the Stx1- or Stx2-specific gold glyconanoparticles. Our results suggest that tailored glyconanoparticles that mimic the natural display of glycans in lipid rafts could serve as potential therapeutics for Stx1 and Stx2. However, a few amino acid changes in emerging Stx2 variants can change receptor specificity, and further research is needed to develop receptor mimics for the emerging variants of Stx2.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of the TonB-dependent haem outer membrane transporter ShuA from Shigella dysenteriae.

As part of efforts towards understanding the crystallization of membrane proteins and membrane transport across the outer membrane of Gram-negative bacteria, the TonB-dependent haem outer membrane transporter ShuA of Shigella dysenteriae bound to heavy atoms was crystallized in several crystallization conditions using detergents. The insertion of a His(6) tag into an extracellular loop of ShuA, instead of downstream of the Escherichia coli peptide signal, allowed efficient targeting to the outer membrane and the rapid preparation of crystallizable protein. Crystals diffracting X-rays beyond 3.5 A resolution were obtained by co-crystallizing ShuA with useful heavy atoms for phasing (Eu, Tb, Pb) by the MAD method at the synchrotron, and the SAD or SIRAS method at the Cu wavelength. The authors collected X-ray diffraction data at 2.3 A resolution using one crystal of ShuA-Pb, and at 3.2 A resolution at an energy remote from the Pb M absorption edges for phasing on PROXIMA-1 at SOLEIL.

[Complete structure of the O-polysaccharide from Shigella dysenteriae type 10].

The structure of the O-specific polysaccharide from Shigella dysenteriae type 10, which has been reported previously in Bioorg. Khim. (1977, vol. 3, pp. 1219-1225), was refined: [Formula: see text].

The O-polysaccharide of Escherichia coli O112ac has the same structure as that of Shigella dysenteriae type 2 but is devoid of O-acetylation: a revision of the S. dysenteriae type 2 O-polysaccharide structure.

The O-antigen structure of Shigella dysenteriae type 2 was reinvestigated using chemical modifications along with high-resolution 2D (1)H and (13)C NMR spectroscopy. The O-antigen was found to contain a pyruvic acid acetal, which was overlooked in an early study, and the following revised structure of the pentasaccharide repeating unit was established: where approximately 70% GlcNAc residues bear an O-acetyl group at position 3. The O-antigen of Escherichia coli O112ac was found to have the same carbohydrate structure but to lack O-acetylation.

[Structures of O-polysaccharides from two Shigella dysenteriae type 8 strains].

The structure of the O-polysaccharide (O-antigen) from Shigella dysenteriae type 8 bacteria (strain 599) was corrected using modern NMR techniques (structure 1). The revisions concerned the position of the Glc residue (in the main, but not the side, chain), the site of its substitution, and the configuration of the O-glycoside linkage of the GlcNAc residue. The S. dysenteriae type 8 bacterium (strain G1221), the second investigated representative, was found to produce another structural variant of the O-polysaccharide. It contains GlcNAc instead of the Glc residue in the main chain (structure 2). This data may lead to approval of division of S. dysenteriae type 8 into two subtypes: [Formula: see text].

[Antigenic polysaccharides of bacteria: 41. Structures of the O-specific polysaccharides of Shigella dysenteriae types 4 and 5 revised by NMR spectroscopy].

The earlier established structures of the acidic O-specific polysaccharides from two typical strains of the Shigella dysenteriae bacterium were revised using modern NMR spectroscopy techniques. In particular, the configurations of the glycosidic linkages of GlcNAc (S. dysenteriae type 4) and mannose (S. dysenteriae type 5) residues were corrected. In addition, the location of the sites of nonstoichiometric O-acetylation in S. dysenteriae type 4 was determined: the lateral fucose residue was shown to be occasionally O-acetylated; also, the position of the O-acetyl group present at the stoichiometric quantity in S. dysenteriae type 5 was corrected. The revised structures of the polysaccharides studied are shown below. The known identity of the O-specific polysaccharide structures of S. dysenteriae type 5 and Escherichia coli O58 was confirmed by 13C NMR spectroscopy and, hence, the structure of the E. coli O58 polysaccharide should be revised in the same manner. [Formula: see text].

[Antigenic polysaccharides of bacteria: 40. The structures of O-specific polysaccharides from Shigella dysenteriae types 3 and 9 and S. boydii type 4 revised by NMR spectroscopy].

The reported structures of O-specific polysaccharides from three standard strains of Shigella bacteria were corrected by modern NMR techniques. The revisions concerned the configuration of the O-glycoside linkage (S. dysenteriae type 3, structure 1), the positions of monosaccharide residue glycosylation and acetylation by pyruvic acid (S. dysenteriae type 9, structure 2), and the attachment position of the side monosaccharide chain (S. boydii type 4, structure 3) [struxture in text].

Experimental In Vivo Models of Bacterial Shiga Toxin-Associated Hemolytic Uremic Syndrome.

Shiga toxins (Stxs) are the main virulence factors expressed by the pathogenic Stx-producing bacteria, namely, Shigella dysenteriae serotype 1 and certain Escherichia coli strains. These bacteria cause widespread outbreaks of bloody diarrhea (hemorrhagic colitis) that in severe cases can progress to life-threatening systemic complications, including hemolytic uremic syndrome (HUS) characterized by the acute onset of microangiopathic hemolytic anemia and kidney dysfunction. Shiga toxicosis has a distinct pathogenesis and animal models of Stx-associated HUS have allowed us to investigate this. Since these models will also be useful for developing effective countermeasures to Stx-associated HUS, it is important to have clinically relevant animal models of this disease. Multiple studies over the last few decades have shown that mice injected with purified Stxs develop some of the pathophysiological features seen in HUS patients infected with the Stx-producing bacteria. These features are also efficiently recapitulated in a non-human primate model (baboons). In addition, rats, calves, chicks, piglets, and rabbits have been used as models to study symptoms of HUS that are characteristic of each animal. These models have been very useful for testing hypotheses about how Stx induces HUS and its neurological sequelae. In this review, we describe in detail the current knowledge about the most well-studied in vivo models of Stx-induced HUS; namely, those in mice, piglets, non-human primates, and rabbits. The aim of this review is to show how each human clinical outcome-mimicking animal model can serve as an experimental tool to promote our understanding of Stx-induced pathogenesis.

Structural relation of the antigenic polysaccharides of Escherichia coli O40, Shigella dysenteriae type 9, and E. coli K47.

O-polysaccharides were isolated from the lipopolysaccharides of Escherichia coli O40 and Shigella dysenteriae type 9 and studied by chemical analyses along with (1)H and (13)C NMR spectroscopy. The following new structure of the O-polysaccharide of E. coli O40 was established: -->2)-beta-D-Galp-(1-->4)-beta-D-Manp-(1-->4)-alpha-D-Galp-(1-->3)-beta-D-GlcpNAc-(1--> TheO-polysaccharide structure of S. dysenteriae type 9 established earlier was revised and found to be identical to the reported structure of the capsular polysaccharide of E. coli K47 and to differ from that of the E. coli O40 polysaccharide in the presence of a 3,4-linked pyruvic acid acetal having the (R)-configuration (RPyr): -->2)-beta-D-Galp3,4(RPyr)-(1-->4)-beta-D-Manp-(1-->4)-alpha-D-Galp-(1-->3)-beta-D-GlcpNAc-(1-->

Synthesis of hexa- to tridecasaccharides related to Shigella dysenteriae type 1 for incorporation in experimental vaccines.

Hexa- to tridecasaccharides corresponding to the O-specific polysaccharide (O-SP) of the Gram-negative bacterium Shigella dysenteriae type 1 were synthesized in solution phase. The syntheses utilized tetra-, octa-, and dodecasaccharide intermediates that represent one to three contiguous tetrasaccharide repeating units of the O-SP [Synlett2003, 743]. These compounds were glycosylated with mono-, di-, and trisaccharide trichloroacetamidates, which were synthesized in this study. The excellent stereodirecting effect of 4,6-O-benzophenone ketals in glycosylation reactions of 2-azido-2-deoxy-glucopyranosyl donors was demonstrated. The free oligosaccharides were characterized by 1H and 13C NMR spectroscopy and by high-resolution mass spectrometry. The oligosaccharides described herein contain the 5-(methoxycarbonyl)pentyl aglycon for eventual attachment to immunogenic carriers using a recently published protocol [J. Org. Chem.2005, 70, 6987].

Structural studies of the O-antigenic polysaccharides from Shigella dysenteriae type 3 and Escherichia coli O124, a reinvestigation.

The structures of the O-antigenic part of the lipopolysaccharides from Shigella dysenteriae type 3 and Escherichia coli O124 have been reinvestigated. (1)H and (13)C NMR spectroscopy in combination with selected 2D NMR techniques were used to determine the O-antigen pentasaccharide repeating units with the following structure: [see text]. From biosynthetic considerations this should also be the biological repeating unit. The structures of the repeating units also explain the previously observed cross-reactivity between the strains and to E. coli O164, which only differs in the terminal sugar residue that is lacking the (R)-1-carboxyethyl group.

A two-step method for extraction of lipopolysaccharide from Shigella dysenteriae serotype 1 and Salmonella typhimurium: An improved method for enhanced yield and purity.

Bacterial lipopolysaccharide (LPS) has been widely used as an antigen and adjuvant in immunological applications. Amongst the methods developed for extraction of LPS, hot phenol extraction (HPE) method is the gold standard. However, the HPE method provides poor yield of LPS (~4.5% by weight), is associated with relatively higher impurities of proteins and nucleic acids, and the acidic hot phenol can cause a degradative effect on LPS. In this work a two-step extraction (TSE) method was developed using a non-capsulated, [Shigella dysenteriae serotype-1] (Sd1) and capsulated [Salmonella typhimurium type B (StB)] species as model pathogens. The TSE method takes advantage of growth kinetics of bacteria wherein a two-step sequential approach for LPS extraction was employed. In step-1, culture supplemented with CaCl2 during early log phase of growth was induced to release LPS by the effect of EDTA at their late exponential phase of growth. In step-II, cells with left over LPS were subjected to modified HPE method that reduced both the degradative effect of acidic hot phenol and associated impurities. The LPS produced using TSE method enabled not only enhanced yield (~2.78 and ~2.91 fold higher for Sd1 and StB respectively) requiring nearly similar duration of extraction, but also was structurally and functionally comparable with LPS produced using HPE method and commercially procured LPS. Overall, the developed TSE method is relatively more efficient (enhanced yield), clean (healthy extraction with reduced impurities), safe (reduced handling of larger pathogenic culture) and cost-effective for LPS extraction with potential for scale up.

Purification and crystallization of Shiga toxin from Shigella dysenteriae.

The protein toxin produced by Shigella dysenteriae consists of one enzymatically active A subunit of 293 amino acid residues and five B subunits of 69 amino acid residues that are involved with cell attachment. The holotoxin has been purified by blue Sepharose and chromatofocusing column chromatography. Two crystal forms of purified holotoxin have been grown by vapor diffusion. One grows as fine needles, hexagonal in cross-section, which do not diffract well enough to characterize crystallographically. The second grows as thin plates that diffract to at least 3 A resolution. Their space group is P2(1)2(1)2(1) with unit cell dimensions of a = 132.0 A, b = 146.0 A and c = 82.5 A. The asymmetric unit of the crystals is likely to contain two AB5 units.

The use of a genetic algorithm search for molecular mechanics (MM3)-based conformational analysis of oligosaccharides.

We have implemented a system called glygal that can perform conformational searches on oligosaccharides using several different genetic algorithm (GA) search methods. The searches are performed in the torsion angle conformational space, considering both the primary glycosidic linkages as well as the pendant groups (C-5-C-6 and hydroxyl groups) where energy calculations are performed using the MM3(96) force field. The system includes a graphical user interface for setting calculation parameters and incorporates a 3D molecular viewer. The system was tested using dozens of structures and we present two case studies for two previously investigated O-specific oligosaccharides of the Shigella dysenteriae type 2 and 4. The results obtained using glygal show a significant reduction in the number of structures that need to be sampled in order to find the best conformation, as compared to filtered systematic search.

Up-regulation of CD80-CD86 and IgA on mouse peritoneal B-1 cells by porin of Shigella dysenteriae is Toll-like receptors 2 and 6 dependent.

Porin of Shigella dysenteriae type 1 increased the mRNA levels for Toll-like receptor (TLR) 2 and TLR6 by 1.5- and 2.9-fold respectively, of peritoneal cavity B-1a and B-1b cells, implicating that coexpression of TLR2 and TLR6 is essential as a combinatorial repertoire for recognition of porin by the B-1 cells. Among the two key TLRs, TLR2 and TLR4, which are primarily responsible for recognizing majority of the bacterial products, TLR2 and not TLR4, participates in porin recognition. TLR2 got increased on both the B-1 cell populations whereas the TLR4 expression remained unaffected. Besides TLRs, mRNA for MyD88, an effector molecule associated with TLR-mediated response was enhanced by 1.8-fold that suggests of its involvement in the activity of porin. Both of the B-1 cell populations expressed strongly the mRNA for NF-kappaB in the presence of porin, that was 2.4-fold more than untreated control, conforming to the earlier finding that coexpression of TLR2 and TLR6, resulted in robust NF-kappaB activation for signaling. Porin treatment of B-1 cell populations of C57BL/6 mice, and C3H/HeJ mice in particular, selectively up-regulated the expression of the costimulatory molecules. CD80 expression got enhanced on the B-1a cells whereas CD86 got solely expressed on B-1b cells. Porin-induced cell surface expression of IgM and IgA on B-1 cell populations from C57BL/6 mice. The IgA-generating capacity, hallmark of mucosal immune response, was confirmed with B-1 cells of C3H/HeJ, the lipopolysaccharide non-responder mouse, in response to the protein. The porin-mediated induction of IgA was augmented by interleukin-6 on B-1a and B-1b cells, by 2.4- and 2.6-fold, respectively. The IgA expressed on both B-1a and B-1b cell surfaces after 72 h of culture was found to bind to the 38 kDa monomer of porin confirming it to be anti-porin IgA antibody.

Synthesis, Chiral Separation, Absolute Configuration Assignment, and Biological Activity of Enantiomers of Retro-1 as Potent Inhibitors of Shiga Toxin.

The Shiga toxin (Stx) family is composed of related protein toxins produced by the bacteria Shigella dysenteriae and certain pathogenic strains of E. coli. No effective therapies for Stx intoxication have been developed yet. However, inhibitors that act on the intracellular trafficking of these toxins may provide new options for the development of therapeutic strategies. This study reports the synthesis, chromatographic separation, and pharmacological evaluation of the two enantiomers of Retro-1, a compound active against Stx and other such protein toxins. Retro-1 works by inhibiting retrograde transport of these toxins inside cells. In vitro experiments proved that the configuration of the stereocenter at position 5 is not crucial for the activity of this compound. X-ray diffraction data revealed (S)-Retro-1 to be slightly more active than (R)-Retro-1.

Detection of E. coli O157:H7 and Shigella dysenteriae toxins in clinical samples by PCR-ELISA.

Shiga toxin producing bacteria are potential causes of serious human disease such as hemorrhagic colitis, severe inflammations of ileocolonic regions of gastrointestinal tract, thrombocytopenia, septicemia, malignant disorders in urinary ducts, hemolytic uremic syndrome (HUS). Shiga toxin 1 (stx1), shiga toxin 2 (stx2), or a combination of both are responsible for most clinical symptoms of these diseases. A lot of methods have been developed so far to detect shiga toxins such as cell culture, ELISA, and RFPLA, but due to high costs and labor time in addition to low sensitivity, they have not received much attention. In this study, PCR-ELISA method was used to detect genes encoding shiga toxins1 and 2 (stx1 and stx2). To detect stx1 and stx2 genes, two primer pairs were designed for Multiplex-PCR then PCR-ELISA. PCR products (490 and 275, respectively) were subsequently verified by sequencing. Sensitivity and specificity of PCR-ELISA method were determined by using genome serial dilution and Enterobacteria strains. PCR-ELISA method used in this study proved to be a rapid and precise approach to detect different types of shiga toxins and can be used to detect bacterial genes encoding shiga toxins.