Flexneri Type Research Articles

Glycoconjugation of Shigella flexneri type 2a O-polysaccharide with CRM197 as a potential vaccine candidate for shigellosis

Shigellosis, a diarrheal disorder caused by an entero-invasive bacterium Shigella, is a major concern among children often leading to mortality. As most of these strains have developed universal antibiotic resistance, the development of a vaccine is crucial in combating the infection. The O-specific polysaccharide (O-PSs) from S. flexneri type 2a is considered to be the major disease-causing antigen in shigellosis. Therefore, the O-PSs conjugated with carrier proteins, can serve as a potential high molecular weight vaccine candidate. Accordingly, in the present study, O-PS extracted from S. flexneri 2a is conjugated with Cross-Reactive Material (CRM197), a non-toxic mutant of diphtheria toxin. We derivatized CRM197 and O-PS separately with adipic acid dihydrazide (ADH) and reacted with their counterparts to probe the conjugation efficacy. Among the two strategies, the CRM197-ADH treated with O-PS has yielded a stable glycoconjugate of 311 kDa. The conjugation efficiency has been probed by estimating the free protein, free O-PS and O-PS:CRM197 ratio using slot-blot, size exclusion and high-performance anion exchange chromatography techniques. The conjugate exhibited enhanced shelf-life of three months. The cytotoxicity studies with Vero/MRC-5 cells have confirmed the non-toxicity of the conjugate, which makes the glycoconjugate a potential vaccine candidate for shigellosis.

Shigella flexneri Disruption of Cellular Tension Promotes Intercellular Spread

SUMMARYDuring infection, some bacterial pathogens invade the eukaryotic cytosol and spread between cells of an epithelial monolayer. Intercellular spread occurs when these pathogens push against the plasma membrane, forming protrusions that are engulfed by adjacent cells. Here, we show that IpaC, a Shigella flexneri type 3 secretion system protein, binds the host cell-adhesion protein β-catenin and facilitates efficient protrusion formation. S. flexneri producing a point mutant of IpaC that cannot interact with β-catenin is defective in protrusion formation and spread. Spread is restored by chemical reduction of intercellular tension or genetic depletion of β-catenin, and the magnitude of the protrusion defect correlates with membrane tension, indicating that IpaC reduces membrane tension, which facilitates protrusion formation. IpaC stabilizes adherens junctions and does not alter β-catenin localization at the membrane. Thus, Shigella, like other bacterial pathogens, reduces intercellular tension to efficiently spread between cells.

Shigella hijacks the exocyst to cluster macropinosomes for efficient vacuolar escape.

Shigella flexneri invades host cells by entering within a bacteria-containing vacuole (BCV). In order to establish its niche in the host cytosol, the bacterium ruptures its BCV. Contacts between S. flexneri BCV and infection-associated macropinosomes (IAMs) formed in situ have been reported to enhance BCV disintegration. The mechanism underlying S. flexneri vacuolar escape remains however obscure. To decipher the molecular mechanism priming the communication between the IAMs and S. flexneri BCV, we performed mass spectrometry-based analysis of the magnetically purified IAMs from S. flexneri-infected cells. While proteins involved in host recycling and exocytic pathways were significantly enriched at the IAMs, we demonstrate more precisely that the S. flexneri type III effector protein IpgD mediates the recruitment of the exocyst to the IAMs through the Rab8/Rab11 pathway. This recruitment results in IAM clustering around S. flexneri BCV. More importantly, we reveal that IAM clustering subsequently facilitates an IAM-mediated unwrapping of the ruptured vacuole membranes from S. flexneri, enabling the naked bacterium to be ready for intercellular spread via actin-based motility. Taken together, our work untangles the molecular cascade of S. flexneri-driven host trafficking subversion at IAMs to develop its cytosolic lifestyle, a crucial step en route for infection progression at cellular and tissue level.

Convergent Chemoenzymatic Strategy to Deliver a Diversity of Shigella flexneri Serotype-Specific O-Antigen Segments from a Unique Lightly Protected Tetrasaccharide Core.

Progress in glycoscience is strongly dependent on the availability of broadly diverse tailor-made, well-defined, and often complex oligosaccharides. Herein, going beyond natural resources and aiming to circumvent chemical boundaries in glycochemistry, we tackle the development of an in vitro chemoenzymatic strategy holding great potential to answer the need for molecular diversity characterizing microbial cell-surface carbohydrates. The concept is exemplified in the context of Shigella flexneri, a major cause of diarrhoeal disease. Aiming at a broad serotype coverage S. flexneri glycoconjugate vaccine, a non-natural lightly protected tetrasaccharide was designed for compatibility with (i) serotype-specific glucosylations and O-acetylations defining S. flexneri O-antigens, (ii) recognition by suitable α-transglucosylases, and (iii) programmed oligomerization following enzymatic α-d-glucosylation. The tetrasaccharide core was chemically synthesized from two crystalline monosaccharide precursors. Six α-transglucosylases found in the glycoside hydrolase family 70 were shown to transfer glucosyl residues on the non-natural acceptor. The successful proof of concept is achieved for a pentasaccharide featuring the glucosylation pattern from the S. flexneri type IV O-antigen. It demonstrates the potential of appropriately planned chemoenzymatic pathways involving non-natural acceptors and low-cost donor/transglucosylase systems to achieve the demanding regioselective α-d-glucosylation of large substrates, paving the way to microbial oligosaccharides of vaccinal interest.

Activation of Shigella flexneri type 3 secretion requires a host-induced conformational change to the translocon pore.

Type 3 secretion systems (T3SSs) are conserved bacterial nanomachines that inject virulence proteins (effectors) into eukaryotic cells during infection. Due to their ability to inject heterologous proteins into human cells, these systems are being developed as therapeutic delivery devices. The T3SS assembles a translocon pore in the plasma membrane and then docks onto the pore. Docking activates effector secretion through the pore and into the host cytosol. Here, using Shigella flexneri, a model pathogen for the study of type 3 secretion, we determined the molecular mechanisms by which host intermediate filaments trigger docking and enable effector secretion. We show that the interaction of intermediate filaments with the translocon pore protein IpaC changed the pore's conformation in a manner that was required for docking. Intermediate filaments repositioned residues of the Shigella pore protein IpaC that are located on the surface of the pore and in the pore channel. Restricting these conformational changes blocked docking in an intermediate filament-dependent manner. These data demonstrate that a host-induced conformational change to the pore enables T3SS docking and effector secretion, providing new mechanistic insight into the regulation of type 3 secretion.

Mapping of Shigella flexneri's tissue distribution and type III secretion apparatus activity during infection of the large intestine of guinea pigs.

ABSTRACT Shigella spp. are bacterial pathogens that invade the human colonic mucosa using a type III secretion apparatus (T3SA), a proteinaceous device activated upon contact with host cells. Active T3SAs translocate proteins that carve the intracellular niche of Shigella spp. Nevertheless, the activation state of the T3SA has not been addressed in vivo. Here, we used a green fluorescent protein transcription-based secretion activity reporter (TSAR) to provide a spatio-temporal description of S. flexneri T3SAs activity in the colon of Guinea pigs. First, we observed that early mucus release is triggered in the vicinity of luminal bacteria with inactive T3SA. Subsequent mucosal invasion showed bacteria with active T3SA associated with the brush border, eventually penetrating into epithelial cells. From 2 to 8 h post-challenge, the infection foci expanded, and these intracellular bacteria displayed homogeneously high-secreting activity, while extracellular foci within the lamina propria featured bacteria with low secretion activity. We also found evidence that within lamina propria macrophages, bacteria reside in vacuoles instead of accessing the cytosol. Finally, bacteria were cleared from tissues between 8 and 24 h post-challenge, highlighting the hit-and-run colonization strategy of Shigella. This study demonstrates how genetically encoded reporters can contribute to deciphering pathogenesis in vivo.

Antigenic Cross-Reactivity Between Escherichia albertii DM104 and Different Shigella spp.

Environmental Escherichia albertii strain DM104 has been found to induce protective immunity against Shigella dysenteriae in guinea pig model and intranasal immunization showed promising results in terms of antibody response and protective efficacy. For selecting a proper immunodiagnostics marker against shigellosis, the current study investigated the antigenic cross-reactivity between DM104 and four different Shigella spp (S. dysenteriae type 4, S. flexneri type 2a, S. boydii type 15 and S. sonnei). At least six antigenic protein bands (85, 72, 34, 30, 23 and 20 k Da) of the surface components from all these species reacted strongly with both homologous and heterologous antisera, suggesting common distribution of antigenic determinants/epitopes in these bacterial species. This experiment, thus gave a clear idea of the level of antigenic determinants/epitopes sharing and variations between the DM104 and four Shigella spp. Results from this study suggest that the 34, 23 and 20 k Da antigenic proteins may be incorporated as immunodiagnostic marker for the detection of different Shigella spp.
 Bangladesh J Microbiol, Volume 35 Number 1 June 2018, pp 17-21

Spatial, Temporal, and Functional Assessment of LC3-Dependent Autophagy in Shigella flexneri Dissemination.

Shigella flexneri disseminates within the colonic mucosa by displaying actin-based motility in the cytosol of epithelial cells. Motile bacteria form membrane protrusions that project into adjacent cells and resolve into double-membrane vacuoles (DMVs) from which the bacteria escape, thereby achieving cell-to-cell spread. During dissemination, S. flexneri is targeted by LC3-dependent autophagy, a host cell defense mechanism against intracellular pathogens. The S. flexneri type III secretion system effector protein IcsB was initially proposed to counteract the recruitment of the LC3-dependent autophagy machinery to cytosolic bacteria. However, a recent study proposed that LC3 was recruited to bacteria in DMVs formed during cell-to-cell spread. To resolve the controversy and clarify the role of autophagy in S. flexneri infection, we tracked dissemination using live confocal microscopy and determined the spatial and temporal recruitment of LC3 to bacteria. This approach demonstrated that (i) LC3 was exclusively recruited to wild-type or icsB bacteria located in DMVs and (ii) the icsB mutant was defective in cell-to-cell spread due to failure to escape LC3-positive as well as LC3-negative DMVs. Failure of S. flexneri to escape DMVs correlated with late LC3 recruitment, suggesting that LC3 recruitment is the consequence and not the cause of DMV escape failure. Inhibition of autophagy had no positive impact on the spreading of wild-type or icsB mutant bacteria. Our results unambiguously demonstrate that IcsB is required for DMV escape during cell-to-cell spread, regardless of LC3 recruitment, and do not support the previously proposed notion that autophagy counters S. flexneri dissemination.

Antimicrobial Potential of Epiphytic Bacteria Associated With Seaweeds of Little Andaman, India.

Seaweeds of the intertidal regions are a rich source of surface associated bacteria and are potential source of antimicrobial molecules. In the present study, 77 epiphytic isolates from eight different algae collected from Little Andaman were enumerated. On testing for their antimicrobial activities against certain pathogens twelve isolates showed positive and six of them showed significant antimicrobial inhibition zone against Shigella boydii type 1, Shigella flexneri type 2a, Shigella dysenteriae type 5, Enterotoxigenic Escherichia coli O115, Enteropathogenic E. coli serotype O114, Vibrio cholera; O1 Ogawa, Aeromonas hydrophila, Klebsiella pneumoniae, Staphylococcus aureus. Based on the activity these six isolates (G1C, G2C, G3C, UK, UVAD, and Tor1) were identified by 16S rRNA gene sequence and were found to belong to the phyla Firmicutes and Proteobacteria. Purified antimicrobial compounds obtained from these isolates were identified by GC-MS. Furan derivatives were identified from G2C Pseudomonas stutzeri KJ849834, UVAD Alcanivorax dieselolei KJ849833, UK Vibrio sp. KJ849837, Tor1 Exiguobacterium profundum KJ849838. While 2-Pyrrolidinone, Phenol, 2, 4-bis (1, 1-dimethylethyl) were from G3C Vibrio owensii KJ849836 and (1-Allylcyclopropyl) methanol from the extracts of G1C Bacillus sp. KJ849835. The results of the present study shows that these six potent isolates isolated from the seaweeds are found to be a source of antimicrobial compounds.

Antibacterial activity of crude extracts of some South African medicinal plants against multidrug resistant etiological agents of diarrhoea

BackgroundThis study evaluated the antibacterial activity of some plants used in folklore medicine to treat diarrhoea in the Eastern Cape Province, South Africa.MethodsThe acetone extracts of Acacia mearnsii De Wild., Aloe arborescens Mill., A. striata Haw., Cyathula uncinulata (Schrad.) Schinz, Eucomis autumnalis (Mill.) Chitt., E. comosa (Houtt.) Wehrh., Hermbstaedtia odorata (Burch. ex Moq.) T.Cooke, Hydnora africana Thunb, Hypoxis latifolia Wight, Pelargonium sidoides DC, Psidium guajava L and Schizocarphus nervosus (Burch.) van der Merwe were screened against Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, multi-resistant Salmonella enterica serovar Isangi, S. typhi, S. enterica serovar Typhimurium, Shigella flexneri type 1b and Sh. sonnei phase II. A qualitative phytochemical screening of the plants extracts was by thin layer chromatography. Plants extracts were screened for antibacterial activity using serial dilution microplate technique and bioautography.ResultsThe TLC fingerprint indicated the presence of terpenoids and flavonoids in the herbs. Most of the tested organisms were sensitive to the crude acetone extracts with minimum inhibitory concentration (MIC) values ranging from 0.018–2.5 mg/mℓ. Extracts of A. striata, C. uncinulata, E. autumnalis and P. guajava were more active against enteropathogens. S. aureus and Sh. flexneri were the most sensitive isolates to the crude extracts but of significance is the antibacterial activity of A. arborescens and P. guajava against a confirmed extended spectrum betalactamase positive S. enterica serovar Typhimurium.ConclusionThe presence of bioactive compounds and the antibacterial activity of some of the selected herbs against multidrug resistant enteric agents corroborate assertions by traditional healers on their efficacies.

Efficient Iterative Synthesis of O-Acetylated Tri- to Pentadecasaccharides Related to the Lipopolysaccharide of Shigella flexneri Type 3 a through Di- and Trisaccharide Glycosyl Donors.

Protection against bacterial infections, including shigellosis, can be achieved by antibodies against the bacterial surface polysaccharide. In line with our efforts to develop vaccine candidates for shigellosis, we report herein the synthesis of penta-, deca-, and pentadecasaccharides as well as tri-, octa-, and tridecasaccharides as the endchain and intrachain fragments, respectively, of the surface polysaccharide of Shigella flexneri 3 a, a prevalent serotype. The syntheses relied on the efficiency of the trichloroacetimidate glycosylation chemistry, whereby iteration with di- and trisaccharide building blocks provided fragments made of up to three mono-O-acetylated polysaccharide repeating units. Pd(OH)2 -mediated hydrogenation/hydrogenolysis enabled the concomitant removal or conversion of up to 31 protecting groups of 4 different origins to provide the targets as propyl glycosides. Oligosaccharides comprising the octasaccharide segment were shown to display high conformational similarities in solution.

Shigellosis in a Newborn---An Uncommon Case

A new born baby was admitted to the Glocal hospital, Krishnanagore, and presented with loose stools mixed with blood and mucous and abdominal distension for last five days. The baby was crying all through the day; presumably, he had severe tenesmus (a feeling of incomplete sense of defecation with rectal pain). He was diagnosed, clinically, to be suffering from shigellosis, which was confirmed by isolation of Shigella spp; by stool culture. The baby recovered with antibiotic treatment. It is an uncommon case of shigellosis in new born babies. Hand washing practices are recommended to prevent transmission of shigellosis and other diseases as well. Shigellae vaccines are attractive disease prevention strategy. Shigellosis caused by S. flexneri type 2 and S. dysenteria type 1 are the two most common and important serotype candidates against which vaccine development are currently being directed.

Development of a Sandwich ELISA for EHEC O157:H7 Intimin γ1.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a zoonotic pathogen of worldwide importance that causes foodborne infections in humans. Intimin gamma 1 (intimin γ1) is one of the most important outer membrane proteins required for EHEC’s intimate adhesion to epithelial cells. Here, we generated a polyclonal antibody (pAb) and a monoclonal antibody (mAb) against intimin γ1 to develop a double antibody sandwich ELISA (DAS-ELISA) with increased sensitivity and specificity for measuring EHEC O157:H7. To achieve this goal, a rabbit pAb was used as a capture antibody, and a mouse mAb was a detection antibody. No cross-reactivity was observed with the other genera of pathogenic bacteria tested with the DAS-ELISA, which included Salmonella enteritidis, Shigella flexneri type 2, Listeria monocytogenes, Streptococcus suis type 2, and other 18 serotype E. coli. Detection limits of the DAS-ELISA were 1 × 103 CFU/mL for EHEC O157:H7 cultures, 1 × 104 CFU/g before enrichment, and 1 × 102 CFU/g after enrichment of contaminated samples. Field samples (n = 498) were tested using a previously established duplex-PCR method and compared to our DAS-ELISA. The DAS-ELISA had a specificity of 94.4%, a sensitivity of 91.5% and accuracy of 94.0% compared with duplex-PCR. The DAS-ELISA developed here can be applied to EHEC O157:H7 quantification in food, animal, and environmental samples.

Supercharging Reagent for Enhanced Liquid Chromatographic Separation and Charging of Sialylated and High-Molecular-Weight Glycopeptides for NanoHPLC-ESI-MS/MS Analysis.

Recent developments in proteomic techniques have led to the development of mass spectrometry (MS)-based methods to characterize site-specific glycosylation of proteins. However, appropriate analytical tools to characterize acidic and high-molecular-weight (hMW) glycopeptides are still lacking. In this study, we demonstrate that the addition of supercharging reagent, m-nitrobenzyl alcohol (m-NBA), into mobile phases greatly facilitates the analysis of acidic and hMW glycopeptides. Using commercial glycoproteins, we demonstrated that in the presence of m-NBA the charge state of sialylated glycopeptides increased and the chromatographic separation of neutral and acidic glycopeptides revealed a remarkable improvement. Next, we applied this system to the characterization of a glycoconjugate vaccine candidate consisting of a genetically detoxified exotoxin A of Pseudomonas aeruginosa covalently linked to Shigella flexneri type 2a O-antigen (Sf2E) produced by engineered Escherichia coli. The addition of m-NBA, allowed us to identify peptides with glycan chains of unprecedented size, up to 20 repeat units (98 monosaccharides). Our results indicated that incorporation of m-NBA into reversed-phase liquid chromatography (LC) solvents improves sensitivity, charging, and chromatographic resolution for acidic and hMW glycopeptides.

Detailed Investigation of the Immunodominant Role of O-Antigen Stoichiometric O-Acetylation as Revealed by Chemical Synthesis, Immunochemistry, Solution Conformation and STD-NMR Spectroscopy for Shigella flexneri 3a.

Shigella flexneri 3a causes bacillary dysentery. Its O-antigen has the {2)-[α-d-Glcp-(1→3)]-α-l-Rhap-(1→2)-α-l-Rhap-(1→3)-[Ac→2]-α-l-Rhap-(1→3)-[Ac→6]≈40 % -β-d-GlcpNAc-(1→} ([(E)ABAc CAc D]) repeating unit, and the non-O-acetylated equivalent defines S. flexneri X. Propyl hepta-, octa-, and decasaccharides sharing the (E')A'BAc CD(E)A sequence, and their non-O-acetylated analogues were synthesized from a fully protected BAc CD(E)A allyl glycoside. The stepwise introduction of orthogonally protected mono- and disaccharide imidate donors was followed by a two-step deprotection process. Monoclonal antibody binding to twenty-six S. flexneri types 3a and X di- to decasaccharides was studied by an inhibition enzyme-linked immunosorbent assay (ELISA) and STD-NMR spectroscopy. Epitope mapping revealed that the 2C -acetate dominated the recognition by monoclonal IgG and IgM antibodies and that the BAc CD segment was essential for binding. The glucosyl side chain contributed to a lesser extent, albeit increasingly with the chain length. Moreover, tr-NOESY analysis also showed interaction but did not reveal any meaningful conformational change upon antibody binding.

Shigella flexneri suppresses NF-κB activation by inhibiting linear ubiquitin chain ligation.

The Linear Ubiquitin chain Assembly Complex (LUBAC) is a multimeric E3 ligase that catalyzes M1- or linear ubiquitination of activated immune receptor signaling complexes (RSCs). While mutations that disrupt linear ubiquitin assembly lead to complex disease pathologies including immunodeficiency and autoinflammation in both humans and mice, microbial toxins that target LUBAC function have not yet been discovered. Here, we report the identification of two homologous Shigella flexneri Type III Secretion System (T3SS) effector E3 ligases IpaH1.4 and IpaH2.5 that directly interact with LUBAC subunit HOIL-1L (RBCK1) and conjugate K48-linked ubiquitin chains to the catalytic RING-between-RING domain of HOIP (RNF31). Proteasomal degradation of HOIP leads to irreversible inactivation of linear ubiquitination and blunting of NF-κB nuclear translocation in response to TNF, IL-1β, and pathogen associated molecular patterns (PAMPs). Loss of function studies in mammalian cells in combination with bacterial genetics explains how Shigella evades a broad spectrum of immune surveillance systems by cooperative inhibition of receptor ubiquitination, and reveals the critical importance of LUBAC in host defense against pathogens.

Implications of Spatiotemporal Regulation of Shigella flexneri Type Three Secretion Activity on Effector Functions: Think Globally, Act Locally.

Shigella spp. are Gram-negative bacterial pathogens that infect human colonic epithelia and cause bacterial dysentery. These bacteria express multiple copies of a syringe-like protein complex, the Type Three Secretion apparatus (T3SA), which is instrumental in the etiology of the disease. The T3SA triggers the plasma membrane (PM) engulfment of the bacteria by host cells during the initial entry process. It then enables bacteria to escape the resulting phagocytic-like vacuole. Freed bacteria form actin comets to move in the cytoplasm, which provokes bacterial collision with the inner leaflet of the PM. This phenomenon culminates in T3SA-dependent secondary uptake and vacuolar rupture in neighboring cells in a process akin to what is observed during entry and named cell-to-cell spread. The activity of the T3SA of Shigella flexneri was recently demonstrated to display an on/off regulation during the infection. While the T3SA is active when bacteria are in contact with PM-derived compartments, it switches to an inactive state when bacteria are released within the cytosol. These observations indicate that effector proteins transiting through the T3SA are therefore translocated in a highly time and space constrained fashion, likely impacting on their cellular distribution. Herein, we present what is currently known about the composition, the assembly and the regulation of the T3SA activity and discuss the consequences of the on/off regulation of T3SA on Shigella effector properties and functions during the infection. Specific examples that will be developed include the role of effectors IcsB and VirA in the escape from LC3/ATG8-positive vacuoles formed during cell-to-cell spread and of IpaJ protease activity against N-miristoylated proteins. The conservation of a similar regulation of T3SA activity in other pathogens such as Salmonella or Enteropathogenic Escherichia coli will also be briefly discussed.

The Shigella flexneri type 3 secretion system is required for tyrosine kinase-dependent protrusion resolution, and vacuole escape during bacterial dissemination.

Shigella flexneri is a human pathogen that triggers its own entry into intestinal cells and escapes primary vacuoles to gain access to the cytosolic compartment. As cytosolic and motile bacteria encounter the cell cortex, they spread from cell to cell through formation of membrane protrusions that resolve into secondary vacuoles in adjacent cells. Here, we examined the roles of the Type 3 Secretion System (T3SS) in S. flexneri dissemination in HT-29 intestinal cells infected with the serotype 2a strain 2457T. We generated a 2457T strain defective in the expression of MxiG, a central component of the T3SS needle apparatus. As expected, the ΔmxiG strain was severely affected in its ability to invade HT-29 cells, and expression of mxiG under the control of an arabinose inducible expression system (ΔmxiG/pmxiG) restored full infectivity. In this experimental system, removal of the inducer after the invasion steps (ΔmxiG/pmxiG (Ara withdrawal)) led to normal actin-based motility in the cytosol of HT-29 cells. However, the time spent in protrusions until vacuole formation was significantly increased. Moreover, the number of formed protrusions that failed to resolve into vacuoles was also increased. Accordingly, the ΔmxiG/pmxiG (Ara withdrawal) strain failed to trigger tyrosine phosphorylation in membrane protrusions, a signaling event that is required for the resolution of protrusions into vacuoles. Finally, the ΔmxiG/pmxiG (Ara withdrawal) strain failed to escape from the formed secondary vacuoles, as previously reported in non-intestinal cells. Thus, the T3SS system displays multiple roles in S. flexneri dissemination in intestinal cells, including the tyrosine kinase signaling-dependent resolution of membrane protrusions into secondary vacuoles, and the escape from the formed secondary vacuoles.

The immune response of two microbial antigens delivered intradermally, sublingually, or the combination thereof

A key consideration to produce a successful vaccine is the choice of appropriate vaccination route. Though most vaccines are administered parenterally, this route is not effective in producing a robust mucosal or cell-mediated response. Intradermal and sublingual vaccinations have been explored recently as potential needle-free immunization strategies. We explored intradermal and sublingual routes as well as the combination of the two routes in eliciting both systemic and mucosal immune responses. Mice were immunized intradermally or sublingually with dmLT, a mutant of Escherichia coli heat-labile toxin. A systemic IgG response is dominant in intradermal immunization while a mucosal IgA response is dominant in sublingual immunization. When routes were combined, a synergistic response was seen with high titers of anti-dmLT IgG and IgA. IpaB/IpaD antigens of Shigella flexneri type III secretion system, were admixed with dmLT as adjuvant and administered by each route alone or in combination. Again, the intradermal route elicited a systemic response while the sublingual route elicited a mucosal response. When combined, the routes produced a robust synergistic response to both antigens that exhibited a balanced Th1/Th2 response. These results provide a new potential needle-free immunization strategy that will benefit low income countries and increase compliance in industrial countries.

Convergent Synthesis and Conformational Analysis of the Hexasaccharide Repeating Unit of the O‐Antigen of Shigella flexneri Serotype 1d

AbstractA hexasaccharide repeating unit of the O‐antigen of the cell wall of Shigella flexneri type 1d has been synthesized using a stereoselective [3+3] block glycosylation approach. Recently developed glycosylation conditions were used in the synthesis. A thioglycoside was used as an orthogonal glycosyl donor during the synthesis. The synthesized hexasaccharide was subjected to detailed NMR spectroscopic and molecular modeling studies to determine its conformational behavior in water. The NOE‐based two‐dimensional NOSEY experiment and all‐atom explicit molecular dynamics (MD) simulation studies suggest that the oligosaccharide is not very flexible, that it remains rigid with respect to the glycosidic linkages between the sugar moieties.