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Shigellosis is common worldwide, and it causes significant morbidity and mortality mainly in young children in low- and middle- income countries. To date, there are not broadly available licensed Shigella vaccines. A novel type of conjugate vaccine candidate, SF2a-TT15, was developed against S. flexneri serotype 2a (SF2a). SF2a-TT15 is composed of a synthetic 15mer oligosaccharide, designed to act as a functional mimic of the SF2a O-antigen and covalently linked to tetanus toxoid (TT). SF2a-TT15 was recently shown to be safe and immunogenic in a Phase 1 clinical trial, inducing specific memory B cells and sustained antibody response up to three years after the last injection. In this manuscript, we advance the study of B cell responses to parenteral administration of SF2a-TT15 to identify SF2a LPS-specific B cells (SF2a+ B cells) using fluorescently labeled bacteria. SF2a+ B cells were identified mainly within class-switched B cells (SwB cells) in volunteers vaccinated with SF2a-TT15 adjuvanted or not with aluminium hydroxide (alum), but not in placebo recipients. These cells expressed high levels of CXCR3 and low levels of CD21 suggesting an activated phenotype likely to represent the recently described effector memory B cells. IgG SF2a+ SwB cells were more abundant than IgA SF2a + SwB cells. SF2a+ B cells were also identified in polyclonally stimulated B cells (antibody secreting cells (ASC)-transformed). SF2a+ ASC-SwB cells largely maintained the activated phenotype (CXCR3 high, CD21 low). They expressed high levels of CD71 and integrin α4ß7, suggesting a high proliferation rate and ability to migrate to gut associated lymphoid tissues. Finally, ELISpot analysis showed that ASC produced anti-SF2a LPS IgG and IgA antibodies. In summary, this methodology confirms the ability of SF2a-TT15 to induce long-lived memory B cells, initially identified by ELISpots, which remain identifiable in blood up to 140 days following vaccination. Our findings expand and complement the memory B cell data previously reported in the Phase 1 trial and provide detailed information on the immunophenotypic characteristics of these cells. Moreover, this methodology opens the door to future studies at the single-cell level to better characterize the development of B cell immunity to Shigella.
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Vacunas contra la Shigella , Shigella , Preescolar , Humanos , Voluntarios Sanos , Inmunoglobulina A , Inmunoglobulina G , Lipopolisacáridos , Células B de Memoria , Serogrupo , Shigella flexneri , Vacunas Sintéticas , Ensayos Clínicos Fase I como AsuntoRESUMEN
Bacterial natural products containing heptosides such as septacidin represent interesting scaffolds for the development of drugs to combat antimicrobial resistance. However, very few synthetic strategies have been reported to grant access to these derivatives. Here, we have devised a synthetic pathway to l-glycero-l-glucoheptoside, a key building block en route to septacidin, directly from l-glucose. Importantly, we show that carbon homologation at C6, encompassing oxidation of the C6-OH followed by methylenation, is significantly influenced by the nature of the C4-moiety. In order to observe the effect of various patterns, namely azide (N3), p-methoxybenzyloxy (OPMB), and benzyloxy (OBn), a thorough analysis was conducted on the corresponding l-glucosides. The results unveiled a distinct trend where the efficiency of methylenation followed the trend OBn > OPMB > N3. Finally, the C6-alkene was dihydroxylated in the presence of osmium tetroxide to yield the expected l/d-glycero-l-glucoheptosides. The lead building block, which features a C-4 azide, was delivered as a phenyl thioglycoside. Added to the suitable masking of the 6,7-diol, this combination enables further functionalization to achieve versatile compounds of biological interest. The study insights into the interplay between substitution at C-4 and carbon homologation at C-6 provide valuable guidance for future endeavors in the synthesis of these carbohydrate molecules.
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Azidas , Glucosa , Heptosas/metabolismo , CarbonoRESUMEN
We report the syntheses of 1,3,4-tri-O-acetyl-2-amino-2,6-dideoxy-ß-d-glucopyranose and allyl 2-amino-2,6-dideoxy-ß-d-glucopyranoside from d-glucosamine hydrochloride. The potential of these two versatile scaffolds as key intermediates to a diversity of orthogonally protected rare deoxyamino hexopyranosides is exemplified in the context of fucosamine, quinovosamine, and bacillosamine. The critical C-6 deoxygenation step to 2,6-dideoxy aminosugars is performed at an early stage on a precursor featuring an imine moiety or a trifluoroacetamide moiety in place of the 2-amino group, respectively. Robustness and scalability are demonstrated for a combination of protecting groups and incremental chemical modifications that sheds light on the promise of the yet unreported allyl 2,6-dideoxy-2-N-trifluoroacetyl-ß-d-glucopyranoside when addressing the feasibility of synthetic zwitterionic oligosaccharides. In particular, allyl 3-O-acetyl-4-azido-2,4,6-trideoxy-2-trifluoroacetamido-ß-d-galactopyranoside, an advanced 2-acetamido-4-amino-2,4,6-trideoxy-d-galactopyranose building block, was achieved on the 30 g scale from 1,3,4,6-tetra-O-acetyl-ß-d-glucosamine hydrochloride in 50% yield and nine steps, albeit only two chromatography purifications.
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Glucosamina , Azúcares , Oligosacáridos , GalactosaRESUMEN
Shigella is a leading cause of moderate to severe diarrhea worldwide and of diarrhea-associated deaths in children under 5 years of age in low-and middle-income countries. A vaccine against shigellosis is in high demand. SF2a-TT15, a synthetic carbohydrate-based conjugate vaccine candidate against Shigella flexneri 2a (SF2a) was found safe and strongly immunogenic in adult volunteers. Here, SF2a-TT15 at 10 µg oligosaccharide (OS) vaccine dose is shown to induce a sustained immune response in magnitude and functionality in the majority of volunteers followed up 2 and 3 years post-vaccination. High levels of either one of the humoral parameters as well as the number of specific-IgG memory B-cells determined 3 months after vaccination were good predictors of the durability of the immune response. This study is the first to examine the long-term durability of antibody functionality and memory B-cell response induced by a Shigella vaccine candidate.
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Chemical synthesis is a powerful tool to access homogeneous complex glycans, which relies on protecting group (PG) chemistry. However, the overall efficiency of chemical glycan assembly is still low when compared to oligonucleotide or oligopeptide synthesis. There have been many contributions giving rise to collective improvement in carbohydrate synthesis that includes PG manipulation and stereoselective glycoside formation and some of this chemistry has been transferred to the solid phase or adapted for programmable one pot synthesis approaches. However, after all glycoside bond formation reactions are completed, the global deprotection (GD) required to give the desired target OS can be challenging. Difficulties observed in the removal of permanent PGs to release the desired glycans can be due to the number and diversity of PGs present in the protected OSs, nature and structural complexity of glycans, etc. Here, we have reviewed the difficulties associated with the removal of PGs from densely protected OSs to obtain their free glycans. In particularly, this review focuses on the challenges associated with hydrogenolysis of benzyl groups, saponification of esters and functional group interconversion such as oxidation/reduction that are commonly performed in GD stage. More generally, problems observed in the removal of permanent PGs is reviewed herein, including benzyl, acyl (levulinoyl, acetyl), N-trichloroacetyl, N-2,2,2-trichloroethoxycarbonyl, N-phthaloyl etc. from a number of fully protected OSs to release the free sugar, that have been previously reported in the literature.
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Carbohidratos , Polisacáridos , Carbohidratos/química , Glicósidos/química , Oligonucleótidos , Oligosacáridos/química , Polisacáridos/química , AzúcaresRESUMEN
Shigella, the causative agent of shigellosis, is among the main causes of diarrheal diseases with still a high morbidity in low-income countries. Relying on chemical synthesis, we implemented a multidisciplinary strategy to design SF2a-TT15, an original glycoconjugate vaccine candidate targeting Shigella flexneri 2a (SF2a). Whereas the SF2a O-antigen features nonstoichiometric O-acetylation, SF2a-TT15 is made of a synthetic 15mer oligosaccharide, corresponding to three non-O-acetylated repeats, linked at its reducing end to tetanus toxoid by means of a thiol-maleimide spacer. We report on the scale-up feasibility under GMP conditions of a high yielding bioconjugation process established to ensure a reproducible and controllable glycan/protein ratio. Preclinical and clinical batches complying with specifications from ICH guidelines, WHO recommendations for polysaccharide conjugate vaccines, and (non)compendial tests were produced. The obtained SF2a-TT15 vaccine candidate passed all toxicity-related criteria, was immunogenic in rabbits, and elicited bactericidal antibodies in mice. Remarkably, the induced IgG antibodies recognized a large panel of SF2a circulating strains. These preclinical data have paved the way forward to the first-in-human study for SF2a-TT15, demonstrating safety and immunogenicity. This contribution discloses the yet unreported feasibility of the GMP synthesis of conjugate vaccines featuring a unique homogeneous synthetic glycan hapten fine-tuned to protect against an infectious disease.
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This review focuses on the molecular glycovaccine concept, a promising option to develop a Shigella glycoconjugate vaccine. Subsequent to original developments involving, as main vaccine component, the detoxified Shigella lipopolysaccharide randomly conjugated at multiple sites to a carrier protein, novelty stems from the use of rationally designed, well-defined chemically synthesized oligosaccharide haptens conceived as functional surrogates of the main surface antigen, linked via single-point attachment onto a carrier. The concept and design of such a fine-tuned Shigella glycovaccine are presented by way of SF2a-TT15, a neoglycoprotein featuring a synthetic 15-mer oligosaccharide, which constitutes an original vaccine prototype targeting Shigella flexneri 2a, one of the predominant circulating strains in endemic settings. The clinical testing of SF2a-TT15 is summarized with the first-in-human phase I trial in young healthy adults showing a good safety profile and tolerability, while inducing bactericidal antibodies towards S. flexneri 2a bacteria. The proof-of-concept of this novel approach being established, an ongoing phase IIa clinical study in the nine-month-old infant target population in endemic area was launched, which is also outlined. Lastly, some challenges to move forward this original approach toward a multivalent cost-effective Shigella synthetic glycan conjugate vaccine are introduced.
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Carbohydrates, in particular microbial glycans, are highly structurally diverse biomolecules, the recognition of which governs numerous biological processes. Of special interest, glycans of known monosaccharide composition feature multiple possible isomers, differentiated by the anomerism and position of their glycosidic linkages. Robust analytical tools able to circumvent this extreme structural complexity are increasing in demand to ensure not only the correct determination of naturally occurring glycans but also to support the rapid development of enzymatic and chemoenzymatic glycan synthesis. In support to the later, we report the use of complementary strategies based on mass spectrometry (MS) to evaluate the ability of 14 engineered mutants of sucrose-utilizing α-transglucosylases to produce type/group-specific Shigella flexneri pentasaccharide bricks from a single lightly protected non-natural tetrasaccharide acceptor substrate. A first analysis of the reaction media by UHPLC coupled to high-accuracy MS led to detect six reaction products of enzymatic glucosylation out of the eight possible ones. A seventh structure was evidenced by an additional step of ion mobility at a resolving power (Rp) of approximately 100. Finally, a Rp of about 250 in ion mobility made it possible to detect the eighth and last of the expected structures. Complementary to these measurements, tandem MS with high activation energy charge transfer dissociation (CTD) allowed us to unambiguously characterize seven regioisomers out of the eight possible products of enzymatic glucosylation. This work illustrates the potential of the recently described powerful IMS and CTD-MS methods for the precise structural characterization of complex glycans.
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Polisacáridos , Espectrometría de Masas en Tándem , Carbohidratos , Isomerismo , Oligosacáridos/química , Polisacáridos/químicaRESUMEN
Enzyme engineering approaches have allowed to extend the collection of enzymatic tools available for synthetic purposes. However, controlling the regioselectivity of the reaction remains challenging, in particular when dealing with carbohydrates bearing numerous reactive hydroxyl groups as substrates. Here, we used a computer-aided design framework to engineer the active site of a sucrose-active [Formula: see text]-transglucosylase for the 1,2-cis-glucosylation of a lightly protected chemically synthesized tetrasaccharide, a common precursor for the synthesis of serotype-specific S. flexneri O-antigen fragments. By targeting 27 amino acid positions of the acceptor binding subsites of a GH70 branching sucrase, we used a RosettaDesign-based approach to propose 49 mutants containing up to 15 mutations scattered over the active site. Upon experimental evaluation, these mutants were found to produce up to six distinct pentasaccharides, whereas only two were synthesized by the parental enzyme. Interestingly, we showed that by introducing specific mutations in the active site of a same enzyme scaffold, it is possible to control the regiospecificity of the 1,2-cis glucosylation of the tetrasaccharide acceptor and produce a unique diversity of pentasaccharide bricks. This work offers novel opportunities for the development of highly convergent chemo-enzymatic routes toward S. flexneri haptens.
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Glucosa/análisis , Glucosa/química , Oligosacáridos/química , Polisacáridos/química , Sacarasa/química , Biotecnología , Carbohidratos/química , Dominio Catalítico , Cromatografía Líquida de Alta Presión , Diseño Asistido por Computadora , Enzimas/química , Glicosilación , Haptenos , Hidrolasas/metabolismo , Biología Molecular , Mutación , Antígenos O , Ingeniería de Proteínas/métodos , Shigella flexneri , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en TándemRESUMEN
The (chemo-)enzymatic synthesis of oligosaccharides has been hampered by the lack of appropriate enzymatic tools with requisite regio- and stereo-specificities. Engineering of carbohydrate-active enzymes, in particular targeting the enzyme active site, has notably led to catalysts with altered regioselectivity of the glycosylation reaction thereby enabling to extend the repertoire of enzymes for carbohydrate synthesis. Using a collection of 22 mutants of ΔN123-GBD-CD2 branching sucrase, an enzyme from the Glycoside Hydrolase family 70, containing between one and three mutations in the active site, and a lightly protected chemically synthesized tetrasaccharide as an acceptor substrate, we showed that altered glycosylation product specificities could be achieved compared to the parental enzyme. Six mutants were selected for further characterization as they produce higher amounts of two favored pentasaccharides compared to the parental enzyme and/or new products. The produced pentasaccharides were shown to be of high interest as they are precursors of representative haptens of Shigella flexneri serotypes 3a, 4a and 4b. Furthermore, their synthesis was shown to be controlled by the mutations introduced in the active site, driving the glucosylation toward one extremity or the other of the tetrasaccharide acceptor. To identify the molecular determinants involved in the change of ΔN123-GBD-CD2 regioselectivity, extensive molecular dynamics simulations were carried out in combination with in-depth analyses of amino acid residue networks. Our findings help to understand the inter-relationships between the enzyme structure, conformational flexibility and activity. They also provide new insight to further engineer this class of enzymes for the synthesis of carbohydrate components of bacterial haptens.
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Proteínas Bacterianas , Haptenos/biosíntesis , Oligosacáridos/biosíntesis , Ingeniería de Proteínas , Shigella flexneri/metabolismo , Sacarasa , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Haptenos/genética , Oligosacáridos/genética , Shigella flexneri/genética , Sacarasa/genética , Sacarasa/metabolismoRESUMEN
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.
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Antígenos O , Shigella flexneri , Secuencia de Carbohidratos , Oligosacáridos , SerogrupoRESUMEN
Shigella sonnei surface polysaccharides are well-established protective antigens against this major cause of diarrhoeal disease. They also qualify as unique zwitterionic polysaccharides (ZPSs) featuring a disaccharide repeating unit made of two 1,2-trans linked rare aminodeoxy sugars, a 2-acetamido-2-deoxy-l-altruronic acid (l-AltpNAcA) and a 2-acetamido-4-amino-2,4,6-trideoxy-d-galactopyranose (AAT). Herein, the stereoselective synthesis of S. sonnei oligosaccharides comprising two, three and four repeating units is reported for the first time. Several sets of up to seven protecting groups were explored, shedding light on the singular conformational behavior of protected altrosamine and altruronic residues. A disaccharide building block equipped with three distinct N-protecting groups and featuring the uronate moiety already in place was designed to accomplish the iterative high yielding glycosylation at the axial 4-OH of the altruronate component and achieve the challenging full deprotection step. Key to the successful route was the use of a diacetyl strategy whereby the N-acetamido group of the l-AltpNAcA is masked in the form of an imide.
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Oligosacáridos , Shigella sonnei , Disacáridos , Glicosilación , PolisacáridosRESUMEN
BACKGROUND: Shigella remains in the top four pathogens responsible for moderate to severe diarrhoea in children below 5 years of age. The shigella O-specific polysaccharide (O-SP) is a promising vaccine target. We developed a conjugate vaccine prototype incorporating a unique well defined synthetic oligosaccharide hapten, chemically designed for optimal antigenic, conformational, structural, and functional mimicry of the O-SP from Shigella flexneri 2a (SF2a). We aimed to assess the safety, tolerability, and immunogenicity of this original synthetic oligosaccharide-based vaccine candidate, SF2a-TT15, conceived to drive the antibody response towards the key protective determinants of the native lipopolysaccharide antigen, in a first-in-human phase 1 study. METHODS: We did a first-in-human, dose-escalating, single-blind, observer-masked, randomised, placebo-controlled study at the Clinical Research Center of Tel Aviv Sourasky Medical Center (Israel). Participants were healthy adults aged 18-45 years with low titres of serum SF2a-specific IgG antibodies. 64 eligible participants were assigned to one of two cohorts. 32 participants in each of the two cohorts were randomly assigned via computer-generated algorithm in a stepwise manner to receive the 2 µg (cohort 1) and 10 µg oligosaccharide dose (cohort 2) of the SF2a-TT15 vaccine candidate non-adjuvanted or adjuvanted with aluminium hydroxide (alum) or matching placebos. The vaccine was administered as three single intramuscular injections into the arm, 28 days apart. The primary outcome was the incidence and severity of adverse events, which were assessed in the intention-to-treat safety population analysis including all participants who were randomly assigned and received at least one vaccine or placebo injection. The immunogenicity endpoints were secondary outcomes and were analysed in all participants who were randomly assigned, received all of the assigned injections before the time of the immunogenicity assessment, and provided blood samples for immunological follow-up (per-protocol immunogenicity analysis). The study is registered with ClinicalStudies.gov, NCT02797236 and is completed. FINDINGS: Of 203 volunteers initially screened, 64 participants were enrolled between Sept 20, 2016, and Sept 26, 2017. In each of the two cohorts, 12 participants received the adjuvanted vaccine, 12 received the non-adjuvanted vaccine and eight received the matching placebo (four each). The SF2a-TT15 glycoconjugate was well tolerated at both doses. No serious or severe adverse events occurred. Overall, seven (88%) of eight to 12 (100%) of 12 in each group of volunteers had one adverse event or more after receiving the study agents with the majority of adverse events, 300 (98%) of 307, considered mild in intensity. Of the seven adverse events defined as moderate in severity, one (nausea) was suspected to be related to the vaccine candidate. At all post-immunisation days and for both oligosaccharide doses, whether adjuvanted or not, SF2a-TT15 induced significantly higher serum IgG anti-SF2a lipopolysaccharide geometric mean titres (GMTs) as compared with baseline or with the corresponding GMTs in placebo recipients (p<0·01). After one injection, the non-adjuvanted 10 µg oligosaccharide dose induced a 27-times increase in IgG GMT (5080 vs 189) and the non-adjuvanted 2 µg oligosaccharide dose induced a five-times increase (1411 vs 283), compared with baseline. Alum enhanced the specific IgG response at 2 µg oligosaccharide dose after the third injection (GMTs 3200 vs 1176, p=0.045). INTERPRETATION: SF2a-TT15 was safe and well tolerated and induced high titres of anti-SF2a LPS IgG antibodies. These results support further evaluation of this original synthetic oligosaccharide-protein conjugate vaccine candidate for safety, immunogenicity, and protective efficacy in target populations. FUNDING: The European Union Seventh Framework Programme.
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Disentería Bacilar/prevención & control , Inmunogenicidad Vacunal , Vacunas contra la Shigella/efectos adversos , Shigella flexneri/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/efectos adversos , Adolescente , Adulto , Hidróxido de Aluminio/administración & dosificación , Hidróxido de Aluminio/efectos adversos , Hidróxido de Aluminio/inmunología , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Relación Dosis-Respuesta Inmunológica , Disentería Bacilar/inmunología , Disentería Bacilar/microbiología , Femenino , Voluntarios Sanos , Humanos , Inyecciones Intramusculares , Masculino , Persona de Mediana Edad , Antígenos O/genética , Antígenos O/inmunología , Vacunas contra la Shigella/administración & dosificación , Vacunas contra la Shigella/genética , Vacunas contra la Shigella/inmunología , Método Simple Ciego , Vacunas Conjugadas/administración & dosificación , Vacunas Conjugadas/efectos adversos , Vacunas Conjugadas/genética , Vacunas Conjugadas/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/efectos adversos , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Adulto JovenRESUMEN
Shigella are gram-negative bacteria that cause severe diarrhea and dysentery, with a high level of antimicrobial resistance. Disease-induced protection against reinfection in Shigella-endemic areas provides convincing evidence on the feasibility of a vaccine and on the importance of Shigella lipopolysaccharides as targets of the host humoral protective immune response against disease. This article provides an overview of the original and current strategies toward the development of a Shigella glycan-protein conjugate vaccine that would cover the most commonly detected strains. Going beyond pioneering "lattice"-type polysaccharide-protein conjugates, progress, and challenges are addressed with focus on promising alternatives, which have reached phases I and II clinical trial. Glycoengineered bioconjugates and "sun"-type conjugates featuring well-defined synthetic carbohydrate antigens are discussed with insights on the molecular parameters governing the rational design of a cost-effective glycoconjugate vaccine efficacious in preventing diseases caused by Shigella in the most at risk populations, young children living in endemic areas.
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Disentería Bacilar/prevención & control , Glicoconjugados/inmunología , Vacunas contra la Shigella/química , Vacunas contra la Shigella/inmunología , Shigella/inmunología , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/inmunología , Carbohidratos/inmunología , Ensayos Clínicos como Asunto , Glicoconjugados/administración & dosificación , Humanos , Shigella/química , Vacunas Conjugadas/inmunologíaRESUMEN
During an infection, the detection of pathogens is mediated through the interactions between pathogen-associated molecular patterns (PAMPs) and pathogen recognition receptors. ß-Heptose 1,7-bisphosphate (ßHBP), an intermediate of the lipopolysaccharide (LPS) biosynthesis pathway, was recently identified as a bacterial PAMP. It was reported that ßHBP sensing leads to oligomerization of TIFA proteins, a mechanism controlling NF-κB activation and pro-inflammatory gene expression. Here, we compare the ability of chemically synthesized ßHBP and Shigella flexneri lysate to induce TIFA oligomerization in epithelial cells. We find that, unlike bacterial lysate, ßHBP fails to initiate rapid TIFA oligomerization. It only induces delayed signaling, suggesting that ßHBP must be processed intracellularly to trigger inflammation. Gene deletion and complementation analysis of the LPS biosynthesis pathway revealed that ADP-heptose is the bacterial metabolite responsible for rapid TIFA oligomerization. ADP-heptose sensing occurs down to 10-10 M. During S. flexneri infection, it results in cytokine production, a process dependent on the kinase ALPK1. Altogether, our results rule out a major role of ßHBP in S. flexneri infection and identify ADP-heptose as a new bacterial PAMP.
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Adenosina Difosfato/metabolismo , Heptosas/metabolismo , Moléculas de Patrón Molecular Asociado a Patógenos/metabolismo , Shigella flexneri/metabolismo , Células HeLa , Heptosas/síntesis química , Heptosas/química , Humanos , Neisseria , Proteínas Quinasas/metabolismo , Multimerización de Proteína , Espectroscopía de Protones por Resonancia MagnéticaRESUMEN
Bacteriophage research is gaining more importance due to increasing antibiotic resistance. However, for treatment with bacteriophages, diagnostics have to be improved. Bacteriophages carry adhesion proteins, which bind to the bacterial cell surface, for example tailspike proteins (TSP) for specific recognition of bacterial O-antigen polysaccharide. TSP are highly stable proteins and thus might be suitable components for the integration into diagnostic tools. We used the TSP of bacteriophage Sf6 to establish two applications for detecting Shigella flexneri (S. flexneri), a highly contagious pathogen causing dysentery. We found that Sf6TSP not only bound O-antigen of S. flexneri serotype Y, but also the glucosylated O-antigen of serotype 2a. Moreover, mass spectrometry glycan analyses showed that Sf6TSP tolerated various O-acetyl modifications on these O-antigens. We established a microtiter plate-based ELISA like tailspike adsorption assay (ELITA) using a Strep-tag®II modified Sf6TSP. As sensitive screening alternative we produced a fluorescently labeled Sf6TSP via coupling to an environment sensitive dye. Binding of this probe to the S. flexneri O-antigen Y elicited a fluorescence intensity increase of 80% with an emission maximum in the visible light range. The Sf6TSP probes thus offer a promising route to a highly specific and sensitive bacteriophage TSP-based Shigella detection system.
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Técnicas de Tipificación Bacteriana , Bacteriófagos/química , Bioensayo , Antígenos O/química , Podoviridae/química , Shigella flexneri/aislamiento & purificación , Proteínas de la Cola de los Virus/química , Bacteriófagos/genética , Bacteriófagos/metabolismo , Secuencia de Carbohidratos , Clonación Molecular , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Colorantes Fluorescentes/química , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Glicósido Hidrolasas , Modelos Moleculares , Antígenos O/metabolismo , Oxadiazoles/química , Podoviridae/genética , Podoviridae/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serogrupo , Shigella flexneri/química , Shigella flexneri/metabolismo , Coloración y Etiquetado/métodos , Proteínas de la Cola de los Virus/genética , Proteínas de la Cola de los Virus/metabolismoRESUMEN
Pseudomonas aeruginosa and Aspergillus fumigatus are the two microorganisms responsible for most of the chronic infections in cystic fibrosis patients. P. aeruginosa is known to produce quorum-sensing controlled rhamnolipids during chronic infections. Here we show that the dirhamnolipids secreted from P. aeruginosa (i) induce A. fumigatus to produce an extracellular matrix, rich in galactosaminogalactan, 1,8-dihydroxynaphthalene (DHN)- and pyo-melanin, surrounding their hyphae, which facilitates P. aeruginosa binding and (ii) inhibit A. fumigatus growth by blocking ß1,3 glucan synthase (GS) activity, thus altering the cell wall architecture. A. fumigatus in the presence of diRhls resulted in a growth phenotype similar to that upon its treatment with anjpegungal echinocandins, showing multibranched hyphae and thicker cell wall rich in chitin. The diRhl structure containing two rhamnose moieties attached to fatty acyl chain is essential for the interaction with ß1,3 GS; however, the site of action of diRhls on GS is different from that of echinocandins, and showed synergistic anjpegungal effect with azoles.
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Aspergillus fumigatus/metabolismo , Glucosiltransferasas/antagonistas & inhibidores , Glucolípidos/metabolismo , Glucolípidos/farmacología , Pseudomonas aeruginosa/metabolismo , Aspergillus fumigatus/citología , Pared Celular , Quitina/metabolismo , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Glucosiltransferasas/metabolismo , Glucolípidos/genética , Hifa/metabolismo , Polisacáridos , Pseudomonas aeruginosa/citología , Percepción de Quorum/efectos de los fármacosRESUMEN
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.
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Lipopolisacáridos/química , Polisacáridos/síntesis química , Shigella flexneri/química , Acetilglucosamina/química , Conformación de Carbohidratos , Disacáridos/química , Glicosilación , Hidrazinas/química , Polisacáridos/química , Trisacáridos/químicaRESUMEN
Shigella flexneriâ 3a causes bacillary dysentery. Its O-antigen has the {2)-[α-d-Glcp-(1â3)]-α-l-Rhap-(1â2)-α-l-Rhap-(1â3)-[Acâ2]-α-l-Rhap-(1â3)-[Acâ6]≈40 % -ß-d-GlcpNAc-(1â} ([(E)ABAc CAc D]) repeating unit, and the non-O-acetylated equivalent defines S. flexneriâ 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 typesâ 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.
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Antígenos O/química , Shigella flexneri/química , Animales , Inmunoquímica , Ratones , Ratones Endogámicos BALB CRESUMEN
Conjugation chemistry is among the most important parameters governing the efficacy of glycoconjugate vaccines. High robustness is required to ensure high yields and batch to batch reproducibility. Herein, we have established a robust bioconjugation protocol based on the thiol-maleimide addition. Major variables were determined and acceptable margins were investigated for a synthetic pentadecasaccharide-tetanus toxoid conjugate, which is a promising vaccine candidate against Shigella flexneri serotype 2a infection. The optimized process is applicable to any thiol-equipped hapten and provides an efficient control of the hapten:carrier ratio. Moreover, comparison of four S. flexneri 2a glycoconjugates only differing by their pentadecasaccharide:tetanus toxoid ratio confirmed preliminary findings indicating that hapten loading is critical for immunogenicity with an optimal ratio here in the range of 17 ± 5. In addition, the powerful influence of alum on the immunogenicity of a Shigella synthetic carbohydrate-based conjugate vaccine candidate is demonstrated for the first time, with a strong anti-S. flexneri 2a antibody response sustained for more than one year.