The neurotoxins of Clostridium botulinum and tetanus bind to gangliosides as a first step of their toxin activity. Identifying suitable receptors that compete with gangliosides could prevent toxin binding to the neuronal cells. A possible ganglioside-binding site of the botulinum neurotoxin B (BoNT/B) has already been proposed and evidence is now presented for a drug binding to botulinum neurotoxin B from structural studies. Doxorubicin, a well known DNA intercalator, binds to the neurotoxin at the receptor-binding site proposed earlier. The structure of the BoNT/B-doxorubicin complex reveals that doxorubicin has interactions with the neurotoxin similar to those of sialyllactose. The aglycone moiety of the doxorubicin stacks with tryptophan 1261 and interacts with histidine 1240 of the binding domain. Here, the possibility is presented of designing a potential antagonist for these neurotoxins from crystallographic analysis of the neurotoxin-doxorubicin complex, which will be an excellent lead compound.

Bacterial Proteins, chemistry, DNA, analysis, molecular, X-ray, gangliosides, proteins, models, Clostridium, Crystallography, Binding Sites, toxins, Clostridium botulinum, Crystallization, Protein Conformation, neurotoxin B, botulism, doxorubicin, Botulinum Toxins, Histidine, Neurotoxins, Tryptophan

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

C[C@@H]1O[C@@H](O)C[C@H](N)[C@@H]1O

147.174 g/mol (C₆H₁₃NO₃)

Bacteria possess an extraordinary repertoire of cell envelope glycans that have critical physiological functions. Pathogenic bacteria have glycans that are essential for growth and virulence but are absent from humans, making them high-priority targets for antibiotic, vaccine, and diagnostic development. The advent of metabolic labeling with bioorthogonal chemical reporters and small-molecule fluorescent reporters has enabled the investigation and targeting of specific bacterial glycans in their native environments. These tools have opened the door to imaging glycan dynamics, assaying and inhibiting glycan biosynthesis, profiling glycoproteins and glycan-binding proteins, and targeting pathogens with diagnostic and therapeutic payload. These capabilities have been wielded in diverse commensal and pathogenic Gram-positive, Gram-negative, and mycobacterial species-including within live host organisms. Here, we review the development and applications of chemical reporters for bacterial glycans, including peptidoglycan, lipopolysaccharide, glycoproteins, teichoic acids, and capsular polysaccharides, as well as mycobacterial glycans, including trehalose glycolipids and arabinan-containing glycoconjugates. We cover in detail how bacteria-targeting chemical reporters are designed, synthesized, and evaluated, how they operate from a mechanistic standpoint, and how this information informs their judicious and innovative application. We also provide a perspective on the current state and future directions of the field, underscoring the need for interdisciplinary teams to create novel tools and extend existing tools to support fundamental and translational research on bacterial glycans.

biosynthesis, glycoconjugates, glycan, polysaccharides, Gram-negative bacteria, gram-positive bacteria

There are 2 chemically distinct structures. Please, select:
Me(1-2)x?Glu?(5-5)[Ac(1-7)]aXLegp(2-3)xDThr? Me(1-2)x?Glu?(5-5)[Ac(1-7)]aXLegp(2-3)xLThr?

SVGMWSMILES3D molIonize

 

CNC(CCC(=O)N[C@H]1[C@H]([C@H](NC(C)=O)[C@@H](C)O)O[C@@](O[C@@H](C)[C@@H](N)C(=O)O)(C(=O)O)C[C@@H]1O)C(=O)O

536.535 g/mol (C₂₁H₃₆N₄O₁₂)

Flagellins from Clostridium botulinum were shown to be post-translationally modified with novel glycan moieties by top-down MS analysis of purified flagellin protein from strains of various toxin serotypes. Detailed analyses of flagellin from two strains of C. botulinum demonstrated that the protein is modified by a novel glycan moiety of mass 417 Da in O-linkage. Bioinformatic analysis of available C. botulinum genomes identified a flagellar glycosylation island containing homologs of genes recently identified in Campylobacter coli that have been shown to be responsible for the biosynthesis of legionaminic acid derivatives. Structural characterization of the carbohydrate moiety was completed utilizing both MS and NMR spectroscopy, and it was shown to be a novel legionaminic acid derivative, 7-acetamido-5-(N-methyl-glutam-4-yl)-amino-3,5,7,9-tetradeoxy-d-glycero-α-d-galacto-nonulosonic acid, (αLeg5GluNMe7Ac). Electron transfer dissociation MS with and without collision-activated dissociation was utilized to map seven sites of O-linked glycosylation, eliminating the need for chemical derivatization of tryptic peptides prior to analysis. Marker ions for novel glycans, as well as a unique C-terminal flagellin peptide marker ion, were identified in a top-down analysis of the intact protein. These ions have the potential for use in for rapid detection and discrimination of C. botulinum cells, indicating botulinum neurotoxin contamination. This is the first report of glycosylation of Gram-positive flagellar proteins by the 'sialic acid-like' nonulosonate sugar, legionaminic acid

legionaminic acid, glycosylation, Flagellin, Clostridium botulinum, protein glycosylation

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6	C7	C8	C9
3,5,2	Me	33.1
3,5	x?Glu	174.6	64.4	26.1	32.9	176.0
3,7	Ac	175.2	23.3
3	aXLegp	174.6	101.8	40.7	69.9	53.3	73.0	55.2	68.4	19.4
	x?Ser


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6	H7	H8	H9
3,5,2	Me	2.73
3,5	x?Glu	-	3.61	2.12	2.38	-
3,7	Ac	-	2.02
3	aXLegp	-	-	1.68-2.71	3.59	3.69	3.92	3.85	3.95	1.16
	x?Ser


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6	C7/H7	C8/H8	C9/H9
3,5,2	Me	33.1/2.73
3,5	x?Glu		64.4/3.61	26.1/2.12	32.9/2.38	
3,7	Ac		23.3/2.02
3	aXLegp			40.7/1.68-2.71	69.9/3.59	53.3/3.69	73.0/3.92	55.2/3.85	68.4/3.95	19.4/1.16
	x?Ser

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CNC(CCC(=O)N[C@H]1[C@H]([C@H](NC(C)=O)[C@@H](C)O)O[C@@](OCC(N)C(=O)O)(C(=O)O)C[C@@H]1O)C(=O)O

522.508 g/mol (C₂₀H₃₄N₄O₁₂)