Simple and practical sialoglycan encoding system reveals vast diversity in nature and identifies a universal sialoglycan-recognizing probe derived from AB5 toxin B subunits.

Vertebrate sialic acids (Sias) display much diversity in modifications, linkages, and underlying glycans. Slide microarrays allow high-throughput explorations of sialoglycan-protein interactions. A microarray presenting ~150 structurally defined sialyltrisaccharides with various Sias linkages and modifications still poses challenges in planning, data sorting, visualization, and analysis. To address these issues, we devised a simple 9-digit code for sialyltrisaccharides with terminal Sias and underlying two monosaccharides assigned from the nonreducing end, with 3 digits assigning a monosaccharide, its modifications, and linkage. Calculations based on the encoding system reveal >113,000 likely linear sialyltrisaccharides in nature. Notably, a biantennary N-glycan with 2 terminal sialyltrisaccharides could thus have >1010 potential combinations and a triantennary N-glycan with 3 terminal sequences, >1015 potential combinations. While all possibilities likely do not exist in nature, sialoglycans encode enormous diversity. While glycomic approaches are used to probe such diverse sialomes, naturally occurring bacterial AB5 toxin B subunits are simpler tools to track the dynamic sialome in biological systems. Sialoglycan microarray was utilized to compare sialoglycan-recognizing bacterial toxin B subunits. Unlike the poor correlation between B subunits and species phylogeny, there is stronger correlation with Sia-epitope preferences. Further supporting this pattern, we report a B subunit (YenB) from Yersinia enterocolitica (broad host range) recognizing almost all sialoglycans in the microarray, including 4-O-acetylated-Sias not recognized by a Yersinia pestis orthologue (YpeB). Differential Sia-binding patterns were also observed with phylogenetically related B subunits from Escherichia coli (SubB), Salmonella Typhi (PltB), Salmonella Typhimurium (ArtB), extra-intestinal E.coli (EcPltB), Vibrio cholera (CtxB), and cholera family homologue of E. coli (EcxB).

In silico and in vivo approaches to recombinant multi-epitope immunogen of GroEL provides efficient cross protection against S. Typhimurium, S. flexneri, and S. dysenteriae.

OBJECTIVES: Stress or Heat Shock Proteins (HSPs) have been included in various operations like protein folding, autophagy, and apoptosis. HSP families recognize as protective antigens in a wide range of bacteria because they have been conserved through evolution. Due to their homology as well as antigenicity they are competent for applying in cross-protection against bacterial diseases. METHODS: In the present study, bioinformatics approaches utilized to design epitope-based construction of Hsp60 (or GroEL) protein. In this regard, potential B-cell and T-cell epitopes except for allergenic sequences were selected by immunoinformatic tools. The structural and functional aspects of the DNA, RNA, and protein levels were assessed by bioinformatics software. Following in silico investigations, recombinant GroEL multi-epitope of Salmonella typhi was expressed, purified, and validated. Mouse groups were immunized with recombinant protein and humoral immune response was measured by enzyme linked immunosorbent assay (ELISA). Animal challenge against Salmonella Typhimurium, Shigella flexneri, and Shigella dysenteriae was evaluated. RESULTS: recombinant protein expression and purification with 14.3 kilodaltons (kDa) was confirmed by SDS-PAGE and western blotting. After animal administration, the immunoglobulins evaluated increase after each immunization. Immunized antisera exhibited 80%, 40%, and 40% protection against the lethal dose infection by S. Typhimurium, S. flexneri, and S. dysenteriae respectively. Passive immunization conferred 50%, 30%, and 30% protection in mice against S. Typhimurium, S. flexneri and S. dysentery respectively. In addition, bacterial organ load had exhibited a significant decrease in colony forming unit (CFU) in the liver and spleen of the immunized mice compared to the control. CONCLUSION: Our study demonstrates the efficacy of S. Typhi recombinant GroEL multi-epitope to consider as a universal immunogen candidate versus multiple bacterial pathogens.

Directional conformer exchange in dihydrofolate reductase revealed by single-molecule nanopore recordings.

Conformational heterogeneity is emerging as a defining characteristic of enzyme function. However, understanding the role of protein conformations requires their thermodynamic and kinetic characterization at the single-molecule level, which remains extremely challenging. Here we report the ligand-induced conformational changes of dihydrofolate reductase (DHFR) by measuring the modulation of the nanopore currents. The long observation time of the electrical recordings enabled the detection of rare conformational transitions hidden in ensemble measurements. We show that DHFR exists in at least four ground-state configurations or conformers with different affinities for its ligands. Unliganded DHFR adopted low-affinity conformers, whereas the binding of substrates promoted the switch to the high-affinity conformer. Conversion between the conformers was accelerated by molecules that stabilized the transition state of DHFR, which suggests that the reaction lowers the energy barrier for conformer exchange and thus facilitates product release. This mechanism might be a general feature in enzymatic reactions affected by product inhibition or when the release of products is the rate-limiting step.

Spectroscopic characterisation of a series of Salmonella Typhi Vi-diphtheria toxoid glycoconjugate antigens differing in polysaccharide-protein ratio.

Glycoconjugate vaccines consisting of the Salmonella enterica subsp. enterica serovar Typhi (S. Typhi) Vi capsular polysaccharide (PS) covalently attached to a suitable carrier protein have become available to support mass paediatric vaccination campaigns against typhoid. One developmental vaccine from the International Vaccine Institute (IVI) uses diphtheria toxoid (DTx) as the carrier protein. Several investigational conjugates with different PS:protein ratios were prepared, as previously reported by the IVI group, for physicochemical and immunochemical characterisation. We describe here the further spectroscopic characterisation of this series of glycoconjugate immunogen bulks using NMR spectroscopy, circular dichroism and absorption spectroscopy. We have used several mathematical approaches to extract information from the spectroscopic data not previously applied to glycoconjugates. These complementary approaches provide information on (i) the integrity of the carrier protein, (ii) consistency between batches of vaccine components, (iii) the polysaccharide: protein ratio (iv) the O-acetylation of the Vi in the conjugate (v) the stability of the O-acetylation of the Vi, and (vi) the presence of residual process reagents in the bulk. The utility of the data analysis approaches is discussed. Together, these analytical methods provide important characterisation of Vi-DTx conjugates to support development and quality control of commercial products.

Total Synthesis of an Immunogenic Trehalose Phospholipid from Salmonella Typhi and Elucidation of Its sn-Regiochemistry by Mass Spectrometry.

Diphosphatidyltrehalose (diPT) is an immunogenic glycolipid, recently isolated from Salmonella Typhi. Despite rigorous structure elucidation, the sn-position of the acyl chains on the glycerol backbone had not been unequivocally established. A stereoselective synthesis of diPT and its regioisomer is reported herein. Using a hybrid MS3 approach combining collisional dissociation and ultraviolet photodissociation mass spectrometry for analysis of the regioisomers and natural diPT, the regiochemistry of the acyl chains of this abundant immunostimulatory glycolipid was established.

Structural and Functional Variation in Outer Membrane Polysaccharide Export (OPX) Proteins from the Two Major Capsule Assembly Pathways Present in Escherichia coli.

Capsular polysaccharides (CPSs) are virulence factors for many important pathogens. In Escherichia coli, CPSs are synthesized via two distinct pathways, but both require proteins from the outer membrane polysaccharide export (OPX) family to complete CPS export from the periplasm to the cell surface. In this study, we compare the properties of the OPX proteins from the prototypical group 1 (Wzy-dependent) and group 2 (ABC transporter-dependent) pathways in E. coli K30 (Wza) and E. coli K2 (KpsD), respectively. In addition, we compare an OPX from Salmonella enterica serovar Typhi (VexA), which shares structural properties with Wza, while operating in an ABC transporter-dependent pathway. These proteins differ in distribution in the cell envelope and formation of stable multimers, but these properties do not align with acylation or the interfacing biosynthetic pathway. In E. coli K2, murein lipoprotein (Lpp) plays a role in peptidoglycan association of KpsD, and loss of this interaction correlates with impaired group 2 capsule production. VexA also depends on Lpp for peptidoglycan association, but CPS production is unaffected in an lpp mutant. In contrast, Wza and group 1 capsule production is unaffected by the absence of Lpp. These results point to complex structure-function relationships between different OPX proteins.IMPORTANCE Capsules are protective layers of polysaccharides that surround the cell surface of many bacteria, including that of Escherichia coli isolates and Salmonella enterica serovar Typhi. Capsular polysaccharides (CPSs) are often essential for virulence because they facilitate evasion of host immune responses. The attenuation of unencapsulated mutants in animal models and the involvement of protein families with conserved features make the CPS export pathway a novel candidate for therapeutic strategies. However, appropriate "antivirulence" strategies require a fundamental understanding of the underpinning cellular processes. Investigating export proteins that are conserved across different biosynthesis strategies will give important insight into how CPS is transported to the cell surface.

Conservation of the OmpC Porin Among Typhoidal and Non-Typhoidal Salmonella Serovars.

Salmonella enterica infections remain a challenging health issue, causing significant morbidity and mortality worldwide. Current vaccines against typhoid fever display moderate efficacy whilst no licensed vaccines are available for paratyphoid fever or invasive non-typhoidal salmonellosis. Therefore, there is an urgent need to develop high efficacy broad-spectrum vaccines that can protect against typhoidal and non-typhoidal Salmonella. The Salmonella outer membrane porins OmpC and OmpF, have been shown to be highly immunogenic antigens, efficiently eliciting protective antibody, and cellular immunity. Furthermore, enterobacterial porins, particularly the OmpC, have a high degree of homology in terms of sequence and structure, thus making them a suitable vaccine candidate. However, the degree of the amino acid conservation of OmpC among typhoidal and non-typhoidal Salmonella serovars is currently unknown. Here we used a bioinformatical analysis to classify the typhoidal and non-typhoidal Salmonella OmpC amino acid sequences into different clades independently of their serological classification. Further, our analysis determined that the porin OmpC contains various amino acid sequences that are highly conserved among both typhoidal and non-typhoidal Salmonella serovars. Critically, some of these highly conserved sequences were located in the transmembrane ß-sheet within the porin ß-barrel and have immunogenic potential for binding to MHC-II molecules, making them suitable candidates for a broad-spectrum Salmonella vaccine. Collectively, these findings suggest that these highly conserved sequences may be used for the rational design of an effective broad-spectrum vaccine against Salmonella.

In silico epitope identification of unique multidrug resistance proteins from Salmonella Typhi for vaccine development.

Typhoid fever is a multisystemic illness caused by Salmonella enterica serovars Typhi and is resistant to most antibiotics and drugs. The resistance is conferred through multidrug resistance (MDR) proteins, which efflux most antibiotics and other drugs. We predicted potential candidate B-cell and T-cell epitopes using bio- and immune-informatics tools in the 11 MDR proteins - EmrA, EmrB, EmrD, MdtA, MdtB, MdtC, MdtG, MdtH, MdtK, MdtL and TolC. The antigenic potential of the MDR proteins was calculated using VaxiJen server. The B-cell and T-cell epitopes of the MDR proteins were predicted using BCPred and ProPredI and ProPred respectively. The binding affinities of the predicted T-cell epitopes were estimated using T-epitope designer and MHCPred tools. 10, 7, 5, 12, 14, 21, 26, 3, 3 and 3 B-cell epitopes were identified in EmrA, EmrB, EmrD, TolC, MdtA, MdtB, MdtC, MdtG, MdtH and MdtL respectively. We predicted 9 T-cell epitopes - YVSRRAVQP (EmrA), FGVANAISI (EmrB), MVNSQVKQA and YQGGMVNSQ (TolC), WDRTNSHKL (MdtA), FLRNIPTAI (MdtB), YVEQLGVTG (MdtG), VKWMYAIEA (MdtH) and LAHTNTVTL (MdtL) capable of eliciting both humoral and adaptive immune responses. These T-cell epitopes specifically bind to HLA alleles - DRB1*0101 and DRB1*0401. This is the first report of epitope prediction in the MDR proteins of S. Typhi. Taken together, these results indicate the MDR proteins - EmrA, MdtA and TolC are the most suitable vaccine candidates for S. Typhi. The findings of our study on the MDR proteins prove to be useful in the development of peptide-based vaccine for the prevention and/or treatment of typhoid fever.

Introducing a cost-effective method for purification of bioactive flagellin from several flagellated gram-negative bacteria.

The objective of this study was to introduce a simple and cheap method for purification of flagellin. So, flagellin proteins of Salmonella typhimurium (S. typhimurium), Escherichia coli (E. coli), Citrobacter freundii (C. freundii) and Salmonella typhi (S. typhi) were purified by a modified simple method. Bacterial cultures were precipitated by centrifugation. Precipitates were washed twice and flagellin proteins were detached by shaking vigorously (in PBS pH = 2), and then flagellin proteins were precipitated by ammonium sulfate saturation. Evaluation of purification efficiency and concentration were examined by SDS-PAGE and Bradford assay. Polyclonal antibodies were produced against S. typhimurium FliC and cross-reactivity of anti-S. typhimurium was assessed against other flagellins. Bioactivity of flagellins was evaluated by cell proliferation and IL-8 protein expression assay in HEK293 cells, and also, IL-6 and TNF-α genes expression in chicken cells. Results showed a single band for flagellin proteins of all bacteria on %10 SDS-PAGE, which concentration ranged from 150 to 400 µg/mL. All flagellin proteins increased cell proliferation, and IL-8 levels were increased after treatment by flagellins and levels of IL-6 and TNF-α were increased after treatment with S. typhimurium FliC. All flagellin proteins showed cross-reactivity with antibodies. Findings showed that application of our method, not only reduced time and cost, but also, the purified flagellin proteins had acceptable bioactivity.

Modular Pore-Forming Immunotoxins with Caged Cytotoxicity Tailored by Directed Evolution.

Immunotoxins are proteins containing a cell-targeting element linked to a toxin that are under investigation for next-generation cancer treatment. However, these agents are difficult to synthesize, chemically heterogeneous, expensive, and show toxicity toward healthy cells. In this work, we describe the synthesis and characterization of a new type of immunotoxin that showed exquisite selectivity toward targeted cells. In our construct, targeting molecules were covalently attached or genetically fused to oligomeric pore-forming toxins. The activity of the immunotoxin was then caged by fusing a soluble protein to the transmembrane domain and activated via cleavage with furin, which is a protease that is overexpressed in many cancer cells. During the several coupling steps, directed evolution allowed the efficient synthesis of the molecules in E. coli cells, as well as selection for further specificity toward targeted cells. The final construct showed no off-target activity, while acquiring an additional degree of specificity toward the targeted cells upon activation. The pore-forming toxins described here do not require internalization to operate, while the many protomeric subunits can be individually modified to refine target specificity.

Immunogenicity and Induction of Functional Antibodies in Rabbits Immunized with a Trivalent Typhoid-Invasive Nontyphoidal Salmonella Glycoconjugate Formulation.

Typhoid fever due to Salmonella Typhi and invasive nontyphoidal Salmonella (iNTS) infections caused by serovars Enteritidis (SE) and Typhimurium (STm) are major pediatric health problems in sub-Saharan Africa. Typhoid has high complication rates, and iNTS infections have high case fatality rates; moreover, emerging antimicrobial resistance is diminishing treatment options. Vi capsule-based typhoid conjugate vaccine (Typbar-TCV™), licensed in India and pre-qualified by the World Health Organization, elicits durable immunity when administered to infants, but no iNTS vaccines are licensed or imminent. We have developed monovalent SE and STm glycoconjugate vaccines based on coupling lipopolysaccharide-derived core-O polysaccharide (COPS) to phase 1 flagellin protein (FliC) from the homologous serovar. Herein, we report the immunogenicity of multivalent formulations of iNTS COPS:FliC conjugates with Typbar-TCV™. Rabbits immunized with the trivalent typhoid-iNTS glycoconjugate vaccine generated high titers of serum IgG antibody to all three polysaccharide antigens for which anti-COPS IgG antibodies were directed primarily against serogroup-specific OPS epitopes. Responses to SE and STm FliC were lower relative to anti-COPS titers. Post-vaccination rabbit sera mediated bactericidal activity in-vitro, and protected mice after passive transfer against challenge with virulent SE or STm Malian blood isolates. These results support accelerated progression to clinical trials.

A simplified globally affordable experimental setup for monitoring DNA diagnosis by a QD-based technique.

The unavailability of simple, quick, and sensitive genetic-based molecular diagnostic techniques has become the main driving force for inventing new approaches in the era of quantum dots (QDs): a new class of fluorescent probes with fascinating optical electronic properties. Using the unique size-dependent light-emitting properties of QDs, we have developed a QD-based ultrasensitive technique which removes the necessity for the genetic amplification step required in almost all types of molecular-based diagnostic techniques. The selectivity of the new approach is warranted by the careful design of a pair of specific oligonucleotide probes, chemically modified at their 5'-ends. Our results indicated the selective detection of Salmonella typhi in an assay time of 50 min with a limit of detection (LOD) of 2 CFU/mL. The rapidity, selectivity, and sensitivity and the low assay cost make the new diagnostic technique a promising new tool for laboratory and field-based approaches to molecular diagnosis of health-threatening pathogens. Graphical abstract.

In vivo tropism of Salmonella Typhi toxin to cells expressing a multiantennal glycan receptor.

Typhoid fever is a life-threatening disease, but little is known about the molecular bases for its unique clinical presentation. Typhoid toxin, a unique virulence factor of Salmonella Typhi (the cause of typhoid fever), recapitulates in an animal model many symptoms of typhoid fever. Typhoid toxin binding to its glycan receptor Neu5Ac is central, but, due to the ubiquity of Neu5Ac, how typhoid toxin causes specific symptoms remains elusive. Here we show that typhoid toxin displays in vivo tropism to cells expressing multiantennal glycoprotein receptors, particularly on endothelial cells of arterioles in the brain and immune cells, which is in line with typhoid symptoms. Neu5Ac displayed by multiantennal N-glycans, rather than a single Neu5Ac, appears to serve as the high-affinity receptor, as typhoid toxin possesses five identical binding pockets per toxin. Human counterparts also express the multiantennal Neu5Ac receptor. Here we also show that mice immunized with inactive typhoid toxins and challenged with wild-type typhoid toxin presented neither the characteristic in vivo tropism nor symptoms. These mice were protected against a lethal-dose toxin challenge, but Ty21a-vaccinated mice were not. Cumulatively, these results reveal remarkable features describing how a bacterial exotoxin induces virulence exclusively in specific cells at the organismal level.

Evolution of host adaptation in the Salmonella typhoid toxin.

The evolution of virulence traits is central for the emergence or re-emergence of microbial pathogens and for their adaptation to a specific host 1-5 . Typhoid toxin is an essential virulence factor of the human-adapted bacterial pathogen Salmonella Typhi 6,7 , the cause of typhoid fever in humans 8-12 . Typhoid toxin has a unique A2B5 architecture with two covalently linked enzymatic 'A' subunits, PltA and CdtB, associated with a homopentameric 'B' subunit made up of PltB, which has binding specificity for the N-acetylneuraminic acid (Neu5Ac) sialoglycans 6,13 prominently present in humans 14 . Here, we examine the functional and structural relationship between typhoid toxin and ArtAB, an evolutionarily related AB5 toxin encoded by the broad-host Salmonella Typhimurium 15 . We find that ArtA and ArtB, homologues of PltA and PltB, can form a functional complex with the typhoid toxin CdtB subunit after substitution of a single amino acid in ArtA, while ArtB can form a functional complex with wild-type PltA and CdtB. We also found that, after addition of a single-terminal Cys residue, a CdtB homologue from cytolethal distending toxin can form a functional complex with ArtA and ArtB. In line with the broad host specificity of S. Typhimurium, we found that ArtB binds human glycans, terminated in N-acetylneuraminic acid, as well as glycans terminated in N-glycolylneuraminic acid (Neu5Gc), which are expressed in most other mammals 14 . The atomic structure of ArtB bound to its receptor shows the presence of an additional glycan-binding site, which broadens its binding specificity. Despite equivalent toxicity in vitro, we found that the ArtB/PltA/CdtB chimaeric toxin exhibits reduced lethality in an animal model, indicating that the host specialization of typhoid toxin has optimized its targeting mechanisms to the human host. This is a remarkable example of a toxin evolving to broaden its enzymatic activities and adapt to a specific host.

A recombinant protein of Salmonella Typhi induces humoral and cell-mediated immune responses including memory responses.

Gram negative enteric bacteria, Salmonella enterica serovar Typhi (S. Typhi), the etiological agent of typhoid fever is a major public health problem in developing countries. While a permanent solution to the problem would require improved sanitation, food and water hygiene, controlling the infection by vaccination is urgently required due to the emergence of multidrug resistant strains in multiple countries. The currently licensed vaccines are moderately efficacious with limited applicability, and no recommended vaccines exist for younger children. We had previously reported that a candidate vaccine based on recombinant outer membrane protein (rT2544) of S. Typhi is highly immunogenic and protective in mice. Here we show that rT2544-specific antiserum is capable of mediating bacterial lysis by the splenocytes through Antibody-Dependent Cellular Cytotoxicity (ADCC). Increased populations of rT2544-specific IgA and IgG secreting plasma cells are found in the spleen, mesenteric lymph nodes and peyer's patches. Cell-Mediated Immune Responses (CMIR) induced by rT2544 consist of Th1 cell differentiation and generation of cytotoxic T lymphocytes (CTL), which produce IFN-γ and are capable of destroying cells displaying T2544-derived antigens. rT2544 elicits pro-inflammatory cytokines (TNF-α, IL-6) from Bone Marrow-Derived Dendritic cells (BMDCs), while in vitro re-stimulation of rT2544-primed CD4+ T cells induces cell proliferation and generates higher amounts of Th1 cytokines, such as IFN-gamma, TNF-α and IL-2. Finally, the candidate vaccine induces immunological memory in the form of memory B and T lymphocytes. Taken together, the study further supports the potential of rT2544 as a novel and improved vaccine candidate against S. Typhi.

Insights into PG-binding, conformational change, and dimerization of the OmpA C-terminal domains from Salmonella enterica serovar Typhimurium and Borrelia burgdorferi.

Salmonella enterica serovar Typhimurium can induce both humoral and cell-mediated responses when establishing itself in the host. These responses are primarily stimulated against the lipopolysaccharide and major outer membrane (OM) proteins. OmpA is one of these major OM proteins. It comprises a N-terminal eight-stranded ß-barrel transmembrane domain and a C-terminal domain (OmpACTD ). The OmpACTD and its homologs are believed to bind to peptidoglycan (PG) within the periplasm, maintaining bacterial osmotic homeostasis and modulating the permeability and integrity of the OM. Here we present the first crystal structures of the OmpACTD from two pathogens: S. typhimurium (STOmpACTD ) in open and closed forms and causative agent of Lyme Disease Borrelia burgdorferi (BbOmpACTD ), in closed form. In the open form of STOmpACTD , an aspartate residue from a long ß2-α3 loop points into the binding pocket, suggesting that an anion group such as a carboxylate group from PG is favored at the binding site. In the closed form of STOmpACTD and in the structure of BbOmpACTD , a sulfate group from the crystallization buffer is tightly bound at the binding site. The differences between the closed and open forms of STOmpACTD , suggest a large conformational change that includes an extension of α3 helix by ordering a part of ß2-α3 loop. We propose that the sulfate anion observed in these structures mimics the carboxylate group of PG when bound to STOmpACTD suggesting PG-anchoring mechanism. In addition, the binding of PG or a ligand mimic may enhance dimerization of STOmpACTD , or possibly that of full length STOmpA.

Vacuolation Activity and Intracellular Trafficking of ArtB, the Binding Subunit of an AB5 Toxin Produced by Salmonella enterica Serovar Typhi.

Various Salmonella enterica serovars, including S. enterica serovar Typhi, encode an AB5 toxin (ArtAB), the A subunit of which is an ADP-ribosyltransferase related to the S1 subunit of pertussis toxin. However, although the A subunit is able to catalyze ADP-ribosylation of host G proteins, a cytotoxic phenotype has yet to be identified for the holotoxin. Here we show that its B subunit pentamer (ArtB) binds to receptors on the surface of Vero (African green monkey kidney) cell, CHO (Chinese hamster ovary) cell, U937 (human monocyte) cell, and HBMEC (human brain microvascular endothelial cell) lines. Moreover, ArtB induced marked vacuolation in all cell lines after 4 h of incubation. Further studies in Vero cells showed that vacuolation was inhibited by bafilomycin A1 and was dependent on the clathrin-mediated uptake of ArtB. Vacuolation was also inhibited by treatment of cells with neuraminidase, indicating that sialylated glycans are functional receptors for ArtB. Confocal colocalization studies indicated that after cell binding and internalization, ArtB undergoes retrograde transport via early endosomes, the trans-Golgi network, and the Golgi apparatus, reaching the endoplasmic reticulum (ER) after approximately 2 h. The onset of vacuolation also coincided with gross cytoskeletal reorganization. At later time points, ArtB colocalized with ER-Tracker Red in the vacuolar membrane, implying that vacuolation is a consequence of ER disorganization. Thus, the isolated B subunit of this cryptic AB5 toxin has significant effects on target cells with the potential to contribute directly to pathogenesis independently of the catalytic A subunit.

NMR line-fitting quantification of polysaccharide N-acylurea-based modification in glycoconjugates of Salmonella Typhi Vi polysaccharide.

The polysaccharides modification via carbodiimide reaction is one of the most applied methods for obtaining conjugated vaccines against Salmonella enterica. However, N-acylurea carbodiimide adduct generated in the process is a critical impurity in carbohydrate-based vaccines. A quantitative NMR method was developed for assessing the N-acylurea carbodiimide adduct impurity. The procedure was based on line-fitting facilities for processing the NMR signals on complex spectra. The method showed good linearity, accuracy and precision under inter-operator variation (relative standard deviation <5%). Copyright © 2017 John Wiley & Sons, Ltd.

Development of recombinant vaccine candidate molecule against Shigella infection.

Shigellosis is an acute bacillary diarrheal disease caused by the gram negative bacillus Shigella. The existence of multiple Shigella serotypes and their growing resistance to antibiotics stress the urgent need for the development of vaccine that is protective across all serotypes. Shigella's IpaB antigen is involved in translocon pore formation, promotes bacterial invasion and induces apoptosis in macrophages. S. Typhi GroEL (Hsp 60) is the immunodominant antigen inducing both arms of immunity and has been explored as adjuvant in this study. The present study evaluates the immunogenicity and protective efficacy of recombinant IpaB domain-GroEL fusion protein in mice against lethal Shigella infection. The IpaB domain and GroEL genes were fused using overlap extension PCR and cloned in pRSETA expression vector. Fused gene was expressed in Escherichia coli BL-21 cells and the resulting 90 KDa fusion protein was purified by affinity chromatography. Intranasal (i.n.) immunization of mice with fusion protein increased the IgG and IgA antibody titers as compared to the group immunized with IpaB and GroEL and control PBS immunized group. Also IgG1 and IgG2a antibodies induced in fusion protein immunized mice were higher than co-immunized group. Significant increase in lymphocyte proliferation and cytokine levels (IFN-γ, IL-4 and IL-10), indicates induction of both Th1 and Th2 immune responses in both immunized groups. Immunization with fusion protein protected 90-95% of mice whereas 80-85% survivability was observed in co-immunized group against lethal challenge with S. flexneri, S. boydii and S. sonnei. Passive immunization conferred 60-70% protection in mice against all these Shigella species. Organ burden and histopathology studies also revealed significant decrease in lung infection as compared to the co-immunized group. Since IpaB is the conserved dominant molecule in all Shigella species, this study will lead to an ideal platform for the development of safe, efficacious and cost-effective recombinant vaccine against Shigella serotypes.

Gold nanoparticle-DNA aptamer conjugate-assisted delivery of antimicrobial peptide effectively eliminates intracellular Salmonella enterica serovar Typhimurium.

Antimicrobial peptides (AMPs) are a promising new class of antibacterial compounds. However, their applications in the treatment of intracellular pathogenic bacteria have been limited by their in vivo instability and low penetrating ability into mammalian cells. Here, we report that gold nanoparticles conjugated with DNA aptamer (AuNP-Apt) efficiently delivered AMPs into mammalian living systems with enhanced stability of the AMPs. C-terminally hexahistidine-tagged A3-APO (A3-APO(His)) AMPs were loaded onto AuNPs conjugated with His-tag DNA aptamer (AuNP-Apt(His)) by simple mixing and were delivered into Salmonella enterica serovar Typhimurium (S. Typhimurium)-infected HeLa cells, resulting in the increased viability of host cells due to the elimination of intracellular S. Typhimurium cells. Furthermore, the intravenous injection of AuNP-Apt(His) loaded with A3-APO(His) into S. Typhimurium-infected mice resulted in a complete inhibition of S. Typhimurium colonization in the mice organs, leading to 100% survival of the mice. Therefore, AuNP-Apt(His) can serve as an innovative platform for AMP therapeutics to treat intracellular bacterial infections in mammals.