Host Adaptation of a Bacterial Toxin from the Human Pathogen Salmonella Typhi

SummarySalmonella Typhi is an exclusive human pathogen that causes typhoid fever. Typhoid toxin is a S. Typhi virulence factor that can reproduce most of the typhoid fever symptoms in experimental animals. Toxicity depends on toxin binding to terminally sialylated glycans on surface glycoproteins. Human glycans are unusual because of the lack of CMAH, which in other mammals converts N-acetylneuraminic acid (Neu5Ac) to N-glycolylneuraminic acid (Neu5Gc). Here, we report that typhoid toxin binds to and is toxic toward cells expressing glycans terminated in Neu5Ac (expressed by humans) over glycans terminated in Neu5Gc (expressed by other mammals). Mice constitutively expressing CMAH thus displaying Neu5Gc in all tissues are resistant to typhoid toxin. The atomic structure of typhoid toxin bound to Neu5Ac reveals the structural bases for its binding specificity. These findings provide insight into the molecular bases for Salmonella Typhi’s host specificity and may help the development of therapies for typhoid fever

Studies on the Detection, Expression, Glycosylation, Dimerization, and Ligand Binding Properties of Mouse Siglec-E

© 2017 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. This is an Open Access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/)CD33-related Siglecs are a family of proteins widely expressed on innate immune cells. Binding of sialylated glycans or other ligands triggers signals that inhibit or activate inflammation. Immunomodulation by Siglecs has been extensively studied, but relationships between structure and functions are poorly explored. Here we present new data relating to the structure and function of Siglec-E, the major CD33-related Siglec expressed on mouse neutrophils, monocytes, macrophages, and dendritic cells. We generated nine new rat monoclonal antibodies specific to mouse Siglec-E, with no cross-reactivity to Siglec-F. Although all antibodies detected Siglec-E on transfected human HEK-293T cells, only two reacted with mouse bone marrow neutrophils by flow cytometry and on spleen sections by immunohistochemistry. Moreover, whereas all antibodies recognized Siglec-E-Fc on immunoblots, binding was dependent on intact disulfide bonds and N-glycans, and only two antibodies recognized native Siglec-E within spleen lysates. Thus, we further investigated the impact of Siglec-E homodimerization. Homology-based structural modeling predicted a cysteine residue (Cys-298) in position to form a disulfide bridge between two Siglec-E polypeptides. Mutagenesis of Cys-298 confirmed its role in dimerization. In keeping with the high level of 9-O-acetylation found in mice, sialoglycan array studies indicate that this modification has complex effects on recognition by Siglec-E, in relationship to the underlying structures. However, we found no differences in phosphorylation or SHP-1 recruitment between dimeric and monomeric Siglec-E expressed on HEK293A cells. Phylogenomic analyses predicted that only some human and mouse Siglecs form disulfide-linked dimers. Notably, Siglec-9, the functionally equivalent human paralog of Siglec-E, occurs as a monomer.Peer reviewedFinal Published versio

Photochemical Surface Functionalization : Synthesis, Nanochemistry and Glycobiological Studies

This thesis mainly deals with the development of photochemical approaches to immobilize carbohydrates on surfaces for glycobiological studies. These approaches have been incorporated into a number of state-of-the-art nanobio-platforms, including carbohydrate microarrays, surface plasmon resonance (SPR), quartz crystal microbalance (QCM), atomic force microscopy (AFM), and glyconanomaterials. All the surfaces have displayed good binding capabilities and selectivities after functionalization with carbohydrates, and a range of important data have been obtained concerning surface characteristics and carbohydrate-protein interactions, based on the platforms established. Besides, a variety of non-carbohydrate and carbohydrate-based molecules have been synthesized, during which process the mutarotation of 1-glycosyl thiols and the stereocontrol in 1-S-glycosylation reactions have been thoroughly studied.QC 2011100

Exploration of sialic acid diversity and biology using sialoglycan microarrays.

Sialic acids (Sias) are a group of α-keto acids with a nine-carbon backbone, which display many types of modifications in nature. The diversity of natural Sia presentations is magnified by a variety of glycosidic linkages to underlying glycans, the sequences and classes of such glycans, as well as the spatial organization of Sias with their surroundings. This diversity is closely linked to the numerous and varied biological functions of Sias. Relatively large libraries of natural and unnatural Sias have recently been chemically/chemoenzymatically synthesized and/or isolated from natural sources. The resulting sialoglycan microarrays have proved to be valuable tools for the exploration of diversity and biology of Sias. Here we provide an overview of Sia diversity in nature, the approaches used to generate sialoglycan microarrays, and the achievements and challenges arising

Control of the ambident reactivity of the nitrite ion.

In previous studies, it was reported that a neighboring equatorial ester group is essential for a good yield of nitrite-mediated triflate inversion, whereas with neighboring benzyl ether groups or axial ester groups, mixts. are generally produced. The origin of this difference was addressed. The ambident reactivity of the nitrite ion is the cause of the complex product formation obsd., which can be controlled by a neighboring equatorial ester group. Both N-attack and O-attack occur in the absence of the ester group, whereas O-attack is favored in its presence. A neighboring group assistance mechanism is proposed, in addn. to steric effects, based on secondary interactions between the neighboring ester group and the incoming nucleophile. High-level quantum mech. calcns. were carried out to delineate this effect. The theor. results are in excellent agreement with expts., and suggest a catalytic role for the neighboring equatorial ester group. [on SciFinder(R)