Membrane lipid remodeling eradicates Helicobacter pylori by manipulating the cholesteryl 6'-acylglucoside biosynthesis.

BACKGROUND: Helicobacter pylori, the main cause of various gastric diseases, infects approximately half of the human population. This pathogen is auxotrophic for cholesterol which it converts to various cholesteryl α-glucoside derivatives, including cholesteryl 6'-acyl α-glucoside (CAG). Since the related biosynthetic enzymes can be translocated to the host cells, the acyl chain of CAG likely comes from its precursor phosphatidylethanolamine (PE) in the host membranes. This work aims at examining how the acyl chain of CAG and PE inhibits the membrane functions, especially bacterial adhesion. METHODS: Eleven CAGs that differ in acyl chains were used to study the membrane properties of human gastric adenocarcinoma cells (AGS cells), including lipid rafts clustering (monitored by immunofluorescence with confocal microscopy) and lateral membrane fluidity (by the fluorescence recovery after photobleaching). Cell-based and mouse models were employed to study the degree of bacterial adhesion, the analyses of which were conducted by using flow cytometry and immunofluorescence staining, respectively. The lipidomes of H. pylori, AGS cells and H. pylori-AGS co-cultures were analyzed by Ultraperformance Liquid Chromatography-Tandem Mass Spectroscopy (UPLC-MS/MS) to examine the effect of PE(10:0)2, PE(18:0)2, PE(18:3)2, or PE(22:6)2 treatments. RESULTS: CAG10:0, CAG18:3 and CAG22:6 were found to cause the most adverse effect on the bacterial adhesion. Further LC-MS analysis indicated that the treatment of PE(10:0)2 resulted in dual effects to inhibit the bacterial adhesion, including the generation of CAG10:0 and significant changes in the membrane compositions. The initial (1 h) lipidome changes involved in the incorporation of 10:0 acyl chains into dihydro- and phytosphingosine derivatives and ceramides. In contrast, after 16 h, glycerophospholipids displayed obvious increase in their very long chain fatty acids, monounsaturated and polyunsaturated fatty acids that are considered to enhance membrane fluidity. CONCLUSIONS: The PE(10:0)2 treatment significantly reduced bacterial adhesion in both AGS cells and mouse models. Our approach of membrane remodeling has thus shown great promise as a new anti-H. pylori therapy.

Tunable Strategy for the Asymmetric Synthesis of Sulfoglycolipids from Mycobacterium tuberculosis To Elucidate the Structure and Immunomodulatory Property Relationships.

We developed a versatile asymmetric strategy to synthesize different classes of sulfoglycolipids (SGLs) from Mycobacterium tuberculosis. The strategy features the use of asymmetrically protected trehaloses, which were acquired from the glycosylation of TMS α-glucosyl acceptors with benzylidene-protected thioglucosyl donors. The positions of the protecting groups at the donors and acceptors can be fine-tuned to obtain different protecting-group patterns, which is crucial for regioselective acylation and sulfation. In addition, a chemoenzymatic strategy was established to prepare the polymethylated fatty acid building blocks. The strategy employs inexpensive lipase as a desymmetrization agent in the preparation of the starting substrate and readily available chiral oxazolidinone as a chirality-controlling agent in the construction of the polymethylated fatty acids. A subsequent investigation on the immunomodulatory properties of each class of SGLs showed how the structures of SGLs impact the host innate immunity response.

Enhanced enzymatic production of cholesteryl 6'-acylglucoside impairs lysosomal degradation for the intracellular survival of Helicobacter pylori.

BACKGROUND: During autophagy defense against invading microbes, certain lipid types are indispensable for generating specialized membrane-bound organelles. The lipid composition of autophagosomes remains obscure, as does the issue of how specific lipids and lipid-associated enzymes participate in autophagosome formation and maturation. Helicobacter pylori is auxotrophic for cholesterol and converts cholesterol to cholesteryl glucoside derivatives, including cholesteryl 6'-O-acyl-α-D-glucoside (CAG). We investigated how CAG and its biosynthetic acyltransferase assist H. pylori to escape host-cell autophagy. METHODS: We applied a metabolite-tagging method to obtain fluorophore-containing cholesteryl glucosides that were utilized to understand their intracellular locations. H. pylori 26695 and a cholesteryl glucosyltransferase (CGT)-deletion mutant (ΔCGT) were used as the standard strain and the negative control that contains no cholesterol-derived metabolites, respectively. Bacterial internalization and several autophagy-related assays were conducted to unravel the possible mechanism that H. pylori develops to hijack the host-cell autophagy response. Subcellular fractions of H. pylori-infected AGS cells were obtained and measured for the acyltransferase activity. RESULTS: The imaging studies of fluorophore-labeled cholesteryl glucosides pinpointed their intracellular localization in AGS cells. The result indicated that CAG enhances the internalization of H. pylori in AGS cells. Particularly, CAG, instead of CG and CPG, is able to augment the autophagy response induced by H. pylori. How CAG participates in the autophagy process is multifaceted. CAG was found to intervene in the degradation of autophagosomes and reduce lysosomal biogenesis, supporting the idea that intracellular H. pylori is harbored by autophago-lysosomes in favor of the bacterial survival. Furthermore, we performed the enzyme activity assay of subcellular fractions of H. pylori-infected AGS cells. The analysis showed that the acyltransferase is mainly distributed in autophago-lysosomal compartments. CONCLUSIONS: Our results support the idea that the acyltransferase is mainly distributed in the subcellular compartment consisting of autophagosomes, late endosomes, and lysosomes, in which the acidic environment is beneficial for the maximal acyltransferase activity. The resulting elevated level of CAG can facilitate bacterial internalization, interfere with the autophagy flux, and causes reduced lysosomal biogenesis.

Cascade In Situ Phosphorylation and One-Pot Glycosylation for Rapid Synthesis of Heptose-Containing Oligosaccharides.

We report a one-pot glycosylation strategy for achieving rapid syntheses of heptose (Hep)-containing oligosaccharides. The reported procedure was designed to incorporate an in situ phosphorylation step into an orthogonal one-pot glycosylation. Hep-containing oligosaccharides were assembled directly from building blocks with minimal effort expended on manipulation of protecting and aglycone leaving groups. The utility of our one-pot procedure was illustrated by synthesizing partial core oligosaccharide structure present in the lipopolysaccharide of Ralstonia solanacearum.

Sub-stoichiometric reductive etherification of carbohydrate substrates and one-pot protecting group manipulation.

In this study, we report a new reductive etherification procedure for protection of carbohydrate substrates and its application for one-pot preparation of glycosyl building blocks. The reported procedure features the use of polymethylhydrosiloxane (PMHS) as a sub-stoichiometric reducing agent, which prevents the transilylation side reaction and improves the efficiency of the reductive etherification method. Application of the PMHS reductive etherification procedure for one-pot protecting group manipulation are described.

Diverse Synthesis of Natural Trehalosamines and Synthetic 1,1'-Disaccharide Aminoglycosides.

A general strategy for the diverse synthesis of ten disaccharide aminoglycosides, including natural 2-trehalosamine (1), 3-trehalosamine (2), 4-trehalosamine (3), and neotrehalosyl 3,3'-diamine (8) and synthetic aminoglycosides 4-7, 9, and 10, has been developed. The aminoglycoside compounds feature different anomeric configurations and numbers of amino groups. The key step for the synthesis was the glycosylation coupling of a stereodirecting donor with a configuration-stable TMS glycoside acceptor. Either the donor or acceptor could be substituted with an azido group. The aminoglycosides prepared in the present study were characterized by 1D and 2D NMR spectroscopy.

Unusually Stable Picoloyl-Protected Trimethylsilyl Glycosides for Nonsymmetrical 1,1'-Glycosylation and Synthesis of 1,1'-Disaccharides with Diverse Configurations.

Nonsymmetrical 1,1'-disaccharides and related derivatives constitute structural components in various glycolipids and natural products. Some of these compounds have been shown to exhibit appealing biological properties. We report a direct yet stereoselective 1,1'-glycosylation strategy for the synthesis of nonsymmetrical 1,1'-disaccharides with diverse configurations and sugar components. The strategy is based on the joined forces of a new class of configurationally stable glycoside acceptors and stereodirecting thioglycoside donors. The new glycoside acceptors feature a picoloyl (Pico) protecting group at the remote C4/C3 position that confers unusual stability on TMS glycosides under acidic conditions.

A flexible 1,2-cis α-glycosylation strategy based on in situ adduct transformation.

A flexible 1,2-cis α-selective glycosylation strategy for a wide range of glycosyl donors and acceptors has been developed, which is based on an in situ adduct transformation protocol. Based on this strategy, both NFM-derived and iodide covalent adducts can be accessed for glycosylation. Using low temperature NMR spectroscopy, the aforementioned glycosyl adducts were detected.

Development of a novel engineered antibody targeting Neisseria species.

OBJECTIVES: A Neissaria bacterial pilus sugar, bacillosamine, was synthesized and, for the first time, used as a probe to screen a single-chain variable fragment (scFv). RESULTS: Four Neisseria, Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria sicca and Neisseria subflava, and two negative controls, Streptococcus pneumoniae and Escherichia coli, were tested through ELISA, immunostaining and gold nanoparticle immunological assay. All results indicated that the selected scFv is feasible for the specific detection of Neisseria species via the recognition of bacillosamine. CONCLUSIONS: The recombinant scFv could detect Neisseria strains at 106 CFU/ml.

Magnetic Nanoparticle-Based Platform for Characterization of Shiga-like Toxin 1 from Complex Samples.

Foodborne illness outbreaks resulting from contamination of Escherichia coli O157:H7 remain a serious concern in food safety. E. coli O157:H7 can cause bloody diarrhea, hemolytic uremic syndrome, or even death. The pathogenicity of E. coli O157:H7 is mainly caused by the expression of Shiga-like toxins (SLTs), i.e., SLT-1 and SLT-2. SLTs are pentamers composed of a single A and five B subunits. In this study, we propose a magnetic nanoparticle (MNP)-based platform to rapidly identify SLT-1 from the complex cell lysate of E. coli O157:H7. The core of the MNPs is made of iron oxide, whereas the surface of the core is coated with a thin layer of alumina (Fe3O4@Al2O3 MNPs). The Fe3O4@Al2O3 MNPs are functionalized with pigeon ovalbumin (POA), which contains Gal-α(1→4)-Gal-ß(1→4)-GlcNAc termini that can bind SLT-1B selectively. Furthermore, POA is a phosphate protein. Thus, it can be easily immobilized on the surface of the Fe3O4@Al2O3 MNPs through aluminum phosphate chelation under microwave heating within 1.5 min. The generated POA-Fe3O4@Al2O3 MNPs are capable of effectively enriching SLT-1B from complex cell lysates simply by pipetting 20 µL of the sample in and out of the tip in a vial for ∼1 min. To release SLT-1 from the MNPs, Gal-α(1→4)-Gal disaccharides were used for displacement. The released target species are sufficient to be identified by matrix-assisted laser desorption/ionization mass spectrometry. Although the sample volume used in this approach is small (20 µL) and the enrichment time is short (1 min), the selectivity of this approach toward SLT-1B is quite good. We have demonstrated the effectiveness of this approach for rapid determination of the presence of SLT-1 from complex cell lysates and ham/juice samples based on the detection of SLT-1B.

A general synthetic strategy and the anti-proliferation properties on prostate cancer cell lines for natural phenylethanoid glycosides.

A general strategy for the synthesis of phenylethanoid glycosides (PhG) including echinacoside 1, acteoside 2, calceolarioside-A 3 and calceolarioside-B 4 is reported. The strategy features the application of low substrate concentration glycosylation and N-formyl morpholine modulated glycosylation methods for the construction of 1,2-trans ß- and α-glycosidic bonds. The reported strategy does not invoke the use of the participatory acyl protecting function, which is incompatible with the ester function present in target PhG compounds. A preliminary study of the anti-proliferation properties of the PhG compounds 1­4 was performed; the acteoside 2 exhibited the best inhibition on the prostatic cancer cell proliferation.

Modulating glycosylation with exogenous nucleophiles: an overview.

The major challenge in carbohydrate synthesis is stereochemical control of glycosidic bond formation. Different glycosylation methods have been developed that are based on the modulation effect of external nucleophiles. This review highlights the development, synthetic application, challenges and outlook of the modulated glycosylation methods.

Sulfoglycolipids and Related Analogues of Mycobacterium tuberculosis: Chemical Synthesis and Immunological Studies.

Mycobacterium tuberculosis (Mtb) causes tuberculosis as one major threat to human health, which has been deteriorated owing to the emerging multidrug resistance. Mtb contains a complex lipophilic cell wall structure that is important for bacterial persistence. Among the lipid components, sulfoglycolipids (SGLs), known to induce immune cell responses, are composed of a trehalose core attached with a conserved sulfate group and 1-4 fatty acyl chains in an asymmetric pattern. At least one of these acyl chains is polymethylated with 3-12 methyl branches. Although Mtb SGL can be isolated from bacterial culture, resulting SGL is still a homologous mixture, impeding accurate research studies. This up-to-date review covers the chemical synthesis and immunological studies of Mtb SGLs and structural analogues, with an emphasis on the development of new glycosylation methods and the asymmetric synthesis of polymethylated scaffolds. Both are critical to advance further research on biological functions of these complicated SGLs.

Orthogonal Glycosylation with Phosphate Acceptors for Expeditious Synthesis of Bacterial Inner Core Oligosaccharides.

We report a practical one-pot glycosylation strategy for synthesis of bacterial inner core oligosaccharides that composed of unavailable L-glycero-D-manno and D-glycero-D-manno-heptopyranose components. The glycosylation method features a new orthogonal glycosylation procedure; whereby a phosphate acceptor is coupled with a thioglycosyl donor producing a disaccharide phosphate, which can be engaged in another orthogonal glycosylation procedure to couple with a thioglycosyl acceptor. The phosphate acceptors used in above one-pot procedure are directly prepared from thioglycosyl acceptors via the in-situ phosphorylation. Such phosphate acceptor preparation protocol eliminates the traditional protection and deprotection procedures. Based on the new one-pot glycosylation strategy, two partial inner core structures of Yersinia pestis lipopolysaccharide and Haemophilus ducreyi lipooligosaccharide were acquired.

Neighboring-group participation by C-2 ether functions in glycosylations directed by nitrile solvents.

Ether-protecting functions at C-2 hydroxy groups have been found to play participating roles in glycosylations when the reactions are conducted in nitrile solvent mixtures. The participation mechanism is based on intramolecular interaction between the lone electron pair of the oxygen atom of the C-2 ether function and the nitrile molecule when they are positioned in a cis configuration. A 1,2-cis glycosyl oxazolinium intermediate is formed. This participation, in conjunction with the anomeric effect of the glycosyl donor, confers high 1,2-trans selectivities on glycosylations. Further application of this concept has led to efficient preparations of α-(1→5)-arabinan oligomers.

Total synthesis of landomycins Q and R and related core structures for exploration of the cytotoxicity and antibacterial properties.

Herein, we report the total synthesis of landomycins Q and R as well as the aglycone core, namely anhydrolandomycinone and a related core analogue. The synthesis features an acetate-assisted arylation method for construction of the hindered B-ring in the core component and a one-pot aromatization-deiodination-denbenzylation procedure to streamline the global functional and protecting group manuipulation. Subsequent cytotoxicity and antibacterial studies revealed that the landomycin R is a potential antibacterial agent against methicillin-resistant Staphylococcus aureus.

Cholesteryl α-D-glucoside 6-acyltransferase enhances the adhesion of Helicobacter pylori to gastric epithelium.

Helicobacter pylori, the most common etiologic agent of gastric diseases including gastric cancer, is auxotrophic for cholesterol and has to hijack it from gastric epithelia. Upon uptake, the bacteria convert cholesterol to cholesteryl 6'-O-acyl-α-D-glucopyranoside (CAG) to promote lipid raft clustering in the host cell membranes. However, how CAG appears in the host to exert the pathogenesis still remains ambiguous. Herein we identified hp0499 to be the gene of cholesteryl α-D-glucopyranoside acyltransferase (CGAT). Together with cholesteryl glucosyltransferase (catalyzing the prior step), CGAT is secreted via outer membrane vesicles to the host cells for direct synthesis of CAG. This significantly enhances lipid rafts clustering, gathers adhesion molecules (including Lewis antigens and integrins α5, ß1), and promotes more bacterial adhesion. Furthermore, the clinically used drug amiodarone was shown as a potent inhibitor of CGAT to effectively reduce the bacterial adhesion, indicating that CGAT is a potential target of therapeutic intervention.

Identification of essential residues of human alpha-L-fucosidase and tests of its mechanism.

Fucosylated glycoconjugates have critical roles in biological processes, but a limited availability of alpha-l-fucosidase has hampered research on this human enzyme (h-Fuc) at a molecular level. After overexpressing h-Fuc in Escherichia coli as an active form, we investigated the catalytic function of this recombinant enzyme. Based on sequence alignment and structural analysis of close homologues of h-Fuc, nine residues of glutamate and aspartate in h-Fuc were selected for mutagenic tests to determine the essential residues. Among the mutants, D225N, E289Q, and E289G lost catalytic activity significantly; their k(cat) values are 1/5700, 1/430, and 1/340, respectively, of that of the wild-type enzyme. The Brønsted plot for k(cat)/K(m) for the E289G mutant is linear with beta(lg) = -0.93, but that for k(cat) is biphasic, with beta(lg) for poor substrates being -0.88 and for activated substrates being -0.11. The small magnitude of beta(lg) for the activated substrates may indicate that the rate-limiting step of the reaction is defucosylation, whereas the large magnitude of the latter beta(lg) value for the poor substrates indicates that the rate-limiting step of the reaction becomes fucosylation. The kinetic outcomes support an argument that Asp(225) functions as a nucleophile and Glu(289) as a general acid/base catalyst. As further evidence, azide significantly reactivated D225G and E289G, and (1)H NMR spectral analysis confirmed the formation of beta-fucosyl azide and alpha-fucosyl azide in the azide rescues of D225G and E289G catalyses, respectively. As direct evidence to prove the function of Glu(289), an accumulation of fucosyl-enzyme intermediate was detected directly through ESI/MS analysis.

Low-concentration 1,2-trans beta-selective glycosylation strategy and its applications in oligosaccharide synthesis.

This study develops an operationally easy, efficient, and general 1,2-trans beta-selective glycosylation reaction that proceeds in the absence of a C2 acyl function. This process employs chemically stable thioglycosyl donors and low substrate concentrations to achieve excellent beta-selectivities in glycosylation reactions. This method is widely applicable to a range of glycosyl substrates irrespective of their structures and hydroxyl-protecting functions. This low-concentration 1,2-trans beta-selective glycosylation in carbohydrate chemistry removes the restriction of using highly reactive thioglycosides to construct 1,2-trans beta-glycosidic bonds. This is beneficial to the design of new strategies for oligosaccharide synthesis, as illustrated in the preparation of the biologically relevant beta-(1-->6)-glucan trisaccharide, beta-linked Gb(3) and isoGb(3) derivatives.

Identification of Pseudomonas aeruginosa using functional magnetic nanoparticle-based affinity capture combined with MALDI MS analysis.

PA-IL is a galactophilic lectin that is found on the outer membrane of Pseudomonas aeruginosa. Pigeon ovalbumin (POA), a phosphoprotein, contains high levels of terminal Gal alpha(1-->4)Gal units. Thus, magnetic nanoparticles with immobilized POA can be used as affinity probes for P. aeruginosa, functioning via the recognition of galactophilic PA-IL. We fabricated POA-bound nanoparticles (NPs) by immobilizing POA onto the surface of core/shell magnetic iron oxide/alumina NPs via metal-phosphate chelation. We then used the generated NPs to probe target bacteria from complex samples. The trapped bacterial cells were characterized based on their mass peak profiles obtained from MALDI MS analyses. In addition, we confirmed the determination of P. aeruginosa using a proteomic strategy: combining the resultant MALDI MS/MS spectra of its tryptic digest with protein database searching. The feasibility of using this approach to rapidly characterize P. aeruginosa from clinical samples without the need to perform culturing steps was also demonstrated.